estudio de la dinámica microbiana durante la fase de
TRANSCRIPT
Iria Villar Comesaña
2017
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TESE DE DOUTORAMENTO
Estudio de la dinámica microbiana durante
la fase de maduración del compostaje de
residuos orgánicos.
Vermicompostaje como alternativa de
tratamiento
Est
udi
o de
la d
inám
ica m
icro
biana d
ura
nte
la f
ase
de
madu
raci
ón d
el c
ompos
taje
de
resi
duos
org
ánic
os.
Ver
mic
ompos
taje
com
o alt
ernati
va d
e tr
ata
mie
nto
2017
Escola Internacional de Doutoramento
Iria Villar Comesaña
TESE DE DOUTORAMENTO
Estudio de la dinámica microbiana durante la fase de maduración
del compostaje de residuos orgánicos. Vermicompostaje como
alternativa de tratamiento
Dirixida polo doutor:
Salustiano Mato de la Iglesia
2017
O meu neno e a miña avoa
AGRADECIMIENTOS
Mi agradecimiento más sincero a Salustiano Mato por compaginar su exigente trabajo de
rector con la dirección de esta tesis dándome la oportunidad de conocer el mundo de la
investigación, ayudándome y destinando su tiempo libre a diseñar, discutir y enriquecer los
estudios de esta tesis.
A Josefina Garrido por escuchar y recibirme en su despacho siempre con una sonrisa y un
buen consejo.
A todos los compañeros del laboratorio, que han sido muchos, y a los que les debo un poco de
lo aprendido en todos estos años: Pachila, Jorge, Velando, Juan, Cereijo, Emma, Elisa, Elena,
Rubén, Salomé, Andrea, Manuel, Cristóbal, Alberto L., María, Cristina, Pablo, Jessica, Lorena,
Hugo, César, Amaia, Sarr, Álex, Yeon, Álvaro, Alberto DS y Javi. Gracias a todos.
A Emilio por echar una mano siempre que lo he necesitado y, de manera muy especial, a
Domingo por sus clases magistrales de informática y automática y todo su apoyo, consejo y
ayuda. Gracias a él sé diferenciar una sonda termopar de una pt100.
Gracias al personal de los servicios del CACTI, del departamento de Ecología y Biología
Animal, de Leboriz SA. y de FEUGA.
A todas las empresas, instituciones y personas con las que he trabajado todos estos años en
este mundo de la gestión de residuos: Vigozoo, ROS ROCA S.A., Complejo Medioambiental
Sierra del Barbanza, Codisoil S.A., Applus Agroambiental S.A., Compost Galicia S.A., Leal &
Soares S.A., Ayuntamiento de Allariz, Cofradía de Pescadores de Vilaboa, Ecocelta, Ramón
Plana, CETMAR, REGATA y Eurofins Agroambiental S.A.
Gracias a Jose de la depuradora de Cangas do Morrazo, a la empresa FRINOVA S.A., a la Granja
El Pego y Compost Galicia S.A. por suministrar los residuos y materiales para la presente
tesis.
Mil gracias a David por acompañarme y animarme en este periplo, por tus críticas, tus
consejos y tus elogios. Sin ti nada de esto hubiera sido posible. Siempre a mi lado.
Al pequeño Edu, mi mayor alegría. Todo es posible a tu lado mi niño.
A mi queridísima abuela, a mis padres Carlos y Rosa y mis hermanas Ana y Olalla, por ser mi
ejemplo a seguir y por vuestra inmensa paciencia y apoyo. A Roberto, Rubén, Iker y Carlota. A
Suso, Fini y Jacobo por recibirme en vuestra casa. Gracias familia.
A mis amigas Noe y Patri por haber estado siempre a mi lado a pesar de no poder vernos
tanto. A Lorena SC por nuestras charlas de café.
A todos y todas los que habéis colaborado con un granito en esta pila de compost.
Gracias
ÍNDICE
Introducción………………………………………………………………………………………………………………………………. 1
1. Degradación de compuestos orgánicos………………………………………………………………………... 3
2. Compostaje…………………………………………………………………………………………………………………. 6
2.1 Generalidades…………………………………………………………………………………………………… 6
2.2 Fases del proceso de compostaje………………………………………………………………………... 7
2.3 Condiciones previas y control del proceso………………………………………………………….. 8
2.4 Microorganismos implicados en el compostaje………………………………............................ 17
2.5 Sistemas de compostaje……………………………………………………………………………………... 20
3. Vermicompostaje………………………………………………………………………………………………………… 25
2.1 Generalidades…………………………………………………………………………………………………… 25
2.2 Organismos implicados en el vermicompostaje………………………………………………….. 25
2.3 Condiciones previas y control del proceso………………………………………………………….. 30
2.4 Sistemas de vermicompostaje……………………………….............................................................. 41
Objetivos…………………………………………………………………………………………………………………………………..... 47
Capítulo 1…………………………………………………………………………………………………………………………………… 51
“Evolution of microbial dynamics during the maturation phase of the composting of different
types of waste”
Capítulo 2…………………………………………………………………………………………………………………………………… 65
“Seafood-processing sludge composting: changes to microbial communities and physico-chemical
parameters of static treatment versus for turning during the maturation stage”
Capítulo 3…………………………………………………………………………………………………………………………………… 85
“Changes in microbial dynamics during vermicomposting of fresh and composted sewage sludge”
Capítulo 4…………………………………………………………………………………………………………………………………… 113
“Product quality and microbial dynamics during vermicomposting and maturation of compost
from pig manure”
Discusión general……………………………………………………………………………………………………………………... 145
Conclusiones………………………………………………………………………………………………………………………………. 157
Bibliografía………………………………………………………………………………………………….......................................... 161
INTRODUCCIÓN
Introducción
3
1. DEGRADACIÓN DE COMPUESTOS ORGÁNICOS
Como consecuencia de las actividades habituales del ser humano se generan una gran
cantidad y volumen de residuos de diferentes orígenes y que varían en composición y
peligrosidad. En 2014, la cantidad total de residuos generados en la EU-28 por la totalidad de
actividades económicas y hogares ascendió a 2598 millones de toneladas. La cantidad media
generada por habitante en España, en ese mismo año, ascendió a 2387kg habitante-1
descendiendo el ratio a 1435kg habitante-1 excluyendo los principales residuos minerales
(Eurostat, 2017). Una parte importante de estos residuos son materiales biodegradables que
presentan diferentes características y composición dependiendo, fundamentalmente, de su
origen biológico: agrícola, ganadero, forestal, agroindustrial o urbano. En general, estos
residuos no deben ser vertidos al suelo de manera directa debido a que su descomposición no
controlada y su toxicidad pueden generar efectos perjudiciales. Estos residuos
biodegradables poseen materia orgánica no estabilizada, por lo que presentan un elevado
nivel de fitotoxicidad y, frecuentemente, una alta carga de agentes patógenos, como virus,
bacterias, hongos y parásitos, tanto para los seres humanos como para los animales y plantas,
y siendo, por lo tanto, un riesgo para el medioambiente y para la salud. De esta manera, los
residuos orgánicos deben ser estabilizados y acondicionados reduciendo la emisión de olores
y los posibles riesgos anteriormente citados antes de su disposición al suelo.
La materia orgánica presente en el suelo de manera natural está formada,
principalmente, por la biomasa viva y por los restos vegetales y animales muertos que se
depositan en el suelo. Esta biomasa está sometida a una sucesión de transformaciones físicas,
químicas y biológicas. La descomposición en el suelo de los compuestos orgánicos se
desarrolla a través de la degradación gradual de componentes como glúcidos, polisacáridos,
proteínas, lípidos, otros materiales alifáticos y ligninas, llevada a cabo por la acción
enzimática de la macrofauna, mesofauna y microfauna que habitan en el suelo en una
interacción constante. Esta degradación de la materia orgánica conlleva un proceso de
mineralización hasta obtener nutrientes que puedan ser asimilados por las plantas. A su vez,
la materia orgánica no mineralizada sufre un proceso de transformación que, junto con
moléculas orgánicas sintetizadas por el metabolismo anabólico, forman los compuestos
húmicos, es decir, polímeros orgánicos de difícil degradación. Además, de estos procesos de
mineralización y humificación, se sucede también la síntesis de secreciones diversas como
vitaminas, fitoreguladores, antibióticos, etc. (Senesi, 1989). La velocidad del proceso de
mineralización de la materia orgánica en el suelo está condicionada por factores internos
propios de su naturaleza y otros externos como la temperatura, la humedad, la luz, etc.
Introducción
4
(Curtin et al., 1998; Kirschbaum, 1995; Leirós et al., 1999). Los descomponedores primarios
del suelo son los principales responsables de la rotura física, dispersando y mezclando la
materia orgánica por el suelo, alterando enzimáticamente los componentes, excretando heces
y fluidos, ingiriendo la propia biomasa microbiana del suelo y, como consecuencia,
exponiendo las moléculas orgánicas a los descomponedores secundarios. Dentro de la
mesofauna y macrofauna del suelo se encuentran especies de miriápodos, arácnidos, insectos,
crustáceos, anélidos, etc, que se alimentan directamente de la materia orgánica y de otra
fauna vinculada al suelo. La descomposición secundaria es desarrollada por bacterias y
hongos que producen las enzimas responsables de la degradación de la materia orgánica. Los
microorganismos del suelo sintetizan y liberan enzimas extracelulares para hidrolizar
sustratos de peso molecular elevado hasta que sean lo suficientemente pequeños para ser
metabolizados por sus células (Coleman et al., 2004; Lavelle and Spain, 2001; Senesi et al.,
2009). Así, durante las primeras fases de la descomposición de los materiales orgánicos,
aparece la microbiota asociada a la degradación de los compuestos solubles de fácil
asimilación y de bajo peso molecular. Por lo general, se trata de microorganismos estrategas
de la r con ventajas competitivas debido a sus altas tasas de crecimiento y reproducción que
dan lugar a una importante proliferación microbiana en los sustratos colonizados cuando los
recursos son abundantes. En consecuencia, los sustratos fácilmente asimilables se reducen y
las poblaciones colonizadoras iniciales decrecen, pudiendo desarrollarse formas de
resistencia. La microfauna especializada en degradar compuestos más complejos continúa la
descomposición de la materia orgánica a un ritmo más lento. Los hongos son los más
eficientes en la degradación de compuestos de lignina y celulosa, especialmente, hongos de la
podredumbre blanca y parda (tanto basidiomicetes como ascomicetes). Además, suelen ser
dominantes en condiciones ácidas debido a la falta de competencia microbiana por los
recursos disponibles (Boer et al., 2005; Senesi et al., 2009). La mayoría de los
microorganismos del suelo son aerobios, por lo que, cuando las condiciones de oxígeno son
limitantes, la actividad microbiana se ve afectada y se produce un cambio hacia poblaciones
microbianas con actividad anaerobia o fermentativa que son procesos menos eficientes en la
degradación de la materia orgánica (Coyne, 1999). En el caso de que las condiciones
conlleven una falta de oxígeno, las bacterias son las principales responsables de la
degradación.
Killham (1994) expone que en un suelo de pradera la biomasa aproximada de bacterias
y hongos es de 1-2 y 2-5 t ha-1, respectivamente. Las bacterias que se encuentran con mayor
frecuencia pertenecen a los géneros Bacillus y Pseudomonas, siendo Streptomyces,
Arthrobacter, Nocardia y Actinoplanes algunas de las actinobacterias aisladas más
Introducción
5
importantes. Dentro de los géneros más comunes de hongos aislados se encuentran
Mortariella, Mucor, Chaetomium y Penicillium, por ejemplo. El tipo y abundancia de bacterias
y hongos depende de muchos factores como la disponibilidad de nutrientes y las condiciones
medioambientales en el suelo (Pierzynski et al., 2005). Como anteriormente se ha comentado,
los microorganismos son los principales descomponedores de la materia orgánica pero no los
únicos. La accesibilidad del sustrato para los microorganismos puede ralentizar la tasa de
descomposición, por lo que la acción de la restante fauna del suelo tiene un papel
fundamental. Las lombrices de tierra son, sustancialmente, el grupo más importante de la
fauna presente en el suelo, destacando su papel en términos de formación y mantenimiento
de la estructura del medio, además de mejorar la fertilidad del suelo. Aunque no son
dominantes en número, su tamaño hace que sean uno de los mayores contribuidores en la
biomasa de invertebrados del suelo y su actividad favorece al mantenimiento de la fertilidad
en distintos tipos de suelo (Edwards, 2004).
Puesto que los residuos orgánicos son ricos en nutrientes y materia orgánica, es
razonable que sean devueltos al suelo en condiciones óptimas para garantizar su fertilidad y
completar el ciclo natural. Del mismo modo que sucede en la naturaleza con los ciclos de
nutrientes, los residuos orgánicos pueden sufrir procesos de biodegradación, es decir, la
rotura de los componentes más complejos en compuestos simples mediante la acción de
organismos vivos. Así mismo, la acción de los organismos provoca la síntesis desde moléculas
simples a moléculas más complejas de mayor estabilidad. Dentro de los procesos que pueden
ser empleados para la estabilización de compuestos orgánicos se encuentran el compostaje y
el vermicompostaje que se describirán en los siguientes apartados de la presente tesis.
Introducción
6
2. COMPOSTAJE
2.1. Generalidades
Desde un punto de vista etimológico compostaje deriva del latín compositum que
significa compuesto o mezcla. Así, el compostaje hace referencia a la biodegradación de una
mezcla de sustratos en estado sólido y en condiciones aerobias llevada a cabo por
comunidades microbianas compuestas por diversas poblaciones. Es un proceso espontáneo y
exotérmico que provoca la liberación de energía en forma de calor causando la elevación de la
temperatura hasta condiciones termófilas (Insam and de Bertoldi, 2007). Durante el proceso
de compostaje se generan diferentes condiciones térmicas que llevan a la sucesión de
distintas comunidades microbianas, así mismo, a lo largo de esta biodegradación se produce
la liberación de sustancias tóxicas (fitotoxinas) como los compuestos intermediarios
amoníaco, ácidos orgánicos de bajo peso molecular y el óxido de etileno. La definición que
Zucconi and De Bertoldi (1987) ofrecieron a la comunidad científica es una de las
descripciones más completas de compostaje: “proceso biooxidativo controlado, en el que un
sustrato orgánico heterogéneo en estado sólido experimenta una etapa termofílica y una
liberación transitoria de fitotoxinas, obteniéndose como productos dióxido de carbono, agua,
minerales y materia orgánica estabilizada denominada compost”. Los términos que destacan
en esta definición son: sólido, aerobio, termófilo y controlado. Estos cuatro términos son
básicos y necesarios para que un proceso de degradación biológica de material orgánico sea
considerado como compostaje. Estos autores incluyeron en la definición el término
controlado, haciendo alusión a la necesidad de monitorización de los parámetros tales como
la temperatura, oxígeno, humedad, etc. a lo largo del proceso, así como, de controlar la
composición y naturaleza del sustrato heterogéneo en fase de compostaje. De esta manera, se
diferencia un proceso natural de degradación de sustratos orgánicos o una putrefacción que
se sucede habitualmente en acumulaciones sobre el suelo, pilas de estiércol o vertederos,
frente al proceso de compostaje. En la Figura 1 se pueden observar los distintos productos
que se generan durante el proceso de compostaje.
Introducción
7
Figura 1. Esquema del proceso de compostaje (elaboración propia).
Los cambios fisicoquímicos y biológicos que se suceden durante el compostaje dan
como resultado la generación de calor, la formación de dióxido de carbono, agua y, como
principal producto, el compost formado por tres componentes básicos: materia orgánica
estabilizada, materia mineral y microbiota. Por lo tanto, la principal finalidad del compostaje
es la obtención de un producto final de uso agronómico a partir del tratamiento de residuos
orgánicos y, por lo tanto, es una tecnología de valorización medioambientalmente respetuosa
al transformar desechos en productos aplicables al suelo posibilitando el cierre del ciclo de
los nutrientes y el desarrollo de la economía circular de la región.
2.2. Fases del proceso de compostaje
Uno de los factores principales y determinantes del compostaje es la temperatura que
conduce el desarrollo del proceso. Básicamente, se considera que el compostaje en óptimas
condiciones puede dividirse en 4 fases:
Fase mesófila inicial (desde la temperatura ambiental hasta los 45 °C): en esta etapa
comienza la degradación de los compuestos más fácilmente biodegradables, como azúcares,
grasas, almidón y proteínas, que tiene como consecuencia el aumento de la actividad
microbiana y la elevación de la temperatura. Esta etapa puede durar desde horas hasta varios
días.
Fase termófila (45-70ºC): a medida que se eleva la temperatura los organismos
adaptados a temperaturas altas reemplazan a los organismos mesófilos. De esta manera,
continúa la degradación de los compuestos más lábiles desarrollándose una actividad
metabólica elevada que genera la prolongación de las altas temperaturas. En esta etapa
comienza la degradación de los compuestos más complejos y resistentes por parte de la
Introducción
8
microbiota termófila. Debido a las elevadas temperaturas se produce la higienización del
sustrato al eliminarse los patógenos, parásitos y semillas de malas hierbas. Esta etapa puede
durar desde varios días a incluso meses dependiendo del tipo de residuo, las características
del sistema y el control sobre el proceso.
Fase de enfriamiento: el agotamiento de los materiales fácilmente biodegradables
conlleva la disminución de la actividad microbiana y, con ello, el descenso térmico hasta
temperaturas ambientales, produciéndose la recolonización por parte de la microbiota
mesófila con capacidad para degradar compuestos como la celulosa, hemicelulosa y otros
polímeros. Esta fase puede durar desde varios días a semanas por eso, en ocasiones, la fase de
enfriamiento y maduración se consideran como una única etapa del proceso.
Fase de maduración: el material orgánico se estabiliza, predominando los procesos de
degradación de los compuestos más recalcitrantes y la polimerización de sustancias similares
al humus. Esta etapa del proceso suele requerir varios meses.
Dependiendo de la naturaleza del material de partida, las condiciones ambientales, el
sistema de compostaje, las operaciones realizadas durante el proceso y la calidad de compost
requerida, la duración del compostaje es variable y puede prolongarse desde los 3-4 meses
hasta el año.
2.3. Condiciones previas y control de proceso
El proceso de compostaje es dirigido por la actividad de los microorganismos, por lo
que los factores que puedan limitar el desarrollo de la microbiota particular en cada fase,
afectarán al desarrollo del proceso de compostaje. Por ello, el compostaje requiere una serie
de requisitos previos y el control de las condiciones que se suceden a lo largo del mismo.
2.3.1. Acondicionamiento
Antes de proceder a compostar un residuo ha de tenerse en cuenta una serie de
parámetros previos que van a condicionar el desarrollo de la actividad microbiana. Las
características del material inicial determinan la evolución del proceso de compostaje, su
eficacia y la calidad del compost. A continuación se presentan algunos de los parámetros más
importantes:
Introducción
9
� Espacio de aire libre y tamaño de partícula
La matriz de compostaje es una masa de partículas sólidas que debe contener
suficientes poros e intersticios para posibilitar que se desarrolle un proceso aerobio, de
manera que, el aire circule por el interior de la masa aportando una concentración óptima de
oxígeno, eliminando el dióxido de carbono y el exceso de humedad y limitando la
acumulación excesiva de calor (Haug, 1993). Por lo tanto, si el residuo no presenta la
estructura óptima, como ocurre, por ejemplo, con residuos pastosos como los lodos de
depuradora urbana o los purines de cerdo, es necesario adicionar un material que aporte
estructura. Por lo general, se emplean como agentes estructurantes materiales de naturaleza
orgánica como los residuos agroforestales. En la Figura 2 se exponen imágenes de materiales
estructurantes.
Figura 2. A la izquierda madera triturada y a la derecha restos de paja que pueden ser
empleados como agente estructurante.
La porosidad o espacios de aire en la matriz de compostaje puede ser medida mediante
diversos métodos. Uno de los más utilizados es la medición del espacio libre de aire (free air
space, FAS). Los valores mínimos de FAS en un proceso de compostaje para asegurar la
actividad biológica se sitúan en torno al 30%, mientras que, los valores del 60-70% parecen
ser excesivos para alcanzar temperaturas termófilas en residuos con bajo contenido de
materia orgánica biodegradable (Haug, 1993; Ruggieri et al., 2009). El uso de agentes
estructurantes posibilita alcanzar valores de FAS óptimos en el sustrato inicial, aunque estos
agentes también afectan a los balances de nutrientes y humedad, como se verá a
continuación.
Se han realizado múltiples investigaciones que han estudiado el ratio residuo/agente
estructurante para determinar su influencia en el proceso de compostaje así como el tipo de
material estructurante y su tamaño de partícula (Tabla 1). El ratio óptimo para el desarrollo
del compostaje va a depender de factores como las condiciones de proceso (sistema de
compostaje, volumen, tiempo, etc.), el residuo (pastosidad, biodegradabilidad, etc.), el origen
Introducción
10
del agente estructurante, el tipo de estructurante (fresco o recirculado), el tamaño de
partícula, las condiciones de la mezcla (FAS, relación C/N, humedad), etc.
Tabla 1. Referencias de investigaciones acerca del material estructurante tipología, ratio e
influencia sobre el proceso de compostaje
Residuo Agente estructurante Sistema de compostaje
Referencia
Estiércol
Paja picada de cebada 1:2 (v:v)
Turba Sphagnum y paja en proporciones de
130: 20: 5 (f.w.)
Tambor
horizontal
giratorio 25m3
(Vuorinen,
2000)
Lodo de
depuradora
urbana
Astillas de madera
Tamaños partícula 20,10,5mm
Proporción 1:1, 2:1, 4:1 (v:v)
Vasos Dewar 45L
Reactor estático
100L
(Gea et al.,
2007)
Lodo de
depuradora
urbana
Triturado Acacia sp 40 mm
5 proporciones (w:w):1/0, 1/1, 1/2, 1/3
Reactor
35kg
(Yañez et al.,
2009)
Lodo de
depuradora
matadero
Residuos verdes y pallets madera triturados.
1/6 (d.w.)
5 tamaños partícula 8, 12, 20, 30, 40 mm
Reactor
cilíndrico 300L
(Trémier et
al., 2009)
Residuos de
alimentos
Cáscara de arroz, serrín y salvado de arroz
Ajuste ratio en función de la humedad, C/N y
espacio aire
Reactor
cilíndrico 180L
(Chang and
Chen, 2010)
Estiércol de
cerdo
Serrín (4:1 w:w), desechos verdes triturados
(4:1 w:w), paja picada (4:1 w:w)
astillas de madera (4:1 w:w)
Pilas volteadas
47.5kg
(Nolan et al.,
2011)
Lodo de
depuradora
urbana
Pallets de madera frescos y recirculados
1/3 (v/v)
Tamaños partícula: >20,<20 mm
2 humedades 50% 65%
Reactores
cilíndricos 47L
(Huet et al.,
2012)
Digestato de
purín de cerdo
Paja de trigo, bagazo, podas de vid, poda de
pimiento; 10 mm
Distintas proporciones (d.w.)
Reactor 350L con
volteos
(Bustamante
et al., 2013)
Residuos sólidos
municipales
Astillas de madera (1:1, v:v)
Tubos de polietileno (1:1, v:v)
Reactor aireación
forzada 50L
(Maulini-
Duran et al.,
2014)
Los microorganismos acceden al residuo y se alimentan de él a una velocidad que va a
depender de la relación superficie/volumen de las partículas de la matriz de compostaje. De
esta manera, partículas de tamaño elevado requerirán más tiempo de ataque microbiano y
ralentizarán el proceso de compostaje mientras que partículas pequeñas reducen la
porosidad, y aumentan la compactación y la posibilidad de anaerobiosis. Según Diaz et al.
(2007) el tamaño de partícula depende de la naturaleza física del residuo que, para
materiales que no se compactan con facilidad, se considera entre 1-5 cm. Por ejemplo,
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11
Hamoda et al. (1998) mostraron que para el compostaje de residuo sólido municipal el
tamaño de partícula óptimo es de 4 cm.
� Humedad
Debido a que el compostaje es un proceso de degradación biológica, es importante que
el contenido de agua disponible sea suficiente para hacer frente a los requerimientos
fisiológicos de la microbiota. El agua actúa como medio de transporte, no sólo para las
sustancias solubles con las que se alimentan los microorganismos sino también, para eliminar
los productos de desecho resultado del metabolismo celular. El contenido óptimo de
humedad al inicio del proceso depende del tipo de material a compostar, pero varía entre 50–
70% (Bernal et al., 2009; de Bertoldi et al., 1996; Richard et al., 2002). Contenidos mayores de
humedad pueden provocar que el agua ocupe los microporos de la mezcla dificultando la
oxigenación, mientras que, contenidos menores provocan el descenso de la actividad
biológica. En la Tabla 2 se presentan las humedades máximas recomendadas para distintos
tipos de residuos. Así mismo, la adición de materiales estructurantes afecta a la humedad,
generalmente, provocando la absorción de agua del sustrato si se emplean estructurantes
porosos y no saturados en agua (Haug, 1993).
Tabla 2. Contenidos de humedad recomendados para el compostaje. Modificado de Haug,
(1993) y Diaz et al. (2002).
Tipo de residuo Humedad (%)
Paja 70-85
Madera en serrín o pequeñas virutas 80-90
Cáscara de arroz 70-85
Residuo sólido urbano 55-65
Estiércoles 55-65
Lodo digerido o fresco 55-60
Desechos húmedos (recortes de hierba, basura, etc) 50-55
� Relación C/N
Se considera que los microorganismos vivos emplean para su metabolismo 30 partes
de carbono por cada parte de nitrógeno, requiriendo carbono como fuente de energía y como
componente de las células y nitrógeno para la síntesis proteica y los ácidos nucleicos. Se
considera que la matriz de compostaje es adecuada cuando presenta un ratio inicial de C/N
entre 25-35 (Bernal et al., 2009; Diaz and Savage, 2007; Haug, 1993; Poincelot, 1972). Valores
elevados del ratio C/N, como ocurre en residuos agroforestales con altos contenidos de
carbono, implican un descenso de la actividad biológica por falta de nitrógeno para el
Introducción
12
metabolismo y, por lo tanto, una ralentización del proceso. Valores bajos del ratio C/N, como
ocurre en el compostaje de lodos de depuradora de aguas residuales urbanas, sufren un
exceso de nitrógeno que se puede perder por volatilización en forma de amoníaco o por
lixiviación en forma de amonio. La adicción de material estructurante ayuda a mejorar este
ratio al proveer de carbono orgánico a la mezcla. Sin embargo, el ratio C/N óptimo está
condicionado por la naturaleza del residuo, por lo que, si el carbono forma parte de
compuestos de difícil degradación, como la lignina, estará lentamente disponible para los
microorganismos (Diaz and Savage, 2007). Por ello es importante considerar el ratio en
función del C y N más asimilable o biológicamente disponible.
Por lo tanto para obtener una matriz en la que se suceda un proceso de compostaje
optimizado, es necesario acondicionar el residuo tanto física, química como
termodinámicamente (Haug, 1993).
− Físico o estructural: acondicionar el residuo para eliminar limitaciones causadas por
la falta de humedad o porosidad.
− Químico: acondicionar el residuo para eliminar limitaciones causadas por la falta de
nutrientes u otros químicos desbalanceados.
− Termodinámico: acondicionar la mezcla para asegurar la disponibilidad de energía
suficiente para conducir el proceso.
En ocasiones, se utilizan materiales estructurantes recirculados, es decir, ya empleados
en otros ciclos de compostaje y recuperados, que van a actuar como inóculo microbiano,
además de la mejora física y química que ejercen.
En las plantas de compostaje no siempre es posible acondicionar el residuo de la
manera más idónea y, en muchas ocasiones, los niveles de nutrientes, en concreto el ratio
C/N, no se encuentran en los valores considerados óptimos, por lo que el proceso ha de ser
controlado para reducir al máximo las pérdidas de nitrógeno.
2.3.2. Seguimiento del proceso
Una vez establecidos y corregidos los parámetros relativos a la naturaleza del sustrato,
es necesario el seguimiento y control del proceso dentro de valores adecuados para cada fase
del compostaje.
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13
Temperatura
La evolución de la temperatura a lo largo del tiempo es un parámetro clave a
consecuencia de la elevada actividad microbiana y, por lo tanto, con relación directa en la
degradación de la materia orgánica. Un proceso de compostaje se desarrolla en tres fases
térmicas: mesófila inicial, termófila y regreso a valores mesófilos. En la Figura 3 se pueden
observar dos ejemplos de evoluciones de temperatura en distintos sistemas y materiales. Los
diferentes perfiles observables son fruto de la distinta naturaleza del sustrato, del volumen
de residuo tratado y del sistema de compostaje empleado.
Figura 3. A la izquierda, evolución de la temperatura en una pila volteada de estiércol de 40m3
durante 230 días y a la derecha, evolución de la temperatura en un reactor estático de 600L con
ventilación forzada durante 22 días.
Para una correcta higienización del material y la obtención de un compost libre de
parásitos y semillas de malas hierbas se han expuesto, por ejemplo, que se han de mantener
en continuo temperaturas por encima de 55ºC durante 15 días (European Commission,
2001). Siendo el exceso de temperatura por encima de los 70ºC no deseable ya que puede
provocar la muerte de la mayoría de los microorganismos, retardar la colonización en las
fases posteriores y, como consecuencia, retrasar la degradación del residuo. De esta manera,
la temperatura durante el proceso debe ser controlada asegurando que se alcancen
temperaturas termófilas y que se sostengan en el tiempo lo suficiente para garantizar la
higienización pero sin exceder de los 70ºC. El control de temperatura se puede realizar o
bien, para enfriar la masa o bien, para reactivar el proceso y se suele efectuar mediante
(Bernal et al., 2009):
− Un sistema de control de retroalimentación de temperatura, mediante el cual se
introduzca aire a la masa en compostaje por ventilación controlada.
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14
− Operaciones de volteo que consisten en la mezcla de la masa en compostaje mediante
medios mecánicos que provocan la dispersión del calor y el enfriamiento pero que
también pueden reactivar el proceso al redistribuir los nutrientes.
− Control del tamaño y la forma de la masa en proceso de compostaje.
Oxígeno
Mantener los niveles de oxígeno es un factor clave para que se desarrolle un proceso
biológico aerobio. La concentración de oxígeno en la masa de compostaje no debe ser inferior
al 5% (Epstein, 2011), ya que se produciría una sucesión hacia microorganismos anaerobios
y, por lo tanto, hacia procesos de fermentación no deseables y la generación de malos olores.
Para el control de los niveles de oxígeno durante el compostaje ha de controlarse la aireación
de la masa. Además de satisfacer la demanda de oxígeno para la descomposición orgánica, la
aireación también favorece la regulación de excesos de agua y ayuda a mantener la
temperatura en valores adecuados (Haug, 1993). La aireación puede ser proporcionada
mediante sistemas de ventilación, y operaciones de volteo o ambos. Así mismo, también se
puede producir la aireación pasiva de manera natural cuando tanto la estructura y porosidad
de la mezcla, así como, la forma y tamaño de la pila posibilitan el intercambio de gases. Esta
aireación natural pasiva se produce cuando el incremento de temperatura provoca el
calentamiento del aire, por lo que la diferencia entre la densidad del aire exterior menos
húmedo con el aire del interior de la pila más húmedo y caliente, genera un ascenso del aire
hacia la parte superior y la entrada de aire frío desde la parte inferior (Figura 4). Los volteos
u otras acciones mecánicas incrementan el aire libre, disminuyen el tamaño de partícula y
minimizan los efectos de la compactación posibilitando condiciones en la pila que mejoran la
ventilación natural.
Figura 4. Esquema de la ventilación natural en una pila de compostaje (Haug, 1993).
Introducción
15
Humedad
El proceso de compostaje tiene como efecto asociado la pérdida de agua como
consecuencia de su evaporación debido a las altas temperaturas, de manera que, los valores
son más altos al inicio y decrecen a medida que el proceso avanza. El requerimiento de
humedad es mayor durante las fases iniciales cuando la actividad es alta, considerando un
valor límite de 30-35% por debajo del cual se inhibe el proceso. Existen una serie de
actuaciones que provocan el descenso de la humedad como son, la aireación forzada de la
masa y los volteos, que pueden utilizarse para descender la humedad en caso de exceso (de
Bertoldi et al., 1996). Así mismo, debe incorporarse agua al proceso en caso de que los
valores de humedad sean limitantes. Es importante destacar que usualmente es ventajoso
obtener un compost con niveles de humedad bajos que faciliten el ensacado y el transporte,
de manera que los valores oscilen entre 35-40%. El Real Decreto de fertilizantes y afines
(Boletín Oficial del Estado, 2013) establece un contenido máximo del 40% para considerar un
compost como enmienda orgánica.
pH
La mayoría de los microrganismos no sobreviven a pH inferiores a 3, ni superiores a 11,
por lo que residuos entre 3-11 pueden considerarse como potencialmente compostables.
Usualmente, el pH no es un factor clave para el compostaje ya que muchos residuos se
encuentran en rangos de pH entre 5.5-8.0 que son estimados como óptimos (de Bertoldi et al.,
1983). Sin embargo, pH bajos provocan la inhibición de los microorganismos termófilos y,
por lo tanto, un retraso en el paso de condiciones mesófilas a termófilas (Sundberg et al.,
2004) y pH básicos, junto con condiciones de elevadas temperaturas, provocan la
volatilización del amonio. En general, se considera que el pH sigue una evolución en fases, por
lo que puede ser considerado un buen indicador de la degradación de la materia orgánica:
condiciones ácidas durante los primeros días, como consecuencia de la degradación de
polisacáridos fácilmente hidrolizados y nueva síntesis de ácidos orgánicos simples; a medida
que la temperatura aumenta, el pH aumenta hasta 8-9, principalmente debido a la
degradación metabólica de los ácidos orgánicos o las pérdidas por volatilización y la
liberación de compuestos amoniacales; finalmente, el pH disminuye ligeramente durante la
fase de enfriamiento y maduración a valores entre 7 y 8 debido a la formación de compuestos
húmicos con efecto tampón (Iglesias Jiménez and Pérez García, 1991). De manera que, el
seguimiento del pH informa de los procesos de degradación, de manera que pH ácidos
pueden ser indicativos de anaerobiosis o de que el material no está suficientemente maduro.
Sin embargo, el pH no suele considerarse como un indicador de estabilidad ya que existen
Introducción
16
excepciones a la curva estándar y, por ejemplo, residuos ácidos pueden generar compost
ácidos aunque las condiciones de proceso sean óptimas (Epstein, 1996).
Materia orgánica y nutrientes
A lo largo del proceso de compostaje la materia orgánica desciende debido a su
mineralización y a la pérdida de carbono en forma de dióxido de carbono como resultado de
la respiración microbiana, sobre todo, en la fase biooxidativa. Mientras que, en la fase de
maduración prevalecen los procesos de síntesis de compuestos húmicos frente a los procesos
de mineralización. A medida que el proceso de compostaje avanza, el contenido de carbono se
reduce y la producción de dióxido de carbono decrece. La actividad respiratoria bien
mediante la medición del dióxido de carbono producido o el oxígeno consumido por los
microorganismos heterótrofos aerobios son indicadores de la evolución de la actividad
biológica y, consecuentemente, de la estabilidad de un compost. La actividad respiratoria
también se puede medir indirectamente mediante la determinación del calor liberado, por
ejemplo, mediante el test de autocalentamiento.
El nitrógeno orgánico está fácilmente disponible cuando se encuentra en las formas
proteínicas, peptídicas o en forma de aminoácidos libres. Por otro lado, debido a la resistencia
de las moléculas de quitina y lignina al ataque microbiano, la pequeña cantidad de nitrógeno
en ellas se libera lentamente (Diaz et al., 2002). Así, el nitrógeno orgánico es mineralizado a
amoníaco mediante procesos de amonificación. En disolución el amonio se puede o bien,
transformar en nuevos compuestos orgánicos por la microbiota o bien, transformarse en
nitrato mediante bacterias nitrificantes que presentan valores óptimos de temperatura por
debajo de 40ºC y condiciones aerobias. Además, a pH altos y temperaturas elevadas el
amoníaco puede volatilizarse. Por lo general, el nitrógeno decrece a lo largo del compostaje,
el amonio desciende y el nitrato aumenta, de manera que, la evolución del ratio
amonio/nitrato proporciona información sobre cómo se ha llevado a cabo el proceso de
compostaje y puede ser empleado como indicador de madurez.
Durante el compostaje, la transformación del sustrato está condicionada por la
naturaleza del material orgánico de acuerdo con su degradabilidad (Haug, 1993). Los
almidones, azúcares y grasas se descomponen o mineralizan mucho más rápido que las
proteínas o celulosa, mientras que la lignina es muy resistente a la mineralización (Epstein,
2011). Barrington et al. (2002) estudiaron las pérdidas de carbono y nitrógeno tras el
compostaje de agentes estructurantes observando que las pérdidas de carbono variaron
entre el 14-52% y las pérdidas de nitrógeno entre el 38-69%. Bernal et al. (2009) mostraron
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17
las pérdidas de carbono y nitrógeno de distintos procesos de compostaje de estiércoles,
observando pérdidas de carbono de hasta 72% y de nitrógeno del 60% durante el compostaje
de estiércol de cerdo. Este estudio mostró que el sistema y las condiciones de compostaje, las
características tanto del material de cama como del agente estructurante añadido para el
compostaje e incluso las condiciones ambientales de la estación del año ejercen una gran
influencia sobre la mineralización de la materia orgánica durante el compostaje.
