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Fermentation

... when there is no external terminal electron acceptor!

Substrate-level phosphorylation

Pyruvate2

Glycolysis

Fig.: Brock (mod.)


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glucose

2 pyruvate

GTP

NADH

ATP

NADH

FADH2

ATP

CO2, NADH

CO2

reduction

equivalentsrespiratory chain

The calvin cycle

glucose

2 pyruvate

GTP

NADH

ATP

NADH

FADH2

ATP

CO2, NADH

CO2

reduction


The general priciple of fermentation

The problem Regeneration of

NADH2 to NAD+

The solutionTransfer of reduction equivalents [H] on intermediates

(e.g. pyruvate) or co-substrates


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Drawback Excretion of energy rich (reduced) substrates (e.g. ethanol)

reduced

products

organic substrate

[H]

ATP

degradation

intermediates

oxidisedproducts


Bacterial fermentations are named by

their characteristical end productsalcohol (Ethanol) lactic acidbutyric acid propionic acidmixture of different acids

Conservation of energy not by

chemiosmotic mechanisms (proton gradient)

but by

Substrate-level phosphorylation

low ATP- and growth yield!

Example alcoholic fermentation: little biomass, a lot of alcohol



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e.g.Lactobacillus spec.

Lactobacteriaceaehomolactic fermentation

Photo: M. Dykstra, R. Barrangou,R. Sanozky-Dawes, and T. R. Klaenhammer

The easiest fermentative pathway

... a bit more complicated:

heterolactic fermentation

The microbiologcal garden

www.mikrobiological-garden.net


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Natural occurance

Milk and milk products, fruit juice,

plant products, intestine, mucosa

Lactobacteriaceae

gram positive rods or cocci obligate fermenters (no respiratory chain)

catalase negative (often aerotolerant)

www.microbiological-garden.net

Play an important role for

production of curdled milk products

also: Sauerkraut and salami

Lactobacteriaceae classified by:

shape (cocci or rods) and type of fermentation

homolactic

cocci rods

Lactococcus Lactobacillus

L. lactis L. plantarumL. casei L. bulgaricus

L. acidophilusEnterococcus

E. faecalis

StreptococcusS. thermophilus

S. salivarius

S. mutans

S. pyogenes

mainly lactate

heterolactic

cocci rods

Leuconostoc Lactobacillus

L. mesenteroides L. brevisL. dextranicum L. kandleri

different fermentation products


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glucose

2 pyruvate

GTP

NADH

ATP

NADH

FADH2

ATP

CO2, NADH

CO2

reduction


reducedproducts

organic substrate

[H]

ATP

degradation

intermediates

oxidised

products


glucose 2 pyruvate

2 NADH2 NAD+

ATP6

COOH

C O

CH3

Lactate dehydrogenase2 lactate

COOH

HC OH

CH3

Homolactic fermentation


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Fig.: Schlegel. (1992)

Heterolactic fermentation

Mixed acids fermentation

Products after fermentation of glucose (e.g. E. coli)

mol per100 mol Glucose

2,3-Butanediol CH3-CHOH-CHOH-CH3 0

Ethanol CH3-CH2OH 42

Succinate COOH-CH2-CH2-COOH 29

Lactate CH3-CHOH-COOH 84

Acetate CH3-COOH 44

Formiate HCOOH 2

Hydrogen H2 43

Carbon dioxide CO2 44 after: Thimann (1955)


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Ethanol CH3-CH2OH

Succinate COOH-CH2-CH2-COOH

Lactate CH3-CHOH-COOH

Acetate CH3-COOH

Formiate HCOOH

Hydrogen H2

Carbon dioxide CO2

glucoseglykolysis

pyruvate lactate

acetyl~CoA

formiate

+

ethanol

acetate

CO2

H2

CO2

succinate

Mixed acids fermentation

Fig.: Brock (mod.)

The horror scheme

Fig.: Brock


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Where can we find fermenters in nature?

the anaerobic food web

The anaerobicfood web

CH4, CO2CO2

secundary fermenters, syntrophs

methanogens

sulfate reducers

primary fermenters

formiate, H2,CO2, methanol

fatty acids, succinate,

alckohols, lactate

acetate

polymers

monomes


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Where can we find fermenters in nature?

alimentary systems

mouth stomachhindgut or colon rectumoesophagus duodenum

rumen,pre gastricfermentation chamber

cecum, post gastricfermentation chamber

Herbivoric vertebrates fermentation chamber for plant material

Ruminants (cow, sheep, camel) fermentation chamber (rumen) in front of the

stomach

Other herbivors (e.g. rodents, horse) between duodenum and colon

Some omnivors (e.g. human) strongly reduced (appendix)

General structure of the vertebrate alimentary system


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Can we live without microbes?

Experiments on animal without intestinal flora

aseptic breeding, no developement of gut flora

high dosage of antibiotics, destruction of gut flora

Why?

As a general rule signs of strong underfeeding, often lethal herbivors cant live at all without their gut flora

Vitamine excretionThiamine, Riboflavine, Pyridoxine, Vit. B12 und Kessential amino acids, ...

Homo sapiens

stomachpH 1,5

duodenumpH 2-5

colonpH 7

normaly free of bacteria

102-103 cellsml-1 in initial partprimarily Lactobacillussp. andEnterococcussp.

1-31011 cellsml-1

e.g. Bacteroides, Bifidobacterium,Enterococcus, Bifidobacterium,Peptococcus, Enterobacteriaceae, ...

Human faeces up to 