Durante la fase de maduración los compuestos más resistentes a los ataques
microbianos y parcialmente transformados, participan en la formación de los compuestos de
naturaleza húmica. Las fracciones húmicas condicionan la calidad del compost final obtenido
y determinan el grado de estabilización y madurez de la materia orgánica del compost. (Sequi
et al., 1986) propusieron un índice de humificación, calculado como el cociente entre la
fracción de carbono orgánico no humificado y el de la fracción humificada, que se basa en que
los materiales no fenólicos (no humificados) se descomponen en materiales polifenólicos
(humificados). En la actualidad se utilizan técnicas que ofrecen un conocimiento más
detallado de la composición y estructura de la fracción húmica de la materia orgánica como
técnicas electroforéticas, técnicas cromatográficas, espectroscopia IR y FTIR, técnicas de
resonancia magnética nuclear, etc. El estudio del progreso de la humificación mediante
diferentes índices de humificación puede ofrecer una valiosa información acerca de la
transformación de la materia orgánica durante el compostaje (Ciavatta et al., 1993; Sequi et
al., 1986).
2.4. Microorganismos implicados en el compostaje
En condiciones aerobias el principal factor selectivo de las poblaciones microbianas es
la temperatura que determina la tasa de las actividades metabólicas y define también las
fases que comúnmente se desarrollan durante el compostaje (Ryckeboer et al., 2003) y que
fueron expuestas en el apartado anterior. Así mismo, la naturaleza de los sustratos orgánicos
es también un factor importante para determinar la dinámica y la diversidad microbianas
durante el compostaje (Klammer et al., 2008; Ryckeboer et al., 2003; Vargas-García et al.,
2010). Los residuos orgánicos poseen diferentes nutrientes en distintas formas disponibles y
que han sido obtenidos, almacenados o tratados mediante procesos que pueden provocar
cambios en sus poblaciones microbianas. Al comienzo del proceso de compostaje, la adición y
mezcla con distintos sustratos colaboran en incorporar microbiota y diferentes fuentes de
nutrientes, más o menos disponibles, así como, modificaciones físicas que también afectan a
la microbiota. Al inicio del proceso se produce la colonización por parte de los
microorganismos que son capaces de alimentarse de los nutrientes disponibles, en las formas
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18
en las que se encuentran y que se van a desarrollar de manera prolífica, siendo estos
microorganismos principalmente bacterias. Esto se debe a que las bacterias presentan mayor
ventaja competitiva que los hongos ya que utilizan una amplia gama de enzimas para
degradar químicamente una gran variedad de compuestos orgánicos y su ciclo de
reproducción y desarrollo es más corto. Las comunidades bacterianas se adaptan a los
cambios que se suceden rápidamente en la disponibilidad de sustrato y a otros factores como
la humedad y temperatura. En consecuencia, las bacterias son responsables de la mayor parte
de la descomposición inicial y la generación de calor durante el proceso de compostaje,
siempre que se cumplan los principales requisitos de crecimiento como humedad, pH,
aireación, etc. (Ryckeboer et al., 2003). El otro grupo representativo son los hongos que
presentan una menor tolerancia a las altas temperaturas y un crecimiento más lento, por lo
que son desplazados por las bacterias en condiciones termófilas. Sin embargo, en las fases
finales los hongos, como consecuencia del descenso de humedad, toman ventaja. Cuando las
temperaturas exceden los valores por encima de los 50-60ºC se produce una selección
favorable para las bacterias frente a los hongos, en especial, hacia las bacterias Gram-
positivas del género Bacillus. Por encima de valores superiores a 70ºC se produce la muerte
de la mayoría de las bacterias pudiendo permanecer en el sustrato los hongos en forma
esporulada y especies de bacterias Gram-negativas Thermus e microorganismos del reino
Archaea. Strom (1985) mostró que la temperatura máxima deseable para el compostaje, de
manera que se mantenga una alta diversidad de especies para la estabilidad de la población y
la versatilidad metabólica, es de 60ºC. Dentro de las bacterias Gram-positivas cabe destacar
las actinobacterias que son termotolerantes y termofílicas, crecen mejor cuando el sustrato
está húmedo y correctamente oxigenado y presentan preferencias por pH neutros y alcalinos.
También es destacable que en condiciones termófilas, con valores entorno a los 45-55ºC, y
una humedad baja, puede aumentar la presencia de hongos termófilos como Aspergillus sp. y
Mucor sp. Los hongos son, en general, más importantes para la degradación de la celulosa que
las bacterias, lo cual es especialmente significativo cuando la celulosa está incrustada con
lignina, por ejemplo, en madera o paja (Insam and de Bertoldi, 2007). Durante la fase de
enfriamiento o maduración de los compost, se observa una gran diversidad metabólica de
bacterias mesófilas que juegan una función esencial para la estabilización del compost (Beffa
et al., 1996).
El estudio de la microbiología del proceso de compostaje se realiza desde los años 50-
60 y, debido a su complejidad, continúa en la actualidad. (Ryckeboer et al., 2003) presentaron
un estudio detallado sobre las bacterias y hongos identificados en distintos trabajos de
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19
investigación durante el compostaje de diferentes residuos. Así en la Tabla 3 se presenta un
resumen de algunas de los géneros más representativos:
Tabla 3. Ejemplos de organismos identificados durante el compostaje (Ryckeboer et al., 2003).
Género/especie Tipología Fases predominantes
Bacillus sp. Bacteria Gram+ Termofílica y mesofílica
Brevibacillus sp. Bacteria Gram+ Termofílica y mesofílica
Clostridium sp. Bacteria Gram+ Termofílica y mesofílica
Enterobacter sp. Bacteria Gram- Termofílica y mesofílica
Flavobacterium sp. Bacteria Gram- mesofílica
Methylobacterium sp. Bacteria Gram- mesofílica
Micromonospora sp. Bacteria Gram+ Termofílica
Nocardia sp. Bacteria Gram+ Termofílica y mesofílica
Paenibacillus sp. Bacteria Gram+ Termofílica y mesofílica
Pseudomonas sp. Bacteria Gram- mesofílica
Serratia sp. Bacteria Gram- mesofílica
Streptomyces sp. Bacteria Gram+ Termofílica y mesofílica
Thermoactinomyces sp. Bacteria Gram+ Termofílica
Thermopolyspora sp Bacteria Gram+ Termofílica
Thermus sp. Bacteria Gram- Termofílica
Acremonium sp. Hongos Mesofílica
Aspergillus sp. Hongos Mesofílica
Aspergillus fumigatus Hongos Termofílica
Chaetomium thermophile Hongos Termofílica
Coprinus sp. Hongos Mesofílica
Doratomyces sp. Hongos Mesofílica
Fusarium sp. Hongos Mesofílica
Gliocladium sp. Hongos Mesofílica
Humicola sp. Hongos Termofílica y mesofílica
Mortierella sp. Hongos Mesofílica
Mucor sp. Hongos Mesofílica
Paecilomyces sp. Hongos Mesofílica
Penicillium sp. Hongos Mesofílica
Scopulariopsis sp. Hongos Mesofílica
Talaromyces sp. Hongos Termofílica
Thermomyces sp. Hongos Termofílica
Trichoderma sp. Hongos Termofílica
Muchos de los trabajos citados por estos autores están basados en técnicas de cultivo. A
lo largo de los años se han mejorado las técnicas y ampliado los estudios sobre distintos
residuos y distintas tecnologías de proceso. Existen en los últimos 10 años numerosos
artículos que estudian los microorganismos durante el proceso de compostaje existentes
mediante diversas técnicas, tanto innovadoras como conservadoras, que proporcionan
información de perfiles de la comunidad microbiana y/o de especies concretas. En la Tabla 4
se presentan algunos ejemplos.
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20
Tabla 4. Algunas referencias de investigaciones de la microbiología del proceso de compostaje
en los últimos años.
Residuo Técnica de estudio Sistema y tiempo de proceso
Referencia
Residuo sólido municipal Cultivo Pila volteada
26 semanas
(Rebollido et
al., 2008)
Lodo de refinería de aceite
vegetal y basura doméstica Análisis de PLFAs
Pila volteada
5 meses
(Amir et al.,
2010)
Biorresiduos municipales e
industriales
Análisis de PLFAs
Extracción de ADN y análisis
PCR con clonación y
secuenciación
2 tambores de
160m3 3 túneles de
150m3
Tambor a escala
piloto 5m3
(Hultman et
al., 2010)
Residuos hortícolas, lodo de
depuradora y residuos
urbanos
Cultivo Pilas volteadas
1.5m3 185-125 días
(Vargas-
García et al.,
2010)
Residuos de aceitunas
Aislamiento en medio de
cultivo e identificación
mediante 16S sRNA
Extracción de ADN y análisis
PCR-DGGE
Pila volteada 3.5m3
140 días
(Federici et
al., 2011)
Plantas de tomate sin fruto
y virutas de pino
Aislamiento en medio de
cultivo y secuenciación parcial
16S rRNA
Pilas con aireación
forzada y volteos
189 días
(López-
González et
al., 2015)
Estiércoles. Tallos de
tomate y residuos de col.
Residuos verde.
Desperdicios de cocina
Residuos sólidos urbanos
Extracción de ADN y análisis
PCR-DGGE
Pilas 3m3
40 días
(Wang et al.,
2015)
Residuos de castaño Extracción de ADN y análisis
PCR-DGGE
Pilas en contenedor
de 4.05m3
345 días
(Ventorino et
al., 2016)
Alperujo y estiércol de
cerdo
Extracción de ADN,
cuantificación
PCR y pirosecuenciación
Pilas volteadas 10t
22 semanas
(Tortosa et
al., 2017)
2.5. Sistemas de compostaje
De manera general, la elección de un sistema o tecnología de compostaje depende de
varios factores como pueden ser: tipología y cantidad de residuos, consideraciones
económicas, aspectos legales, localización, aspectos ambientales y calidad del producto, entre
otros (Epstein, 2011). En la Tabla 5 se presentan algunos de los sistemas más comunes,
clasificados según su relación con el medio y la mezcla o cambio de posición.
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Tabla 5. Clasificación de los sistemas de compostaje más comúnmente empleados (Diaz et al.,
2007; Epstein, 2011; Moreno and Moral, 2008).
Sistemas abiertos Sistemas Semiabiertos Sistemas cerrados
Estáticos Montones/Pilas/
Mesetas con aireación
pasiva o forzada
Pilas con cubierta
semipermeable aireadas
Contenedores/Túneles
aireados
Dinámicos Pilas volteadas
Mesetas volteadas
Trincheras
Pilas/Mesetas en naves
cerradas volteadas
Túneles dinámicos
Tambores
Diaz et al. (2007) establecieron una diferenciación de los sistemas de compostaje en
dos grandes grupos “windrow”, que hace referencia al acumulo del material a compostar en
pilas o hileras más o menos alargadas, e “in vessel” donde el material se dispone confinado
dentro de un reactor.
Desde un punto de vista operativo, es importante destacar que en muchos casos se
considera que el proceso de compostaje se puede diferenciar únicamente en dos etapas. Una
etapa biooxidativa o intensiva que se corresponde principalmente con las 2 primeras fases
del proceso de compostaje, mesófila inicial y termófila, que se caracteriza por altas
temperaturas, elevado consumo de oxígeno y la producción de emisiones gaseosas y líquidas
(Haug, 1993). Esta fase biooxidativa está condicionada por los procesos de descomposición
de la materia orgánica y, por lo general, en las plantas industriales se desarrolla con una
tecnología concreta y cuya duración va desde los pocos días hasta meses. La segunda fase se
corresponde esencialmente con la etapa de enfriamiento y maduración. Suele tener una
duración superior a la fase biooxidativa aunque el tiempo de residencia en esta fase está
condicionado por las características del material de partida y las condiciones
medioambientales y de operación de la planta de compostaje (Diaz et al., 2002). En general,
presenta un menor control de proceso y, en muchos casos, se limita erróneamente al
almacenamiento del compost para su ensacado (Rynk, 2000). Así una fase biooxidativa corta
y/o en un material energético puede reactivarse en su estancia en la zona de maduración, por
lo que la falta de control puede conllevar problemas de anaerobiosis, lixiviación, olores,
ralentización del proceso y descenso de la calidad del compost (formación de metabolitos
secundarios no deseables y compost con mal olor). La fase biooxidativa se realiza mediante
alguna de las tecnologías de la Tabla 5, mientras que la estancia en maduración se lleva a cabo
en pilas o mesetas, con mayor o menor intervención dependiendo de la idiosincrasia de la
planta de tratamiento o del espacio disponible. Particularmente, la mayoría de las plantas de
Introducción
22
compostaje que emplean como tecnología los sistemas “in vessel” implican el uso de mesetas
para madurar el compost (Diaz et al., 2007).
De manera resumida, se exponen algunas de las características principales de los
sistemas de compostaje:
Las pilas o mesetas pueden desarrollarse con mayor o menor tecnología, en
condiciones más o menos protegidas del exterior y diferenciándose principalmente según su
método de aireación: volteado y aireación forzada o estática. La manera más sencilla consiste
en la formación de una pila y su mantenimiento en condiciones estáticas mediante la
realización de una buena mezcla inicial con el material estructurante y una forma de la pila
adecuada que faciliten la aireación pasiva. Es una tecnología de bajo coste pero puede
presentar problemas para asegurar la reducción de los patógenos por debajo de los límites
legales al no producirse la mezcla del material. Las pilas estáticas también pueden airearse de
manera forzada mediante dos configuraciones diferenciadas: modelo Betsville y modelo
Rutgers. El modelo Betsville fue el primero en desarrollarse y consiste en la colocación de
tuberías por el suelo y encima la mezcla a compostar. Se genera presión negativa en la masa a
compostar mediante succión con un ventilador centrífugo según concentración de oxígeno,
entrando el aire exterior en la pila y recogiéndose el aire del interior del material en un
biofiltro para reducir los olores. En el modelo Rutgers el aire es forzado a pasar a través de la
pila mediante presión positiva lo cual permite garantizar la oxigenación y mayor control de la
temperatura de la pila según las necesidades de proceso. En ambos sistemas la formación de
la mezcla es esencial requiriéndose unas condiciones de porosidad y humedad que permitan
crear las condiciones adecuadas para que la aireación mantenga la aerobiosis en la pila. El
sistema de pila aireada puede ser abierto o semiabierto mediante el uso de lonas o la
realización de las pilas bajo cubierta o con muros. Las mesetas o pilas volteadas son uno de
los sistemas más ampliamente utilizados debido a su baja inversión y a su aplicabilidad en
una gran variedad de residuos. El volteado consiste en la destrucción-reconstrucción de la
pila y en la mayoría de las plantas se emplean palas o volteadoras mediante las cuales el
material se agita y/o mezcla volviendo a depositar el material en una forma semejante a la de
inicio (Figura 5). La dimensión de las mesetas o pilas varían en función de la tecnología de
volteo y a la envergadura de la maquinaria empleada. La frecuencia de los volteos es variable
siendo función de las variables de proceso (oxígeno, temperatura, humedad, etc) y las
variables técnicas y económicas de la planta. Al igual que para el sistema estático, pueden ser
abiertos o semiabiertos mediante el uso de lonas o la realización de las pilas bajo cubierta.
Introducción
23
Figura 5. Imagen de una pila de compostaje a la izquierda y detalle de un volteo con pala a la
derecha.
En cuanto a los sistemas “in vessel” destaca el sistema estático en túneles,
contenedores, reactor estático o similar que mantienen la masa a compostar en un sistema
cerrado en el cual la aireación se realiza mediante aireación forzada. Este método requiere
mayor inversión pero presenta un mayor control del proceso: tratamiento de gases, recogida
de lixiviados, sistema de toma de datos de variables básicas mediante sensores en paredes,
riego por aspersión, etc. El tiempo de residencia habitual en el sistema de túneles se sitúa en
torno a los 14 días. Los reactores estáticos son sistemas que permite la monitorización del
proceso y, por lo tanto, se han empleado en investigación experimental (Figura 6). Otro
sistema “in vessel” es el reactor rotatorio que se diferencia de otros sistemas cerrados en que,
además de la posibilidad de introducir la ventilación forzada, el material gira debido a la
propia rotación del reactor, evitando su compactación y disminuyendo su estratificación
gracias a la homogenización provocada por el giro del tambor.
Las trincheras son un sistema semiabierto que se puede considerar tanto “in vessel”
como “in windrow”. Puede combinar la ventilación forzada, mediante la inclusión de suelo
perforado por donde se introduce el aire, y el sistema de volteos a lo largo de la trinchera
mediante el empleo de volteadoras tipo puente. Esta volteadora circula sobre raíles
dispuestos en los muros longitudinales que delimitan el material (Figura 6).
Introducción
24
Figura 6. A la izquierda, bioreactor de residuos orgánicos (BIORESOR) del Equipo de
Biotecnología Ambiental de la Universidad de Vigo (Patente 009900981) y a la derecha, detalle
de una trinchera de compostaje.
Introducción
25
3. VERMICOMPOSTAJE
3.1. Generalidades
El vermicompostaje es una técnica de degradación de sustratos orgánicos en la cual se
emplean lombrices de tierra para obtener un producto de alto valor agronómico denominado
vermicompost. Esta tecnología utiliza la actividad de algunas especies de lombrices de tierra
que en presencia de residuos orgánicos, directamente mediante su capacidad detritívora y, de
manera indirecta, mediante su interacción con la microbiota, provocan una aceleración en la
descomposición de la materia orgánica. Las lombrices de tierra se alimentan de los residuos,
digiriéndolos y triturándolos y, por lo tanto, provocando cambios físicos, químicos y
biológicos en las propiedades del residuo, debido a la interacción con la propia microbiota
presente en el intestino de las lombrices y porque algunos microorganismos presentes en los
residuos forman parte de su dieta (Edwards and Fletcher, 1988). Domínguez (2004) definió
al vermicompostaje como un proceso biooxidativo en el que los lombrices interaccionan con
los microorganimos y otra fauna descomponedora, acelerando la degradación y estabilización
de la materia orgánica.
3.2. Organismos implicados en el vermicompostaje
En un proceso de vermicompostaje la descomposición de la materia orgánica está
gobernada principalmente por las lombrices de tierra y los microorganismos. Se puede
encontrar otra fauna asociada como cochinillas, tijeretas, larvas de moscas, enquitreidos,
nematodos, ácaros, etc. aunque su efecto en el proceso es menor si éste se desarrolla de
manera controlada y bajo un correcto manejo.
3.2.1. Lombrices de tierra
Las lombrices de tierra son anélidos oligoquetos clitelados que habitan en los suelos y
cuya actividad ejerce un efecto importante en la estructura y en los servicios ecosistémicos
(Blouin et al., 2013). En la actualidad, se considera que existen más de 8000 especies
descritas de Oligochaeta de las cuales la mitad son lombrices terrestres (Reynolds and
Wetzel, 2016) y representan la mayor biomasa animal de la superficie del suelo en la mayoría
de ecosistemas terrestres (Lavelle and Spain, 2001). De acuerdo con Bouché (1977), las
lombrices de tierra ocupan diferentes nichos ecológicos y presentan distintos ciclos vida,
procesos de movimiento y estrategias de alimentación, por lo que se pueden diferenciar en
epigeas, endogeas y anécicas. En la Tabla 6 se presentan las principales características de las
tres categorías ecológicas.
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26
Tabla 6. Principales características de las tres categorías ecológicas de las lombrices de tierra
(Benckiser, 1997; Coleman et al., 2004; Edwards and Bohlen, 1996; Karaca, 2010).
Epigeas Endogeas Anécicas
Hábitat Horizonte orgánico. Horizonte mineral Horizonte orgánico y
mineral.
Fuente de alimento Hojarasca en
descomposición
Suelo mineral rico en
materia orgánica
Hojarasca en
descomposición
Estrategia alimentación
Detritívora Geófaga Detritívora
Galerías Ninguna
Continuas y
horizontales a 10-15
cm
Verticales desde capa
mineral a la superficie.
Grandes y permanentes
Musculatura Poco desarrollada Desarrollada Bien desarrollada
Tasa de reproducción Alta Limitada Limitada
Tamaño adultos Pequeño Mediano Grande
Pigmentación Uniforme e intensa Ausente o levemente
pigmentada
Dorsalmente y anterior.
Moderada
Movilidad Rápida en respuesta a
perturbaciones Lenta
Moderada en las
galerías
Condiciones adversas (sequedad)
Estado de capullo Diapausa Inactividad
Ejemplos Eisenia fetida,
Eisenia andrei
Dendrobaena octaedra
Aporrectodea
calaginosa Allobophora
chlototica
Lumbricus terrestris
Apporeoctodea longa
De la totalidad de lombrices descritas sólo algunas de ellas se emplean actualmente en
vermicompostaje, siendo la mayoría de ellas pertenecientes a la clase ecológica epigea. Ello se
debe a que las lombrices deben cumplir una serie de requisitos para que su cultivo y su uso
controlado en el proceso de vermicompostaje sea posible:
− Alta capacidad de colonización de distintos residuos orgánicos.
− Elevada capacidad de consumir y digerir sustratos orgánicos.
− Alta capacidad reproductiva y viabilidad de capullos.
− Rápido crecimiento y ciclo de vida corto.
− Rango de tolerancia a variaciones ambientales amplio.
− Elevada resistencia a la manipulación.
Las especies de lombrices de tierra más frecuentemente empleadas en procesos de
vermicompostaje son Eisenia andrei y Eisenia fetida. Se consideraron la misma especie
debido a que comparten parecido morfológico, ciclos de vida similares y forman colonias
mixtas en pilas de estiércol (Bouché, 1972). Sin embargo, se han demostrado sus diferencias
biológicas, reproductivas y filogenéticas (Domínguez et al., 2005; Pérez-Losada et al., 2005) y
Introducción
27
se ha constatado que bajo condiciones controladas, es decir, en procesos de vermicompostaje,
E. andrei puede llegar a desplazar a E. fetida, debido a su mayor tasa de crecimiento y
reproducción. Ambas se caracterizan por poseer un ciclo de vida más corto en comparación
con otras especies de lombrices de tierra empleadas en procesos de vermicompostaje (Figura
7). Así mismo, consumen importantes cantidades de residuos, toleran un amplio rango de
temperatura y humedad y son fácilmente manejables (Domínguez and Edwards, 2011). Se
conocen habitualmente con el nombre de lombriz roja californiana (E. andrei), por su
pigmentación homocrómica rojo oscuro, y lombriz tigre (E. fetida), debido a su color marrón
con bandas intersegmentarias amarillentas. Ambas son especies de climas templados aunque
se han convertido en especies cosmopolitas tanto por su uso en vermicompostaje y
vermicultura como por su empleo en técnicas de ecotoxicología (Domínguez and Edwards,
2011; Voua Otomo et al., 2013).
Figura 7. Ciclo de vida de Eisenia andrei y Eisenia fetida. Modificado de Domínguez and Pérez-
Díaz (2011)
Otras especies de climas templados empleadas en vermicompostaje son Dendrobaena
venetta, Dendrodrilus rubidus y Lumbricus rubellus. Esta última, presenta gran interés en
vermicultura debido a su tamaño, lo cual la hace útil como cebo de pesca y en producción de
harina de lombriz. En cuanto a las especies de lombriz de clima tropical, las más empleadas
en vermicompostaje son Eudrilus eugeniae y Perionyx excavatus. Ambas requieren que el
vermicompostaje se desarrolle bajo control de la temperatura debido a sus estrechos
márgenes de tolerancia a la temperatura ambiental. E. eugeniae es la lombriz de tierra más
grande empleada en procesos de vermicompostaje con un peso medio de 3.5g (Domínguez
and Edwards, 2011; Edwards and Arancon, 2004)
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28
3.2.2. Microorganismos
Los microorganimos juegan un papel crucial en la descomposición de la materia
orgánica durante el vermicompostaje. En el proceso se ven involucrados una gran cantidad de
microorganismos tanto bacterias, hongos, actinomicetos como algas y protozoos. A diferencia
del compostaje, donde se produce una sucesión microbiana entre mesófilos y termófilos, el
vermicompostaje es un proceso que se desarrolla en condiciones mesofílicas.
Como muchos autores han expuesto anteriormente (Aira et al., 2002; Brown, 1995;
Domínguez, 2011; Lavelle and Spain, 2001) las lombrices de tierra ejercen un efecto directo e
indirecto sobre las comunidades microbianas. En la Figura 8 se exponen las interacciones
ejercidas por las lombrices sobre la microbiota.
Figura 8. Esquema de las interacciones de las lombrices de tierra sobre los microorganismos
(elaboración propia)
Las lombrices de tierra se alimentan de los sustratos orgánicos que, al pasar a través de
la molleja, se fragmentan, aumentando la relación superficie/volumen de las partículas y
falicitando a los microorganimos el acceso al alimento y, por lo tanto, a su colonización. Así
mismo, el alimento ingerido por las lombrices sufre procesos de degradación enzimática a
causa de la actividad de las propias enzimas segregadas en el mucus y por la pared intestinal
de las lombrices, por la actividad de las enzimas segregadas por los microorganismos
endosimbiontes del intestino y por la actividad de la microbiota ingerida propia del sustrato.
De esta manera, el material orgánico digerido se dispersa en forma de excrementos, o casts,
en coexistencia con el sustrato no ingerido. Estos casts recién excretados contienen
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29
nutrientes y microbiota diferente al material previamente ingerido y son dispersados por el
sustrato al desplazarse las lombrices. Además de los casts, las lombrices de tierra secretan al
exterior mucus y otras sustancias como urea y amonio que constituyen un aporte de
nutrientes fácilmente asimilables para los microorganimos (Domínguez et al., 2011). La
microbiota del residuo forma parte de la dieta de las lombrices, de manera que se ha
demostrado que se alimentan principalmente de hongos, tanto de manera selectiva como
generalista. Algunas especies de bacterias pueden ser digeridas, otras pueden sobrevivir al
paso a través del tracto intestinal y otras bacterias pueden crecer y volverse más activas.
También pueden constituir parte de la dieta de las lombrices de tierra los protozoos, algas y
nematodos, bien mediante una ingesta accidental o por preferencia. La cantidad de
microorganismos consumidos y la capacidad de las lombrices para digerirlos varían con la
especie de lombriz de tierra, su categoría ecológica, el tipo de alimento y las condiciones
ambientales, de ahí que el papel de los microorganismos como fuente de nutrición de las
lombrices no esté claro (Brown and Doube, 2004; Curry and Schmidt, 2007). La actividad de
las lombrices a través del residuo provoca modificaciones físicas, como consecuencia de su
movimiento y excavación, como son la aireación y homogeneización del sustrato, lo cua,
favorece la actividad microbiana y mejora la descomposición del residuo (Domínguez, 2004).
Además, existe una competencia entre las lombrices y la microbiota por los recursos del
sustrato. Tiunov and Scheu (2004) observaron que la presencia de lombrices incrementaba la
limitación en carbono para los microorganismos del suelo a causa del aumento de nitrógeno y
fósforo en los casts o a un agotamiento directo de los recursos de carbono fácilmente
disponibles.
En la Tabla 7 se presentan investigaciones que estudian las comunidades microbianas
durante el vermicompostaje o en el vermicompost.
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30
Tabla 7. Referencias de investigaciones de la microbiología del proceso de vermicompostaje.
Residuo/ sistema Lombriz de tierra Técnica de estudio Referencia
Residuo fresco de aceituna y
estiércol de oveja. Camas 1.5 m2
E. fetida
5g/100 g ps
Extracción ADN y análisis
PCR-DGGE y PCR tiempo
real
(Vivas et al.,
2009)
Purín de cerdo. Módulos 1,5kg o
3kg. Recipientes 200g
E. fetida
500 indiv/módulo
50indiv/recipiente
Coliformes por filtración y
cultivo
(Monroy et al.,
2009)
Residuo de la industria azucarera.
Recipiente 2,5 kg
E. eugeniae
15g/kg
Extracción ADN y análisis
PCR-DGGE
(Sen and
Chandra,
2009)
Purín de cerdo.
Módulos 1,5kg o 3 kg
E. fetida
500 indiv.
maduros/módulo
Análisis PLFAs
(Gómez-
Brandón et al.,
2011a)
Vermicompost a partir de
distintos residuos y sistemas de
vermicompostaje.
E. fetida Extracción ADN y análisis
PCR-DGGE, Microarray
(Fernández
Gómez et al.,
2012)
Residuos agroindustriales de
vino, aceituna y estiércol. Cajas
2,4m2
E. fetida biomasa
equiv. 3% m.o.
Extracción ADN y análisis
PCR-DGGE y PCR tiempo
real
(Castillo et al.,
2013)
Residuos vegetales.
Reactores 200g
E. fetida
10 g/reactor
Extracción ADN y análisis
PCR-DGGE
(Huang et al.,
2013)
Residuos frutas y verduras.
Contenedores 2kg
E. fetida 100
indiv/contenedor
PCR tiempo real, PCR-
DGGE, pirosecuenciación
(Huang et al.,
2014)
Hojarasca de maíz y excrementos
de pollo. Recipientes 200g
E. fetida
6 indiv/recipiente
Extracción ADN y análisis
PCR, pirosecuenciación
(Chen et al.,
2015b)
3.3. Condiciones previas y control de proceso
El vermicompostaje es un proceso biotecnológico y, como tal, presenta unos
requerimientos iniciales impuestos por la propia biología de la microbiota y la fauna
implicada. Requiere un control del proceso para que los organismos se desarrollen
adecuadamente en condiciones y tiempo que permitan la obtención del producto en apto
para su uso posterior.
3.3.1. Condiciones previas
Como se comentó anteriormente, las lombrices implicadas en procesos de
vermicompostaje presentan diferentes rangos de tolerancia a las condiciones ambientales.
Estos factores deben ser tenidos en cuenta cuando se busca tratar un residuo orgánico. A
continuación se exponen algunos de los parámetros más importantes:
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31
Humedad
Las lombrices de tierra están protegidas del exterior con una fina cutícula a través de la
cual se efectúa el intercambio gaseoso. Su epidermis cuenta con glándulas mucosas que
excretan distintos fluidos que las mantienen húmedas, de modo que el oxígeno se disuelve en
esta película superficial (Edwards and Bohlen, 1996). Las condiciones de sequedad pueden
provocar la muerte o la migración de las lombrices hacia zonas más favorables, por lo que, el
mantenimiento de condiciones de humedad óptimas permite aumentar la eficacia en el
tratamiento de los residuos. Los requerimientos de humedad varían dependiendo de la
especie de lombriz y del residuo orgánico a tratar, aunque de manera general se establece un
rango entre 50-90%. A modo de ejemplo, Domínguez and Edwards (1997) observaron que E.
andrei crece y madura en estiércol de cerdo entre 65-90% de contenido de humedad siendo
el óptimo 85%.
Temperatura
Las distintas especies de lombriz difieren en cuanto a los rangos tolerables de
temperatura. Loehr et al. (1985) y Neuhauser et al. (1988) observaron que Dendrobaena
veneta, Eisenia fetida, Eudrilus eugeniae, Perionyx excavatus and Pheretima hawayana
presentan temperaturas óptimas para su crecimiento y reproducción en torno a 20-25ºC. A
temperaturas inferiores a 15ºC sólo E. fetida produce capullos mientras que a 30ºC sólo P.
excavatus los produce. Sin embargo, Edwards (1988) observó que a temperaturas inferiores a
9ºC y superiores a 30ºC tanto P. excavatus y E. eugeniae presentaban una mortalidad elevada
siendo su temperatura óptima 25ºC. Estos estudios observaron que las temperaturas óptimas
para la producción de capullos fueron más bajas que las más adecuadas para el crecimiento
de las especies estudiadas. En general, la temperatura óptima para un adecuado proceso de
vermicompostaje se sitúa entre los 10-35ºC.
Estructura física y aireación
Como se ha comentado, la respiración cutánea de las lombrices de tierra exige una
correcta difusión del oxígeno con el medio a través de la pared del cuerpo. Por lo tanto, el
residuo debe poseer una estructura suficientemente porosa para posibilitar concentraciones
de oxígeno óptimas para las lombrices. Condiciones de humedad excesiva, residuos con un
alto contenido graso o compactación del material pueden provocar situaciones de
anaerobiosis afectando al desarrollo de las lombrices por falta de oxígeno y formación de
compuestos tóxicos. Además, la estructura del residuo debe posibilitar el adecuado
movimiento de las lombrices, así mismo, las lombrices, con su propio desplazamiento,
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32
contribuyen al aumento de la aireación. Otro parámetro que afecta a la porosidad y
estructura es el tamaño de partícula. El residuo debe poseer partículas con superficie
área/volumen accesible a las lombrices. Lowe and Butt (2005) establecieron que el tamaño
de partícula afecta a las tasas de crecimiento y reproducción de lombrices, requiriendo las
lombrices más pequeñas tamaños más reducidos (<1mm).
Concentración de sales y amonio
La mayor parte de la absorción y pérdida de agua en las lombrices se produce a través
de la pared del cuerpo observándose que las lombrices pueden mantener relativamente
constante su presión osmótica interna en soluciones diluidas pero son incapaces de
mantenerla en las más concentradas (Edwards and Bohlen, 1996). Por ello, un exceso de sales
inorgánicas y altos niveles de amonio en el medio resultan altamente tóxicos para las
lombrices inhibiendo su desarrollo y causando la muerte. En el caso de E. fetida y E. andrei se
ha visto que conductividades eléctricas superiores a 8dS m-1 y contenidos de amonio
superiores a 0.5 mg g-1 se consideran tóxicos (Edwards, 1988).
Otros parámetros físico-químicos a considerar son el pH del residuo y sustancias
tóxicas tanto orgánicas como inorgánicas presentes en el sustrato. Las lombrices de tierra
presentan un rango de tolerancia amplio de pH y pueden sobrevivir en condiciones desde
ácidas a alcalinas (pH 5-9) aunque el pH óptimo depende de la especie de lombriz. Por otro
lado, la presencia en el medio de sustancias como son los plaguicidas, antibióticos,
hidrocarburos o metales pesados puede causar efectos adversos en el desarrollo,
supervivencia y actividad de las lombrices, ello dependerá de la dosis de contaminante y la
especie de lombriz (Benitez et al., 1999a; Fernández Gómez et al., 2011; Reinecke et al., 2001;
Thiele-Bruhn, 2003)
La naturaleza y propiedades del residuo orgánico condicionan el desarrollo adecuado
de las lombrices de tierra y, por lo tanto, la eficacia del proceso de vermicompostaje. De esta
manera, en muchos casos es necesario acondicionar el residuo que se pretende tratar de
manera que se posibilite la supervivencia y desarrollo de las lombrices. Para ello se pueden
realizar distintas acciones de manera independiente o en conjunto:
• Mezclas con otros residuos: la finalidad de la mezcla es mejorar las propiedades de los
residuos modificando positivamente su estructura, pH, salinidad, relación C/N, etc.
Por ejemplo, Domínguez et al. (2000) mezclaron lodo de depuradora con diferentes
estructurantes observando mejoras en la reproducción frente al lodo sin estructurar y
Elvira et al. (1998) mejoraron el vermicompostaje de lodos de depuradora de
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33
industrias del papel y lecherías mediante la adicción de estiércol de vaca. De la misma
manera, Kaushik and Garg (2004) vermicompostaron lodos de la industria textil al
mezclarlos con estiércol de vaca.
• Pre-compostaje: realizar un compostaje previo a la inoculación de las lombrices de
tierra tiene varios objetivos:
− Asegurar la eliminación de patógenos y semillas de malas hierbas al
desarrollarse altas temperaturas durante el compostaje del residuo.
− Eliminar posibles tóxicos para las lombrices de tierra que presentan los
residuos orgánicos frescos.
− En otros casos, los residuos presentan altos contenidos de sustancias
fácilmente asimilables sobre los que puede proliferar de manera importante la
microbiota, aumentando la temperatura e impidiendo la supervivencia de las
lombrices, por lo que una fase previa de pre-compostaje degrada la materia
orgánica más lábil
− Desde una escala industrial, el pre-compostaje facilita el manejo del residuo
para su distribución en los sistemas de vermicompostaje.
Tras un proceso de compostaje puede ser necesario airear el material para enfriarlo o
permitir la liberación de sustancias como el amonio. Diversas investigaciones han
realizado pre-compostaje previamente al vermicompostaje (Tabla 8).
• Otros procesos necesarios para que el residuo pueda ser tratado mediante las
lombrices de tierra incluyen la trituración y la humectación.
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Tabla 8. Referencias de investigaciones que emplean el pre-compostaje previo al
vermicompostaje.
Residuo Tiempo pre-compostaje/Óptimo
Especie de lombriz
Referencia
Residuos de jardín 0, 2, 4, 6 semanas/Limitar al
mínimo E. andrei
(Frederickson et
al., 1997)
Biosólidos y papel 28 días E. fetida (Ndegwa and
Thompson, 2001)
Estiércol de ganado 0, 1, 2, 3, 4, 5
semanas/Limitar al mínimo E. fetida
(Gunadi et al.,
2002)
Residuos de cocina, recortes
de césped, papel triturado
0, 6, 9, 12, 15 días/
mejores resultados 9 días
L. rubellus
E. fetida (Nair et al., 2006)
Estiércol de ganado 15 días E. andrei (Lazcano et al.,
2008)
Residuos industria azucarera 60 días E. eugeniae (Sen and Chandra,
2009)
Estiércol y papel 28 días E. fetida (Mupondi et al.,
2010)
Residuos de tomate y
cáscaras de almendra 63 días
E. fetida
E. andrei
(Fornes et al.,
2012)
Estiércol pato con paja y
zeolita 45 días E. fetida
(Wang et al.,
2014)
A estos parámetros hay que añadir los efectos de la densidad de población de
lombrices. Dependiendo de la especie de lombriz de tierra, la calidad del alimento y la
superficie del sistema de vermicompostaje empleado, existe unos valores óptimos para la
densidad de población. Un exceso de densidad puede provocar competencias por el alimento
mientras que un defecto de individuos puede generar problemas en la reproducción. Del
mismo modo, el objetivo perseguido también puede variar la densidad necesaria, ya que si se
busca reproducción, crecimiento o el tratamiento del residuo, las densidades pueden diferir.
Domínguez and Edwards (1997) encontraron que en condiciones óptimas de humedad 8
individuos de E. andrei en 43.61g de purín (masa seca) era la densidad ideal para el
crecimiento y maduración de esta especie. Ndegwa et al. (2000) determinaron que, variando
la tasa de alimentación, la densidad de 1.60kg lombrices m-2 muestra la mayor bioconversión
de biosólidos en biomasa de lombrices y las mejores producciones de vermicompost.
3.3.2. Fases del proceso
Durante el proceso de vermicompostaje se pueden distinguir de manera general dos
fases (Lazcano et al., 2008):
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Fase activa: caracterizada por la actividad de las lombrices que se desplazan por el
residuo y se alimentan de los sustratos orgánicos digiriéndolos, asimilándolos y excretando
casts, lo cual, provoca cambios en las propiedades fisicoquímicas y microbianas del residuo.
El vermicompost resultante es una mezcla de casts y material no procesado por las lombrices.
La duración de esta fase no es fija y depende de la densidad de población, de las especies
empleadas y de la tasa de ingestión y procesado del residuo (Domínguez, 2004)
Fase de maduración: las lombrices se desplazan hacia materiales más frescos, por lo que
los microorganismos asumen el control de la descomposición del material procesado.
Domínguez (2004) explica que los casts están sujetos a un proceso de maduración y a la
acción de los microorganismos y microinvertebrados presentes en el sustrato.
Otra consideración a tener en cuenta del proceso de vermicompostaje es la etapa de
adaptación de las lombrices al residuo. Cuando se dispone en contacto un nuevo residuo con
la población de lombrices que lo va a digerir puede existir un período de baja alimentación o
incluso mortalidad de parte de la población (Elvira et al., 1998; Gajalakshmi et al., 2002;
Haimi and Huhta, 1986). Este período es más habitual en microcosmos experimentales o
sistemas de vermicompostaje de pequeño volumen debido a que las lombrices no encuentran
hábitats ecológicos adecuados y pueden sufrir estrés (Domínguez, 2004). La etapa de
adaptación se puede minimizar de diferentes maneras; comenzando directamente el
vermicompostaje con las lombrices en el propio sustrato de maternidad o cultivo y añadiendo
capas de residuo sobre este sustrato; aclimatando las lombrices mediante la incorporación de
dosis bajas de residuo mezclado con el alimento durante la elaboración del stock de
lombrices; emplear sustratos de escape, como la vermiculita, de manera que las lombrices se
vayan alimentando lentamente del residuo y puedan migrar hacia el sustrato en caso de
requerirlo.
3.3.3. Cambios y control del proceso de vermicompostaje
El vermicompostaje es una tecnología, en general, sencilla y de bajo coste que
aprovecha la capacidad de las lombrices de digerir sustratos orgánicos para bioestabilizar un
residuo orgánico transformándolo en vermicompost. Este proceso, a priori sencillo, tiene
unos requerimientos iniciales, ya presentados en el epígrafe 3.3.1, y también conlleva,
necesariamente, un control del proceso, de manera que las lombrices se desarrollen
adecuadamente y el proceso sea efectivo. A continuación se presentan los parámetros de
seguimiento más ampliamente utilizados:
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Parámetros fisicoquímicos
Condiciones aerobias: la propia actividad de las lombrices posibilita el mantenimiento
de los niveles de oxígeno adecuados a lo largo del proceso de vermicompostaje. Sin embargo,
durante la adición de residuo en sistemas de vermicompostaje de alimentación continua debe
asegurarse que la capa dispuesta presenta un grosor óptimo para evitar fermentaciones y,
con ello, todos los problemas asociados (metabolitos indeseables, anaerobiosis) para las
lombrices de tierra.
pH: en general, el vermicompostaje cambia el pH hacia la neutralidad, con
independencia del pH inicial de los sustratos orgánicos (Pramanik et al., 2007). Si bien, el pH
desciende levemente durante el vermicompostaje como consecuencia de la mineralización
del nitrógeno a nitratos, la formación de ortofosfatos, y la mineralización de materia orgánica
a dióxido de carbono y a otros compuestos orgánicos ácidos intermedios. Singh et al. (2005)
observaron un aumento en el pH en la fase inicial del proceso de vermicompostaje que
atribuyeron al metabolismo microbiano aerobio que tiene como resultado la formación de
hidróxidos básicos en el sustrato en descomposición. Ndegwa et al. (2000) observaron que
distintos sustratos pueden producir diferentes especies intermedias, por lo que, diferentes
residuos muestran un comportamiento distinto en la evolución del pH. Así, autores como Vig
et al. (2011) y Bhat et al. (2013) observaron aumentos en el pH de varios vermicomposts de
distinto origen sugiriendo que podría ser causado por el exceso de nitrógeno orgánico no
requerido por los microorganismos que se libera como amoníaco, se disuelve en agua e
incrementa el pH del vermicompost.
Carbono: durante el vermicompostaje se produce la mineralización del carbono y su
pérdida en forma de dióxido de carbono. Las lombrices y los microorganismos emplean el
carbono como fuente de energía y en su crecimiento celular. Las tasas de reducción del
carbono dependen del residuo, la especie de lombriz, su densidad y el tipo de
vermicompostaje realizado. En experimentos en macetas con E. fetida durante 100 días, Garg
et al. (2006) observaron reducciones del carbono orgánico total (COT) superiores al 50% en
agroresiduos y residuos de cocina, en torno al 40% en residuos municipales y 32% de
reducción en residuo de la industria textil. Elvira et al. (1998) encontraron reducciones del
COT entre 20-43% durante el vermicompostaje en literas de distintos residuos con E. andrei.
Se han visto reducciones en torno al 25% y 31% del COT durante el vermicompostaje en
contenedores de lodo de depuradora con E. fetida en 90 días (Li et al., 2011) y con P.
excavatus en 45 días (Khwairakpam and Bhargava, 2009), respectivamente. Durante el
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vermicompostaje de estiércol de vaca y alperujo con E. andrei, Plaza et al. (2008) obtuvieron
una reducción del 58% del COT en 8 meses.
Nitrógeno: las variaciones en la concentración de nitrógeno en el proceso de
vermicompostaje son variables observándose tanto descensos como aumentos que dependen
del residuo y de las particularidades del proceso. Las lombrices mejoran la mineralización del
nitrógeno y lo consumen empleándolo en su construcción celular. Por otro lado, las lombrices
enriquecen el residuo con nitrógeno mediante la excreción de moco, enzimas y por la propia
descomposición de lombrices muertas. Además, el descenso en contenidos de carbono
orgánico durante el vermicompostaje permite un aumento de la concentración de nitrógeno
(Suthar, 2007). Cambios en el pH hacia condiciones ácidas pueden ser también un factor
importante en la retención de nitrógeno ya que en condiciones alcalinas el amoníaco es
volátil (Hartenstein and Hartenstein, 1981). Por lo tanto, el contenido de nitrógeno en el
vermicompostaje depende del nitrógeno inicial en el residuo y de los procesos de asimilación
y descomposición por parte de las lombrices.
Otros macronutrientes: en general, la concentración de nutrientes aumenta durante el
proceso de vermicompostaje como consecuencia de la mineralización de la materia orgánica
debido a un efecto de concentración. Autores como Elvira et al. (1996), Garg and Kaushik
(2005) o Yadav and Garg (2009) observaron un aumento en el contenido de fósforo, potasio y
calcio tras el vermicompostaje de diferentes residuos. Sin embargo, Orozco et al. (1996) y
Nogales et al. (2005) observaron un descenso en el potasio durante el vermicompostaje de
pulpa de café y residuos vinícolas, respectivamente, debido a su lixiviación por exceso de
agua. Benitez et al. (1996) encontraron que los lixiviados recogidos durante el proceso de
vermicompostaje presentan concentraciones elevadas de potasio.
Metales pesados: los metales pesados tales como Cd, Cu, Ni, Pb, Zn o Hg tienden a
aumentar su concentración total como consecuencia de la mineralización de la materia
orgánica, sin embargo, se ha observado que las formas disponibles tiende a disminuir. Por
ejemplo, Elvira et al. (1995) observaron un descenso en la capacidad de extracción de Mn, Cu
y Zn durante el vermicompostaje de lodos industriales. Singh and Kalamdhad (2013)
observaron que el vermicompostaje era muy eficaz para reducir las fracciones más
biodisponibles de los metales pesados, y que la biodisponibilidad dependía de propiedades
fisicoquímicas y biológicas del vermicompost, siendo el pH uno de los principales factores. El
vermicompostaje tiende a estabilizar los metales pesados redistribuyéndolos desde estados
relativamente lábiles a más inmovilizados (Morgan, 2011), de manera que, tienden a formar
complejos con los ácidos húmicos y las fracciones orgánicas más polimerizadas (Domínguez,
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38
2004). A este cambio en la disponibilidad de los metales pesados a lo largo del proceso, hay
que añadir que las lombrices de tierra son capaces de acumular un número esencial y no
esencial de metales en sus tejidos. La dinámica de los metales pesados durante el proceso de
vermicompostaje es complejo, posiblemente debido a especificidades del metal y las
interacciones con la matriz (Morgan, 2011).
Humificación: en diversas investigaciones se han observado descensos en contenidos
de carbono solubles y totales y aumentos en sustancias húmicas y , por lo tanto, en las tasas
de humificación tras el vermicompostaje de distintos residuos orgánicos (Elvira et al., 1998,
1996; Plaza et al., 2008; Romero et al., 2007). Domínguez (2004) expuso que las lombrices de
tierra aceleran y mejoran el proceso de humificación debido a la fragmentación de la materia
orgánica, al aumento de la actividad microbiana dentro del intestino de las lombrices y a la
aireación y los volteos del material como consecuencia de la alimentación y el movimiento de
las lombrices.
Parámetros biológicos
Población y estado de las lombrices: tanto la densidad de lombrices como su estado debe
ser controlado a lo largo del proceso de vermicompostaje prestando atención a si las
lombrices se alimentan del residuo, crecen y se reproducen, así como, a la viabilidad de los
nuevos individuos. Cuando se pretende vermicompostar un nuevo residuo es conveniente
conocer sus propiedades, de manera que, se cumplan los requerimientos para el
vermicompostaje (pH, humedad, amonio,…), pero también, realizar estudios de supervivencia
de lombrices de tierra con la especie de interés y la densidad de población que se va a utilizar
posteriormente en el proceso. Al inicio del vermicompostaje pueden producirse mortalidades
elevadas de la población de lombrices causadas por su adaptación a la matriz y a las
condiciones del proceso, así mismo, a medida que el residuo se consume puede aumentar la
mortalidad por falta de alimento. Elvira et al. (1997) mostraron mortalidades altas en
distintas mezclas con lodo de depuradora de una industria de fabricación de papel,
mostrando que en varias de las mezclas la mortalidad no ocurrió por falta de alimento sino
por cambios en las características del sustrato al producirse la degradación de la materia
orgánica.
Las lombrices de tierra alcanzan su madurez sexual después de un período que varía
según la especie de lombriz y las condiciones ambientales. El estudio de la presencia de
individuos tanto clitelados como inmaduros, así como, juveniles y capullos, ofrece
información sobre si las condiciones del residuo y del proceso son favorables. Se ha visto que
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la tasa de producción de capullos está relacionada con la calidad del residuo que es uno de los
factores que determinan el tiempo necesario para alcanzar la madurez sexual y para el inicio
de la reproducción (Edwards et al., 1998).
Actividades microbianas: el análisis de la actividad microbiana aporta información
sobre la degradación de la materia orgánica. Estas actividades se miden, principalmente,
mediante técnicas respiratorias y la determinación de actividades enzimáticas. Las
actividades respiratorias se relacionan con el metabolismo microbiano, de manera que, en la
fase inicial del vermicompostaje, cuando se degradan los compuestos fácilmente asimilables,
la tasa respiratoria es alta y desciende a lo largo del proceso de vermicompostaje como
consecuencia de la reducción de la disponibilidad de recursos para las comunidades
microbianas (Domínguez, 2011). Tanto los microorganismos del residuo como los
endosimbiontes del intestino de las lombrices, producen enzimas tanto intracelulares como
extracelulares que provocan la descomposición de los distintos sustratos orgánicos.
Numerosos autores han estudiado las actividades enzimáticas durante el vermicompostaje de
distintos residuos: purín de cerdo (Aira et al., 2007a, 2007b, 2006), estiércol de ganado
(Lazcano et al., 2008), lodo de depuradora (Benitez et al., 1999b), residuos de aceituna
(Benitez et al., 2005; Melgar et al., 2009; Vivas et al., 2009), residuos vitivinícolas (Nogales et
al., 2005), residuos vegetales de invernadero (Fernández Gómez et al., 2013, 2010a, 2010b),
residuos de la industria azucarera y cenizas (Pramanik and Chung, 2011), residuos
agroindustriales lignocelulósicos (Castillo et al., 2013), residuos de maíz (Chen et al., 2015a),
lodo de panadería (Yadav et al., 2015), residuos de curtiduría (Ravindran et al., 2014), etc. Las
principales enzimas analizadas son la actividad deshidrogenasa y las actividades hidrolíticas
como la β-glucosidasa, proteasa, fosfatasas, celulasas y ureasas, entre otras. Al igual que para
la actividad respiratoria, las fases iniciales presentan, en general, mayor actividad enzimática
reduciéndose a medida que avanza el vermicompostaje. Sin embargo, la interpretación de los
datos enzimáticos es complicado ya que las actividades dependen de numerosos factores y de
la diferente localización de las enzimas que contribuyen a la medida enzimática en el sistema
estudiado (Nannipieri et al., 2002).
Estructura de la comunidad microbiana: es aconsejable estudiar las distintas
poblaciones microbianas durante el vermicompostaje para establecer los efectos que las
lombrices de tierra ejercen sobre los microorganismos, ya que si las lombrices estimulan o
reducen la microbiota, o modifican la estructura y función de las comunidades microbianas,
pueden tener efectos muy diferentes sobre las tasas y forma de descomposición de la materia
orgánica (Domínguez, 2011). Gómez-Brandón et al. (2011b) mostraron que el paso de
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distintos estiércoles a través del intestino de la lombriz E. fetida provocaba la reducción de la
biomasa bacteriana y Fernández Gómez et al. (2010a) observaron que la estructura de las
comunidades fúngicas difería en la etapa de máxima biomasa de lombrices durante el
vermicompostaje de residuos de fruta, sugiriendo ambos trabajos un fuerte efecto asociado al
paso del residuo a través del intestino de las lombrices. Huang et al. (2013) observaron una
reducción de la biomasa microbiana en el vermicompostaje de residuos vegetales pero un
aumento de la diversidad microbiana. Sin embargo, en un estudio posterior, Huang et al.
(2014) mostraron un aumento de las poblaciones bacteriana y fúngica durante el
vermicompostaje lo cual sugiere que los diferentes tipos de sustratos, modos de operación y
condiciones ambientales utilizadas en los experimentos, pueden influir en el crecimiento y
reproducción de lombrices y la microbiota (Gunadi and Edwards, 2003). Además, el tipo de
método empleado para el análisis y seguimiento de la estructura microbiana aporta distinta
información sobre la diversidad y las funciones biólogicas de las distintas comunidades
microbianas implicadas en el vermicompostaje.
Los vermicompost presentan distinta diversidad y carga microbiana que afectarán
positivamente (estímulación de la microbiota del suelo, supresión de patógenos, degradación
de plaguicidas y otros compuestos orgánicos, etc.) o negativamente (inoculación de
patógenos al suelo, exceso de biomasa microbiana y competencia por nutrientes, etc) al
crecimiento de las plantas. Por lo que el estudio de la microbiota es esencial no sólo para la
mejora del proceso de vermicompostaje, sino también para la obtención de un producto de
valor añadido con diferentes usos.
Patógenos: a diferencia del compostaje, durante el vermicompostaje no se alcanzan las
temperaturas que aseguran la eliminación de patógenos, si bien, en diversos residuos se
utiliza el pre-compostaje como acción previa a la inoculación de las lombrices, por lo que este
problema se reduce. En caso del vermicompostaje sobre residuos frescos, se ha visto que las
lombrices reducen la carga patogénica del residuo. Se ha constatado que el vermicompostaje
es efectivo para reducir y eliminar distintos patógenos en biosólidos y lodos (Eastman et al.,
2001; Rodríguez-Canché et al., 2010). Así mismo, Monroy et al. (2009, 2008) observaron
reducciones superiores al 98% de la población de coliformes como resultado del paso de
purín de cerdo a través del intestino en distintas especies de lombriz. Sin embargo, debido a
las diferencias fisicoquímicas y biológicas de los distintos residuos, el sistema de
vermicompostaje y las condiciones de proceso, la reducción de patógenos durante el
vermicompostaje puede diferir. Hénault-Ethier et al. (2016) encontraron que la reducción de
E. coli durante el vermicompostaje de residuos orgánicos separados en origen es causado
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fundamentalmente por la microbiota antagonista, mientras que el efecto de las lombrices
sobre este patógeno es menor. Aira et al. (2011) mostraron que, en un vermirreactor de
alimentación continua con estiércol de vaca, el efecto de las lombrices depende del tipo de
patógeno considerado.
Además, durante el vermicompostaje debe vigilarse la presencia de predadores que
puedan causar daños a las lombrices como aves, ratones, topos, hormigas, etc. El mayor o
menor riesgo vendrá asociado al tipo de sistema de vermicompostaje implantado y al lugar
donde se realice (outdoor or indoor).
3.4. Sistemas de vermicompostaje
Se pueden diferenciar los sistemas de vermicompostaje en función de diferentes
argumentos, por ejemplo:
− Según el sistema de alimentación: discontinua, donde el alimento o residuo orgánico
se añade una única vez y es separado de las lombrices una vez digerido, o continua, el
alimento se añade por tandas, de manera que, cuando una cantidad de residuo es
procesado por las lombrices se añade nuevo residuo.
− Según el nivel de tecnología: tradicional, que hace referencia a sistemas sencillos no
automatizados y de fácil uso, o moderno con sistemas más tecnificados y que
requieren una mayor especialización en el uso.
− Según la escala: doméstica, de un tamaño pequeño para hacer frente a la generación
del residuo orgánico en el hogar; pequeña o mediana escala, para la obtención de
residuo dentro de la propia explotación o instalación de manera que el residuo
orgánico generado se trata y el vermicompost se emplea como fertilizante en la
misma instalación; escala industrial, cuyo objetivo es el tratamiento del residuo y la
producción de vermicompost. Un sistema industrial de vermicompostaje exige que la
planta de procesado este diferenciada en zonas: área de pre-procesamiento o
acondicionamiento del residuo, área de cría o maternidad de lombriz, área de
procesamiento del residuo y área de maduración y almacenamiento del
vermicompost.
A continuación, se presentan, brevemente, los sistemas de vermicompostaje en función
de su tipología, es decir, según los distintos modelos de sistemas empleados o presentes en el
mercado.
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Literas, camas, pilas o zanjas
Es el más similar al proceso natural de degradación, debido a su sencillez y se basa en el
apilamiento del residuo orgánico en espacios más o menos rectangulares o piramidales
(Figura 9). Suelen delimitarse con maderas, ladrillos, bloques, etc., de manera que, se
contenga el residuo y se evite la fuga de las lombrices. Los apilamientos no deben ser
excesivamente altos para evitar el autocalentamiento del residuo y/o compactaciones
excesivas del material.
Figura 9. Foto de una litera tomada de la Junta de Andalucía (izquierda) y litera experimental
del Equipo de Biotecnología Ambiental (derecha)
La alimentación puede ser realizada en una única vez o ser dispuesta en capas
superiores o frontales para que las lombrices avancen horizontal o verticalmente. De manera
habitual, se cubren para protegerlas de las condiciones ambientales y de los depredadores.
Una vez que se alcanza la capacidad de tratamiento es necesario separar el vermicompost de
la población de lombrices, para lo cual se suelen emplear trampas con residuo fresco hacia
donde se muevan las lombrices. Las literas y similares son, en general, sistemas sencillos y
económicos, aunque para la producción de vermicompost a gran escala es necesaria la
utilización de una superficie extensa (Tabla 9).
Tabla 9. Ventajas y desventajas del sistema de vermicompostaje en literas, modificado de
Edwards (2010).
Ventajas Baja inversión económica
Manejo sencillo
Desventajas Mano de obra intensiva
Espacio necesario elevado
Tiempo de proceso lento
Considerables pérdidas de nutrientes por volatilización y
lixiviación
Difícil recoger el vermicompost sin lombrices
Introducción
43
Algunas de estas desventajas se reducen al realizar las pilas bajo techo y en condiciones
ambientales controladas de manera que las condiciones estacionales (temperaturas altas o
bajas, lluvias, insolación, etc.) tengan menor afectación sobre el proceso.
Contenedores
Su uso se emplea tanto a nivel doméstico como industrial. Pueden ser de dos tipos,
modulares y no modulares. Los modulares consisten en el uso de recipientes, bandejas o
similar, apilables y agujereados (Figura 10). Cuando el residuo de un recipiente es consumido
por las lombrices de tierra, se añade residuo fresco en un nuevo recipiente que se deposita
sobre el anterior. Las lombrices pueden moverse a través de los orificios que comunican los
recipientes, accediendo al nuevo recipiente con alimento fresco. El vermicompost se
almacena en los módulos inferiores gracias al desplazamiento vertical de las lombrices,
pudiendo retirarse cuando el vermicompost alcance la madurez deseada, sin interferir en la
continuidad del proceso. Un sistema industrial con contenedores modulares exige una
cantidad importante de módulos, lo cual conlleva requerimientos importantes de personal.
Figura 10. Sistema de vermicompostaje vertical modular doméstico Can-o-worm (izquierda) y
sistema de vermicompostaje en cajas modulares de Ecocelta Galicia S.L.(derecha).
En cuanto a los no modulares, al igual que los anteriores, el residuo se deposita
periódicamente en la superficie del contenedor o recipiente. Las lombrices se mueven en
vertical hacia el alimento fresco dejando el vermicompost en la zona inferior del contenedor
pudiendo recoger el humus líquido en la parte inferior del mismo. A diferencia de los
modulares, el vaciado del vermicompost debe realizarse mediante una puerta inferior o
lateral o, mediante volcado del contenedor con previa retirada de las lombrices mediante el
uso de trampas. A escala industrial se requieren un número importante de contenedores pero
en menor cantidad que las modulares, ya que se pueden emplear recipientes de mayor
volumen.
Introducción
44
Ambos sistemas permiten la recogida de fertilizante líquido y las lombrices están más
protegidas de las condiciones ambientales y de depredadores que el sistema de literas. Así
mismo, la superficie necesaria para un sistema industrial es menor que las literas (Tabla 10).
Tabla 10. Ventajas y desventajas del sistema de vermicompostaje en literas, modificado de
Edwards (2010).
Ventajas Mayor protección de las condiciones ambientales
Menor necesidad de espacio que las literas
Desventajas Gastos considerables en contenedores, recipientes y equipos
de movimiento de contenedores
Dificultad para mantener condiciones óptimas de humedad
Mano de obra intensiva
Dificultades para recoger el vermicompost sin lombrices en
no modulares
Lechos de flujo continuo
En general, son sistemas mecanizados que se emplean a escala media e industrial, y
requieren una inversión inicial mayor. Consisten en lechos o contenedores elevados por
encima del suelo con la base perforada y que presentan una descarga mecánica de las capas
inferiores de vermicompost (Figura 11).
Figura 11. Foto de un vermireactor diseñado por el Equipo de Biotecnología Ambiental de la
Universidad de Vigo (izquierda) y foto de una planta de producción de vermicompost de la
empresa Vermigrand (derecha).
Las capas de residuo se disponen periódicamente en la parte superior del lecho, de
manera que, las lombrices ascienden al alimento fresco y el vermicompost libre de lombrices
de tierra se acumula en la parte inferior. Esta separación se debe a que las lombrices epigeas
habitan la superficie, en torno a 10-15cm, y se mueven constantemente hacia la nueva fuente
de alimento, por lo que el sistema de flujo continuo elimina la necesidad de separar las
lombrices del vermicompost. La adicción de alimento puede ser mecanizada, así como, la
Introducción
45
recogida del vermicompost en la base del lecho. Al estar elevado del suelo está protegido de
depredadores y pueden mantenerse bajo cubierta para controlar las condiciones ambientales
(Tabla 11).
Tabla 11. Ventajas y desventajas del sistema de vermicompostaje en literas, modificado de
Edwards (2010).
Ventajas Alta protección de las condiciones ambientales y
depredadores
Menor necesidad de mano de obra
Mayor control de las condiciones de proceso
Posibilidad de automatización
Desventajas Importante inversión económica
OBJETIVOS
Objetivos
49
El objetivo general de este estudio es conocer la evolución de las actividades y las
comunidades microbianas en la fase de maduración del proceso de compostaje. Además, se
estudia esa misma dinámica en el proceso de vermicompostaje y se comparan en los dos
procesos de forma paralela y combinada. Las investigaciones se han enfocado en la fase de
maduración del compostaje con la finalidad de mejorar el proceso mediante el empleo de
distintas prácticas, entre ellas la inoculación de lombrices de tierra, y a la evaluación de la
estabilidad de los productos obtenidos a partir de los diferentes procesos. En consecuencia,
los objetivos específicos pueden resumirse como:
1. Obtener información que permita mejorar y optimizar el proceso de
compostaje a través del estudio de la estancia en maduración de material pre-compostado de
diferentes residuos y mediante distintas intervenciones (capítulos 1, 2, 3 y 4).
2. Estudiar el efecto que presenta el tipo de residuo sobre la evolución de la
dinámica microbiana, tanto de las comunidades microbianas como las actividades
enzimáticas implicadas en los procesos hidrolíticos de degradación de los compuestos
orgánicos, durante la estancia en maduración del proceso de compostaje (capítulo 1).
3. Establecer si la realización de prácticas de volteo sobre el material pre-
compostado en fase de maduración de un residuo energético es más efectivo para la
obtención de un producto es mejores condiciones de estabilidad y/o en menor tiempo que el
mantenimiento estático del compost (capítulo 2).
4. Ampliar el estudio sobre un residuo poco investigado como es el lodo de
depuradora proveniente de la elaboración de alimentos precocinados y ultracongelados del
mar (capítulos 1 y 2).
5. Determinar la viabilidad de la inoculación de lombrices de tierra epigeas en
residuos frescos y pre-compostados de diferente origen (capítulos 3 y 4).
6. Determinar cómo influye en la calidad de los productos y la evolución de los
procesos el empleo de lombrices de tierra tanto en el tratamiento de residuo orgánico fresco
como en la maduración del residuo pre-compostado a través del estudio de la dinámica
microbiana (capítulo 3 y 4).
7. Establecer si la maduración de estiércol pre-compostado mediante prácticas
dinámicas por medio de la inoculación de lombrices de tierra es más efectivo para la
obtención de un producto en mejores condiciones de estabilidad y/o en menor tiempo que
mediante maduración estática (capítulo 4).
Objetivos
50
8. Estudiar si las relaciones entre las actividades enzimáticas y las comunidades
microbianas permiten determinar hacia donde evolucionan los distintos tratamientos
estudiados y si permiten establecer las condiciones de estabilidad de los procesos orgánicos
(capítulos 1, 2, 3 y 4).
CAPÍTULO 1
Evolution of microbial dynamics during the maturation
phase of the composting of different types of waste
Villar I., Alves D., Garrido J., Mato S., 2016. Evolution of microbial dynamics during the
maturation phase of the composting of different types of waste. Waste Manag. 54, 83–
92. http://doi: 10.1016/j.wasman.2016.05.011.
Estado: publicado
Permiso: pdf editor
Capítulo 1
53
RESUMEN
Durante el compostaje, las instalaciones suelen ejercer un mayor control sobre la fase
bio-oxidativa del proceso, para la cual se utiliza una tecnología específica y, generalmente,
tiene una duración establecida. Tras esta fase, el material se deposita a madurar con menos
control. Si bien ha habido un estudio considerable de los parámetros biológicos durante la
fase termofílica, hay menos investigación sobre la fase de estabilización y maduración. Este
estudio evalúa los efectos del tipo de material de partida sobre la evolución de la dinámica
microbiana durante la fase de maduración del compostaje. Se utilizaron tres tipos de
residuos: lodo de la industria de transformación de pescado, lodo de aguas residuales
municipales y estiércol de cerdo, cada uno independientemente mezclado con madera de
pino triturada como agente estructurante. El sistema de compostaje, para cada residuo,
comprendió un reactor estático de 600L de capacidad para la fase bio-oxidativa seguida por
la fase de estabilización y maduración en triplicado en cajas de 200L durante 112 días. Los
ácidos grasos fosfolípidos, las actividades enzimáticas y los parámetros físico-químicos
fueron determinados a lo largo de la fase de maduración. La evolución de la biomasa
microbiana total, bacterias Gram +, bacterias Gram −, hongos y enzimas (β - glucosidasa,
celulasa, proteasa, fosfatasa ácida y alcalina) dependió significativamente del tipo de residuo
(p < 0.001). La comunidad microbiana predominante para cada tipo de residuo permaneció
durante todo el proceso de maduración, lo que indica que el tipo de residuo determina los
microorganismos que pueden desarrollarse en esta etapa. Mientras que los hongos
predominaron durante la maduración del lodo de pescado, el estiércol y el lodo municipal se
caracterizaron por una mayor proporción de bacterias. Tanto la estructura de la comunidad
microbiana como las actividades enzimáticas proporcionaron información importante para
monitorear el proceso de compostaje. Debe prestarse más atención a la fase de maduración
para optimizar el proceso de compostaje.
Palabras claves: actividad enzimática, ácidos grasos fosfolípidos (PLFA), comunidad
microbiana, compostaje, maduración
Waste Management 54 (2016) 83–92
Contents lists available at ScienceDirect
Waste Management
journal homepage: www.elsevier .com/locate /wasman
Evolution of microbial dynamics during the maturation phase of thecomposting of different types of waste
http://dx.doi.org/10.1016/j.wasman.2016.05.0110956-053X/� 2016 The Authors. Published by Elsevier Ltd.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
⇑ Corresponding author.E-mail address: [email protected] (I. Villar).
Iria Villar ⇑, David Alves, Josefina Garrido, Salustiano MatoDepartment of Ecology and Animal Biology, University of Vigo, 36310 Vigo, Spain
a r t i c l e i n f o
Article history:Received 30 December 2015Revised 9 May 2016Accepted 10 May 2016Available online 25 May 2016
Keywords:Enzyme activityPhospholipid fatty acid (PLFA)Microbial communityCompostingMaturation
a b s t r a c t
During composting, facilities usually exert greater control over the bio-oxidative phase of the process,which uses a specific technology and generally has a fixed duration. After this phase, the material isdeposited to mature, with less monitoring during the maturation phase. While there has been consider-able study of biological parameters during the thermophilic phase, there is less research on the stabiliza-tion and maturation phase. This study evaluates the effects of the type of starting material on theevolution of microbial dynamics during the maturation phase of composting. Three waste types wereused: sludge from the fish processing industry, municipal sewage sludge and pig manure, each indepen-dently mixed with shredded pine wood as bulking agent. The composting system for each waste typecomprised a static reactor with capacity of 600 L for the bio-oxidative phase followed by stabilizationand maturation phase in triplicate 200 L boxes for 112 days. Phospholipid fatty acids, enzyme activitiesand physico-chemical parameters were measured throughout the maturation phase. The evolution of thetotal microbial biomass, Gram + bacteria, Gram � bacteria, fungi and enzymatic activities (b-glucosidase,cellulase, protease, acid and alkaline phosphatase) depended significantly on the waste type (p < 0.001).The predominant microbial community for each waste type remained present throughout the maturationprocess, indicating that the waste type determines the microorganisms that are able to develop at thisstage. While fungi predominated during fish sludge maturation, manure and municipal sludge were char-acterized by a greater proportion of bacteria. Both the structure of the microbial community and enzy-matic activities provided important information for monitoring the composting process. Moreattention should be paid to the maturation phase in order to optimize composting.� 2016 The Authors. Published by Elsevier Ltd. This is anopenaccess article under the CCBY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Composting is a process of biological degradation of solidorganic substrates under aerobic conditions through the action ofdifferent microbial populations, yielding a stable, humidified andsuitable product to add to the soil (Insam and de Bertoldi, 2007).The organic material goes through different phases: a mesophilicphase, characterized by the proliferation of the microbiota, a ther-mophilic phase where a high rate of biodegradation, the growth ofthermophilic organisms and the inhibition of non-thermotolerantorganisms occur and the final phase that includes a period of cool-ing, stabilization and maturation, characterized by the growth ofmesophilic organisms and the humification of the compost(Ryckeboer et al., 2003b). In the composting facilities the matura-tion phase is usually carried out with less control and monitoringthan the bio-oxidative phase. The duration of the bio-oxidative
phase that is carried out in bioreactors depends upon the type ofsubstrate that is used but generally lasts from 7 to 15 days. Afterthis phase, the material that exits the reactor generally is placedin windrows for a curing phase (Diaz et al., 2007). The timerequired for the maturation phase is a function of the substrateand environmental and operating conditions of the facility andcan range from a few weeks to a year or two (Diaz et al., 2002). Thislack of control over the process may cause environmental prob-lems such as odours and leachates, in addition to adversely affect-ing the quality of the compost.
The maturation phase has mainly been studied in terms of thephysico-chemical and biological parameters in order to determinewhen compost is mature enough to be added to the soil by estab-lishing maturity and stability criteria and indexes of the final pro-duct (Bernal et al., 2009; Insam and de Bertoldi, 2007; Paradeloet al., 2010). In terms of biological parameters, several enzymaticstudies have been carried out to determine microbiological activityduring composting and provide indicators of the stability of differ-ent composts (Cayuela et al., 2008; Ros et al., 2006). Castaldi et al.
Table 1Physico-chemical characteristics of the wastes used in the composting experiments:sludge from fish processing industry (FI), municipal sewage sludge (MSS) and pigmanure (PM).
FI MSS PM
Moisture (%) 65.4 87.0 82.8Organic matter (%) 93.0 73.1 79.6Total carbon (mg g�1 dw) 532.5 364.0 402.1Total nitrogen (mg g�1 dw) 26.8 46.5 31.9Water soluble carbon (mg g�1 dw) 20.50 2.54 19.78Dissolved organic nitrogen (mg g�1 dw) 10.05 9.07 10.18Total phosphorus (mg g�1 dw) 7.16 20.01 16.07Fats (% dw) 22.3 6.0 15.6
dw: dry weight.
84 I. Villar et al. /Waste Management 54 (2016) 83–92
(2008) proposed that the study of dynamics of certain enzymaticactivities, without single point determinations, could be a suitableindicator of stability, although it was not possible to establish athreshold value. However, the study of enzyme activities providesinformation on the breakdown of organic matter and the metabolicprocesses that take place during composting and, therefore, onproduct stability.
Most studies on the structure of the microbial community incomposting have specifically focused on the early stages of the pro-cess because, under aerobic conditions, temperature is the biggestselective factor of microbial populations (Ryckeboer et al., 2003b).Likewise, the nature of the organic substrates is also an importantfactor in determining the dynamics and microbial diversityduring composting (Klammer et al., 2008; Ryckeboer et al.,2003b; Vargas-García et al., 2010). Ishii and Takii (2003) showedthat the main factor affecting microbial communities was theconcentration of dissolved organic substances, which dependedon the type of starting material. López-González et al. (2015)showed that the composition of fresh materials and operatingconditions determine how the microbiota behaves, as well as itsstructure and its biodiversity.
Phospholipid fatty acid (PLFA) profiles analysis is a techniquethat provides information about the structure of the microbialcommunity and how it changes during composting. The totalamount of PLFAs can be used as an indicator of viable microbialbiomass. Furthermore, some PLFAs are specific to certain livingorganisms (e.g. bacteria, fungi, actinomycetes and plants) whichmeans they can be used as biomarkers for the presence and abun-dance of specific microbial groups (Zelles, 1999). There have beenstudies of the evolution of PLFAs during the composting of differ-ent wastes, with greatest emphasis on the initial stages of the pro-cess and some authors including on time sampling during thematuration phase (Amir et al., 2010; Eiland et al., 2001;Hellmann et al., 1997; Klamer and Bååth, 1998). Jindo et al.(2012) found that after 12 weeks of composting, factor analysisbased on the relative abundance of individual PLFAs revealedchanges in the structure of the microbial community thatdepended on the original organic waste. Boulter-Bitzer et al.(2006) studied the microbial community of different compostsduring maturation and storage, noting that PLFA analysis was avaluable method for characterizing the microbial communitystructure during this phase of the composting process.
The study of biological parameters during stabilization andmaturation and the influence of the source material can helpimprove the quality of compost and optimize the composting pro-cess, meaning that a better understanding of changes in the micro-bial dynamics is necessary for the maturation phase of composting.The objectives of this research were: (1) to study the developmentand structure of the microbial community using PLFAs and enzy-matic activities during the maturation phase of the compostingprocess; (2) evaluate the effect of the type of waste on the micro-bial structure and activity; and (3) improve and optimize the com-posting process by providing more information on the maturationphase.
2. Materials and methods
2.1. Composting materials
Composting experiments were performed using three differentwaste types whose main physico-chemical properties are detailedin Table 1:
– Sewage sludge from the food industry (FI), from precooked andfrozen fish and cephalopods, obtained after separation of fatsand treatment with coagulants and flocculants.
– Manure from a pig-breeding farm (PM), collecting the solidfraction of slurry after storage in manure pits.
– Municipal sewage sludge wastewater (MSS) obtained afteraerobic digestion in lagoons and dewatering with band filter.
Shredded pine wood passed through a 3 cm sieve was used as abulking agent and each waste type was mixed with this agent toobtain a ratio 1:2 (v/v), a free air space (FAS) of 30–40% and a mois-ture content of around 60–70%.
2.2. Experimental design
After applying the bulking agent, each waste type was subjectedto a composting process in which the bio-oxidative phasetook place in a static reactor with forced ventilation and thestabilization and the maturation phase (hereinafter referred to asthe maturation phase) were carried out in triplicate in 200 Lbatches (Fig. 1).
The adiabatic composting reactor had an effective volume of600 L, a perforated floor and a ventilation system with the abilityto introduce fresh or recirculated air through the top and bottomof the reactor. The temperature and oxygen level were recordedevery minute using a Eurotherm controller with three temperatureprobes at different depths and a gas probe inside the mass with anoxygen sensor. A feedback loop of oxygen and temperature (venti-lation when temperatures exceeded 60 �C or oxygen fell below 5%)and a time controller were used for aeration. The material was keptin the composting reactor until the temperature fell below 35 �C,requiring a total of 20, 18 and 17 days for FI, PM and MSS, respec-tively. After emptying the reactor, each waste type was mixed andplaced in triplicate in maturation systems of 200 L. Wooden boxesof 70 � 54 � 54 cm with a perforated base and open top were usedfor the maturation systems to allow gas exchange with the outside,attempting to simulate the inside of a maturation pile by maintain-ing some isolation from the outside. Similarly, to take placecorrectly, composting requires moisture balanced conditions toprevent the occurrence of water stress that might generate biolog-ical inactivity and false compost stabilization. A layer of bulkingagent was placed at the top and bottom of the box to provide ther-mal insulation for the waste and prevent moisture loss. Mesh wasplaced between the composted material and the bulking agent toprevent them mixing.
The beginning of the maturation process (day 0) started afteremptying the reactor. Maturation systems were emptied andmixed by hand at 14, 28, 42, 56, 70 and 91 days to simulate thedynamics of turning a pile of compost under maturation. A com-posite sample was taken of each system during each turning. Thetotal volume of the composite sample was 1500 mL. This wassieved through a 1 cm mesh to remove the bulking material. Thetemperature and oxygen level was monitored daily for the first42 days and three times per week until the end of the process at112 days. The moisture level was maintained above 60%, except
Centrifugal fan
Control andregistrationsystem
Temperature probesGas intake probe
plenum chamber
Ventilation system
Biofilter
bulking agent
compost
mesh
perforatedbase
woodenbox
Fig. 1. Scheme of the reactor and the maturation boxes used during the composting process for each waste type.
I. Villar et al. /Waste Management 54 (2016) 83–92 85
for FI, which required periodic rewetting in the first weeks of theprocess related to its highly hydrophobic nature due to a high lipidcontent (Table 1) and the higher temperatures reached at the startof the maturation phase.
2.3. Chemical analysis
The moisture and organic matter contents of the samples weredetermined after drying at 105 �C until constant weight and ashingat 550 �C for 4 h, respectively. Fresh samples were extracted with0.5 M K2SO4 in a ratio 1:10 (w/v) for analyses of inorganic nitrogen(N-NH4
+ and N-NO3�) (Sims et al., 1995) and dissolved organic nitro-
gen content (DON) (Cabrera and Beare, 1993). Total nitrogen con-tent (TN) and total carbon content (TC) were determined bycombustion of dried samples using a LECO 2000 CN elemental ana-lyzer. Water soluble carbon content (WSC) was analyzed in aque-ous extracts 1:5 (w/v) by dichromate oxidation in sulfuric acidsolution (Sims and Haby, 1971). Electrical conductivity was deter-mined in aqueous extracts 1:10 (w/v) using a conductivimeter Cri-son CM 35.
2.4. Biological and biochemical analysis
The microbial community composition and biomass weredetermined by phospholipid fatty acid (PLFA) analysis followingthe method described by Gómez-Brandón et al. (2010) for organicsamples. Briefly, total lipids were extracted by stirring from200 mg of each freeze-dried sample with 60 mL of chloroform–methanol (2:1, v/v). Phospholipid fraction was obtained after sep-aration on silicic acid columns and was subjected to derivatizationwith trimethylsulfonium hidroxyde (TMSH). Fatty acid methylesters (FAMEs) obtained were analyzed by gas chromatographyand mass spectrometry (GC–MS). GC–MS analysis was performedon a column CP-Select FAME, 100 m � 0.25 mm. FAMEs wereidentified by comparison of their retention time and mass spectrawith known standards (Larodan Fine Chemicals AB, Malmo,Sweden). The quantification was performed with methylnonadecanoate fatty acid (C19:0) as internal standard. PLFAs wereused to estimate the biomass of specific microbial groups:gram-positive bacteria (i14:0, i15:0, a15:0, i16:0, a17:0),gram-negative bacteria (16:1x7, 17:1x7, 18:1x7, cy19:0) andfungi (18:2x6, 18:1x9, 20:1x9). The total amount of PLFAsidentified (totPLFAs) was used as an indicator of the viable micro-bial biomass (Zelles, 1999).
b-glucosidase was estimated by incubating 1 g of fresh samplewith 1 mL of p-nitrophenyl-b-D-glucopiranoside (0.025 M) for 1 hat 37 �C and subsequent colorimetric measurement ofp-nitrophenol released (Eivazi and Tabatabai, 1988). Alkaline andacid phosphatase were measured by incubating 0.5 g of fresh sam-ple with 1 mL of p-nitrophenylphosphate (0.015 M) for 1 h at 37 �Cand subsequent colorimetric measurement of p-nitrophenolreleased (Eivazi and Tabatabai, 1977). Protease was measured bycolorimetric determination of the amino acids released, after theincubation of 1 g of fresh sample with 5 mL of sodium caseinate(2%) for 2 h at 50 �C, using Folin-Ciocalteu reagent (Ladd andButler, 1972). Cellulase was assessed by colorimetric determina-tion of reducing sugars released after incubation of 5 g of freshsample with 15 mL of carboxymethyl cellulose sodium salt (0.7%)for 24 h at 50 �C (Schinner and von Mersi, 1990).
Germination index (GI) was calculated according to Zucconiet al. (1981) by determining seed germination and root length ofLepidium sativum growing in 2 mL of aqueous extracts 1:5 (w/v)in Petri dishes lined with paper filter during 48 h.
Static respiration rate (SR) was measured using manometricrespirometers by OxiTop� system (WTW GmbH, Weilheim,Germany). Briefly, fresh weight equivalent to 4 g of dried samplewas placed in a hermetic container with a 1 M NaOH trap tocapture CO2, and the pressure drop, due to microbial oxygenconsumption, was recorded during 24 h at constant temperature.The self-heating test was carried out using 2 L Dewar flask for10 days at room temperature (TMECC, 2002).
2.5. Statistical analysis
All statistical tests were performed using R software (RDevelopment Core Team, 2014). The physico-chemical data wassubjected to principal component analysis (PCA) after normaliza-tion to zero mean and unit variance, and the analysis wasperformed on the correlation matrix. The principal componentswith eigenvalues greater than one were retained. The PCA wasperformed using the prcomp function and the package ggbiplot(Vu, 2011). Enzyme and PLFA data was analyzed with linearmixed-effects models using the nlme package (Pinheiro et al.,2015). The waste type and time were fixed factors and the repeatedmeasurement throughout time in each maturation box was treatedas a random effect to address the non-independence of samples.Logarithmic and square root transformations of the data were nec-essary to ensure the normality and homogeneity of the variance of
86 I. Villar et al. /Waste Management 54 (2016) 83–92
residuals of models. For post hoc comparison between treatments,Tukey tests were carried out using the glht function of the mult-comp package (Hothorn et al., 2008). Correlation analyses werealso carried out to examine the relationships between PLFAs andenzyme activities with cor function of the stats package. All statis-tical tests were evaluated at the 95% confidence level and valuesare given as the mean ± standard error.
3. Results
3.1. Temperature evolution
The temperature inside the reactor increased rapidly at thebeginning of the process (Fig. 2), reaching over 45 �C on days oneand two for FI and PM, respectively, and both wastes maintainedthermophilic temperatures for more than 10 days. For MSS, ther-mophilic temperatures were reached inside the reactor on day fourand were maintained for seven days.
At the beginning of the maturation phase, the temperature of FIincreased to 60 �C, falling on day four and reactivating after turningat 14 and 28 days, although in the latter cases, the temperatureremained below 40 �C. Both PM and MSS generally remainedbelow 25 �C during the maturation phase. In terms of the oxygenlevels during the maturation phase, measurements remainedabove 17% for all waste types.
3.2. Composting parameters
The principal component analysis of the physico-chemical vari-ables is presented in Fig. 3 and shows the three separate groups forthe three different waste types. The samples taken during matura-tion of FI exhibited the greatest dispersion along the axes. Theparameters responsible for the differences between waste typesalong principal component one (PC1) were WSC (r = �0.97,p < 0.0001) and moisture (r = 0.89, p < 0.0001), differentiating sam-ples during the maturation of FI, PM and MSS. During the process,FI maintained a high concentration of WSC, with a maximum of25.6 mg g�1 at the beginning of the maturation phase and a mini-mum of 9.1 mg g�1 at 112 days. Both PM and MSS reached a max-imum of around 7.5 mg g�1, with a drop throughout thematuration process to achieve significantly lower values in PMthan MSS (Table 2). In terms of moisture, PM and MSS remainedat around 60–70% throughout the process, while FI required peri-odic rewetting during the first seven weeks, obtaining minimumvalues of 35% by day 28 and remaining between 45% and 55% fromday 42 until the end of the process. The parameter with the biggestcontribution to the separation of the waste along principal compo-nent two (PC2) was the ratio C/N (r = 0.92, p < 0.0001), primarily
Fig. 2. Temperature evolution during the reactor phase and the maturation phasefor sludge from fish processing industry (FI), municipal sewage sludge (MSS) andpig manure (PM). Arrows correspond to the turnings after emptying the reactor(time 0) and during the maturation phase (14, 28, 42, 56, 70, 91 days).
differentiating between PM and MSS. In the latter one, values ofC/N > 12 were not detected along the maturation phase, whilePM and FI reached around 18 at the beginning of maturation, withsignificantly lower values for FI at the end of the process (Table 2).
3.3. Microbial community
Microbial biomass, Gram + bacteria, Gram � bacteria and fungiwere significantly different between wastes (p < 0.001). Likewise,significant differences caused by time (p < 0.0001) and significantinteractions between time and waste type (p < 0.0001) for allmicrobial biomass and microbial groups were observed.
The highest concentration of microbial biomass was observed inMSS at the beginning of maturation and fell progressively overtime (Fig. 4). This reduction was also present in the group indica-tors, falling to about 97.8% in Gram + bacteria, 99.5% in Gram �bacteria and 99.3% in fungi. At the beginning of the process theproportion of PLFAs of a specific group with respect to total PLFAsgroup indicators was higher in Gram� bacteria (>40%), whereas bythe end, the greater proportion was in Gram + bacteria (>60%).Hence, more PLFAs characteristic of bacteria than fungi ones wereobserved during the process. Finally, the microbial biomass of MSSduring maturation was correlated with all microbial groups(r > 0.98, p = 0.000).
In the case of FI, there was an increase in microbial biomassduring the first weeks of the process, declining in the followingsamplings and showing a significant increase during the last21 days of maturation. The microbial biomass was strongly corre-lated with the concentration of fungi (r = 0.915, p = 0.000). In thiscase, the predominance of PLFAs characteristic of fungi was main-tained at over 55% during the process.
Furthermore, PM presented less microbial biomass than theother ones in the first samplings, gradually falling until day 56and later recovering to values slightly below the initial value. Ahigh correlation between microbial biomass and the PLFAs charac-teristic of Gram + and Gram � bacteria (r > 0.93, p < 0.0001) wereobserved. During the maturation phase, bacteria predominated(values above 75%), especially Gram + bacteria.
3.4. Enzyme activities
The type of waste significantly affected the evolution of allhydrolytic activities studied (p < 0.001). Also, significant differ-ences caused by time (p < 0.0001) and interaction between timeand waste (p < 0.0001) were also observed for all the enzymes.
After intensive composting in the reactor, MSS presented thehighest activity of b-glucosidase (Fig. 5a), which fell significantlyin the first 14 days and remained stable from 70 days, resultingin a final reduction of 85%. In contrast, at the start of the matura-tion phase, PM and FI showed similar values of b-glucosidase,but from day 56, PM had significantly higher values than the otherwaste types.
With respect to cellulase (Fig. 5b), increased activity wasobserved in PM, except for the last sampling where similar resultswere found in FI. In the case of MSS, a continued reduction in cel-lulase activity was detected, while it increased slightly for FI in thefinal maturation sampling.
The acid and alkaline phosphatase activities exhibited similartrends for the same waste type (Fig. 5c and d, respectively), exceptthe initial samplings of PM. Acid phosphatase activity was similarto b-glucosidase activity, with higher levels in MSS for initial sam-plings and high activity at the end of the process for PM. However,FI remained significantly lower throughout maturation. Despitethe different activity levels observed at the reactor outlet, FI andMSS showed similar trends of alkaline phosphatase enzymes, withactivity peaking on day 42, followed by a sharp decline to similar
Fig. 3. Correlation biplot of principal component analysis where vectors correspond to the variables that define the components and points correspond to sampling duringthe maturation phase for sludge from fish processing industry (FI), municipal sewage sludge (MSS) and pig manure (PM).
Table 2Parameters of composts after 112 days of maturation phase for sludge from fishprocessing industry (FI), municipal sewage sludge (MSS) and pig manure (PM).
FI MSS PM
Ratio C/N 12.25 ± 0.11a 11.53 ± 0.20b 16.31 ± 0.31c
Ratio NH4+/NO3
� 0.24 ± 0.03a 0.09 ± 0.01b 0.56 ± 0.03c
SR (mg O2 g�1 OM h�1) 0.53 ± 0.01a 0.33 ± 0.02b 0.78 ± 0.01c
Self-heating test Class V Class V Class VGI (%) 91.3 ± 2.1a 99.5 ± 1.1b 94.1 ± 1.4a
WSC (mg g�1 dw) 9.11 ± 0.15a 3.50 ± 0.20b 2.67 ± 0.15c
SR: static respiration, GI: germination index, WSC: water soluble carbon, dw: dryweight.In each parameter the different letters indicate significant differences betweencomposts (Tukey post hoc test p < 0.05).
I. Villar et al. /Waste Management 54 (2016) 83–92 87
values for both waste types at the end of the process. Similar toacid phosphatase and b-glucosidase, PM showed significantlyhigher values of alkaline phosphatase than MSS and FI in the finalsamplings.
Only the protease enzyme (Fig. 5e) exhibited similar trends inall wastes, with reduction rates of 80%, 72% and 22% for PM, MSSand FI respectively. In all cases, activity stabilized in the finalsamplings.
As shown in Table 3, microbial groups for MSS were positivelycorrelated with all enzymatic activities. In contrast, in FI, onlyPLFAs characteristic of fungi were positively correlated with theb-glucosidase and alkaline phosphatase enzymes. With respect tobacterial biomass for PM, both Gram + and Gram � bacteria werecorrelated with alkaline phosphatase, protease and cellulase activ-ities, while fungal biomass was positively correlated with cellulase.
4. Discussion
4.1. Temperature evolution
Unlike FI, PM and MSS exhibited biological degradation oforganic matter prior to composting, the former during storage inseptic tanks for solid-liquid separation of the pig slurry and the lat-
ter during treatment of wastewater in aeration tanks. Conse-quently, both had a lower content of organic matter than FI(Table 1), allowing the full thermophilic phase to take place inthe static reactor. The materials did not require turning to reacti-vate the process, showing that the forced ventilation was effective,and both fresh composts matured in boxes with stable and envi-ronmental temperatures. In the case of FI, however, turningfavoured the increase in temperature at the beginning (day 0),and at 14 and 28 days, meaning that forced ventilation was not suf-ficient to allow full development of the bio-oxidative phase of thecomposting process in the reactor. Alburquerque et al. (2009)showed that forced ventilation was effective when performedtogether with mechanical turning during alperujo (olive waste)composting, concluding that turning improved the porosity andhelped distribute the moisture, substrates and microorganisms.During treatment of wastewater from fish processing a digestionprocess was not performed, meaning that the organic load of thesludge was high (Table 1), and turning and watering during thefirst weeks allowed re-heating to provide biodegradable substratesfor the microorganisms from the outside to the inside of the box.Despite this re-heating, FI kept the characteristics of a maturationprocess with temperatures similar to MSS and PM from the firstmonth. So, in a composting facility, this highly organic wasteshould be monitored when it is disposed to mature, to preventfalse stabilizations of compost, odours and other environmentalproblems.
4.2. Multivariate analysis
Multivariate analysis shows that the source material deter-mined the physico-chemical development of the stabilization andmaturation process for composting. Several authors have foundthat composts obtained from different organic waste types differin their physico-chemical composition and, hence, in terms of theirstabilities and qualities, depending on the composition of sourcematerial used in the composting (Bernal et al., 1998; Ranalliet al., 2001). Similarly, the physico-chemical properties during
Fig. 4. Changes in (a) microbial biomass, (b) fungi, (c) Gram + bacteria and (d) Gram � bacteria estimated by phospholipid fatty acid (PLFA) analysis during the maturationphase for sludge from fish processing industry (FI), municipal sewage sludge (MSS) and pig manure (PM).
88 I. Villar et al. /Waste Management 54 (2016) 83–92
the maturation phase determined the separation of the three wastetypes and affected the final composition of the composts. FIshowed the greatest changes in physico-chemical compositionduring maturation, possibly due to its initial re-activation causedby its high organic load, as observed in the temperature profile.The highest respiratory activities and contents of C and N formsoccurred in FI, meaning more time may be required for stabiliza-tion. The maturation process of PMwas characterized by maintain-ing the C/N ratio at a low value (Bernal et al., 2009), suggesting apossible stabilization of the degradation process of organic matter.However, Fialho et al. (2010) have shown that the C/N ratio is not agood method for monitoring the composting process and thatthere is not an optimal ratio that characterizes humified compost.Finally, MSS was characterized by high levels of ammonia due tothe low C/N ratio present throughout the whole process. DeGuardia et al. (2008) have shown that an excess of aeration afterthe thermophilic phase could be responsible for the loss of N. Thus,stabilization of the composting parameters of MSS suggested thatthere were too much turnings or time processing for this type ofwaste, with the potential to increase the volatility and loss of Nduring the maturation phase.
4.3. Compost quality
All compost types presented parameters of stability and matu-rity as were highest rating during the self-heating test (class V,mature compost) and values of GI above 80% indicative of absenceof phytotoxic substances for plant growth (Riffaldi et al., 1986).However, MSS showed a higher degree of maturity and stabilitybecause it had a respiratory rate below 0.5 mg O2 g�1 SV h�1
(Iannotti et al., 1993), a ratio C/N below 12 and an ammonia/nitrateratio below 0.16 (Bernal et al., 1998). Likewise, Garcia et al. (1991)proposed the use of WSC content as a useful method for determin-ing the maturity of a compost, with a value below 5 mg g�1 formature compost. In this experiment, FI exhibited higher values ofthis content. Both PM and FI may require more time to achieve
similar maturity and stability parameters to MSS, even thoughboth were classified as ‘‘mature” according to the rates in TMECC(TMECC, 2002). Although the waste types studied underwent thesame treatment in composting, they exhibited different stabilityand maturity levels. As such, it is important to design and controlthe composting process as a whole, paying particular attention tothe maturation phase, where it is possible to design an ad hoc pro-cess to yield higher quality compost in less time, depending on thephysico-chemical properties of each type and its evolution overtime.
4.4. Microbiological evolution
Changes in both enzyme activities and the microbial commu-nity during the maturation phase were determined by the individ-ual features of organic waste types.
The decline in microbial biomass in MSS was consistent withprevious studies (Garcia et al., 1992; Klamer and Bååth, 1998;Mondini et al., 2004; Ros et al., 2006) using different methods ofquantification (Biolog, ATP, fumigation-extraction, PLFA) andmicrobial monitoring during the maturation phase. As mentioned,previous research has focused on the most intensive phase of com-posting, although some studies include occasional samplings dur-ing the maturation phase. Garcia et al. (1992) observed that thecontinued decrease in microbial biomass during the compostingof municipal solid waste could be attributed to the slow andincomplete stabilization of organic matter. Here it should be notedthat the low C/N ratio observed throughout the maturation of MSScould cause a shortage of available carbon, leading to a progressivedecline in microbial biomass, slowing down the process for thedegradation of organic matter. However, the results in Table 1show optimal properties for compost, suggesting the drop inmicrobial biomass was the result of the maturity and stability ofthe organic matter. In terms of the structure of the microbial com-munity, the abundance of PLFAs characteristic of bacteria detectedthroughout the maturation phase was high and could be attributed
Fig. 5. Changes in (a) b-glucosidase, (b) cellulase, (c) acid phosphatase, (d) alkaline phosphatase and (e) protease during the maturation phase for sludge from fish processingindustry (FI), municipal sewage sludge (MSS) and pig manure (PM).
I. Villar et al. /Waste Management 54 (2016) 83–92 89
to the origin and physico-chemical properties of the waste. Themain and most important microbial population that develops inwastewater treatment systems is bacteria, especially Gram � bac-teria such as Proteobacteria and Bacteroidetes, and Gram + bacteriasuch as Actinobacteria (Wagner and Loy, 2002). Furthermore, fol-lowing the phase of thermophilic composting, the recolonizationof the compost with mesophilic microorganisms of the environ-ment occurs, dominated by the bacteria of the phylum Bacteroide-tes (Insam and de Bertoldi, 2007; Ryckeboer et al., 2003b). Eilandet al. (2001) have found that bacteria dominated the microbialcommunity throughout the composting process when C/N waslow, although all treatments studied showed a fungi/bacteria ratioof less than 0.5. The waste used (straw with various additions ofslurry) was a predominantly bacterial medium for all tested mix-tures and throughout composting. Ishii and Takii (2003) observedsimilar bacterial communities in different composting sewagesludge processes, including Bacillus, Actinobacteria and Gram �bacteria. These authors have suggested that microorganisms thatproliferate in composting processes adapt to the composting envi-ronment and are selected by factors within the composting mate-rials. Furthermore, in a previous study using sewage sludge fromthe same wastewater plant, a predominance of PLFAs typical of
bacteria were observed in both vermicomposting and the com-bined process composting-vermicomposting (Villar et al., 2016).So, the physico-chemical factors exhibited by MSS characterizedthe microbial biomass that developed during the maturationprocess.
The microbial biomass for FI developed in a similar way to pre-vious studies on PLFAs (Boulter-Bitzer et al., 2006; Hellmann et al.,1997), with an increase in the abundance of PLFAs in the finalstages of composting. The microbial biomass growth in FI wascaused by an increase in PLFA fungi biomarkers. It is normal forfungi to increase during the maturation phase of composting as aresult of the breakdown of substrates of difficult degradation andless aggressive environmental factors (Albrecht et al., 2010;Hassen et al., 2001) and there is evidence of an increase in fungaldiversity (Shemekite et al., 2014). However, FI showed greaterabundance of PLFA fungi biomarkers than bacterial ones through-out the maturation process. The lipidic nature of the starting mate-rial and its highWSC content throughout maturation might involvethe proliferation of saprophytic sugar fungi, such as Zygomycetesspecies, in early stages of the maturation and the proliferation ofcellulolytic fungi during the final phase (Richardson, 2009;Ryckeboer et al., 2003b). Similarly, Amir et al. (2010) found that
Table 3Correlation matrix between enzyme activities and PLFAs during the maturation phasefor sludge from fish processing industry (FI), municipal sewage sludge (MSS) and pigmanure (PM).
totPLFAs Gram + Gram � Fungi
F1b-Glucosidase 0.358* NS �0.531** 0.384*
Cellulase NS �0.411* NS NSAcid phosphatase NS NS NS NSAlkaline phosphatase NS NS �0.421* 0.409*
Protease NS NS �0.627** NSMSSb-Glucosidase 0.921** 0.887** 0.900** 0.911**
Cellulase 0.892** 0.850** 0.889** 0.888**
Acid phosphatase 0.549** 0.582** 0.464** 0.564**
Alkaline phosphatase 0.702** 0.704** 0.634** 0.719**
Protease 0.821** 0.827** 0.756** 0.831**
PMb-Glucosidase NS NS NS NSCellulase 0.581** 0.459* 0.506** 0.590**
Acid phosphatase NS NS NS �0.401*
Alkaline phosphatase 0.644** 0.715** 0.633** NSProtease 0.720** 0.702** 0.601** NS
NS: not significant.* Indicates significance at the 0.05 probability level.
** Indicates significance at the 0.01 probability level.
90 I. Villar et al. /Waste Management 54 (2016) 83–92
the presence of fungi was greater in waste with a high fatty acidcontent, which impeded the stabilization and maturation of thecompost. Hence, the maintenance of high content of fungi couldbe indicative of the lack of stabilization during the maturationphase of this type of waste.
For PM, the stabilization of microbial biomass at the end of mat-uration suggested nutrients were available to the microorganisms,probably due to the high content of cellulosic materials, such asstraw and remnants of seeds that are normally present in pig slurryand degrade more slowly. Tiquia et al. (2002a) found ATP stabiliza-tion during pruning composting, indicating the maturity of thecompost and suggesting a change in the microbial community tomore specialized microbial groups, such as fungi and Actino-mycetes. However, as observed for MSS, bacterial biomass wasgreater than fungal biomass during the maturation process, partic-ularly PLFA biomarkers of Gram + bacteria. Elouaqoudi et al. (2015)have suggested that the increase in Gram + bacteria in the finalphase of composting indicates the availability of organic substratesdue to the breakdown of lignocellulosic compounds. Moreover, theorigin of waste, which characterizes its physico-chemical proper-ties, may influence the development of the microbial communityduring the maturation phase, since pig manure is an environmentrich in bacteria with a high content of fermentative microbialgroups, especially Gram + bacteria, and bacteria dominate duringthe initial phase of the decomposition of manure due to the highavailability of water and compounds that can be easily brokendown (Domínguez et al., 2010; Snell-Castro et al., 2005).
The change in the microbial community during the maturationprocess appears to be affected by the adaptation of microorgan-isms to mesophilic conditions and the different physico-chemicalproperties of different sources of waste, meaning there is a contin-uous turnover of the microbial groups while maintaining the influ-ence of the nature of the starting material and the predominantmicrobial groups.
The study of enzymatic activities during maturation showed thedynamics of the metabolic processes of the carbon, nitrogen andphosphorus cycle and thus hydrolytic enzymes were indicative ofthe evolution of organic matter and the biological activity duringthe final phase of the composting process.
In terms of MSS, the high correlation between all enzymes andmicrobial biomass indicates the enzyme activities during
maturation were a direct result of the microbial community, bothbacterial and fungal. The sharp decline of enzymes and PLFA con-tent over time suggests a decrease of available substrates formicroorganisms. At the beginning of the process, MSS had thehighest b-glucosidase and phosphatase activities. The low C/N ofthis waste may require the synthesis of b-glucosidase, since thelimitation of carbon with respect to the available nitrogen andphosphorus provides a strong incentive for microorganisms toinvest in the acquisition of carbon (Allison and Vitousek, 2005).Likewise, García et al. (1993) indicated that high values ofphosphatase activity in sewage sludge can be induced by thepresence of phosphate compounds from detergents in wastewater.b-glucosidase, protease and acid phosphatase enzymes showedstabilization in the final samplings while alkaline phosphatasecontinued to decline and cellulase increased slightly in the finalsampling. Castaldi et al. (2008) observed a decrease in bothenzyme activities and water-soluble fractions during compostingof the organic fraction of municipal solid waste with plant waste,indicating a stabilization of hydrolytic enzymes and hence theorganic matter during the maturation phase.
In contrast to MSS, no correlation was observed between themeasure of microbial biomass and enzymes, except forb-glucosidase during maturation of FI, suggesting the enzymeactivities were not directly associated with the development ofthe microbial biomass. Burns (1982) proposed that the enzymesmay be located in dead cells, cell debris or stabilized in organiccomplexes and remain as active hydrolyzing substrates. A furtherstudy by Mondini et al. (2004) found no correlation between themicrobial biomass carbon and different enzyme activities duringthe composting of different wastes, indicating stabilization ofextracellular enzymes due to the formation of complexes withhumic substances. During maturation, FI exhibited low enzymeactivities, compared to MSS and PM. The high content of nutrientsdirectly available for microorganisms, such as WSC and DON, couldinhibit the production of enzymes. Cellulase and protease activitiesincreased at the end of the process, indicating that the fall insoluble organic matter is accompanied by an increase in thehydrolysis of more complex organic compounds (Tiquia et al.,2002b).
In terms of the hydrolytic activity of PM, particularly high cellu-lase activity was observed throughout the maturation. However,this was in line with expectations of high cellulosic activity duringcomposting of pig manure due to the predominance of celluloseand hemicellulose in this type of waste (Iannotti et al., 1979). Bothbacteria and fungi determined cellulase activity and the predomi-nance of bacteria throughout the process resulted in the presenceof a significant cellulolytic bacterial community during maturationof PM, as was observed by Ryckeboer et al. (2003a) during the com-posting of vegetable, fruit and garden waste. Moreover, b-glucosidase and phosphatases increased at the end of the matura-tion process, with a notable progressive increase in alkaline phos-phatase over time. The highest level of enzymatic activity in thePM compost was similar to that shown by other authors(Cayuela et al., 2008; Tiquia and Tam, 1998) and was indicativeof the presence of available nutrients for the microbial biomass,especially bacteria, and/or the protection of enzymes in humiccomplexes, as indicated by the lack of correlation between b-glucosidase and acid phosphatase with the microbial community.The protease enzyme was of particular importance, since itdecreased during the maturation of PM, FI and MSS and stabilizedat the end of the process. This was the only enzyme to be positivelycorrelated with WSC (p < 0.01) in the three waste types, suggestingthe availability of carbon regulates the synthesis of proteases(Allison and Vitousek, 2005). Indeed, Lazcano et al. (2008) sug-gested that protease may be indicative of the degradation oforganic matter because of an extreme dependence on the availabil-
I. Villar et al. /Waste Management 54 (2016) 83–92 91
ity of substrate. Hence, the increased availability of substrates andtherefore the higher protease activity at the end of the process wasobserved in FI, PM and finally MSS.
The different correlation between the microbial groups andenzymatic activities during maturation provided informationon the state of the degradation of organic matter andcompost quality, as well as the requirement for more or lessprocessing time and handling. The authors recommend moreresearch on different maturation systems and waste typeswith particular attention to biological parameters during thematuration phase.
5. Conclusions
Taken together, the structure of the microbial community andenzymatic activities provide important information for monitor-ing the composting process and on the stability and maturity ofcompost. Low enzymatic activity is not indicative of stabilization,as observed by the high microbial community present in sludgefrom the fish processing industry, although a decrease in bothenzymatic activity and microbial community may indicate stabil-ity, as was the case during the maturation of municipal sewagesludge. The predominant microbial community for each type ofwaste remained present during the maturation process, indicatingthat the different origin of the waste and the different physico-chemical properties determine the microorganisms that are ableto develop at this stage. Although waste types were subjectedto the same composting process, the level of stability andmaturity was different and this represents an important factorfor designing ad hoc processes and controlling the compostingprocess according to waste type, paying particular attentionto the process as a whole, including maturation. It is importantto monitor microbial communities and their activity overtime to determine if and when compost is stable enough to beapplied to soil, or whether more time or alternative processmanagement is required.
Acknowledgments
This study was financially supported by the Xunta de Galicia(Regional Autonomous Government of Galicia) (09MDS024310PR).The authors thank the research support services of the Universityof Vigo (CACTI) for the carbon and nitrogen analysis. The authorsalso thank Emilio Rodríguez Cochón and Domingo Pérez Díaz fortechnical support.
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CAPÍTULO 2
Seafood-processing sludge composting: changes to
microbial communities and physico-chemical parameters
of static treatment versus for turning during the
maturation stage
Villar I., Alves D., Mato S., 2016. Seafood-Processing Sludge Composting: Changes to
Microbial Communities and Physico-Chemical Parameters of Static Treatment versus
for Turning during the Maturation Stage. PLoS ONE 11(12): e0168590.
https://doi.org/10.1371/journal.pone.0168590
Estado: publicado
Permiso: pdf editor
Capítulo 2
67
RESUMEN
En general, en las plantas de compostaje la fase activa o intensiva se realiza
separadamente de la fase de maduración, usando una tecnología y tiempo específicos. El
material pre-compostado a ser madurado puede presentar suficientes sustratos
biodegradables que provoquen la proliferación microbiana y la consecuente reactivación de
la temperatura. Por lo tanto, la falta de control sobre la estancia en maduración durante la
gestión del residuo a nivel industrial puede resultar en situaciones no deseables. La principal
hipótesis de esta investigación es que el control de la fase de maduración mediante volteos
conlleva una optimización del proceso de compostaje en comparación con una maduración
estática. El residuo empleado fue lodo de depuradora de una industria de procesado de
productos del mar mezclado con madera triturada (1:2, v/v). El sistema de compostaje
consistió en una fase bio-oxidativa en reactor estático de 600L seguido de una fase de
maduración por triplicado en cajas de 200L durante 112 días. Se realizaron dos pruebas con
el mismo proceso en reactor y diferentes tratamientos en cajas: maduración estática y
maduración volteada cuando las temperaturas superaron los 55ºC. Se midieron
periódicamente PLFAs, materia orgánica, pH, conductividad eléctrica, formas de nitrógeno,
formas de carbono, enzimas hidrolíticas y actividad respiratoria. Los volteos aumentaron
significativamente la duración de la fase termófila con el consecuente aumento de la
degradación de la materia orgánica. El PCA diferenció significativamente los dos tratamientos
en función de los parámetros de seguimiento, especialmente, pH, carbono total, formas de
nitrógeno y ratio C/N. En consecuencia, se alcanzaron valores óptimos de madurez y
estabilidad en menor tiempo en el compost con volteos. Mientras que el tratamiento volteado
mostró una estabilización de los grupos microbianos y un bajo ratio mono/sat, el tratamiento
estático presentó mayor variabilidad en los grupos microbianos y un alto ratio mono/sat. La
presencia de sustratos degradables causa cambios en las comunidades microbianas y su
estudio durante la maduración ofrece una aproximación al estado de degradación de la
materia orgánica. La obtención de un compost de calidad y la optimización del proceso
requiere del control mediante volteos durante la etapa de maduración.
RESEARCH ARTICLE
Seafood-Processing Sludge Composting:
Changes to Microbial Communities and
Physico-Chemical Parameters of Static
Treatment versus for Turning during the
Maturation Stage
Iria Villar*, David Alves, Salustiano Mato
Department of Ecology and Animal Biology, University of Vigo, Vigo, Pontevedra, Spain
Abstract
In general, in composting facilities the active, or intensive, stage of the process is done sep-
arately from the maturation stage, using a specific technology and time. The pre-composted
material to be matured can contain enough biodegradable substrates to cause microbial
proliferation, which in turn can cause temperatures to increase. Therefore, not controlling
the maturation period during waste management at an industrial level can result in unde-
sired outcomes. The main hypothesis of this study is that controlling the maturation stage
through turning provides one with an optimized process when compared to the static
approach. The waste used was sludge from a seafood-processing plant, mixed with shred-
ded wood (1:2, v/v). The composting system consists of an intensive stage in a 600L static
reactor, followed by maturation in triplicate in 200L boxes for 112 days. Two tests were car-
ried out with the same process in reactor and different treatments in boxes: static maturation
and turning during maturation when the temperature went above 55˚C. PLFAs, organic mat-
ter, pH, electrical conductivity, forms of nitrogen and carbon, hydrolytic enzymes and respi-
ratory activity were periodically measured. Turning significantly increased the duration of
the thermophilic phase and consequently increased the organic-matter degradation. PCA
differentiated significantly the two treatments in function of tracking parameters, especially
pH, total carbon, forms of nitrogen and C/N ratio. So, stability and maturity optimum values
for compost were achieved in less time with turnings. Whereas turning resulted in microbial-
group stabilization and a low mono/sat ratio, static treatment produced greater variability in
microbial groups and a high mono/sat ratio, the presence of more degradable substrates
causes changes in microbial communities and their study during maturation gives an
approach of the state of organic-matter degradation. Obtaining quality compost and optimiz-
ing the composting process requires using turning as a control mechanism during
maturation.
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 1 / 15
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OPENACCESS
Citation: Villar I, Alves D, Mato S (2016) Seafood-
Processing Sludge Composting: Changes to
Microbial Communities and Physico-Chemical
Parameters of Static Treatment versus for Turning
during the Maturation Stage. PLoS ONE 11(12):
e0168590. doi:10.1371/journal.pone.0168590
Editor: Andrew C Singer, Natural Environment
Research Council, UNITED KINGDOM
Received: June 23, 2016
Accepted: December 2, 2016
Published: December 21, 2016
Copyright: © 2016 Villar et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by Xunta de
Galicia, 09MDS024310PR. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Food-processing industries in the European Union generate a large quantity of waste-prod-
ucts, estimated to be 35 megatonnes per year; 60% of this consists of organic material [1]. The
seafood-processing industry generates effluent with a high content of fish remains and oils,
which means wastewater with a high organic load [2]. For the management of organic sludge
from wastewater treatment, one of the most widely-used techniques is composting. Compost-
ing is a process of biologically degrading solid organic substrates under aerobic conditions
through the action of diverse microbial populations; through this process one obtains a stable-
humified-product that is suitable for land application as a soil-improver and source of organic
matter and nutrients [3]. The elevated availability of nourishment in organic waste causes
microbial growth and with it an elevation in temperature and consequently the succession of
different microbial communities which appear at different stages of the composting process.
In the first phase, known as the mesophilic phase, proliferate mesophiles decompose the most
basic organic compounds, causing the temperature to exceed 45˚C. This increase in tempera-
ture results in the growth of thermophilic organisms and the inhibition of thermally-intolerant
ones in what is known as the thermophilic phase. The final phases consist of a cooling period,
which is characterized by the growth of mesophilic organisms, and a maturation period during
which the organic material is stabilized and turned into humus, obtaining a product suitable
for use as a soil amendment [4].
Generally speaking, industrial composting facilities differ in two different areas: the first
one where the material undergoes intensive decomposition (corresponding to the initial meso-
philic and thermophilic phases) and the second one where stabilization and maturation phases
should take place. The intensive stage is characterized by high temperatures, elevated oxygen
consumption, and the production of gaseous and liquid emissions [5] and, therefore, compost-
ing facilities pay special attention to this part of the process, carried out with a specific tech-
nique or technology, with greater control and tracking than the second one. When this stage is
thought to be finished, or the established time of the intensive process ends, the pre-composted
material is deposited in the maturation area where the degradation and polymerization of
organic substances under mesophilic conditions should continue. Nevertheless, the material
obtained after the intensive stage may not be sufficiently decomposed and may undergo reacti-
vation during the maturation stage. For example, in facilities with composting tunnels, the
most active phase is often limited to a specific time (a two-to-three week stay) after which the
content removed from the tunnels is deposited in piles to mature [6]. The limited duration
and the lack of mixture of the waste during the time in the tunnel usually cause the reactivation
of the material in the maturation area and the amount of attention given to this phase depends
on the composting facility, but typically the maturation area is simply considered to be a stor-
age space for compost [7]. The lack of control during this phase can cause environmental
problems such as smells and leachates; it can also lower the quality of the compost. In order to
obtain stable and matured compost, composting-facility operation and design should integrate
both phases [5]. In many cases it is not possible to make changes a posteriori on the technique
used in the intensive stage but it is possible to act on the pre-composted material disposed in
the maturation area. So, it is important to know how the management and control of the mate-
rial deposited in maturation conditions similar to composting facilities affects the physical-
chemical and biological parameters and if this management allows a reduction of time and/or
quality improvement of compost.
Turning is usually employed as a means to aerate, homogenize, and control the temperature
during composting [8]. The effects of turning, along with how often it occurs, have been stud-
ied by a wide range of authors, who have observed changes to physical, chemical, and
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 2 / 15
biological characteristics which affect the rate of decomposition and consequently the time
needed to achieve maturity and high-quality compost [9–12]. Nevertheless, information about
the possible effects of dynamic or static management of the pre-composted waste has not been
found; this is due to the fact that turning is generally carried out during the most active part of
the process, or scheduled throughout the entire process.
Therefore, the intention of this study is to understand, at a pilot scale, the maturation pro-
cess carried out at composting facilities, examining the effects of both static management and
turning on temperature control during this stage of the process. Furthermore, there are few
studies related to seafood-processing sludge composting and even fewer dealing with the mat-
uration phase. In a previous experiment, Villar et al. [13] studied the maturation of this type of
waste product in comparison with pig manure and sludge from an urban wastewater treatment
plant. The research demonstrates that the seafood-processing sludge used contains a high level
of biodegradable substrates, which cause it to reactivate when left to mature after an intensive
period in a static reactor. This made it ideal to be used in this study.
The objectives of this research were, firstly, to study whether static management or turning
for temperature control influence the physico-chemical and biological parameters and the
microbial-community structure throughout the maturation phase and, secondly, to determine
if controlling the maturation phase produced a more stable product in less time. This study
addresses the hypothesis that controlling by turning the pre-composted sludge allows for an
improvement in compost quality, a reduction in the time process to obtain appropriate stabil-
ity and maturity parameters and a microbial-community profile different to the static treat-
ment one.
Materials and Methods
Composting experiment
Composting substrates. For this experiment, the wastewater sludge was from a purifier
from a plant that produces pre-cooked and flash-frozen fish and cephalopods; it was obtained
after fats had been separated from the sludge and the waste product had been treated with
coagulants and flocculants; pinewood shred to smaller than 3cm was used as a bulking agent.
The initial characteristics of these materials can be seen in Table 1. Both materials were mixed
in a volumetric ratio 1:2, respectively, as in the previous study [13].
Composting design. Composting was carried out in two stages, the objective being to
simulate waste-product management as seen in industrial composting facilities. The compost-
ing system has been described in detail in Villar et al. [13]. In brief, the sludge-and-bulking-
Table 1. Physico-chemical composition of the materials used in the composting experiments.
Seafood sludge Bulking agent
Moisture content (%) 61.8 ± 0.4 41.4 ± 0.2
Organic matter (% dw) 87.7 ± 0.3 93.9 ± 0.4
pH 4.90 ± 0.04 6.66 ± 0.01
Electrical conductivity (mS cm-1) 0.55 ± 0.00 0.29 ± 0.01
Total carbon (mg g-1 dw) 513.7 ± 3.2 558.2 ± 2.8
Total nitrogen (mg g-1 dw) 19.24 ± 0.16 12.80 ± 0.29
C/N ratio 26.7 ± 0.2 43.6 ± 0.5
Fat content (% dw) 19.8 ± 0.8 N.D.
dw: dry weight, N.D: not detected
doi:10.1371/journal.pone.0168590.t001
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 3 / 15
agent mixture was composted in a 600-liter-capacity static reactor; aeration was achieved
through programmed forced ventilation of 400 m3/h by a periodic flow rate of 30 seconds
every 90 min and an alarm flow for temperature and oxygen control. The material was kept in
the reactor, where it underwent the intensive stage, until the temperature had dropped to
below 35˚C. The reactor was emptied and the homogenized material placed in triplicate in
200-liter-capacity wooden boxes where the cooling, stabilization, and maturation stage took
place (henceforth called the maturation phase). The top parts of these boxes were open, their
bottom part perforated; inside them, the material rested between two layers of bulking agent to
lend some thermal insulation to the pre-composted material.
Two composting tests were carried out, using the same mixture and handling in the reactor;
each test, however, received a different treatment in boxes, either static treatment or turning
treatment for temperature control. The boxes that received the static treatment were left for
112 days without being homogenized, whereas the ones that received the turning treatment
were mixed when temperatures went above 55˚C to aerate, avoid excess temperature and
homogenize the compost. The turnings were carried out in order to simulate industrial-level
compost turning by emptying completely each box, thoroughly mixing and placing it again
inside the box. The compost was moistened when moisture levels fell below 40% during both
types of treatment. The boxes underwent oxygen and temperature control daily for the first 42
days and three times a week afterwards until the end of the 112-day process. 1500-milliliter of
composite samples were taken from each box on day 0 (when the reactor was emptied) and on
the 14th, 28th, 42nd, 56th, 70th, and 91st days of being in the box; they were screened through
a one-centimeter sieve in order to remove the bulking agent. Once 112 days had passed, the
boxes were emptied and all the compost screened. The samples were divided in three parts.
One part was dried for two days at 40˚C and ground in order to determine the total amount of
carbon and nitrogen. Another part was freeze-dried and ground using mortar-and-pestle in
order to analyze phospholipid fatty acids (PLFAs). The remaining part was kept fresh for the
rest of the analytical determinations.
Composting parameters
Moisture and organic matter contents of samples were calculated gravimetrically after drying
at 105˚C until constant weight and combustion at 550˚C for 4 h, respectively. Total carbon
(TC) and total nitrogen (TN) contents were determined by combustion using a LECO 2000
CN elemental analyzer. Inorganic nitrogen (N-NH4+ and N-NO3
−) was determined in 0.5 M
K2SO4 extracts (1:10, w/v) using the modified indophenol blue colorimetric method [14].
Total extractable nitrogen was determined in the same extracts after oxidation with K2S2O8, as
described by Cabrera and Beare [15], and dissolved organic nitrogen content (DON) was cal-
culated as (total extractable N)–(inorganic N). Water soluble carbon content (WSC) was ana-
lyzed in aqueous extracts (1:5, w/v) by dichromate oxidation in sulfuric acid solution.
Electrical conductivity and pH were determined in aqueous extracts (1:10, w/v) using a pH
meter Crison Basic 20 and a conductivimeter Crison CM 35. Fat content was determined in
samples from day 56 and 112 by Soxhlet method using n-hexane as organic solvent [16].
The microbial community composition and biomass were determined by PLFA analysis
following the method described by Gomez-Brandon et al. [17] for organic samples. The analy-
sis was performed with a CP-Select FAME, 100m x 0.25mm in a gas chromatograph-mass
spectrometer (GC-MS). Identification was done by comparison of retention times and mass
spectra with known external standards (Larodan Fine Chemicals AB, Malmo, Sweden) and
quantification was performed with methyl nonadecanoate fatty acid (C19:0) as internal stan-
dard. PLFAs were used to estimate the biomass of specific microbial groups: Gram-positive
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 4 / 15
bacteria (i14:0, i15:0, a15:0, i16:0, a17:0), Gram-negative bacteria (15:1ω5, 16:1ω7, 17:1ω7,
18:1ω7) and fungi (18:2ω6, 18:1ω9, 20:1ω9). The total amount of PLFAs identified (totPLFAs)
was used as an indicator of the viable microbial biomass [18]. The ratio of monounsaturated
PLFAs to saturated PLFAs (mono/sat) was used as an indicator of physiological or nutritional
stress in microbial communities [19].
β-glucosidase was estimated by the colorimetric measurement of p-nitrophenol released
after incubation 1 g of fresh sample with 1 mL of p-nitrophenyl-β-D-glucopiranoside
(0.025 M) for 1 h at 37˚C [20]. Alkaline phosphatase was measured by incubating 0.5 g of fresh
sample with 1 mL of p-nitrophenylphosphate (0.015 M) for 1 h at 37˚C and subsequent colori-
metric measurement of p-nitrophenol released [21]. Protease activity was measured after the
incubation of 1 g fresh sample with 5 mL of sodium caseinate (2%) for 2 h at 50˚C and subse-
quent determination of the amino acids released using Folin-Ciocalteu reagent [22].
Static respiration rate (SR) was measured using manometric respirometers by OxiTop1
system (WTW GmbH, Weilheim, Germany). Briefly, fresh weight equivalent to 4 g of dry sam-
ple was placed in a hermetic container with a 1 M NaOH trap to capture CO2, the pressure
drop, due to microbial oxygen consumption, was recorded during 24 h at constant tempera-
ture. Germination index (GI) was calculated on day 56 and 112 by determining seed germina-
tion and root length of Lepidium sativum growing in 2 mL of aqueous extracts (1:5, w/v) in
Petri dishes lined with paper filter during 48 h [23]. The self-heating test was carried out in the
final compost using 2 L Dewar flask for 10 days at room temperature.
Statistical analysis
All statistical tests were performed using R software [24]. Principal component analysis (PCA)
was performed on the correlation matrix after normalization to zero mean and variance unit
of the variables. The PCA was conducted using the prcomp function and the package factoex-
tra [25]. Mixed models were fitted with the nlme package [26] to evaluate the differences
between treatments. The waste type and time were fixed factors and the repeated measurement
throughout time in each maturation box was treated as a random effect to address the non-
independence of samples. The best model was selected according to Akaike Information Crite-
rion (AIC). Logarithmic and square root transformations of the data were necessary to ensure
the normality and homogeneity of the variance of residuals of models. Student’s t tests were
performed to determine the difference between compost. PLFAs data of each treatment were
subjected to cluster analysis with the hclust function to determine the differences in the struc-
ture of the microbial community according to time. All statistical tests were evaluated at the
95% confidence level and values are given as the mean ± standard error.
Results
Temperature evolution
The temperature profile during the reactor phase was similar in both tests, as can be seen in
Fig 1; there were no significant differences between them (p> 0.05). The temperature slowly
increased at the start of process, getting above 45˚C starting on the 5th day. Both tests main-
tained thermophilic temperatures for 28 days. After emptying the reactors, reactivation of the
process with thermophilic temperatures was quickly reached in the box tests.
The temperature evolutions of the two treatments during maturation were significantly dif-
ferent (p< 0.05). Static treatment saw temperatures maintained constantly in excess of 45˚C
for ten days, with a second peak temperature being registered on the 23rd day at no higher
than 45˚C; on the 14th day it was necessary to remoisten the material in order to keep moisture
levels above 40%. Turning treatment saw the material in the boxes being turned seven times
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 5 / 15
over a period of 20 days; the material was remoistened during three of these turns and thermo-
philic temperatures were maintained for 26 days. Both treatments saw oxygen levels superior
to 14% during the material’s time in boxes.
Composting parameters
The PCA was undertaken with the physico-chemical and biological variables shown in Fig 2.
Three separate groups were yielded; they were the one formed at the start of the maturation
phase of both tests, in other words when the materials left the reactor (time 0), the turning-
treatment sample group, and the static-treatment sample group. The principal component
1 (PC1) accounted for 51.2% of the variance and significantly separated the three groups
(F2, 45 = 337.95, p< 0.0001). The parameters responsible for the differences between the
groups through PC1 were, fundamentally, pH (r = 0.96, p<0.0001), TC (r = -0.94, p<0.0001),
TN (r = 0.92, p<0.0001), and the C/N ratio (r = -0.97, p<0.0001).
The parameters that contributed the most to separating the groups during the principal
component 2 (PC2), which accounted for 29% of the variance, were DON (r = 0.88, p
<0.0001), and ammonium (r = 0.90, p<0.0001); the three groups were significantly different
from each other (F2, 45 = 29.09, p<0.0001). The initial material of the maturation stage con-
tained high amounts of TC, OM, SR, and totPLFAs and low amounts of pH, TN, β-glucosidase
and protease activity. Static treatment was characterized by higher levels of ammonium
(p<0.0001), EC (p<0.0001), DON (p<0.0001), OM (p<0.0001), pH (p<0.0001), alkaline
phosphatase (p<0.0001), protease (p<0.0001), and TN (p<0.0001) during the maturation
process than those obtained via the turning treatment; static treatment produced lower levels
Fig 1. Temperature evolution during the reactor stage and the maturation stage for the turning treatment and static treatment.
doi:10.1371/journal.pone.0168590.g001
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 6 / 15
of β-glucosidase (p<0.0001) than that achieved via turning. Organic-matter-loss at the end of
maturation during the box phase was greater in the case of the turning treatment (42.5%) than
the static one (35.6%); likewise, the percentage of carbon reduction was greater in the turned
boxes (17.4%) than in the static ones (10.6%). Both treatments presented significantly different
parameters in the middle of the process (56th day); these differences, with the exception of the
C/N ratio, which was present at similar levels in both types of compost (Table 2), were main-
tained up to, and including, the final sampling.
Fig 2. A) Principal component analysis (PCA) of the physico-chemical and biological variables of the turning treatment and static treatment B)
Correlation circle showing the variables that define the components. CN: carbon-to-nitrogen ratio, DON: dissolved organic nitrogen, EC: electrical
conductivity, OM: organic matter, SR: static respiration, TC: total carbon, TN: total nitrogen, totPLFAs: total amount of PLFAs, WSC: water soluble carbon.
doi:10.1371/journal.pone.0168590.g002
Table 2. Compost characteristics after 56 and 112 days of maturation in the turning treatment and static treatment.
56 days 112 days
Turning Static Turning Static
Organic Matter (%) 74.3 ± 0.3 76.8± 0.8* 70.1 ± 0.5 75.7 ± 0.5*
pH (mS cm-1) 5.99 ± 0.03 6.61± 0.03* 6.01 ± 0.04 5.98 ± 0.05
ratio C/N 15.1 ± 0.5 12.7± 0.2* 13.8 ± 0.2 13.1 ± 0.2
NH4+/NO3
- 0.17 ± 0.02 0.23 ± 0.01* 0.11 ± 0.01 0.21 ± 0.01*
SR (mg O2 g-1OM h-1) 0.48 ± 0.03 0.64 ± 0.02* 0.36 ± 0.02 0.47± 0.02*
Sel-heating test - - clase V clase V
GI(%) 82.7 ± 1.5 58.3 ± 2.1* 122.9± 6.9 77.3 ± 5.7*
Fat (%) 6.03 ± 0.03 9.14 ± 0.03* 4.21 ± 0.02 8.00 ± 0.01*
* indicates that samples for the same time between treatments are significantly different (Student t-test, p < 0.05) (SR: static respiration, GI: germination
index).
doi:10.1371/journal.pone.0168590.t002
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 7 / 15
Microbial community
Cluster analysis based on PLFA profiles for turned boxes and static boxes show two clusters
for each of the treatments. In the case of the turned boxes (Fig 3A), the samples on day 0, the
14th day, and the 28th day formed a cluster that was separate from the rest of the samplings,
although the distance between day 0, the 14th day, and the 28th day was high; this suggests
that the PLFA profiles were different. In the case of the second group (the 42nd day, 56th day,
72nd day, 91st day, and the 112th day), the similarities between the samples within the cluster
were greater, with little distance between the samplings. With regards to static boxes (Fig 3B),
the samples on day 0 and the 14th day formed a cluster, although the distance between one
another was high; this suggests important differences between both samplings. Significant
increases in bond distance were observed in the second cluster (the 28th day, 42nd day, 56th
day, 70th day, 91st day, and the 112th day), indicating a low level of similarity between the
samplings, especially between the 28th-day sampling and the rest, and between 70th-day-to-
112th-day subgroup and the 56th-day-and-42nd-day-and-91st-day subgroup.
Table 3 shows the evolution of microbial groups during their time in boxes. The
treatment performed on the material of the boxes significantly affected microbial-biomass evo-
lution (p<0.0001), Gram + bacteria (p<0.0001), Gram—bacteria (p<0.0001), and fungi
(p<0.0001). Likewise, significant differences caused by time (p<0.0001) and significant inter-
action between time and treatment (p<0.0001) were observed in the microbial biomass and
all microbial groups. Both maturation treatments showed a high concentration of PLFA fungal
biomarkers for the first samplings, with a decrease over time; this was especially true for the
turning treatment (88.9% reduction in the case of turning versus 60.7% in the case of static
treatment). Fungi were the main microbial group (averaging 48%) for practically the entire
process, with the exception of the 28th-day-and-42nd-day-turning-treatment samplings in
which Gram + bacteria were dominant. Gram + bacteria were reduced by 81% and 61% during
the turning treatment and static treatment, respectively. The PLFA-Gram-negative-bacteria
Fig 3. Dendrograms of cluster analysis based on PLFA profiles during the maturation stage in boxes of A) turning treatment and B) static
treatment. Dendrograms were done with the data of 8 samplings taken during the maturation stage (day 0 to the 112th day) and were drawn based on
Ward’s method on the Euclidean distance.
doi:10.1371/journal.pone.0168590.g003
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 8 / 15
indicators showed a high increase on the 28th day of the static-treatment process; much higher
levels than the turning treatment were maintained until the end of the process; static treatment
showed an increase of 33%, whereas the turning treatment showed a 74% reduction. The com-
post produced by static treatment had a higher-level concentration of all the PLFA-group indi-
cators than the turned compost.
The mono/sat ratio achieved by static treatment was greater than that achieved by turning
starting from the 28th day, the latter maintaining stable levels starting from the 70th day.
Discussion
The temperature profiles of both tests while in the reactor, corresponding to the intensive
stage, showed patterns typical of the composting process; in other words, temperatures
increasing to thermophilic levels followed by maintaining said temperature and a subsequent
decline in temperature until reaching mesophilic levels. Despite thermophilic conditions being
reached slowly, compost hygienization was ensured by continuously maintaining tempera-
tures above 55˚C for more than 15 days [27]. Similar temperature profiles have been observed
by other authors during the composting of fatty wastes [8,28,29], therefore, a high level of fat
causes the compost to reach thermophilic temperatures more slowly but once reached they
cause the compost to maintain thermophilic temperatures for longer due to the fact that the
lipids present provide a greater amount of energy than other organic compounds. The sludge
used in the research came from the purification of wastewater generated by the production of
precooked and frozen foods, such as breaded and battered fish and cephalopods, so the lipid
content of the waste was high (Table 2). Thus, the sludge used had an optimum amount of fats
for microbial degradation through composting since the appropriate values for this process
should not exceed 20–25% according to studies carried out by Fernandes et al. [28] for urban,
agricultural and industrial waste. Likewise, the low pH of the sludge, with values less than 5,
was able to inhibit thermophilic microorganisms and slow the transition from mesophilic to
thermophilic temperatures during the initial composting reactor stage, as observed by Sund-
berg et al. [30]. The lack of difference between the temperature profiles of both tests in the
reactor makes it possible to carry out statistical analysis conducive to checking the current
study’s objectives by submitting the material to the same processing conditions during the
intensive phase.
After the static-reactor stage, the pre-composted material was homogenized and placed in
boxes, resulting in a quick reactivation for both treatments, indicating that the material had
Table 3. Changes in microbial groups: bacteria Gram +, bacteria Gram—and fungi and ratio of monounsaturated to saturated (mono/sat) PLFAs
during the maturation stage of the turning treatment and static treatment.
Time (days) Gram + (μg g-1dw) Gram–(μg g-1dw) Fungi (μg g-1dw) mono/sat
Turning Static Turning Static Turning Static Turning Static
0 139.9 ± 3.9 176.1 ± 8.9* 44.5 ± 4.1 39.0 ± 1.3 265.5 ± 2.1 276.4 ± 14.9 1.61 ± 0.10 1.25 ± 0.01*
14 119.8 ± 4.3 117.8 ± 5.7 20.0 ± 1.2 22.5 ± 1.4 244.9 ± 12.0 147.6 ± 10.3* 1.32 ± 0.07 0.99 ± 0.02*
28 122.4 ± 7.7 91.3 ± 1.9* 23.4 ± 1.6 90.6 ± 5.5* 109.5 ± 6.6 106.8 ± 8.5 1.04 ± 0.06 1.50 ± 0.09*
42 41.0 ± 5.9 57.5 ± 3.1 13.3 ± 0.8 52.8 ± 6.6* 29.8 ± 3.1 76.6 ± 8.3* 0.78 ± 0.06 1.22 ± 0.06*
56 26.4 ± 2.1 60.2 ± 3.9* 8.1 ± 1.0 52.9 ± 5.4* 27.4 ± 3.5 79.2 ± 3.8* 0.59 ± 0.09 1.27 ± 0.10*
70 39.4 ± 3.1 37.1 ± 1.9 10.4 ± 2.3 26.5 ± 3.8* 45.6 ± 4.9 76.3 ± 7.0* 0.78 ± 0.06 1.11 ± 0.06*
91 41.2 ± 4.2 61.4 ± 2.0* 14.5 ± 1.0 46.1 ± 4.3* 45.6 ± 2.3 94.7 ± 8.7* 0.83 ± 0.05 1.18 ± 0.05*
112 26.6 ± 2.0 68.5 ± 4.5* 11.7 ± 0.7 25.8 ± 0.4* 29.4 ± 0.6 108.7 ± 6.9* 0.79 ± 0.03 1.38 ± 0.06*
* indicates that samples for the same time between treatments are significantly different (Student t-test, p < 0.05) dw: dry weight
doi:10.1371/journal.pone.0168590.t003
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 9 / 15
enough biodegradable substrates to allow it to self-heat until it reached thermophilic tempera-
tures. Likewise, turnings carried out during the first several days of the material being in the
boxes facilitated the extension of thermophilic conditions more so than the static approach.
Therefore, turning reactivated the process of incorporating the least-degraded material and, as
a result, made it possible to supply easily assimilable substrates to the microbial biomass, start-
ing from the exterior of the boxes and moving inwards. Ruggieri et al. [8] have suggested that
turning during the composting of fatty wastes is preferable to the static system because it pre-
vents the mixture from forming clumps and compacting. Likewise, Albuquerque et al. [31]
have observed that, during alperujo composting, forced-air ventilation is only effective when
done along with turning because it improves the substrate’s porosity. It has also showed that
turning, after static-reactor composting seafood sludge with forced ventilation, makes length-
ening the thermophilic phase possible. At most composting facilities, turnings of the pre-
composted waste in the maturation stage are not based on the biological process, but on indus-
trial criteria (increase space, move the material to another location, dry the material. . .) so, the
implementation of protocols for the maturation stage is considered important to improve the
composting process as has been observed.
According to multivariate analysis, the treatment applied while the material is in the boxes
determines its physico-chemical, and biological, development vis-à-vis compost stabilization
and maturation. Turning the material during the maturation stage allows for compost to
degrade to a greater degree, suggesting that the time needed to achieve stability is reduced
when controlling the process is done under dynamic conditions. Other authors have found
similar results with regards to all stages of the composting process and not only the maturation
stage in particular. For example, Brito et al. [32] have found that turning increases the degrada-
tion rate during the composting of cattle slurry; statically-treated piles require more processing
time to reach organic-material values similar to that obtained in compost that had been
turned. Nikaeen et al. [33] have also pointed out that the time needed to stabilize organic com-
pounds in static piles is greater due to the lack of heat exchange. Furthermore, it has been
stated that turning and how often it occurs affects the EC, pH, TC, TN, C/N ratio, GI, and tem-
perature during the composting of different types of waste products [9–12,34]. Similar results
have been obtained from this study, showing that turning pre-composted waste in line with
temperature criteria following static-reactor composting affected the pH, electrical conductiv-
ity, ammoniacal nitrogen, dissolved organic nitrogen, organic matter, enzymatic activities,
total nitrogen and total carbon; this led to the compost becoming stable in less time than
through static conditions for the maturation stage. Turning-treatment samples starting from
the 56th day showed stable conditions; some parameters, like the respiration rate and the ger-
mination index for example, were improved by prolonging the process. Nonetheless, the stati-
cally-treated compost had insufficient quality parameters on the 56th day; there were also
significant variations throughout the entire process. The analysis of the compost after 112 days
in the maturation boxes showed parameters that indicated stability in both cases; these param-
eters included the maximum rating for the Dewar self-heating test (class V, mature compost)
[35], and a respiration rate that was less than 0.5 mgO2 g-1SV h-1 [36]. Also, both types of treat-
ment produced compost that reached optimum C/N-ratio levels of less than 20 [35]. However,
the turned compost had an ammonium/nitrate ratio of less than 0.16 [37] and a germination
index superior to 80% [38], which showed a greater degree of maturity than the compost that
received the static treatment. Controlling through turning significantly affected physico-
chemical, and biological, characteristics as well as microbial activity; making it possible to
achieve a more stable, and more mature, product in less time than the unhandled material. So,
when a composting facility is decided to work with a static technique for the intensive stage,
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 10 / 15
the control of the maturation by turnings could optimize the process to obtain a compost of
better quality in less time.
It is known that the nature of organic substrates and temperature are the principal factors
that determine the composting process with respect to microbial dynamics [4,39,40]. Due to
the fact that the initial material placed in boxes after having been composted in the reactor
were very similar on a physico-chemical, and biological, level -as observed using multivariate
analysis- the changes in the microbial community’s structure were mainly produced as a con-
sequence of the temperature reached in the boxes during the first weeks of maturation stage.
Bacterial and fungal populations decreased with both treatments, which is an expected decline
as the most easily assimilable substrates are consumed, reducing the available food for micro-
bial growth. Nevertheless, turning produced a less noticeable decrease of Gram + bacteria dur-
ing the first few weeks, which was due to Gram + bacteria, in particular those that belong to
the genus Bacillus, dominating during the thermophilic phase of composting [4] and turning
boxes maintaining thermophilic temperatures for more days. In the same way, it is known that
increasing temperatures caused fungi to decrease [4,41], although in the case of turning the
population stayed the same for the first month when the temperatures were high and this
could mean that were thermophilic or thermo-tolerant fungi present [13,42]. Choi and Park
[43] have also observed high activity of yeast in thermophilic composting of food waste, indi-
cating that the ability of yeast to grow at a lower pH than bacteria could explain their presence.
The pH and the lipid content of the pre-composted sludge allowed for the development of
thermophilic fungi and turnings allowed for the input of food to initially maintain their popu-
lation. So, the cluster obtained by analyzing the PLFAs found in the first two static-treatment
samplings, and those obtained from the first three turning-treatment samplings, are mainly
characterized by the temperature reached during the first few weeks which in turn depended
on the treatment carried out.
It is normal for the number of bacteria to lower in population, but increase in terms of
diversity, and for fungi to increase both in terms of quantity and diversity during the matura-
tion phase itself [4]. Nonetheless, a decrease in temperature did not produce an observable
increase in how abundant fungi were; both treatments, however, were characterized by the
fungal biomass being predominant, particularly in the case of static treatment, throughout the
entire process. Amir et al. [44] found that fungi are more present in wastes with a high level of
fatty acids. Likewise, Villar et al. [13] suggested that the influence the initial material, in this
case lipid in nature and pH, has on the maturation phase is reflected by microbial diversity;
this means that although the waste undergoes significant degradation during the composting
process, the properties of the starting material determine the dominant microbial groups
throughout the maturation stage. Following the high-temperature phase, there was a large
increase in Gram-negative-bacteria PLFA biomarkers with respect to static treatment. Due to
Gram—bacteria’s limited resistance to temperature the normal course of action is for thermo-
philic Gram+ bacteria to give way to mesophilic Gram—bacteria during composting or matu-
ration [45]. It has been confirmed that bacteria belonging to the phylum Bacteroidetes and
class Alphaproteobacteria frequently dominate both in compost after it has reached high tem-
peratures and in compost that has not matured [46–48]. Likewise, other Gram—bacteria like
Gammaproteobacteria are dominant in cured compost [48]. However, the fluctuation of
Gram—bacteria together with the physico-chemical data could indicate a lack of stability in
the static treatment in the final stages of maturation. On the contrary, all the microbial groups
in the turned compost stayed at similar levels following the high-temperature phase; this pro-
duced microbial-community homogeneity as observed in group II of the multivariate analysis.
Microbial-population values in turned compost stayed low once it had reached stability and
maturation parameters, whereas the increase in Gram + bacteria and fungi obtained from
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 11 / 15
static treatment could indicate the availability of biodegradable substrates [49]. Turning the
material made it possible to maintain more stable and more similar microbial diversity after
reaching mesophilic temperatures whereas not homogenizing the boxes maintained a greater
degree of difference between samplings. Cahyani et al. [46] have pointed out that the microbial
community remained stable during the maturation phase of composting rice straw. On the
other hand, Danon et al. [48] observed changes to the microbial communities during the year-
long composting of biosolids. These authors observed that in the case of bacterial populations
specialized in breaking down macromolecules such as lignocellulose, stability was maintained
during the intermediate part of the maturation phase.
Likewise, the mono/sat ratio values after high temperatures in the static treatment indicated
that microbial communities do not have limited resources and there are enough nutrients for
their growth. However, in the compost that received the turning treatment the mono/sat ratio
showed that the microbial communities were subjected to nutritional and physiological stress.
Jindo et al. [50] have suggested that low mono/sat ratio numbers demonstrates the final part of
organic material’s transformation into compost and Bastida et al. [51] have pointed to a greater
degree of carbohydrate-substrate biodegradability when the numbers of the aforementioned
ratio are high. Therefore, mono/sat-ratio stabilization can be indicative of mature compost.
Bacterial-population stabilization, as a result of running out of easily degradable substrates,
can indicate the degradation of more calcitrant compounds, ergo turning the material while it
is in maturation stage allows for a greater degree of organic-material degradation and micro-
bial succession towards a more stable mesophilic community, reflecting compost maturation.
Conclusions
The fresh-compost-turning system made it possible to maintain temperatures and, as a result,
prolonged the thermophilic phase; this allowed for a high level of organic-matter degradation
over a longer period. Controlling the composting of highly-energy- waste through turning
after the most intensive stage in the reactor made it possible to achieve stability and maturity
in a shorter time frame than statically treating pre-composted waste. Studying microbial dyna-
mism has helped to characterize the state of degradation of organic matter as stable compost
showing a stable microbial structure whereas the presence of biodegradable substrates causes
significant changes to microbial populations. Looking at the results, composting plants that
utilize turning during the maturation period optimize the process by cutting the time needed
to reach sufficiently high levels of quality.
Acknowledgments
The authors thank the research support services of the University of Vigo (CACTI) for the car-
bon and nitrogen analysis. The authors also thank Emilio Rodrıguez Cochon, Domingo Perez
Dıaz and Josefina Garrido Gonzalez for their help and technical support.
Author Contributions
Conceptualization: SM.
Data curation: IV.
Formal analysis: IV DA.
Funding acquisition: SM.
Investigation: IV DA SM.
Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 12 / 15
Methodology: IV SM.
Project administration: SM.
Resources: SM.
Supervision: SM.
Writing – original draft: IV DA SM.
Writing – review & editing: IV DA.
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Seafood-Processing Sludge Composting: Static Treatment versus Turning during the Maturation Stage
PLOS ONE | DOI:10.1371/journal.pone.0168590 December 21, 2016 15 / 15
CAPÍTULO 3
Changes in microbial dynamics during vermicomposting of
fresh and composted sewage sludge
Villar, I., Alves, D., Pérez-Díaz, D., Mato, S., 2016. Changes in microbial dynamics
during vermicomposting of fresh and composted sewage sludge. Waste Manag. 48,
409–417. doi:10.1016/j.wasman.2015.10.011
Estado: publicado
Permiso: post print
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RESUMEN
El lodo de depuradora municipal es un residuo con alta carga orgánica que se genera en
grandes cantidades y el cual puede ser tratado mediante técnicas de biodegradación para
reducir su riesgo para el medio ambiente. Esta investigación estudia el vermicompostaje y el
vermicompostaje después del compostaje de lodo de depuradora de aguas residuales con la
especie Eisenia andrei. Para determinar el efecto que las lombrices ejercen sobre la dinámica
microbiana en función del tratamiento, se evaluó la estructura y actividad de la comunidad
microbiana, mediante el análisis de ácidos grasos fosfolípidos y las actividades enzimáticas,
durante 112 días de vermicompostaje de lodo de depuradora fresco y compostado con y sin
lombrices de tierra. La presencia de las lombrices de tierra redujo significativamente la
biomas microbiana y todos los grupos microbianos (bacterias Gram +, bacterias Gram – y
hongos), así como las actividades celulasa y fosfatasa ácida. El tratamiento combinado
compostaje-vermicompostaje mostró un menor desarrollo de lombrices, una mayor biomasa
bacteriana y fúngica que el tratamiento de vermicompostaje, y mayores diferencias
comparado con el control sin lombrices en celulasa, β-glucosidasa, fosfatasa alcalina y ácida.
Ambos tratamientos son adecuados para la estabilización de lodo de depuradora y el
tratamiento combinado compostaje-vermicompostaje puede ser un proceso viable para la
maduración del compost fresco.
Palabras claves: actividades enzimáticas, PLFAs, comunidad microbiana, lombrices de
tierra, residuo orgánico, estabilidad
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ABSTRACT
Municipal sewage sludge is a waste with high organic load generated in large quantities
that can be treated by biodegradation techniques to reduce its risk to the environment. This
research studies vermicomposting and vermicomposting after composting of sewage sludge
with the earthworm specie Eisenia andrei. In order to determine the effect that earthworms
cause on the microbial dynamics depending on the treatment, the structure and activity of the
microbial community was assessed using phospholipid fatty acid analysis and enzyme
activities, during 112 days of vermicomposting of fresh and composted sewage sludge, with
and without earthworms. The presence of earthworms significantly reduced microbial
biomass and all microbial groups (Gram + bacteria, Gram – bacteria and fungi), as well as
cellulase and alkaline phosphatase activities. Combined composting-vermicomposting
treatment showed a lesser development of earthworms, higher bacterial and fungal biomass
than vermicomposting treatment and greater differences, compared with the control without
earthworms, in cellulase, β-glucosidase, alkaline and acid phosphatase. Both treatments were
suitable for the stabilization of municipal sewage sludge and the combined composting-
vermicomposting treatment can be a viable process for maturation of fresh compost.
Keywords: enzyme activities, PLFAs, microbial community, earthworm, organic waste,
stability
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1. INTRODUCTION
Municipal wastewater treatment plants produce significant amounts of sewage sludge,
amount to about 11 million dry tonnes per year in the EU, which needs suitable and
environmentally accepted management before final disposal (Kelessidis and Stasinakis,
2012). Sewage sludge can harm the environment when it is deposited directly on soil due to
its fermentative capacity and the presence of hazardous substances, both organic and
inorganic, including pathogenic organisms and heavy metals (Williams, 2005). Due to its high
organic load, sewage sludge is a suitable waste for being treated by biological techniques
such as composting and vermicomposting aimed at obtaining a stable product with a high
agronomic value.
Vermicomposting is a bio-oxidation and stabilization process of organic matter as a
result of the interaction between microorganisms and earthworms. Microorganisms are
mainly responsible for the degradation of organic material, although earthworms stimulate
microorganisms due to the modification of substrate properties through feeding, aeration
and cast excretion, which leads to the acceleration of mineralization of organic matter and the
improvement of nutrient availability for plants (Domínguez, 2004). Vermicomposting has
been successfully applied on the treatment of municipal sewage sludge. Most research on
sewage sludge vermicomposting has focused on the study of physical-chemical parameters
such as nutrients (Domínguez and Gómez-Brandón, 2013; Fu et al., 2015), humic and fulvic
substances (Zhang et al., 2015) and heavy metals (Suthar, 2010). Nevertheless, less is
reported on the microbiological and biochemical changes that occur during the
vermicomposting of municipal sewage sludge. Benitez et al. (1999) observed a reduction in β-
glucosidase, protease, urease and dehydrogenase activities related to the decline in available
substrates, in the first 6 weeks of vermicomposting of municipal sewage sludge mixed with
paper mill sewage sludge. Domínguez and Gómez-Brandón (2013) found that the presence of
the earthworm Eisenia andrei increased microbial biomass, measured as N-microbial biomass
by fumigation-extraction method, from week 1 to week 16 of vermicomposting of sewage
sludge compared to the control without earthworms. On the contrary, Fu et al. (2015)
observed a decrease of C-microbial biomass in the first 40 days of vermicomposting of
pelletized dewatered sludge, with subsequent low and constant values that indicated the
stability of the final products.
The integration of composting and vermicomposting has been considered a suitable
method for waste management (Ndegwa and Thompson, 2001). The inoculation of
earthworms in the material that passed through the thermophilic phase of composting has
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been used as a pre-treatment before vermicomposting, in order to remove compounds
harmful for earthworms, such as ammonium (Domínguez, 2004). Several authors have
investigated the combined use of composting and vermicomposting for the treatment of
different organic materials, showing that prior composting can accelerate degradation and
improve the stabilization of the final product (Frederickson et al., 1997; Lazcano et al., 2008).
Fornes et al. (2012) studied the evolution of composting, vermicomposting and the combined
composting-vermicomposting process of horticulture waste, focusing their research on the
physical-chemical changes over time. These authors showed that vermicomposts had better
properties as growing media than as compost. Also, Lazcano et al. (2008) compared the
products of these processes, but not the evolution over time, by the study of microbiological
and biochemical parameters, noting that the combined process was the most effective
method for the stabilization of the cattle manure. Hait and Tare (2011) reported that the
combined composting-vermicomposting process of sewage sludge made it possible to obtain
a good quality pathogen-free product. Likewise, Sen and Chandra (2009) showed that
earthworms changed the dynamic of the bacterial community in the combined process
compared with composting of sugar waste, but they did not study the vermicomposting
process. So, no research on microbiological evolution over time of the vermicomposting
process and the combined composting-vermicomposting process for the same waste was
found.
It has been observed that the study of enzyme activities is a reliable index of the
evolution of organic matter during vermicomposting (Benitez et al., 1999; Aira et al., 2007a).
Enzyme activities provide information on the conversion of complex organic compounds into
more readily assimilable substances and, hence, enzymes are of interest to evaluate
stabilization throughout waste biodegradation. Likewise, enzyme activities have been related
to earthworm growth. Thus, they have been proposed as indicators to optimize
vermicomposting process (Fernández-Gómez et al., 2010). Benitez et al. (1999)
demonstrated that hydrolytic enzyme activities tended towards stability in the course of
sewage sludge vermicomposting, but however, the lack of controls makes it difficult to
distinguish between the effect caused by earthworms and the effect of the microbiota present
in the waste.
Conversely, phospholipid fatty acid analysis (PLFAs) is a useful tool for monitoring the
microbial community dynamics. The total amount of PLFAs can be used as an indicator of
viable microbial biomass and some PLFAs are specific to certain living organisms and,
therefore, can be used as biomarkers for the presence and abundance of microbial groups
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(Zelles, 1999). Thus, analysing PLFAs during vermicomposting makes it possible to know the
changes in the microbial community composition over time. Fernández-Gómez et al. (2013)
observed a reduction in total PLFAs of different organic wastes after 24 weeks of
vermicomposting. In the same way, Gómez-Brandón et al. (2011a, 2013) reported that the
activity of earthworms reduced the PLFAs characteristic of bacterial and fungal biomass. This
reduction was more pronounced between week 21 and week 36 of rabbit manure
vermicomposting and pig slurry vermicomposting.
In this work, we studied the microbiological evolution during vermicomposting
compared with vermicomposting after composting, for the treatment and stabilization of
sewage sludge. The main hypothesis was that earthworms cause a different effect on
microbial community structure, depending on whether they feed on fresh or previously
composted material. To this end, enzyme activities (cellulase, β-glucosidase, protease,
alkaline and acid phosphatase) and the structure of the microbial community by analysing
PLFAs were assessed throughout the vermicomposting of fresh and composted sewage
sludge with the earthworm specie Eisenia andrei. In order to discern the effects due to the
different microbiological composition of the waste from the effects caused by earthworms,
the same substrates incubated without earthworms were studied.
2. MATERIALS AND METHODS
2.1. Substrates and earthworms
Sewage sludge was collected from a municipal wastewater treatment plant in Cangas
(Pontevedra, NW Spain) after an aerobic biological treatment and subsequent dehydration.
The sludge was mixed with wood chips as a bulking agent, adjusting the ratio to 1:2 (v/v). A
part of this mixture was used for the vermicomposting treatment (V). Another part of the
mixture was subjected to composting in a static adiabatic reactor with a 600 L capacity and
automatic control of temperature and oxygen. Forced aeration was applied, using a
centrifugal fan intermittently and depending on the controlled variables. The temperature
was maintained above 45°C for 7 days with maximum values of 60°C. The process ended after
15 days when the temperature in the composting mass reached values below 35°C. The fresh
compost was removed from the reactor, mixed and used as a substrate for vermicomposting
in the combined composting-vermicomposting treatment (CV). The earthworm species E.
andrei (Bouché, 1972) was used for the vermicomposting due to its high tolerance to
environmental factors and its high rate of organic matter processing (Domínguez, 2004). In
order to determine if the substrates affected the growth and maturation of the earthworms,
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juvenile specimens with an average weight of 310 ± 25 mg were collected from a laboratory
culture fed with horse manure.
2.2. Experimental design
Vermicomposting was carried out in rectangular culture systems of 14 L capacity,
which were filled with a layer of sieved and moistened vermiculite as refuge for earthworms,
with the advantage of being a biologically inert material. A plastic mesh (5 cm mesh size) was
placed between the vermiculite and the substrate to prevent their mixture and facilitate the
sampling. Two kilograms of substrate sludge or compost (2000 ± 6 g) and 115-120
earthworms according to the feed rate of 0.75 kg feed/kg worm/day (Ndegwa et al., 2000),
were introduced. Each substrate was replicated three times. Controls involving the same
materials (vermiculite, mesh and sludge or compost) incubated without earthworms were
included in triplicate. Culture systems were kept in darkness under the same conditions. The
moisture content was controlled and maintained above 70% by watering throughout the
process. After 70 days, cocoons, earthworms and hatchlings were removed by hand from the
cultures, counted and weighed. The culture systems were maintained until day 112 to enable
the maturation of the vermicompost. Samples were taken at 0, 14, 28, 42, 56, 70, 91 and 112
days. In order to remove the bulking agent, samples were sieved (less than 10 mm) and
several parameters were determined, as detailed below.
2.3. Physical-chemical analysis
Organic matter content was measured by the loss on ignition of dried samples at 550ºC
for 4 hours. Inorganic nitrogen (N-NH4+ and N-NO3−) was determined in 0.5 M K2SO4 extracts
in a ratio of 1:10 (w/v) applying the modified indophenol blue colorimetric method (Sims et
al., 1995). Total extractable nitrogen was determined in the same extracts after oxidation
with K2S2O8, as described by Cabrera and Beare (1993), and dissolved organic nitrogen
content (DON) was calculated as (total extractable N) – (inorganic N). Total nitrogen content
(TN) and total carbon content (TC) were determined by combustion of dried samples using a
LECO 2000 CN elemental analyser. Water soluble carbon content (WSC) was analysed in
aqueous extracts 1:5 (w/v) after oxidation with H2SO4 (96%) and K2Cr2O7 (1N) at 160ºC for
30 minutes and spectrophotometric measurement of reduced chromium. The pH was
determined in aqueous extracts 1:10 (w/v) using a pH meter Crison Basic 20.
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2.4. Biological and biochemical analysis
β-glucosidase was estimated by incubating the sample (1 g fresh weight) with 1 mL of
p-nitrophenyl-β-D-glucopiranoside (0.025 M) for 1 h at 37ºC and subsequent colorimetric
measurement of p-nitrophenol released (Eivazi and Tabatabai, 1988). Alkaline and acid
phosphatase was measured by incubating the sample (0.5 g fresh weight) with 1 mL of p-
nitrophenylphosphate (0.015 M) for 1 h at 37ºC and subsequent colorimetric measurement
of p-nitrophenol released (Eivazi and Tabatabai, 1977). Protease activity was measured by
colorimetric determination of the amino acids released, after the incubation of the sample (1
g fresh weight) with 5 mL of sodium caseinate (2%) for 2 h at 50ºC, using Folin-Ciocalteu
reagent (Ladd and Butler, 1972). Cellulase activity was assessed by colorimetric
determination of reducing sugars released after incubation of the sample (5 g fresh weight)
with 15 mL of carboxymethyl cellulose sodium salt (0.7%) for 24 h at 50ºC (Schinner and Von
Mersi, 1990). Germination index (GI) was calculated according to Zucconi et al. (1981) by
determining seed germination and root length of Lepidium sativum growing in 2 mL of
aqueous extracts 1:5 (w/v) in Petri dishes lined with paper filter during 48 hours.
The microbial community composition and biomass was determined by phospholipid
fatty acid analysis (PLFAs) following the method described by Gómez-Brandón et al. (2010)
for organic samples. Briefly, total lipids were extracted by stirring from 200 mg of each
freeze-dried sample with 60 mL of chloroform–methanol (2:1, v/v) and separated into
neutral lipids, glycolipids and phospholipids on silicic acid columns. The phospholipid
fraction was subjected to derivatization with trimethylsulfonium hidroxyde (TMSH) and fatty
acid methyl esters (FAMEs) obtained were analysed by gas chromatography and mass
spectrometry (GC-MS). GC-MS analysis was performed on a column CP-Select FAME, 100 m x
0.25 mm. FAMEs were identified by comparison of their retention time and mass spectra with
known standards (Larodan Fine Chemicals AB, Malmo, Sweden). The quantification was
performed using internal standard calibration. PLFAs were used to estimate the biomass of
specific microbial groups: gram-positive bacteria (i14:0, i15:0, a15:0, i16:0, a17:0), gram-
negative bacteria (16:1ω7, cy17:0, 17:1ω7, 18:1ω7, cy19:0) and fungi (18:2ω6, 18:1ω9,
20:1ω9)(Frostegård and Bååth, 1996; Zelles, 1997; Madan et al., 2002). The total amount of
PLFAs identified (totPLFAs) was used as an indicator of the viable microbial biomass (Zelles,
1999).
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2.5. Statistical analysis
Microbiological and biochemical data were analysed by repeated measures analysis of
variance (ANOVAR) in which the type of material and the presence or absence of earthworms
were set as between-subjects factors, and time was set as within-subjects factor. Correlation
analyses were carried out to examine the relationships between PLFAs and enzyme activities.
Student's t-tests were performed to determine the difference between the two types of
materials used in vermicomposting and one-way analysis of variance (ANOVA) to determine
the difference between physical-chemical parameters at the end of the vermicomposting
process. For post hoc comparison between groups in the case of a significant effect, HSD
Tukey tests were used. Where the assumptions of normality and variance homogeneity were
not met, data were log-transformed. All statistical tests were evaluated at the 95% confidence
level using the SPSS 20.0 program.
3. RESULTS AND DISCUSSION
3.1. Growth and reproduction of E. andrei
As shown in Table 1, the survival of E. andrei in both treatments V and CV was high (>
96.5% in all culture systems) and no significant differences were found (p > 0.05), so fresh
and composted sewage sludge presented good properties for their management by
vermicomposting.
Table 1. Growth, sexual development and survival of E. andrei after 70 days of vermicomposting
(V) and composting-vermicomposting (CV). Values are mean ± standard error (n = 3).
V CV
Earthworm biomass (g) 54.8 ± 1.2a 45.3 ± 0.6b
Weight gain per earthworm (mg) 169.4 ± 5.1a 80.7 ± 6.2b
Matured earthworms (%) 95.0 ± 1.3a 86.3 ± 2.3b
No. of hatchlings 517 ± 54a 10 ± 1b
No. of cocoons 840 ± 42a 282 ± 11b
Survival (%) 97.7 ± 1.1a 99.4 ± 0.3a
Means with the same letter are not significantly different (paired-
sample Student’s t-test, p < 0.05).
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The growth of E. andrei in V treatment was significantly larger than in VC treatment (t =
9.063, p < 0.05) and earthworm biomass significantly increased relative to the beginning of
the process, about 51.3% in V (t = 16.206, p < 0.01) and 25.6% in CV (t = 14.042, p < 0.01).
The highest number of mature earthworms, cocoons and hatchlings were obtained in V
treatment, with significant differences between treatments (t = 4.706, p < 0.05; t = 9.347, p <
0.05; t = 16.818, p < 0.05, respectively). These results suggested that the vermicomposting of
sewage sludge showed slightly better conditions for the development of E. andrei than
composted sludge. Frederickson et al. (1997) found that the growth rate of the epigeic
earthworm Eisenia fetida was reduced in pre-composted green waste compared with
vermicomposting of fresh material, suggesting that the nutritional content was more rapidly
decreased during the early stages of composting. Gunadi and Edwards (2003) propounded
that pre-composting of cattle manure can reduce bioavailable nutrients for earthworms,
inhibiting the growth rate and the number of cocoons and hatchlings produced by E. fetida.
Similar results were established in this study for municipal wastewater sewage sludge.
Moreover, CV treatment presented greater concentrations of harmful compounds for
earthworms, such as ammonium (Table 2), which could negatively affect earthworm
development.
3.2. Physical-chemical parameters
The physical-chemical properties at the initial and final materials are shown in Table 2.
The organic matter content was significantly lower in the presence of earthworms than in
controls (F1,11 = 62.483, p < 0.0001). After vermicomposting, organic matter decreased
about 14.6% in V treatment with earthworms and 3.4% in treatment without earthworms,
while reductions were about 12.7% and 6.6% in CV treatment with and without earthworms,
respectively. The presence of earthworms accelerated the mineralization of organic matter
(Elvira et al., 1996) so that the highest earthworm biomass in V treatment may have
produced a greater decrease in this measure. A greater reduction was noted in organic matter
in VC controls compared to V controls, which showed a higher loss of organic matter through
microbial breaking down after the passage of sewage sludge at the thermophilic phase of
composting. Ryckeboer et al. (2003) pointed out that the taxonomic and metabolic diversity
of bacteria and available substrates for fungi increase after the thermophilic phase of
composting.
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Table 2. Initial and final physical-chemical properties of vermicomposting (V) and composting-
vermicomposting (CV). Values are means ± standard error (n = 3).
Time V_E.andrei V_control CV_E.andrei CV_control
OM (g) 0 495.0 ± 1.1a 507.4 ± 0.5b
112 421.2 ± 1.0a 480.2 ± 0.7b 443.5 ± 1.1c 473.1 ± 4.1b
pH 0 6.2 ± 0.1a 7.1 ± 0.0b
112 5.7 ± 0.0a 5.6 ± 0.0a 5.9 ± 0.0b 6.0 ± 0.1b
C to N ratio 0 10.2 ± 0.2a 9.6 ± 0.1a
112 9.9 ± 0.3a 10.5 ± 0.3ab 10.5 ± 0.3ab 11.2 ± 0.2b
TC (g kg-1dw) 0 428.0 ± 1.9a 401.8 ± 1.6b
112 319.6 ± 7.1a 350.1± 7.6b 323.4 ± 5.5a 349.2 ± 5.1b
TN (g kg-1dw) 0 39.0 ± 0.6a 38.9 ± 0.3a
112 32.3 ± 0.2ab 33.4 ± 0.5a 30.7 ± 0.4b 31.2 ± 1.0ab
WSC (g kg-1dw) 0 2.64 ± 0.13a 7.53 ± 0.33b
112 2.88 ± 0.11a 4.43 ± 0.10b 3.02 ± 0.10a 3.42 ± 0.05c
DON (g kg-1dw) 0 4.57 ± 0.08a 5.16 ± 0.15b
112 6.56 ± 0.08a 7.88 ± 0.22b 3.84 ± 0.23c 3.95 ± 0.13c
NH4+ (g kg-1dw) 0 0.69 ± 0.04a 2.41 ± 0.14b
112 0.20 ± 0.01a 0.37 ± 0.03b 0.28 ± 0.02c 0.37 ± 0.02b
GI (%) 0 65.8 ± 2.3a 33.3 ± 1.1b
112 92.1 ± 0.4a 76.7 ± 0.9b 97.9 ± 0.4c 80.2 ± 0.5d
OM: organic matter, dw: dry weight, TC: total carbon, TN: total nitrogen, WSC: water soluble carbon,
DON: dissolved organic nitrogen, GI: germination index. Means with different letter in the same row
are significantly different (Tukey HSD, p < 0.05).
All final products had acidic conditions and there were no significant differences
between the presence and absence of earthworms, but V treatment showed lower pH than CV
(F1,11 = 24.666, p < 0.0001). These results agreed with those obtained by other authors
(Ndegwa et al., 2000; Khwairakpam and Bhargava, 2009; Hait and Tare, 2011), who observed
a decrease in pH after vermicomposting of sewage sludge due to the formation of organic
acidic compounds and the mineralization of nitrogen and phosphorus. The decrease in
carbon and nitrogen content, due to the degradation and mineralization of organic matter by
microorganisms, earthworms and the joint action of both, maintained the C to N ratio at a low
level in all treatments, with similar initial and final values. This ratio is extensively used as an
indicator of maturity of organic waste, although Yadav et al. (2010) reported that the C to N
ratio should not be used as a maturity parameter for the vermicomposting if the original
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waste is rich in nitrogen. A significant reduction of TN and TC was observed between initial
and final values with significant differences between all treatments (F3,11 = 153.409, p <
0.0001). With respect to TN, it was reduced by 21% on average in CV and 16% on average in
V, finding significant differences between V and CV treatments (F3,11 = 10.935, p < 0.01). The
low C to N ratio in the substrates and the aeration provided by the bulking agent may cause
the release of ammonia gas and decrease of TN in vermicomposting (Benitez et al., 1999;
Domínguez, 2004). In the case of DON, significant differences between V and CV (F1,11 =
98.193, p <= 0.0001) were detected with a reduction in CV and an increase in V in both
treatments with earthworms and controls. The presence of E. andrei showed lower
concentrations of ammonium (F1,11 = 0.437, p < 0.01), WSC (F1,11 = 16.131, p < 0.01) and CT
(F1,11 = 23.478, p < 0.01) than controls. These results were consistent with the general
hypothesis that earthworms promote carbon and nitrogen mineralization (Domínguez,
2004). Although readily assimilable carbon and nitrogen content increased after composting,
the subsequent process of vermicomposting diminished these parameters, favouring the
stabilization of combined CV treatment. Also, the presence of E. andrei showed significantly
higher GI than controls (F1,11 = 21.023, p < 0.01) and the CV treatment presented the greatest
value (97.9%). According to Zucconi et al. (1985), values of germination index greater than
80% present no phytotoxicity, so that both treatments with earthworms were effective for
eliminating phytotoxic substances, suggesting a suitable level of maturation. The physical-
chemical parameters evaluated and the germination index showed that earthworms
improved the sewage sludge properties, reaching optimal values of stabilization and
maturation in both V and CV treatments.
3.3. Enzyme activities
In general, the five hydrolytic enzymes studied decreased throughout all treatments
(Fig. 1, Fig. 2) accordingly, with metabolic degradation processes of organic matter
diminishing during vermicomposting. Benitez et al. (1999) observed similar trends during
vermicomposting of sewage sludge, suggesting that the decrease in hydrolytic activities
indicated stabilization of organic matter.
Cellulase and β-glucosidase are enzymes of the carbon cycle that play an important role
in the breakdown of organic matter. Cellulases degrade cellulose, releasing reducing sugars
and β-glucosidases catalyze the hydrolysis of β-glycosidic bonds of the carbohydrates (Alef
and Nannipieri, 1995). The cellulase activity decreased after composting and significant
differences were observed between V and CV, both at the outset (t = 4.455, p < 0.05) and
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during vermicomposting (F1,8 = 8.607, p < 0.05) (Fig. 1a and 1b). Owing to cellulose being
broken down by thermophilic organisms (Ryckeboer et al., 2003), the decrease in substrates
after composting may have reduced cellulase activity. In V treatment, with and without
earthworms, cellulase activity diminished in a similar way over the time, whereas in CV
treatment the decline was clearly higher in the presence of earthworms compared to controls
from day 28. Earthworms significantly reduced cellulase activity (F1,8 = 40.530, p < 0.0001)
producing a significant interaction between treatment, time and earthworms
presence/absence (F6,48 = 4.302, p < 0.01). Therefore, not only the presence of E. andrei
resulted in a decrease of cellulase activity, but also the effect caused by time and the type of
substrate, that probably affected the available substrate for this enzyme and cellulolytic
microbial community. Due to the fact that earthworms can feed on fungi (Schönholzer et al.,
1999), the main consumers of cellulose, changes in fungal community by earthworms, as has
been observed in this study by the analysis of PLFAs, could affect the production of cellulases.
Likewise, Gómez-Brandón et al. (2011b) found that earthworm activity reduced cellulase
enzyme after vermicomposting of grape marc, suggesting that the vermicomposted material
reached a high degree of stabilization.
Fig. 1. Changes in cellulase and β-glucosidase activities with and without E. andrei in
vermicomposting (V) (a, c) and composting-vermicomposting (CV) (b, d). Values are mean ±
standard error (n = 3).
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Regarding β-glucosidase, no significant differences between V and CV were detected at
the beginning of vermicomposting (t = -0.251, p > 0.05), these being about 4200 µg PNP g-1dw
h-1 (Fig. 1c and 1d). β-glucosidase activity presented significant differences between V and CV
during vermicomposting (F1,8 = 36.319, p < 0.0001), producing a significant interaction
between treatment, time and earthworms presence/absence (F6,48 = 5.374, p < 0.0001). There
was a gradual decline in enzyme β-glucosidase in V treatment with a minimum at the end of
the experiment (85% reduction), both in the presence and absence of earthworms. In the CV
treatment, the decline in the first sampling involved about 60% reduction of the enzyme
activity, with and without earthworms, and then the activity remained stable from 56 days.
Similar results were obtained by Benitez et al. (1999) during vermicomposting of sewage
sludge, showing a sharp decrease in β-glucosidase activity during the first 6 weeks with a
subsequent stabilization trend as a result of the decrease in available organic substrates.
Moreover, when the availability of C and N required for enzyme synthesis is limiting,
microorganisms can constrain their production of enzymes (Allison and Vitousek, 2005).
Sewage sludge showed a low C to N ratio (Table 2), which may lead to carbon becoming a
limiting substrate for the microbiota. These results showed differences between treatments
for enzymes of the C cycle, indicating that enzyme activities decreased and stabilized before
in combined composting-vermicomposting than in vermicomposting of fresh sewage sludge.
Alkaline and acid phosphatase enzymes catalyze the hydrolysis of organic
phosphomonoester to inorganic phosphorus, differing according to their optimum pH of
activity. At the beginning of vermicomposting, significant differences were observed between
V and CV for both acid phosphatase (t = 7045, p < 0.05) (Fig. 2a and 2b) and for alkaline (t =
3442, p < 0.05) (Fig. 2c and 2d). Differences in the activities were also detected between the
two treatments for the two enzymes throughout the process (F1,8 = 7.147, p < 0.05 for acid
phosphatase; F1,8 = 6.153, p < 0.05 for alkaline phosphatase). Phosphatase activities
decreased with time, but because phosphatases are highly influenced by the pH (Eivazi and
Tabatabai, 1977), a further decrease of the alkaline phosphatase with values greater than
60% reduction was observed. The accumulation of inorganic compounds of phosphorus as a
result of enzymatic activity has been shown to repress phosphatase activity (Alef and
Nannipieri, 1995). Likewise, Aira et al. (2007a) suggested that a decrease in microbial
biomass may cause an increase of available phosphorus and, therefore, a decrease in
phosphatase activity. This drop in phosphatase activity was also observed by other authors in
the vermicomposting of different wastes (Fernández-Gómez et al., 2010, 2013). In V
treatment (Fig. 2a and 2c), phosphatase activities evolved in a similar way, regardless of the
presence or absence of earthworms. However, a greater influence of E. andrei on enzyme
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activity was observed in the CV treatment (Fig. 2b and 2d) with a faster decrease in
phosphatase activities in the presence of earthworms, resulting in significant interaction
between treatment, time and earthworms presence/absence in acid (F6,48 = 10.810, p <
0.0001) and alkaline phosphatase (F6,48 = 1.961, p <0.05). Furthermore, earthworms
significantly affected alkaline phosphatase (F1,8 = 24.109, p < 0.01). Earthworms had a greater
effect on the enzymes of the phosphorus cycle in the combined CV treatment than V
treatment. Microbiota decrease and mineralization increase during composting, with
subsequent activity of earthworms, may have increased the content of orthophosphate and
reduced the activity and synthesis of phosphatase in CV treatment.
Fig. 2. Changes in acid phosphatase, alkaline phosphatase and protease activities with and
without E. andrei in vermicomposting (V) (a, c, e) and composting-vermicomposting (CV) (b, d,
f). Values are mean ± standard error (n = 3).
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Protease enzymes supply a large part of the available nitrogen by catalyzing the
hydrolysis of proteins to peptides and amino acids (Alef and Nannipieri, 1995; Geisseler and
Horwath, 2008). Significant differences between V and CV (t = 11.046, p < 0.01) were
detected at the beginning of vermicomposting (Fig. 2e and 2f). Protease activity decreased in
all treatments with a greater reduction in V compared to CV (F1,8 = 36.986, p < 0.0001 ).
Protease activity is dependent on substrate availability (Aira et al., 2007a), which was
reduced as the vermicomposting process developed. Also, the greater activity in the final
samples of CV treatment could be caused by the formation of complexes between
extracellular enzymes and humic substances because of the previous process of composting
that can increase the formation of these substances compared to vermicomposting
(Campitelli and Ceppi, 2008). The evolution through time in V treatment was similar with and
without earthworms, showing a decrease in the first 14 days of process, remaining stable up
to 70 days and decreasing around 5000 µg tyrosine g-1dw 2h-1 in the final sampling. In CV
treatment without earthworms, protease activity slightly increased over time until it reduced
from 70 days to similar values to those observed at the beginning of the process. In the case
of CV treatment with earthworms, two different stages were observed: a first stage where
activity increased to a maximum at 28 days (17700 µg tyrosine g-1dw 2h-1) and a second
stage, from 42 days to 112 days, where the activity fell below the values noted at the
beginning of the process. No significant differences between treatments with earthworms
and controls were detected, although there was a significant interaction between treatment,
time and earthworms presence/absence (F6,48 = 9.273, p < 0.0001). In general, a reduction in
protease was observed over time, more marked in the treatment V and with a slight negative
effect of earthworms on this enzymatic activity. Several studies have observed reductions of
protease activity in the presence of earthworms in contrast to controls with different organic
substrates (Aira et al., 2007a; Gómez-Brandón et al., 2011b; Fernández-Gómez et al., 2013).
Geisseler and Horwath (2008) suggested that microorganisms regulate protease synthesis,
depending on their needs of carbon and nitrogen, so that the low C to N ratio could cause a
decrease in microbial biomass and enzyme synthesis.
3.4. Dynamic of the microbial community
Microbial biomass presented significant differences between V and CV (t = 21.719, p <
0.01) at the beginning of vermicomposting with a totPLFAs content in the first case of 1280
µg g-1dw, and after the composting process this was reduced up to 895 µg g-1dw. Both
treatments showed a decrease in microbial biomass throughout the vermicomposting
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process (Fig. 3), especially in the first samplings, resulting in a significant interaction between
treatment and time (F6,48 = 70.315, p < 0.0001).
Fig. 3. Changes in microbial biomass, measured as totPLFAs, with and without E. andrei in
vermicomposting (V) and composting-vermicomposting (CV). Bars represent standard errors.
Different letters in the same time of sampling are significantly different (Tukey post hoc test p <
0.05).
This decline in microbial biomass, measured as totPLFAs, was consistent with the
results obtained by other authors during vermicomposting (Fernández-Gómez et al., 2013;
Gómez-Brandón et al., 2013). The sewage sludge used in this experiment was biologically
degraded during processing at the plant, so that the easily degradable nutrients could
exhaust, thereby resulting in a reduction of microbial biomass. Earthworm activity greatly
reduced the abundance of totPLFAs (F1,8 = 117.789, p < 0.0001), although this effect was
more pronounced in V treatment (98.5% reduction from initial) compared with CV (89.4%
reduction from initial), producing a significant interaction between treatment and presence
/absence of earthworms (F1,8 = 6.469, p < 0.05). Because V treatment presented a higher
increase in earthworm population and biomass compared to that observed in CV treatment,
microbial biomass showed a larger decline in totPLFAs. These findings are consistent with
previous observations that suggest that the digestion of organic material by epigeic
earthworms has negative effects on microbial biomass (Gómez-Brandón et al., 2011c). In
addition, Tiunov and Scheu (2004) observed that earthworms can compete with
microorganisms for available carbon resources and cause a sharp decline in microbial
biomass when carbon is limiting. The reduction of available resources due to the action of
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microorganisms, competition for them with earthworms and feeding with bacteria and fungi
by earthworms, may explain the higher decline in biomass in the presence of E. andrei. In V
treatment with earthworms, totPLFAs correlated positively with all enzymes: cellulase (r =
0.787, p <0.0001), alkaline phosphatase (r = 0.832, p < 0.0001), β-glucosidase (r = 0.942, p <
0.0001), acid phosphatase (r = 0.684, p < 0.001) and protease (r = 0.750, p < 0.0001), with a
slightly lower correlation in all enzymes (p < 0.01) in absence of earthworms. In CV
treatment with earthworms, correlation between totPLFAs with cellulase (r = 0.791, p <
0.0001), glucosidase (r = 0.649, p < 0.01), protease (r = 0.481, p < 0.05), alkaline (r = 0.822, p
< 0.0001) and acid phosphatase (r = 0.601, p < 0.001) were observed. No correlations were
found, however, between totPLFAs and acid phosphatase and protease in CV control, whereas
correlations were detected with cellulase (r = 0.533, p < 0.01), alkaline phosphatase (r =
0.763, p < 0.01) and β- glucosidase (r = 642, p < 0.01). Despite the different evolution of
hydrolytic enzymes, the results in general showed that a decrease in microbial biomass was
accompanied by a decrease in enzymes, suggesting that enzyme activities were directly
associated with living microorganisms. However, V treatment with E. andrei showed enzyme
activities very similar to controls, despite having a stronger reduction of microbial biomass.
This may indicate the formation of complexes between enzymes and humic substances
during vermicomposting (Benitez et al., 2005) or the presence of a less efficient microbial
community for organic matter degradation in controls (Aira et al., 2007b). Although
earthworms exerted an important effect on the microbial biomass, the performance of the
enzyme activity was determined by both the type of waste used in the vermicomposting and
the presence or absence of earthworms. Both treatments were suitable for the reduction and
stabilization of microbial biomass of sewage sludge.
Table 3. Changes in PLFAs without earthworms (control) and in the presence of E. andrei in vermicomposting (V) and composting-vermicomposting
(CV). Values are means ± standard error (n = 3).
Time Gram+ bacteria (µg g-1dw ) Gram– bacteria (µg g-1dw ) Fungi (µg g-1dw )
V CV V CV V CV
E.andrei control E.andrei control E.andrei control E.andrei control E.andrei control E.andrei control
0 197.0±8.8a 178.5±7.2a 370.6±14.9a 270.1±16.8b 338.2±15.6a 186±17.2b
14 274.9±12.1a 270.9±16.9a 89.2±10.6b 160.2±5.4c 313.0±14.4a 321.3±13.7a 113.7±11.3b 184.6±13.0c 214.7±11.1a 217.8±15.3a 62.5±8.1b 105.8±10.8c
28 121.3±7.9a 159.2±10.1b 42.6±3.5c 97.7±4.6a 134.2±7.7a 211.0±13.1b 38.4±3.8c 102.3±9.8a 111.1±9.1a 159.9±13.0b 34.6±3.5c 78.4±10.2d
42 38.0±4.8a 98.5±2.5b 28.5±1.9a 59.1±4.4c 28.4±4.7a 144.7±10.2b 26.6±4.0a 57.8±5.2c 34.5±5.1a 115.5±6.5b 26.4±3.6a 39.0±4.6a
56 42.5±1.6a 96.4±9.5b 27.3±4.3c 44.0±5.6ac 41.6±1.2a 107.1±1.9b 14.3±2.7c 52.0±5.0a 46.4±1.8a 95.0±2.6b 19.4±2.5c 38.7±4.0a
70 27.9±2.4a 77.5±2.2b 23.4±2.6a 53.3±3.1c 18.7±3.1a 54.1±4.9b 13.5±3.0a 51.3±2.8b 22.5±4.9ac 69.2±3.1b 17.0±5.4c 43.2±7.0a
91 11.4±1.6a 38.5±4.0b 17.6±3.6a 27.8±0.7b 8.9±1.5a 25.9±2.2b 10.5±1.7a 24.0±0.6b 10.3±1.4a 47.1±3.5b 9.6±1.9a 26.4±0.4c
112 2.8±0.1a 52.4±5.9b 12.6±2.1c 16.8±1.0c 0.4±0.1a 33.3±3.2b 11.0±1.2c 26.5±2.2b 0.9±0.3a 69.8±8.6b 8.1±1.4c 36.6±2.2d
In each parameter different letters in the same time of sampling are significantly different (Tukey post hoc test p < 0.05).
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Earthworm activity had a strong effect on the abundance of PLFAs with a significant
reduction in Gram + bacteria (F1,8 = 134.119, p < 0.0001), Gram – bacteria (F1,8 = 106.862, p <
0.0001) and fungi (F1,8 = 76.260, p < 0.0001) with regard to control during vermicomposting
(Table 3). Similar results have been reported by Gómez-Brandón et al. (2011a) during
vermicomposting of pig slurry with E. fetida, suggesting that this earthworm modified the
structure of microbial communities. In the same way, E. andrei affected the abundance of
bacterial and fungal PLFAs during vermicomposting of fresh and composted sewage sludge.
Previous studies have observed that epigeic earthworms had a greater effect on Gram +
bacteria than Gram – bacteria through the gut associated processes and that Gram – bacteria
can survive the transit through the earthworm gut (Gómez-Brandón et al., 2011c; Williams et
al., 2006). In accordance with this, V treatment with earthworms showed a higher reduction
in Gram + bacteria than Gram – bacteria compared to controls throughout the process.
Likewise, E. andrei had a greater effect on Gram + bacteria in the first samplings of CV
treatment. Also, a significant decrease in the fungal biomass was observed in the presence of
earthworms. This effect on fungal populations was reported by other authors. Huang et al.
(2013) and Fernández-Gómez et al. (2013) found a decrease in fungal biomass in the end
products after vermicomposting with E. fetida. Such decreases may be due to the fact that
earthworms can feed on fungi that provide them an important source of nutrients
(Schönholzer et al., 1999). The decrease of microbial groups was greater in V treatment with
earthworms (> 98.6%) than in CV treatment with earthworms (93% Gram + and 96% for
Gram – and fungi), while the reduction in controls was higher in CV treatment for Gram +
bacteria (90.6%) compared to V treatment (73.4%) and similar for Gram – bacteria (90-91%)
and fungi (79-80%), showing a significant interaction between treatment, time and
earthworms presence/absence for all indicators of specific microbial groups: Gram + (F6,48 =
8.697, p < 0.01), Gram – (F6,48 = 6.216, p < 0.01) and fungi (F6,48 = 4,989, p < 0.01). Regarding
the last samplings, it was observed that treatments with earthworms, both in V and CV, had a
lower concentration of bioindicators of microbial groups than the controls. It has been found
that the degradation and mineralization of organic matter in the controls of vermicomposting
is insufficient compared to treatment with earthworms (Elvira et al., 1996). So, the greatest
microbial load in controls may indicate the presence of organic matter available for the
microorganisms, being lower in CV treatment due to its passage through the thermophilic
phase of composting.
As can be seen in the last sampling, final products presented different microbial
communities. Lores et al. (2006) reported that the fingerprint of the microbial community of
a vermicompost depends on the type of substrate and earthworm species used in the process.
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Fernández-Gómez et al. (2012) reported that vermicomposts produced from wastes of
different nature and origin can contain similar microbial communities, if the same earthworm
species is used. Sen and Chandra (2009) showed divergent bacterial community in compost
and vermicompost obtained from the same initial waste, despite similar changes in their
physico-chemical parameters during the processes. In this case, the microbial community
seemed to differ depending on if V or CV treatment were applied and on the presence or
absence of earthworms. Differences may be attributable to the distinct physicochemical and
microbiological composition of the waste and the interactions of earthworms on microbial
communities.
4. CONCLUSIONS
In this study, the ability of the earthworm Eisenia andrei to degrade fresh and
composted municipal sewage sludge was shown. The PLFA analysis indicated that E. andrei
reduced microbial biomass, both bacterial and fungal, and that the composition of the
microbial community depended on the presence or absence of earthworms as well as the
treatment used. Earthworm incorporation after the thermophilic stage of composting
accelerated the degradation of the waste, with a greater decrease in the phosphorus and
carbon cycling enzymes. Therefore, the combined composting-vermicomposting process can
be a good alternative to the management of sewage sludge.
ACKNOWLEDGMENTS
This study was financially supported by the Xunta de Galicia (Regional Autonomous
Government of Galicia) (09MDS024310PR). The authors thank the wastewater treatment
plant of Cangas for supplying sewage sludge and the research support services of the
University of Vigo (CACTI) for the carbon and nitrogen analysis. The authors also thank
Emilio Rodríguez Cochón for technical support and referees for their valuable comments and
suggestions.
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CAPÍTULO 4
Product quality and microbial dynamics during
vermicomposting and maturation of compost from pig
manure
Revista: Waste Management
Estado: bajo revisión
Permiso: pre print
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RESUMEN
El estiércol de cerdo es un residuo ganadero que se produce en elevadas cantidades y
que presenta una alta carga orgánica pudiendo ser tratado mediante procesos biológicos. El
principal objetivo de este trabajo es estudiar, a través de la dinámica microbiana, el proceso
de vermicompostaje y el empleo de lombrices de tierra en la maduración del compost fresco,
y la influencia de estos procesos en la calidad de los compost y vermicompost. Para
determinar el efecto que las lombrices de tierra causan en la dinámica microbiana, se
evaluaron las actividades enzimáticas y los perfiles de ácidos grasos fosfolípidos a lo largo de
112 días. Así mismo se analizaron parámetros fisicoquímicos y biológicos en los productos
obtenidos. La presencia de lombrices de tierra redujo significativamente la biomasa
microbiana y todos los grupos microbianos (bacterias Gram +, bacterias Gram – y hongos)
tanto en el estiércol fresco como en el pre-compostado. Las actividades enzimáticas (celulasa,
β-glucosidasa, fosfatasa ácida y proteasa) se comportaron de manera significativamente
distinta dependiendo del tratamiento efectuado. La comunidad microbiana tuvo un efecto
directo sobre los procesos degradativos durante la maduración del compost, sin embargo, en
presencia de Eisenia andrei se establecieron complejas interacciones lombrices-microbiota-
sustrato. Así, las actividades enzimáticas en presencia de lombrices no fueron consecuencia
directa de la síntesis de enzimas por parte de la microbiota presente en el sustrato. La
inoculación de lombrices de tierra en el compost mejoró la calidad del producto obtenido por
lo que el proceso combinado compostaje-vermicompostaje es un proceso viable para la
maduración del compost. Aunque el vermicompostaje del estiércol fresco alcanza valores de
calidad, es necesario optimizar el tiempo de la fase de maduración del vermicompost.
Palabras clave: actividades hidrolíticas, PLFAs, evolución microbiana, madurez, curado
del compost, lombrices de tierra.
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ABSTRACT
Pig manure is a livestock waste product which is produced in large quantities and has a
high organic load, which can be treated through biological processes. The main objective of
this research is to study, through microbial dynamics, the vermicomposting process and the
use of earthworms in the maturation of fresh compost, and the influence of these processes
on compost and vermicompost quality. In order to determine the effect that earthworms have
on microbial dynamics, the enzymatic activities and profiles of phospholipid fatty acids
(PLFAs) were evaluated over a 112-day period. The physicochemical and biological
parameters of the obtained products were also analysed. The presence of earthworms
significantly reduced microbial biomass and all the microbial groups (Gram + bacteria, Gram
– bacteria, and fungi), both in fresh and the pre-composted manure. The enzymatic activities
(cellulase, β- glucosidase, acid phosphatise, and protease) behaved in a significantly
distinctive manner, depending on the treatment used. The microbial community had a direct
effect on the degradative processes during compost maturation. However, complex
earthworm-microbiota-substrate interactions were established in the presence of Eisenia
andrei. Likewise, the enzymatic activities in the presence of earthworms were not a direct
consequence of in situ enzyme synthesis by the microbiota present in the substrate. The
inoculation of the earthworms in the compost improved the quality of the product obtained,
which means that the combined process of composting-vermicomposting is a viable process
for maturing compost manure. Although the vermicomposting of fresh manure achieved
quality values, it is necessary to optimize the vermicompost maturation phase period in
terms of time.
Keywords: hydrolytic activities, PLFAs, microbial evolution, mature, compost curing,
earthworms
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1. INTRODUCTION
Spain is the second largest pork producer in the European Union and generates more
than 50 million m3 of pig slurry per year as a result of this activity; this then must be
correctly dealt with (MAGRAMA, 2012). The separation of the slurry’s liquid phase from its
solid one is a treatment process regularly carried out on pig farms (Hjorth et al., 2010). The
solid portion is mainly composed of excrement, straw, and the remains of food with a high
content of organic matter, which allows for it to be treated through biological techniques,
such as vermicomposting and composting. Vermicomposting is a process which bio-oxidizes
and stabilizes the organic material due to the interaction between microorganisms and
earthworms. The microorganisms are the ones principally responsible for degrading the
organic material, although the earthworms stimulate the microorganisms as a result of them
modifying the substrate’s properties through feeding, aeration, and the excretion of casts; all
of this leads to the acceleration of the mineralization of the organic material and an
improvement in the nutrient availability for plants (Domínguez, 2004). The vermicomposting
process can generally be separated into two phases. First, an active phase, characterized by
the earthworms which move through the waste, causing changes to its physiochemical and
microbial properties. This is followed by the second phase, the maturation stage, when the
earthworms move towards fresher material; this results in the microorganisms taking
control of decomposing the processed material (Lazcano et al., 2008). Various authors have
studied the effects of epigeic earthworms have on pig manure. For example, Aira et al. (2007)
studied the interaction between the earthworms and microbial biomass and enzymatic
activity by using continuous feed vermireactors, with different doses of manure in the
presence and absence of Eisenia fetida. In a later study Aira and Domínguez (2009) compared
microbial stabilization and nutrients of the casts produced by E. fetida fed with pig manure
with those fed with cow manure. Similarly, Gómez-Brandón et al. (2011a) studied the impact
the species E. andrei has on the structure and function of the microbial community in the
casts from three types of animal manure, among them pig manure. Monroy et al. (2009)
studied the effect of the E. fetida earthworm on the total number of coliforms in pig manure in
continuous feed vermireactors, observing a reduction of 98% of coliforms density.
Composting is a process of biological degradation of solid organic substrates under
aerobic conditions through the action of different microbial communities, resulting in a stable
and humified product and apt for adding to the soil (Insam and de Bertoldi, 2007). The
organic material goes through different stages: a mesophilic stage, characterized by
microbiota proliferation; a thermophilic stage, which produces a high level of biodegradation,
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the growth of thermophilic organisms and the inhibition of non-thermo-tolerant organisms;
and the final stage, which includes a period of cooling, stabilization, and maturation, and is
characterized by the growth of mesophilic organisms and the humification of the compost
(Ryckeboer et al., 2003b). The inoculation of earthworms in the material after going through
the thermophilic stage has been used as a pre-treatment prior to vermicomposting, with the
aim being to eliminate compounds present in some waste products which are noxious for
earthworm; these include, for example, high levels of ammonia (Domínguez, 2004). For
example, (Chan and Griffiths (1988) faced with the impossibility of vermicomposting the pig
manure without treating it, so prepared it through composting and the addition of calcium
sulphate. The comparison of vermicomposting with regards to composting pig manure was
dealt with by Zhu et al. (2016) who compared both treatments with the objective of studying
the variation of heavy metals and dissolved organic matter. No studies about the inoculation
of earthworms in pig manure after a previous composting with the aim of knowing about the
changes during maturation, with or without epigeic earthworms, and comparing the compost
and vermicompost obtained have been found.
This paper studies the microbial dynamics during the vermicomposting of pig manure
and during the maturation process of pig manure compost, with and without epigeic E. andrei
earthworms. The main hypothesis of this study are 1) that the presence of detritivores
earthworms determines the changes to the microbiota and thus the changes in the enzymatic
activity and that 2) maturing the compost with earthworms improves the product obtained in
terms of physic-chemistry and biology. For this, enzymatic activity (cellulase, β-glucosidase,
protease, and acid phosphatase) and the microbial community’s structure were studied by
analyzing PLFAs throughout the vermicomposting and compost maturation process. The
stability and maturity parameters in the products obtained were also analyzed.
2. MATERIALS AND METHODS
2.1. Substrates and earthworms
The solid fraction of manure was collected from a pig breeding farm after storage in
manure pits for liquid separation. The manure was mixed with shredded wood as a bulking
agent, adjusting the ratio to 1:2 (v/v). The initial properties of this mixture can be seen in
table 1 as fresh pig manure. One part was used for the vermicomposting treatment with
earthworms (V treatment) and the control treatment without earthworms (Vcontrol treatment).
Another part was subjected to composting in a static adiabatic reactor with a 600 L capacity
and automatic control that has been described in detail in Villar et al. (2016a). The
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temperature was maintained above 45°C for 8 days with maximum values of 60°C. The
process ended after 14 days when the temperature in the composting mass reached values
below 35°C. The fresh compost was removed from the reactor, mixed and one part was used
as a substrate for vermicomposting in the combined composting-vermicomposting treatment
(CV treatment) and the other part continued the composting maturation process without
earthworms (C treatment). The initial properties of the pre-composted pig manure can be
seen in the table 1.
Table 1. Physico-chemical characteristics of the substrates used in the treatments
Fresh pig manure
Pre-composted pig
manure
Moisture (%) 76.2 ± 0.5 68.8 ± 0.3*
Organic matter (%) 87.3 ± 0.2 85.7 ± 0.8*
pH 8.40 ± 0.03 6.86 ± 0.03*
Electrical conductivity (mS cm-1) 0.86 ± 0.02 0.64 ± 0.02*
Total carbon (mg g-1dw) 433.1 ± 11.2 419.0 ± 6.9*
Total nitrogen (mg g-1dw) 23.2 ± 0.4 22.5 ± 1.3
P2O5 (mg g-1dw) 11.3 ± 0.3 11.7± 0.4
Ammoniacal nitrogen (mg g-1dw) 3.02 ± 0.10 1.40 ± 0.05*
The asterisk indicates significant differences between substrates (paired-sample
Student’s t-test, p < 0.05) dw: dry weight
Specimens with an average weight of 325 ± 34 mg of the earthworm species E. andrei
(Bouché, 1972) were collected from a laboratory culture fed with horse manure for the CV
and V treatments.
2.2. Experimental design
Vermicomposting treatments were carried out according with previous work (Villar et
al., 2016b). Briefly, rectangular culture systems of 14 L capacity were filled with a layer of
sieved and moistened vermiculite, a plastic mesh of 5 cm mesh size and 2010 ± 8 g of
substrate: pig manure or compost. Each substrate was replicated three times and 115-120
earthworms were introduced in each system. Controls involving the vermiculite, mesh and
fresh pig manure incubated without earthworms were included in triplicate for Vcontrol
treatment. Culture systems were kept in darkness under the same conditions. The moisture
content was controlled and maintained above 70% by watering throughout the process. After
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70 days, cocoons, earthworms and hatchlings were removed by hand from the cultures,
counted and weighed. The culture systems were maintained until day 112 to enable the
maturation of the vermicompost. Maturation of the pre-composted pig manure without
earthworms (C treatment) was done in triplicate in wooden boxes of 200L as has been
described in Villar et al. (2016a). The compost was left for 112 days without being
homogenized and it was not necessary to moisten the material. Therefore, four different
treatments were carried out (Table 2). Samples were taken at 0, 14, 28, 42, 56, 70, 91 and 112
days in composting boxes and vermicomposting systems. In order to remove the bulking
agent, samples were sieved (less than 10 mm) and several parameters were determined, as
detailed in the following sections.
Table 2. Abbreviations, substrates and presence/absence of earthworms from the experimental
design
Treatments Substrate Earthworms
V Fresh pig manure yes
Vcontrol Fresh pig manure no
CV Pre-composted pig manure yes
C Pre-composted pig manure no
2.3. Physicochemical analysis
Organic matter content was measured by the loss on ignition of dried samples at 550 º
C for 4 hours. Total carbon content (TC) and total nitrogen content (TN) were determined by
combustion of dried samples using a LECO 2000 CN elemental analyser. N-NH4+ and N-NO3−
were determined in 0.5 M K2SO4 extracts in a ratio of 1:10 (w/v) according with Sims et al.
(1995). Water soluble carbon content (WSC) was analysed in aqueous extracts 1:5 (w/v) by
dichromate oxidation in sulfuric acid solution and spectrophotometric measurement of
reduced chromium. The pH and electrical conductivity were determined in aqueous extracts
1:10 (w/v) using a pH meter Crison Basic 20 and a conductivimeter Crison CM 35.
2.4. Biological and biochemical analysis
β-glucosidase and acid phosphatase were determined by the colorimetric measurement
of p-nitrophenol released according to the methods reported by (Eivazi and Tabatabai, 1988,
1977). Protease activity was measured by colorimetric determination of the amino acids
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released using Folin-Ciocalteu reagent (Ladd and Butler, 1972). Cellulase activity was
assessed by colorimetric determination of reducing sugars released according with Schinner
and von Mersi (1990). Germination index (GI) was calculated according to Zucconi et al.
(1981) by determining seed germination and root length of Lepidium sativum growing in
aqueous extracts 1:5 (w/v).
Static respiration rate (SR) was measured using manometric respirometers by
OxiTop® system (WTW GmbH, Weilheim, Germany). The microbial community composition
and biomass were determined by phospholipid fatty acid analysis (PLFAs) following the
method described by Gómez-Brandón et al. (2010) for organic samples. The analysis was
performed with a CP-Select FAME, 100m x 0.25mm in a gas chromatograph-mass
spectrometer (GC-MS). Identification was done by comparison of retention times and mass
spectra with known external standards (Larodan Fine Chemicals AB, Malmo, Sweden) and
quantification was performed with methyl nonadecanoate fatty acid (C19:0) as internal
standard. PLFAs were used to estimate the biomass of specific microbial groups: Gram-
positive bacteria (i14:0, i15:0, a15:0, i16:0, a17:0), Gram-negative bacteria (16:1ω7, 17:1ω7,
18:1ω7) and fungi (18:2ω6, 18:1ω9, 20:1ω9). The total amount of PLFAs identified
(totPLFAs) was used as an indicator of the viable microbial biomass (Zelles, 1999).
2.5. Statistical analysis
All statistical tests were performed using R software (R Development Core Team,
2014). Student's t-tests were performed to determine the difference between earthworm-
related parameters and substrates and one-way analysis of variance were performed to
determine difference between final products. Enzyme and PLFA data was analyzed with
linear mixed-effects models using the nlme package (Pinheiro et al., 2015). The type of
substrate and the presence or absence of earthworms were fixed factors and the repeated
measurement throughout time in each maturation box was treated as a random effect to
address the non-independence of samples. Logarithmic and square root transformations of
the data were necessary to ensure the normality and homogeneity of the variance of residuals
of models. For post hoc comparison between treatments, Tukey tests were carried out using
the glht function of the multcomp package (Hothorn et al., 2008). Correlation analyses were
also carried out to examine the relationships between PLFAs and enzyme activities with cor
function of the stats package. All statistical tests were evaluated at the 95% confidence level
and values are given as the mean ± standard deviation.
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3. RESULTS
3.1. Growth and reproduction of E. andrei
Table 3 shows the parameters related to the growth, reproduction, and sexual
development of E. andrei in fresh manure and compost after leaving the reactor. Both mixes
make it possible for high level of earthworms to survive, although there are significant
differences (p < 0.05) due to the higher mortality rate in the earthworm population in the
treatment with compost as the substrate. Earthworm growth, the number of mature
individuals and cocoon production were similar in both substrates, there being no significant
difference between the two (p > 0.05). With respect to the number of juveniles, the V
treatment showed significantly more individuals than the CV treatment (p < 0.05).
Table 3. Growth, sexual development and survival of E. andrei after 70 days of
vermicomposting.
V treatment CV treatment
Earthworm biomass (g) 40.8 ± 0.3 37.0 ± 2.4*
Weight gain per earthworm (mg) 44.6 ± 2.4 43.1 ± 6.9
Mature earthworms (%) 96.8 ± 1.2 94.7± 1.1
No. of hatchlings 1208 ± 102 955 ± 43*
No. of cocoons 548 ± 67 463 ± 59
Survival (%) 98.8 ± 1.3 85.8 ± 3.7*
The asterisk indicates significant differences between substrates (Student’s t-test, p <
0.05)
3.2. Microbial dynamics evolution
3.2.1. Microbial activity
The substrates showed significant differences at 0 time with respect to the enzymes:
greater cellulose (p< 0.0001) and β-glucosidase (p< 0.05) activity and less acid phosphatase
(p < 0.0001) and protease (p < 0.0001) activity in the compost as compared to the fresh
manure. All the enzymatic activity studied (β-glucosidase, cellulase, acid phosphatase, and
protease) showed significant differences (p < 0.01), depending on the treatment; this resulted
in significant interactions between the treatment and time (p <0.0001) (Figure 1).
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Cellulase activity was significantly affected by the presence of earthworms (p < 0.001)
this effect being dependent on the substrate (p < 0.05). The presence of earthworms in fresh
manure caused a decrease in cellulase activity, as compared to the Vcontrol treatment, which
had high cellulase activity during the first 91 days of the process. The presence of E. andrei
caused an initial increase in cellulase activity in the CV treatment up until day 28, but after
that saw a sharp decrease, resulting in values similar to the C treatment at the end of the
process. The C treatment had similar activity values throughout the entire maturation
process. After 112 days, the activities were C, CV > Vcontrol > V.
Fig. 1. Changes in cellulase (a), β-glucosidase (b), acid phosphatase (c) and protease (d)
activities during vermicomposting (V) and control (Vcontrol) treatments of pig manure,
maturation with earthworms (CV) and maturation without earthworms (C) of pre-composted
pig manure.
In terms of β-glucosidase activity, the presence/absence of earthworms had a
significant effect (p < 0.05) this effect being dependent on the substrate (p < 0.0001). In the
case of fresh manure, the inoculation of E. andrei caused a clear decrease in activity as
compared to the control without earthworms: the V treatment had slight fluctuations until
reaching values around 1000µg g-1 in the last samplings, while Vcontrol had large swings
throughout the process, but with values greater than V in every sample. The presence of
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earthworms caused an initial decrease β-glucosidase activity in the CV treatment, and then
increased after the day-28 sampling, showing values which were clearly superior to C
treatment. Despite these differences, after 112 days V and C products showed values that
were similar to each other and less than those found in CV and Vcontrol, which also reached
similar values. No correlations between these two carbon-cycle enzymes were observed.
The presence/absence of earthworms significantly affected acid-phosphatase activity
(p < 0.001), independent of the type of substrate. The species E. andrei in both substrates
increased this enzyme’s activity to a greater degree than the treatments without earthworms.
At the end of the process, the activity in the Vcontrol and CV treatments were very similar, as
occurred with β-glucosidase, the values being higher than the activity in treatment V, while
treatment C showed activity in the last samplings of around 2000µg g-1, which are less than
the rest of the treatments.
The presence of earthworms in the substrates did not affect protease activity, although
significant interaction between the presence/absence and type of substrate was observed (p
< 0.01) as well as significant interaction between the presence/absence and time (p < 0.01).
Compost maturation with and without earthworms showed different evolutions after day 28,
greater activity being observed in CV than in C, while in the fresh manure the effects were to
the contrary and the presence of earthworms caused a decrease in protease activity over
time. In the last sampling, only the compost showed differences, with lower values; as in the
case of β-glucosidase and acid phosphatase, CV y Vcontrol products presented similar values.
The correlations were different according to the treatment, so that cellulase correlated
with protease in the V (r = 0.81, p < 0.0001), Vcontrol (r = 0.74, p < 0.0001), and CV (r = -0.52, p
< 0.001) treatments. Cellulase correlated with acid phosphatase in the V (r = -0.90, p <
0.0001), and Vcontrol (r = -0.81, p < 0.0001) treatments. Acid phosphatase negatively correlated
with protease in the V (r = -0.82, p < 0.0001), Vcontrol (r = -0.63, p < 0.01), and C (r = -0.87, p <
0.0001) treatments. β-glucosidase correlated with acid phosphatase in the CV (r = 0.72, p <
0.001) and C (r = -0.87, p < 0.001) treatments. β-glucosidase correlated with protease in the C
(r = 0.83, p < 0.0001) treatment.
3.2.2. Microbial communities.
The substrates showed significant differences at 0 time in all the assessed parameters;
totPLFAs (p < 0.0001), Gram + bacteria (p < 0.0001) and fungi (p < 0.0001) all had a
significant decrease in concentration after the composting process, while the Gram – bacteria
increased significantly (p< 0.05) after the manure went through the composting reactor. Both
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the microbial biomass and the microbial groups studied over time showed significant
differences, depending on the treatment (p < 0.0001) resulting in significant interactions
between treatment and time (p <0.0001) (Figure 2).
Fig. 2. Changes in a) microbial biomass, b) Gram + bacteria, c) Gram – bacteria and d) fungi
estimated by phospholipid fatty acids (PLFA) analysis during vermicomposting (V) and control
(Vcontrol) treatments of pig manure, maturation with earthworms (CV) and maturation without
earthworms (C) of pre-composted pig manure.
TotPLFAs showed significant differences as a consequence of the presence or absence
of E. andrei (p < 0.001), independent of the type of substrate. The earthworms caused a
decrease in microbial biomass, although there was an increase after their removal at time 70.
In the case of the V treatment, concentration of totPLFAs increased by a factor of 2.8, whereas
the CV treatment saw an increase by a factor of 1.3. Both the Vcontrol and C treatments
maintained stable biomass levels after day 56, with a greater concentration in C (around 120
µg g-1) than in Vcontrol (around 100 µg g-1). So, it has been observed in the C treatment that
the totPLFAs is correlated with every enzyme: positively with β-glucosidase (r = 0.75, p <
0.0001) and protease (r = 0.75, p < 0.0001) and negatively with cellulase (r = -0.58, p < 0.01)
and acid phosphatase (r = -0.79, p < 0.0001). The enzymes in the V treatment did not
correlate with totPLFAs. In the Vcontrol treatment totPLFAs correlated with the protease (r =
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0.56, p < 0.01) and the acid phosphatase (r = -0.48, p < 0.05) and in the CV treatment with the
β-glucosidase (r = -0.46, p < 0.0001) and cellulase (r = 0.57, p < 0.0001).
Likewise, the earthworms caused a decrease in the abundance of the PLFA biomarkers
for Gram + bacteria and, after their removal at time 70, both treatments gradually increased;
V treatment saw an increase of 4.4 times and CV treatment saw an increase of 2.3 times at the
end of the process. In this case, the Vcontrol treatment also increased the abundance of Gram +
bacteria in the final sampling, whereas the C treatment maintained stable levels, around 40
µg g-1, starting from the day 42 sampling. So, significant differences caused the
presence/absence of earthworms were observed (p < 0.01), independent of the type of
substrate. So, it has been observed that in the C treatment the Gram + bacteria correlated
with every enzyme: positively with the β-glucosidase (r = 0.76, p < 0.0001) and the protease
(r = 0.68, p < 0.001) and negatively with the cellulase (r = -0.56, p < 0.01) and the acid
phosphatase (r = -0.73, p < 0.001). The enzymes from the Vcontrol did not correlate with the
Gram + bacteria. The Gram + bacteria correlated with the β-glucosidase (r = 0.46, p < 0.05) in
the V treatment. In the CV treatment the PLFAs characteristic of Gram + bacteria correlated
with the cellulase (r = 0.50, p < 0.05) and the protease (r = -0.54, p < 0.05).
A reduction in the PLFAs characteristic of Gram – bacteria was observed, as a
consequence of the activity of E. andrei earthworms with respect to the treatments without
earthworms (p <0.001). This effect did not depend on the type of substrate, but rather that
the epigeic earthworms reduced to a great degree these PLFA biomarkers in the fresh
manure and after the removal of the earthworms the abundance increased until reaching
levels greater than the other treatments (increased of 3.8 times in V and 1.7 in CV). The
abundance of PLFAs characteristic of Gram – bacteria remained stable from day 56 in the
Vcontrol treatment. So, it has been observed that in the C treatment the Gram – bacteria
correlated with every enzyme: positively with the β-glucosidase (r = 0.81, p < 0.0001) and the
protease (r = 0.75, p < 0.0001) and negatively with the cellulase (r = -0.51, p < 0.05) and the
acid phosphatase (r = -0.75, p < 0.001). The enzymes in the V treatment did not correlate with
the Gram – bacteria. In the CV treatment they correlated with the cellulase (r = 0.50, p < 0.05)
and with the protease (r = -0.44, p < 0.05) in the Vcontrol treatment.
Just as in the previous parameters, significant differences of the abundance of
biomarkers characteristic of fungi in function of the presence/absence of E. andrei
earthworms were observed (p < 0.0001), independent of the type of substrate. The CV
treatment had a gradual decrease over time of the fungal biomass and the V treatment saw a
marked decrease until the removal of the earthworms on day 70, which produced a slight
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increase (2.8 times). Both C and Vcontrol treatments saw decreases with greater fluctuation. So,
it has been observed that the C treatment the fungi correlated with every enzyme: positively
with the β-glucosidase (r = 0.63, p < 0.01) and the protease (r = 0.65, p < 0.001) and
negatively with the cellulase (r = -0.47, p < 0.05) and the acid phosphatase (r = -0.79, p <
0.0001). The PLFAs characteristic of fungi in the V treatment correlated with the cellulase (r =
0.47, p < 0.05), the protease (r = 0.58, p < 0.01) and the acid phosphatase (r = -0.63, p < 0.01).
In the Vcontrol the fungal biomass correlated with the protease (r = 0.55, p < 0.05), the cellulase
(r = 0.56, p < 0.01) and the acid phosphatase (r = -0.71, p < 0.001). In the CV treatment the
fungal biomass correlated with the β-glucosidase (r = -0.55, p < 0.01) and the acid
phosphatase (r = -0.80, p < 0.0001).
3.3. Compost parameters
With the exception of the C/N and WSC ratios, the stability and maturation parameter
results showed significant differences between treatments (p < 0.05) (Table 4).
The result of the individual PLFA cluster analysis (Figure 3) showed two groups clearly
formed by the vermicompost of the V treatment and another group formed by the products
from the other three treatments. In this last group there was the biggest difference between
the CV treatment and the Vcontrol and C treatments, there existing greater similarity between
products that had not been treated with earthworms.
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Table 4. Parameters of stability and maturity of compost and vermicompost after 112 days of
process.
Fresh pig manure Pre-composted pig manure
Earthworms Without Earthworms Without
Organic Matter (%) 71.0 ± 0.6b 76.4± 1.3a 67.4 ± 1.6c 74.5 ± 1.5a
pH 7.06 ± 0.09a 6.51± 0.04c 6.82 ± 0.04b 6.28 ± 0.12d
EC(mS cm-1) 0.41 ± 0.1d 0.54 ± 0.0b 0.45 ± 0.0c 0.70 ± 0.12a
Total carbon (mg g-1dw) 351.2 ± 0.3d 407.5± 6.7a 364.9 ± 4.1c 390.3 ± 6.8b
Total nitrogen (mg g-1dw) 17.8 ± 0.5c 20.1± 1.9a 18.3 ± 0.3b 22.0 ± 1.2a
ratio C/N 19.7 ± 0.5 20.5± 2.2 19.9 ± 0.6 17.8 ± 1.2
WSC (mg g-1dw) 1.74 ± 0.06 2.00 ± 0.10 2.09 ± 0.09 2.21 ± 0.12
NH4+/NO3- 0.08 ± 0.01d 0.19 ± 0.01c 0.15 ± 0.02b 0.65 ± 0.03a
SR (mg O2 g-1OM h-1) 0.57 ± 0.01b 0.54 ± 0.03b 0.45 ± 0.02c 0.69 ± 0.03a
GI (%) 97.9 ± 3.5a 76.5 ± 3.4c 96.6± 3.3a 88.1 ± 9.1b
Parameters with different letters indicate significant differences between products (Tukey test, p <
0.05). Parameters without letters were not significantly different (ANOVA, p < 0.05). (EC: electrical
conductivity, WSC: water soluble carbon, SR: static respiration, GI: germination index).
Fig. 3. Dendrogram of cluster analysis based on PLFA profiles of the products obtained after 112
days of process. Vermicomposting (V) and control (Vcontrol) treatments of pig manure,
maturation with earthworms (CV) and maturation without earthworms (C) of pre-composted
pig manure.
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4. DISCUSSION
4.1. Growth and reproduction of E. andrei
The results show the ability of the E. andrei earthworms to grow and reproduce both in
fresh pig manure and in pre-composted pig manure, which means that vermicomposting is
appropriate for treating pig manure and maturing after previously composting it.
From the point of view of the development and reproduction of E. andrei, maturing the
pre-composted manure with inoculated epigeic earthworms is a viable alternative to
maturing the compost sensu stricto. It has been noted that it is necessary or convenient to
carry out pre-composting for some waste products in order to avoid earthworm death by
passing the waste product through thermophilic temperatures in order to eliminate or
reduce compounds toxic to these detritivore organisms (Garg et al., 2005). In this case, the
pre-composting produced slightly noxious conditions in the substrate which caused a greater
death rate for the E. andrei specimens. Gunadi and Edwards (2003) proposed that the pre-
composting of cattle manure could reduce nutrients bioavailable to the earthworms,
inhibiting the growth and reproduction of E. fetida and Yadav et al. (2015) stated that the
quality and palatability of the food directly affected survival, the growth rate, and the
reproduction potential of the earthworms. Despite the fact that pre-composting reduces the
nutritional content for the earthworms, this reduction was not significant; the population
ended up developing adequately. (Villar et al., 2016b) showed that the presence of toxic
compounds in the pre-composted sewage sludge affected the growth and reproduction of E.
andrei. So, pre-composting created the inconvenience of generating compounds that were
toxic for E. andrei, although the recuperation and establishment of the earthworm population
indicated that these compounds were volatile and/or easily biodegradable, thus reducing
their toxicity within a short period of time.
4.2. Microbial dynamics evolution
The results showed distinct evolutions in the microbial biomass and microbial groups,
depending on the treatment used as reflected to a lesser or greater degree in the distinct
behavior of the enzymatic activity. The complex interactions that occurred between the
earthworm-microbiota-substrate had an effect on the degradative activity of the organic
compounds and the distinct microbial populations. The low rate of correlation observed
between the enzymatic activity and the different microbial groups verifies an important effect
of the synthesized and released enzymes on the environment throughout the process and
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they remained active or inactive, in the medium or attached to colloids, depending on
environmental conditions and, to a lesser extent, as a consequence of enzymatic activity
synthesized in situ by the live microbiota present in the substrate. So, it is known that
enzyme behaviour depends on various factors that interact together as well as the location of
the enzymes which contribute to the activity (Nannipieri et al., 2002).
The hydrolytic activities presented a different behaviour between treatments, meaning
that the degradative activity was different in the vermicomposting and compost maturation,
depending on whether it was with or without earthworms.
The carbon-cycle enzymes studied are those related to the decomposition of cellulolytic
material: cellulase and β-glucosidase activity. The cellulase measurement jointly estimates
both endoclunase activity, which depolymerizes the cellulose, and the β-glucosidase, which
hydrolyzes cellobiose to make glucose. Likewise, β-glucosidase activity was determined
independently. The synthesis of these enzymes implies the induction by the substrate and
repression by an easily-used source of carbohydrates, such as glucose. Pig manure is a
material rich in this type of cellulolytic material, due the high levels of bedding and food
remains which form part of the manure. The earthworms cause a decrease in cellulase
activity both with vermicomposting of fresh manure as well as in compost maturation.
Gómez-Brandón et al. (2011c) observed a reduction in cellulase activity after incubating
grape marc with E. andrei, taking into account that this decrease could be due to the effect the
earthworms have on the microbial biomass, reducing enzyme synthesis, as the decrease of
available nitrogen and carbon compounds affecting enzymatic activity. This effect was
observed in the treatments of fresh manure such that a decrease in fungi cause a decline
cellulase enzyme production, there being greater in the presence of E. andrei, since the fungal
community is the main source of food for earthworms and these enzymes are largely
synthesized by fungi. Nonetheless, epigeic earthworms also affected the fungal community
during compost maturation, but a direct effect of the fungi on the cellulase was not observed.
It is true, in this case, that bacterial evolution, both Gram + and Gram –, affected the activity of
this enzyme. In this respect, Villar et al. (2016a) attributed cellulase activity during pig
manure compost maturation via turning system to the presence of a cellulolitic bacterial
community. In terms of compost maturing under static conditions, the maintenance of the
cellulase activity and its negative correlation with all microbial groups could indicate the
slow degradation of this polymer by a specialized microbial community and the presence of
extracellular free enzymes on the medium or joined to particles of the substrate. Inasmuch as
the variability and concentration of compounds decline during composting, it decreases the
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microbiota; although its diversity may be greater and more effective at degrading the more
complex polymeric compounds (Ryckeboer et al., 2003b). When epigeic earthworms are
inoculated into pre-composted manure the bacteria take control of synthesizing this enzyme,
as already been mentioned, which shows that competition, interaction, and feeding of the
microorganisms on the part of the earthworms causes a selective filter on the microbiota and
that the remaining bacterial biomass is a community specialized in metabolizing cellulolytic
compounds. At the end of the process, the cellulase was greater in compost maturation
treatments; consequently, the passing of manure through the thermophilic stage meant a
short-term degradation of the most available materials, resulting in the concentration of
cellulolytic materials and cellulase synthesis.
The action of E. andrei earthworms on β-glucosidase activity had antagonistic effects,
depending on substrate that it fed from; the earthworms caused a reduction in activity in the
fresh manure, whereas the opposite was true for compost. The greater β-glucosidase activity
in the present of earthworms concurs with the results of other authors (Aira et al., 2007;
Benitez et al., 1999; Benítez et al., 2002; Vivas et al., 2009) who demonstrated an increase in
β-glucosidase activity, due to it being an enzyme inducible by substrate; the combined action
of microorganisms and earthworms can liberate low-molecular weight carbon molecules,
which increase this activity. According to this premise, β-glucosidase should have increased
as well during the vermicomposting of fresh manure, although the opposite effect was
observed. Benitez et al. (1999) and Villar et al. (2016b) showed a decrease in β-glucosidase
enzyme activity during sewage sludge, assuming that the reduction in activity was due to the
depletion of substrates and the low C/N ratio, which limited enzyme production. Nonetheless,
the increase in activity after removing the earthworms on day 70 suggests that the decrease
could be the consequence of the effect or combined interaction of: the consumption by the
earthworms of the substrates that could be hydrolyzed by this enzyme and, as a result, cause
a decline in the microbial population in the pig manure; and the direct consumption of the
microbiota by the earthworms. This interaction corresponds with the pronounced decline of
the microbial biomass and, in particular, of Gram + bacteria, which correlate with β-
glucosidase activity. Unlike that observed in the cellulase enzyme, the evolution of β-
glucosidase during the static maturation of compost was positively related to the evolution of
both the bacterial and fungal biomass. The lack of the influence of epeigeic earthworms on
the microbiota allowed for a direct relation between the synthesis of the β-glucosidase
enzyme by the microorganisms with their hydrolytic activity once compounds that were
easiest to degrade had been consumed in the prior process of composting.
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The enzyme phosphatase is pH dependent and in all the treatments maintained pH
values which were close to neutral, but slightly acidic, despite the fresh manure initially
having an alkaline pH, which means that the increase in the acid phosphatase activity in all
the treatments is connected with the change in pH towards acidic conditions. Garcia et al.
(1992) related the increase of phosphatase activity with the high quantities of phosphorous
compounds present in some waste products. The pig manure used in this study showed total
phosphorous levels greater than the sewage sludge used by Garcia et al. (1992). What pigs
are fed contributes to the increase of the phosphorous contents in their excrement, mainly by
inorganic compounds (von Wandruszka, 2006). As such, the concentration of phosphate
compounds could induce the synthesis of this enzyme. Thus, the acidification of the medium
and the induction by substrate promoted phosphatase activity in every treatment, likewise
the increase in the mineralization of organic phosphorous also contributed to the decrease in
pH. Likewise, the presence of E. andrei caused phosphatase activity to be greater than in
substrates without earthworms. According to Satchell and Martin (1984) the increase of this
enzyme in casts may be due to the enzymes themselves excreted by the earthworms and
indirectly by stimulating the microbiota. Furthermore, Aira and Domínguez (2009) showed
that an increase in the inorganic phosphorous in casts could cause an increase in phosphatase
activity. The presence of E. andrei, both in manure as well as compost, reduced microbial
biomass; this indicates that phosphatase activity may be due to free enzymes, joined to
colloids or humic particles, or promoted by the earthworms, and that the activity persists in
conditions which inhibit the microbial biomass. Likewise, a negative correlation has been
found with the biomarkers for fungi in every treatment. Bacteria are more effective at
dissolving phosphorous than fungi, which means that the decline in the fungal biomass did
not negatively affect the phosphatase enzyme activity, reducing the competition in terms of
the types of phosphorous with the bacteria. Consequently, the smaller microbial biomass
maintained greater activity than the treatments without earthworms, suggesting that the
passage through the intestine could result in microbial population that, while smaller, is
metabolically more active (Gómez-Brandón et al., 2011b).
The enzyme protease is considered indicative of the state of degradation of organic
material due to its extreme dependence on the availability of substrate (Lazcano et al., 2008).
During compost maturation in static conditions both the correlation with the microbial
biomass and maintenance of protease activity over time suggest the stability of degradative
activities of the organic substrates directly acted upon by both the bacterial and fungal
microbiota present in the compost. Voběrková et al. (2017) observed greater protease
activity during fungi inoculation during composting, suggesting that proteolytic activity
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seems to be strongly influenced by the microbial populations and that protein and
polypeptide hydrolysis increases during the maturation stage, which shows incomplete
protein decomposition during the biooxidative stage. It has been noted that the presence of
earthworms tends to reduce protease activity in various waste products (Aira et al., 2007;
Fernández Gómez et al., 2013; Gómez-Brandón et al., 2011b; Villar et al., 2016b). This effect
was clearly observed in the treatment of fresh manure with earthworms, while the
maturation of compost with E. andrei saw greater protease activity than in the compost
without earthworms. Parthasarathi and Ranganathan (2000) observed that protease activity
is greater in casts made by Eudrilus eugeniae and Lampito mauritii than in material that has
not been ingested. Ravindran et al. (2016) observed an increase in proteolytic bacteria in the
first weeks of vermicomposting; these then decreased afterwards as a result of a decline in
proteins. These results seem contradictory, since a greater number of protein compounds are
degraded during the thermophilic stage, decreasing the substrate for this enzyme in the pre-
composted manure. But after the removal of the earthworms, the protease activity in the
vermicompost slightly increased while that in the pre-composted manure decreased, which
could indicate a decline in protein compounds accompanied by a decline in proteolytic
bacteria and fungi.
In compost undergoing maturation, where the more assimilable compounds have
already been degraded in the thermophilic stage, both the bacterial and fungal microbial
biomass directly directs the degradation of the more recalcitrant organic material. The effect
of the earthworms makes it so hydrolytic activities are no directly driven by changes in the
microbial community of the substrate, especially in fresh manure.
The decrease in the microbial biomass, measured in totPLFAs, was consistent with the
results obtained by other authors during vermicomposting (Fernández Gómez et al., 2013;
Gómez-Brandón et al., 2013; Villar et al., 2016b) and composting (Garcia et al., 1992; Ros et
al., 2006; Villar et al., 2016a). The greater reduction of the microbial biomass with the
presence E. andrei is consistent with previous observations, which suggested that the
digestion of organic material by the earthworms has a negative effect on the microbial
communities (Gómez-Brandón et al., 2011a). The reduction in the available resources due to
microorganisms action, the competition for them with earthworms and the feeding of
bacteria and fungi by earthworms, may explain the greater reduction of biomass with the
presence of E. andrei (Villar et al., 2016b). This was demonstrated through removing the
competition and activity exerted by the earthworms on the microbial populations; the PLFA
biomarkers of the microbial biomass and groups increased, showing the presence of nutrition
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available to the microbiota in the vermicomposting of fresh manure. In the maturing
compost, the microbial biomass was maintained from day 28. At this respect, Tiquia et al.,
(2002) found that the stabilization of ATP during composting may indicate the maturity of
the compost and suggest a change in the microbial community to more specialized microbial
groups and Villar et al., (2016a) suggested that the stability of the microbial biomass in the
turned maturation of pig manure could be due to the slow degradation of more recalcitrant
compounds. The stable conditions of the maturation process keep the microbial biomass
stable by slowing degrading the organic compounds present.
The increase of Gram + bacteria after removing the earthworms could suggest the
availability of organic substrates as a consequence of the breakage of lignocellulolytic
compounds (Elouaqoudi et al., 2015), the increase in compost maturation being less due to
the material going through the thermophilic stage that reduced nutritional content. Likewise
Gram – bacteria increased to a greater degree in the fresh manure following the removal of
the earthworms but also in pre-composted material and to a lesser extent in the static
compost. Kato et al. (2005) suggested that the proportion of Gram + PLFA markers could be
used as a tool to determine compost maturity. On the other hand, Danon et al. (2008) showed
that Bacteroidetes dominated at the start of compost maturation, followed by Actinobacteria,
and finally by Gammaproteobacteria. Neher et al. (2013) showed that Chloroflexi and c-
Proteobacteria decreased in relative abundance during maturation and were surpassed in
abundance by Bacteroidetes in the finished composts. Castillo et al. (2013) showed that, at the
end of the vermicompost maturation period, the relative abundance of Betaproteobacteria
and Actinobacteria experienced a reduction, while Gammaproteobacteria saw a significant
increase. Consequently, a non-excessive increase in Gram – bacteria could be used as a
maturity indicator, although the type of bacteria should be expanded and accompanied by
physicochemical and biological maturity criteria.
As previously commented, fungi are the main source of nutrition of epigeic earthworms
which means that their decrease in the presence of E. andrei is to be expected. As example,
Huang et al. (2013) observed that earthworms caused a decrease in fungal density during the
vermicomposting of fruits and vegetables. Nonetheless, after removing the earthworms the
abundance of PLFA markers for fungi increased in fresh manure, suggesting the presence of
substrates available for these organisms, accompanied by an increase in protease and
cellulase activity. The evolution of enzymes and microbial groups in the vermicompost
obtained from pig manure indicates hydrolytic activities after maturing without earthworms
for 42 days, suggesting that a greater aging time for the product may be required. Likewise,
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fungi also increased in compost matured without earthworms, along with cellulase, protease,
and β-glucosidase. Pramanik and Chung (2011) found that the cellulolytic fungi population
increased throughout the vermicomposting of different organic waste mixes, both in the
presence of E. fetida and E. eugeniae. According to Castillo et al. (2013), the abundance of
fungi increases over the maturation period due to these microorganisms having the ability to
grow in recalcitrant compounds after a period of vermicomposting under low humidity
conditions. Also, Ryckeboer et al. (2003a) observed an increase in fungi in the maturation
process of biowaste compost.
4.3. Compost and vermicompost
According to the final parameters analyzed, the treatments with E. andrei reached
maturation and stability levels more suitable. Elvira et al. (1996) showed that the presence of
earthworms accelerates the decomposition of organic material. Consequently, the previous
step through the thermophilic phase of pig manure showed greater degradation of organic
material as a result of microbial activity and the posterior interaction of earthworms and
microbiota with the pre-composted material.
With the exception of the vermicompost obtained from fresh manure, which showed
optimal parameters close to neutrality, the pH of the remaining products achieved slightly
acidic values. Fornes et al. (2012) observed higher pH values in the vermicompost than the
combined compost-vermicompost process during the treatment of biosolids. Likewise, Villar
et al. (2016b) observed lower pH in sewage-sludge compost and vermicompost, suggesting
the accumulation of organic acids and an increase in nitrogen and phosphorus mineralization.
The high activity of acid phosphatase shows in these processes corroborates these results.
Albanell et al. (1988) suggested the formation of fulvic and humic acids during degradation
acidifies the vermicompost, whereas El Fels et al. (2014) show that pH tends to be neutral,
due to the formation of humic compounds which have buffer properties and that the pH
remains stable with an optimal value of 6-8. EC is a direct consequence of the mineralization
of organic material and, as such, the concentration of nutrients, such as nitrate. Although the
greatest degradation of organic material and carbon was in treatments with earthworms, the
compost showed high levels of EC; this could be the result of the greater level of irrigation
found in the vermicomposting system in order to maintain optimal conditions during the
process and a lower output of soluble metabolites, such as ammonium. EC values were not
greater than 3mS cm-1, making it safe to apply to the soil (Lazcano et al., 2008). The C/N
values were lower in the compost, although Fialho et al. (2010) stated that the C/N ratio is
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not a good method for monitoring process and there is no optimal relation which
characterizes humified compost, although it is considered as a given that total C/N values
below 25-20 are necessary for compost to be considered stable or not immature. According
to Zucconi et al. (1985) germination index values greater than 80% are indicative of the
absence of phytotoxic substances for plant growth. Excepting the control, the vermicompost
and compost showed a sufficient level of maturation, the vermicompost being more
appropriate for plant growth. Atiyeh et al. (2000) showed that earthworms play an important
role in processing of cow manure, since the earthworm’s activity accelerates the
decomposition and stabilization process of manure and promotes biochemical characteristics
which are favorable to plant growth. Likewise, the compost showed a lower degree of
maturation and stability due to a respiration rate above 0.5 mg O2 g-1SV h-1 (Iannotti et al.,
1993) and an ammonium/nitrate ratio above 0.16 (Bernal et al., 1998). The inoculation of
earthworms in the maturation of pre-composted manure improved quality parameters of
compost matured under static conditions.
According to the cluster, the PLFA profiles showed that E. andrei earthworms had a
great influence on the microbial community’s structure. The final products had different
microbial communities, there being greater differences between vermicomposts, especially
that arising from fresh manure, and compost treated without earthworms. Fernández Gómez
et al. (2012) reported that vermicomposts produced from waste products of different origins
can contain similar microbial communities if the same species of earthworm is used. Sen and
Chandra (2009) showed a divergent bacterial community in compost and vermicompost
obtained from the same initial waste, despite similar changes in their physicochemical
parameters. The inoculation of E. andrei earthworms made the development of a microbial
structure different to those treatments without those detritivore organisms possible, despite
the similarity in origin, although the former composting process assimilated the microbial
communities; this established greater similarity between the products after compost
maturation without earthworms and maturation via vermicomposting. Villar et al. (2016b)
stated that the differences in microbial communities can be attributed to the microbiological
and physicochemical composition of the waste products and the interaction of the
earthworms in the microbial communities. The physicochemical composition of the initial
materials contributes to a lesser extent to separating the products, which means that the
presence or absence of earthworms determines the PLFA profile.
The structure of the microbial communities through the PLFA study is not considered
to be a quality criterion for compost or vermicompost, but it does help one to understand the
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complex biological processes that occur during the degradation processes, which are
influenced as much by the nature of the substrate, the physicochemical changes which it
undergoes, the interaction between the microbiota and earthworms, and environmental
factors.
5. CONCLUSIONS
From a biological and final product quality standpoint, the aforementioned results
suggest that vermicomposting and compost maturation with earthworms are better
treatment options for pig manure than composting. The presence of earthworms E. andrei are
able to degrade fresh pig manure with optimal quality values, although the tracking of the
microbial community and the enzymes showed an increase of both after 42 days of
vermicompost aging, which is why we consider it necessary to optimize maturation time after
removing the earthworms. The use of earthworms to mature compost allows one to
accelerate the degradation process of the organic material, increasing the degradation of
recalcitrant compounds, in contrast with the compost matured under static conditions. The
stability of enzymatic activity, the maintenance of an abundance of fungi and bacteria, and the
study of the physicochemical parameters show that maturing compost under static
conditions produces slow and continuous degradation, which increases processing time.
Enzymatic activity is complex, which is why it is necessary to accompany them with a
physicochemical study and microbiological measurements, such as PLFAs, DGGE,
microarrays, etc.; these allow one to understand the complexity of the processes which occur
during the treatment of waste via composting and vermicomposting. Likewise, studying the
microbiology of these processes is not advisable if one does not study their activity and the
resulting physicochemical changes.
ACKNOWLEDGMENTS
This study was financially supported by the Xunta de Galicia (Regional Autonomous
Government of Galicia) (09MDS024310PR). The authors thank the research support services
of the University of Vigo (CACTI) for the carbon and nitrogen analysis.
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DISCUSIÓN GENERAL
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Los resultados aportados a lo largo de esta tesis profundizan en la dinámica microbiana
que dirige la transformación de los compuestos orgánicos que se sucede durante la fase de
maduración del proceso de compostaje y durante el vermicompostaje como proceso
alternativo o como proceso complementario de maduración del compost fresco.
La maduración del proceso de compostaje ha sido tratado en la bibliografía científica
generalmente como parte de un proceso íntegro de compostaje en estudios, por ejemplo, de
parámetros fisicoquímicos, (Eklind and Kirchmann, 2000; García et al., 1990; Garcia et al.,
1991), biológicos (Ryckeboer et al., 2003; Wong, 1985) o ambos (Benito et al., 2003;
Vuorinen and Saharinen, 1997) y, en menor medida, se han enfocado los estudios sobre
criterios microbiológicos únicamente durante la fase de maduración del compost (Danon et
al., 2008; Steger et al., 2007). Durante el compostaje en instalaciones industriales es frecuente
realizar un mayor control sobre la fase bio-oxidativa o intensiva del proceso para la cual se
emplea un sistema o tecnología específica y, por lo general, presenta una duración
determinada en función de la capacidad de tratamiento. Tras un tiempo convenido, es
habitual cambiar de posición el material y depositarlo en una zona de maduración donde el
seguimiento del proceso y las intervenciones suelen ser limitadas, por lo que, se requiere
mayor conocimiento del proceso biológico durante la estancia del compost fresco en esta
etapa de maduración, así como, conocer las posibles técnicas o aplicaciones que permitan
mejorar u optimizar el proceso y los productos obtenidos. De esta manera, los capítulos 1 y 2
del presente documento de tesis abarcan el estudio de los cambios microbiológicos y
fisicoquímicos a lo largo de la fase de maduración del compostaje. En el capítulo 1 se compara
la evolución de tres residuos de diferente origen durante la etapa de maduración (tras una
fase bio-oxidativa previa en reactor estático). Los tres residuos (municipal, industrial y
agropecuario) se han seleccionado con el fin de caracterizar de manera representativa los
cambios fisicoquímicos y microbiológicos durante la fase de maduración del proceso de
compostaje de residuos orgánicos de diferente origen. En el capítulo 2 se evalúa la
maduración dinámica con respecto a la maduración estática del residuo más energético, en
este caso, el residuo industrial, mediante la realización de volteos en función de los valores de
temperatura. En los capítulos 3 y 4 se evalúa el proceso de vermicompostaje como técnica
alternativa a la fase de maduración del compost estudiando cómo afecta a la comunidad
microbiana y a los parámetros bioquímicos la introducción de lombrices de tierra epigeas E.
andrei en residuo fresco y pre-compostado. De manera que, el capítulo 3 enfoca el estudio en
el vermicompostaje y el proceso combinado compostaje-vermicompostaje de lodo de aguas
residuales de una depuradora municipal. En el capítulo 4, se compara la maduración del
compost en condiciones estáticas frente a la maduración del compost en presencia de
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lombrices de tierra, incluyendo el vermicompostaje como proceso opcional al compostaje
sensu estricto. Para ello se escogió el estiércol de cerdo por ser un residuo, en general, mucho
más rico, con mayor contenido nutricional, que el lodo de depuradora municipal.
Los parámetros fisicoquímicos analizados en todos los capítulos de esta tesis,
proporcionan información convencional que permite determinar la estabilidad y madurez de
los productos finales de los diferentes procesos. Sin embargo, el estudio de los parámetros de
la dinámica microbiana permite conocer su evolución a lo largo del proceso y explica, en
función de la estabilidad biológica, las diferencias entre los productos finales y el tiempo de
proceso. Por lo tanto, la dinámica microbiana en conjunto con los parámetros fisicoquímicos
ponen de manifiesto la viabilidad de los tratamientos para producir compost y vermicompost
en condiciones adecuadas, así como, determinar las necesidades de optimización de los
procesos, lo cual permite establecer criterios para seleccionar las mejores prácticas
aplicables en una planta de tratamiento de residuos orgánicos.
Caracterización de la maduración del compost de diferentes residuos orgánicos
(capítulo 1)
De la gran variabilidad de residuos orgánicos que se generan en la actualidad se
seleccionó un residuo municipal (lodo de depuradora de aguas residuales municipales), un
residuo agropecuario (estiércol de cerdo), y un residuo industrial (lodo de depuradora de
aguas residuales de la elaboración de precocinados de pescado y cefalópodos) que se
caracterizan por su diferente origen y, por lo tanto, por las diferencias tanto fisicoquímicas
como biológicas que presentan. El estudio de la fase de maduración de estos residuos de
origen tan variado, contribuyó a aportar información útil sobre el comportamiento de sus
comunidades microbianas y su actividad de degradación de los distintos compuestos
orgánicos que componen cada residuo. El paso previo de los tres residuos por la fase más bio-
oxidativa del compostaje, en las mismas condiciones de proceso, permitió la degradación de
los compuestos más fácilmente asimilables y la generación de compuestos intermedios. Los
compost frescos son colonizados por comunidades microbianas que se caracterizan por la
composición de cada compost fresco. Así, tanto el comportamiento de las comunidades
microbianas como la evolución de las actividades enzimáticas durante la maduración fueron
condicionados por el tipo de residuo pre-compostado. A este respecto, Vargas-García et al.
(2010) mostraron que la dinámica y diversidad microbiana a lo largo del estudio de un
proceso de compostaje íntegro dependían de la naturaleza del sustrato orgánico. De la misma
manera, durante la etapa de maduración permanece la influencia ejercida por el distinto
origen que presentan los residuos; ganadero, alimentario y municipal y, por lo tanto, por las
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diferencias tanto fisicoquímicas como biológicas que los caracteriza. El paso a través de la
fase termófila establece variaciones en estos parámetros, sin embargo, el material pre-
compostado mantiene condiciones que diferencian, discriminan o facilitan el desarrollo de
comunidades microbianas particulares. Ishii and Takii, (2003) establecieron que los
microorganismos que proliferan a lo largo del compostaje se adaptan al medio ambiente del
sustrato y son seleccionados por factores del propio material compostado. Así, el lodo
municipal, con una diversidad microbiana inicial elevada, presentó una colonización por
parte de bacterias mesófilas y, en menor medida hongos, que dirigieron las actividades
enzimáticas del ciclo de carbono, nitrógeno y fósforo. El estiércol de cerdo se caracterizó por
una biomasa bacteriana mesofílica Gram +, elevados valores de actividad celulasa durante
toda la maduración y un aumento de las actividades fosfatasas y β-glucosidasa al final del
proceso. Las mayores diferencias en el proceso de maduración se observaron en el lodo
industrial el cual experimentó una reactivación en su estancia en maduración debido a su alto
contenido energético. El carácter lipídico y ácido, además del alto contenido en nutrientes,
que caracteriza al lodo procedente de la elaboración de alimentos precocinados, favorecieron
el desarrollo de una comunidad fúngica a lo largo de la maduración, en mayor medida que el
lodo municipal y el estiércol de cerdo. La evolución microbiana junto con los parámetros
fisicoquímicos permitieron determinar que: el estiércol de cerdo requiere mayor tiempo de
proceso u otro tipo de práctica durante la maduración; el lodo municipal alcanzó valores de
estabilidad por lo que el tiempo de proceso y el sistema de maduración se adaptaron al
residuo; y el lodo industrial requiere un cambio del proceso de maduración para tratar de
mejorar el producto y ajustar la duración del proceso. A la vista de los resultados obtenidos a
partir de la caracterización de la dinámica microbiana y la evolución de los parámetros
fisicoquímicos a lo largo de la fase de maduración, se considera necesario caracterizar los
residuos, previamente a su compostaje en planta, con la finalidad de determinar cuáles
pueden ser sus requerimientos de proceso y las mejores prácticas posibles a adaptar en las
instalaciones de tratamiento.
Influencia del diferente manejo sobre la maduración del compostaje (capítulo 2)
El residuo de la industria alimentaria experimenta un proceso de reactivación en la fase
de maduración, como consecuencia del alto contenido nutricional en compuestos energéticos
como los lípidos, por ello posibilita el estudio de la influencia de los tratamientos de manejo.
Tras la etapa intensiva en el reactor de compostaje, el material pre-compostado conservó los
suficientes nutrientes para desencadenar el crecimiento exponencial de la microbiota hasta
que su actividad propició el desarrollo de comunidades termófilas. La temperatura es uno de
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los principales factores que determina los cambios y sucesiones de las comunidades
microbianas y, por lo tanto, de sus actividades enzimáticas durante el compostaje. En
consecuencia, todos los grupos microbianos se vieron mayormente afectados por el régimen
de temperaturas termófilas, de modo que, las comunidades microbianas evolucionaron de
manera diferente dependiendo del sistema aplicado. Durante la maduración dinámica se
redujo la abundancia de todos los grupos microbianos mientras que esta reducción fue menor
en el tratamiento estático donde aumentó la abundancia de PLFAs biomarcadores de
bacterias Gram – y se observó un mayor ratio monoinsaturados/saturados indicativo de que
las comunidades microbianas no están sometidas a estrés y concluyendo que existe
disponibilidad suficiente de recursos. De la misma manera que en el capítulo 1, se observó la
predominancia de hongos a lo largo de la maduración como consecuencia del efecto del
residuo de origen. Sin embargo, la diferente manipulación a la que se sometió el material pre-
compostado y los cambios hacia valores de madurez provocaron una variación a
comunidades microbianas más diversas si sobre el material se realizan volteos según
criterios de proceso, en este caso, por control de la temperatura. Así, las diferentes prácticas
sobre el material pre-compostado provocaron efectos en la evolución de los grupos
microbiano. Además de los previsibles efectos que la reactivación puede presentar sobre un
proceso a escala industrial, como olores y lixiviados, conocer cómo se suceden los cambios en
la microbiota y su actividad si el material pre-compostado se mantiene en condiciones
estáticas o se voltea según criterios biológicos, permite obtener información para optimizar
el proceso de compostaje de residuos altamente energéticos. De esta manera, los estudios de
esta tesis demostraron que, si cuando se produce un cambio de posición del material pre-
compostado hacia la zona de maduración se monitoriza la temperatura y se ejercen volteos
cuando se sobrepasan valores cercanos a los 55ºC, se alarga la duración de la fase termófila.
La prolongación de las condiciones termofílicas incrementa la degradación de la materia
orgánica alcanzando criterios de maduración y estabilidad en menos tiempo que el
mantenimiento del compost en condiciones estáticas. El empleo de los volteos durante el
compostaje es una práctica esencial para el control de la temperatura y el oxígeno en
diferentes sistemas de compostaje y, en muchos casos, opcional durante la fase de
maduración en función del espacio en planta o de los recursos disponibles. Ruggieri et al.
(2008) destacaron que un residuo altamente lipídico presenta mejores resultados si su
compostaje se realiza mediante medios dinámicos que estáticos, al permitir la
homogenización y rotura de los agregados que se forman durante el compostaje. Los trabajos
de esta tesis demuestran que estos volteos son efectivos durante la fase de maduración tras
un pre-compostaje previo y controlado en un reactor con aireación forzada. Por lo que, se
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considera necesario prestar mayor atención a la estancia en maduración de un material pre-
compostado energético con la finalidad de mejorar el proceso de compostaje. Para una planta
de compostaje que presente una tecnología en túneles para materiales energéticos se
recomienda emplear la maduración volteada por control de temperatura u oxígeno con la
finalidad de reducir el tiempo de proceso y mejorar el producto obtenido.
Debido al auge de los productos procesados, las novedosas investigaciones realizadas
sobre este residuo exponen la viabilidad del compostaje como tratamiento de valorización.
Los resultados expuestos pueden servir como referencia para la aplicación del compostaje en
residuos de origen similar, teniendo en consideración, el aumento en la generación de este
tipo de residuos como consecuencia del incremento en la fabricación de productos
ultracongelados y procesados,
Viabilidad del empleo de lombrices E. andrei para el tratamiento de material fresco
o pre-compostado (capítulo 3 y 4)
Tanto el lodo municipal como el estiércol de cerdo presentaron una maduración en
condiciones mesófilas tras el pre-compostaje en reactor. La elevación térmica afecta a la
supervivencia de las lombrices de tierra que presentan una temperatura de desarrollo óptima
mesófila, por lo tanto, en ambos residuos pre-compostados existe la posibilidad de inocular
lombrices de tierra. De ambos residuos orgánicos existen trabajos previos donde se emplean
lombrices de tierra para su tratamiento biológico como los estudios de Aira et al. (2002)
sobre el vermicompostaje de estiércol de cerdo y Benitez et al. (1999) sobre el
vermicompostaje de lodo de depuradora, ambos con la misma especie Eisenia fetida. En
cuanto a la inoculación de lombrices de tierra en residuo pre-compostado, existen menos
trabajos de investigación, a modo de ejemplo, Chan and Griffiths (1988) realizaron pre-
compostaje de estiércol de cerdo ante la imposibilidad de vermicompostar el residuo fresco y
Rodríguez-Canché et al. (2010) pre-compostaron fangos de tanque séptico para conseguir
mantener una supervivencia máxima de las lombrices de tierra. En los estudios presentados
en esta tesis las lombrices de tierra E. andrei inoculadas en el lodo de depuradora municipal,
tanto fresco como pre-compostado, y en estiércol de cerdo, tanto fresco como pre-
compostado, presentaron valores elevados de supervivencia y, en general, parámetros
adecuados de crecimiento y desarrollo. Estos resultados constatan la viabilidad de las
lombrices de tierra epigeas para alimentarse y desarrollarse en estos residuos frescos y la
posibilidad de emplear estos organismos detritívoros para la maduración de compost fresco
como proceso complementario a la maduración del compost. Si bien, los datos mostraron
mejores condiciones para el desarrollo de estos organismos en los residuos que no fueron
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152
sometidos a compostaje. A este respecto, existen en la bibliografía investigaciones que
sustentan estos resultados como Frederickson et al. (1997), Gunadi and Edwards (2003) o
Yadav et al. (2015), quienes observaron que el pre-compostaje puede reducir los nutrientes
biodisponibles en sustratos tan variados como residuos verdes, estiércoles y residuos de
alimentos. Esta reducción del contenido nutricional en el lodo municipal junto con,
posiblemente, altos contenidos de amonio afectaron a la reproducción y crecimiento de los
organismos. En el caso del estiércol pre-compostado, los resultados mostraron que el
contenido de compuestos lábiles o volátiles pueden ser los causantes de mortalidades
iniciales, ya que una vez que estos compuestos se reducen, el contenido nutricional del
compost fresco permitió el desarrollo de las lombrices de tierra. A este respecto, puede ser
suficiente una mayor aireación del material pre-compostado a su salida del reactor, para la
reducción de los compuestos volátiles, o emplear sistemas de vermicompostaje abiertos en
lugar de sistemas cerrados, como los seleccionados para la realización de los trabajos de
investigación de esta tesis debido a que presentan un mayor control sobre las posibles
contaminaciones externas e imposibilitan la fuga de las lombrices de tierra hacia el exterior.
Determinar el sistema idóneo para cada tipo de residuo, fresco o pre-compostado, se percibe
como un paso previo importante para el diseño de procesos de vermicompostaje a mayor
escala, considerando también la introducción de material de escape y/o refugio para las
lombrices de tierra ante compuestos tóxicos o volátiles que no hayan podido eliminarse en el
acondicionamiento previo.
Efecto de E. andrei sobre la dinámica microbiana en lodo municipal y estiércol de
cerdo (capítulos 3 y 4)
Desde el punto de vista de la evolución de las características microbiológicas de los
sustratos estudiados, se observó que el lodo municipal, a pesar de presentar mayor biomasa
microbiana inicial tanto en fresco como pre-compostado, presentó limitados recursos para el
desarrollo de la microbiota mesófila Esto es consecuencia de la degradación biológica
durante el tratamiento de las aguas residuales en la estación depuradora que reduce el
contenido nutricional del lodo y, posteriormente, como consecuencia del paso del residuo por
la fase termófila del proceso de compostaje. Así, la inoculación de lombrices de tierra provocó
un descenso más rápido en las enzimas celulasa, fosfatasas y proteasa en el material pre-
compostado, mientras que en el lodo fresco las actividades enzimáticas presentaron mayor
similitud al lodo fresco sin lombrices. De modo que, se observó que las lombrices de tierra
ejercen un mayor efecto sobre las actividades degradativas en condiciones más limitantes al
aumentar la competencia por los recursos, consumir la microbiota y reducir los sustratos
Discusión
153
para las enzimas hidrolíticas. Sin embargo, el efecto sobre la biomasa microbiana fue mayor
en el lodo fresco debido a una mayor población de lombrices de tierra juveniles que
compitieron y se alimentaron de la microbiota provocando un descenso notable en las
comunidades microbianas. De esta manera, la microbiota en el lodo fresco vermicompostado
fue más eficiente en la degradación de los compuestos orgánicos al mantener actividades
enzimáticas similares al control con menor biomasa microbiana. Este efecto también se
observó en el estiércol de cerdo donde los controles presentaron una mayor carga
microbiana no siempre acompañada de una mayor actividad enzimática. Estos resultados
indican la existencia de una biomasa menos eficiente en la degradación de la materia orgánica
y mayor disponibilidad de recursos para los microorganismos en los controles, mientras que
en presencia de lombrices de tierra los microorganismos se asimilan más eficientes. Por lo
tanto, dependiendo del tipo de sustrato, puede aparecer, como consecuencia de la acción de
las lombrices de tierra epigeas, una microbiota reducida, pero metabólicamente más activa,
tal y como han señalado otros autores (Gómez-Brandón et al., 2011; Zhang et al., 2000).
Tanto en el lodo pre-compostado como en el estiércol pre-compostado, la actividad de
las lombrices redujo en mayor grado la biomasa fúngica que la bacteriana en comparación
con los controles. Sin embargo, en el lodo fresco las lombrices redujeron mayoritariamente
las bacterias Gram + y los hongos que las bacterias Gram –, mientras que en el estiércol fresco
la retirada de las lombrices de tierra provocó la recuperación de las poblaciones bacterianas
y una leve reducción de hongos en comparación con el control. Estos resultados son
congruentes con estudios previos que apuntan a un efecto selectivo por parte de las
lombrices de tierra epigeas sobre la presencia y la abundancia de determinados grupos
microbianos (Gómez-Brandón et al., 2012). Por lo tanto, las lombrices de tierra ejercen un
efecto selectivo dependiente de sustrato siendo este efecto mayor sobre la comunidad
fúngica, lo cual es debido a que los hongos son parte principal de la dieta de las lombrices de
tierra epigeas (Schonholzer et al., 1999). El estiércol de cerdo presentó altos contenidos de
moléculas complejas que no fueron sometidas a una degradación controlada previa como
ocurrió en los tanques de tratamiento biológico de la planta de aguas residuales. Así, la
biomasa microbiana, de manera más importante en el estiércol fresco, aumentó tras la
retirada de las lombrices de tierra indicando la presencia de suficientes compuestos
degradables para la microbiota. Este efecto de recuperación de las comunidades microbianas
no se observó tras la retirada de las lombrices de tierra en el lodo municipal, indicando un
mayor contenido nutricional en el estiércol. Además, las actividades enzimáticas presentaron,
en general, valores más altos que en el lodo municipal a lo largo de los distintos tratamientos
y en los productos finales, pero, en general, estas actividades hidrolíticas no se
Discusión
154
correlacionaron con los grupos microbianos. La falta de recursos en el lodo municipal
conlleva a que sea la microbiota asociada a cada sustrato la que dirija los procesos de
degradación de la materia orgánica, sin embargo, en el estiércol se sucede una interacción
lombrices-microbiota-sustrato que ejerce complejos efectos sobre las actividades
degradativas, mayor cuanto mayor contenido nutricional presente, es decir, en el proceso de
vermicompostaje con estiércol fresco.
Los vermicomposts de ambos procesos presentaron actividades biológicas y
parámetros fisicoquímicos similares en el lodo municipal y ligeramente mejores en el
vermicompost a partir de estiércol pre-compostado frente al fresco. Por lo que, tanto el
vermicompostaje del sustrato fresco como la maduración del material pre-compostado con E.
andrei son prácticas recomendables para el tratamiento del lodo de depuradora municipal y
el estiércol de cerdo.
Efecto de la inoculación de lombrices de tierra en material pre-compostado como
posible alternativa al compostaje estático de estiércol de cerdo (capítulo 4)
La maduración del compost fresco de estiércol no posibilitó su control mediante
criterios biológicos por aumento de temperatura (capítulo 1), por lo tanto, se comparó la
maduración del compost mediante condiciones estáticas frente a condiciones dinámicas
mediante la inoculación de lombrices de tierra E. andrei. Así, se observó que en el estiércol de
cerdo el compostaje previo y el posterior vermicompostaje favorecen la estabilización de los
materiales y, por lo tanto la mejora de la calidad de los productos obtenidos. El proceso de
vermicompostaje utilizando como sustrato material pre-compostado no es novedoso y ha
sido estudiado por diversos autores aunque, en la mayoría de las investigaciones, los trabajos
se basan en la determinación y seguimiento de parámetros fisicoquímicos o, por ejemplo, de
organismos patógenos. Así, Tognetti et al. (2007) observaron que el vermicompostaje de
residuo orgánico municipal, tras un compostaje previo de 50 días, conducía a un producto
final más rico en nutrientes y materia orgánica que el compostaje mientras que Mupondi et al.
(2010) aconsejan el pre-compostaje para la eliminación de patógenos previo al
vermicompostaje de estiércol. Es posible referenciar más trabajos de investigación realizados
en base a otros objetivos y sobre otro tipo de residuos, sin embargo, estudios comparativos
sobre la maduración del compost con lombrices de tierra que profundicen en la dinámica
microbiana son escasos, por ejemplo, Sen and Chandra (2009). Ambos procesos, compostaje y
vermicompostaje, son procesos biológicos que son dirigidos por los microorganismos
presentes en el sustrato y que se ven afectados tanto por factores fisicoquímicos
(temperatura, humedad, oxígeno,…) como biológicos (lombrices de tierra), por lo que el
Discusión
155
estudio de la microbiota y su actividad se considera sumamente importante para entender y
mejorar estos procesos de biodegradación.
Así, en la maduración del compost sin la adición de lombrices de tierra se observó una
relación clara entre las actividades enzimáticas y la evolución de la biomasa microbiana,
conllevando el mantenimiento estático del compost un proceso lento pero progresivo de
degradación de los compuestos orgánicos. La presencia de lombrices de tierra ejerció
complejas interacciones entre las lombrices-microbiota-sustrato afectando a las actividades
degradativas, de manera que, diversos factores provocaron cambios en las actividades
enzimáticas (actividades intracelulares o extracelulares, enzimas propias del intestino de las
lombrices, formación de complejos, etc) y no únicamente por la síntesis directa de enzimas
por parte de la microbiota presente en el sustrato. La evolución de la dinámica microbiana y
los valores fisicoquímicos del compost de estiércol de cerdo en maduración estática indicaron
que el proceso de degradación se mantuvo constante por lo que se considera necesario
establecer cambios para acelerar la maduración tratando de estimular el proceso como se
observó mediante la inoculación de lombrices de tierra epigeas. Cabe destacar que el paso del
residuo a través de la etapa termófila no únicamente presenta efectos en la abundancia de los
grupos microbianos a lo largo de la maduración sino que también en el perfil de PLFAs. De
manera que, el efecto de las lombrices de tierra epigeas sobre el material pre-compostado
varía el perfil de PLFAs pero en menor medida que sobre los residuos frescos, por lo que, el
compost y el producto combinado compost-vermicompost presentan comunidades
microbianas más similares que el vermicompost a partir de estiércol fresco. Así, Sen and
Chandra (2009) encontraron comunidades divergentes en compost y vermicompost del
mismo residuo y Lores et al. (2006) mostraron que los perfiles de las comunidades varían con
el tipo de sustrato y la especie de lombriz empleada. En este caso, se demuestra que el perfil
de la comunidad microbiana varía en mayor medida por los cambios que las lombrices
ejercen sobre el estiércol fresco mientras que el pre-compostaje asimila los perfiles de PLFAs
en los productos. Como se comentó en el apartado anterior, la retirada de lombrices de tierra
en el estiércol fresco provocó una reducción de la competencia y de depredación sobre la
microbiota, generando aumentos en las comunidades microbianas y las actividades
enzimáticas e indicando presencia de compuestos biodegradables. Por ello, es necesario
alargar el tiempo de proceso para estabilizar las actividades degradativas. A la vista de los
resultados, el estudio de la dinámica microbiana y los parámetros de calidad (ratio C/N, ratio
amonio/nitrato, índice de germinación, etc.) muestran que los vermicompost (a partir de
residuo fresco y pre-compostado) presentaron mejores condiciones que el compost y, por lo
tanto, la inoculación de lombrices de tierra en el material pre-compostado mejora el proceso
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156
de degradación y estabilización del estiércol de cerdo, siendo un proceso alternativo a la
maduración sensu estricto del compostaje.
CONCLUSIONES
Conclusiones
159
Los resultados de esta tesis muestran que las actividades enzimáticas y las
comunidades microbianas son parámetros que deben determinarse conjuntamente ya que, a
pesar de ofrecer conocimiento relevante del proceso por separado, su estudio independiente
no permite una estima adecuada del estado de degradación de la materia orgánica y, por lo
tanto, si un compost o vermicompost ha alcanzado criterios adecuados de madurez o
estabilidad. A la vista de los resultados de los distintos capítulos, no se puede establecer un
nivel base o límite de biomasa microbiana que permita distinguir un compost maduro o
inmaduro y de la misma manera, tampoco se puede establecer un valor límite o adecuado
para las actividades enzimáticas. Sin embargo, el empleo combinado de ambas estimaciones
aporta información sobre la evolución del proceso de maduración y del estado de
degradación de los compuestos orgánicos permitiendo establecer las posibles prácticas para
optimizar el proceso y el tiempo en obtener materiales más estables y maduros.
En base a los objetivos planteados inicialmente en este estudio, las siguientes
conclusiones constituyen una síntesis de los resultados más relevantes obtenidos.
1. De manera general, la comunidad microbiana que es capaz de desarrollarse en
un compost fresco en maduración depende del residuo de origen de manera que los
microorganismos mesofílicos predominantes capaces de recolonizar el compost tras las
condiciones termofílicas son similares a las comunidades microbianas predominantes del
residuo de partida. A pesar de existir un continuo cambio en las poblaciones microbianas se
mantiene la influencia del material originario y de los grupos microbianos predominantes
(capítulo 1)
2. La caracterización del residuo inicial es un proceso previo fundamental para
poder establecer una fase de maduración adaptada a cada tipo de residuo (capítulo 1).
3. El empleo de los volteos como mecanismo de control del proceso de
compostaje en residuos altamente energéticos permite un mayor grado de degradación de la
materia orgánica, mejores valores de estabilidad y madurez en menor tiempo, resultando en
una sucesión microbiana hacia comunidades mesofílicas más estables (capítulo 2).
4. El compostaje es un proceso de valorización apropiado para el lodo de
procesado de productos precocinados y ultracongelados y su estudio es un referente para
residuos con características similares.
Conclusiones
160
5. Ambas, la estructura de la comunidad microbiana y las actividades
enzimáticas, proveen importante información para el monitorio del proceso de compostaje y
el vermicompostaje. La diferente relación entre los grupos microbianos y las actividades
enzimáticas durante los distintos procesos informan del estado de degradación de la materia
orgánica y la calidad de los composts y vermicomposts, así como las necesidades de mayor o
menor tiempo de proceso o de la implantación de diferentes intervenciones sobre el material
(capítulos 1, 2, 3 y 4)
6. La inoculación de las lombrices de tierra epigeas E. andrei en todos los
residuos estudiados mostró condiciones adecuadas de supervivencia y desarrollo que ponen
de manifiesto la viabilidad de su uso para la valorización de sustratos de diferente origen
(capítulos 3 y 4).
7. Las complejas interacciones microbiota-lombrices-residuo afectan a las
actividades degradativas, por lo que el estudio de los grupos microbianos requiere del
estudio de parámetros de actividad, y viceversa, para poder concluir el devenir de los
procesos de biodegradación de los compuestos orgánicos (capítulos 3 y 4)
8. El empleo de lombrices de tierra epigeas E. andrei es recomendable para
mejorar el proceso de degradación biológica de los compuestos orgánicos en contraposición a
la maduración estática (capítulo 4).
9. Debe prestase más atención a la estancia en maduración del proceso de
compostaje y a los manejos a los que los materiales pre-compostados son sometidos,
especialmente, si se trata de residuos con alto contenido orgánico con capacidad de re-
activación. De esta manera, el proceso de compostaje debe ser diseñado y controlado en todas
sus fases, siendo posible establecer diseños ad hoc para obtener compost y vemicompost de
mejor calidad, en menor tiempo, en función del tipo de residuo y su evolución microbiana
(capítulos 1, 2, 3 y 4).
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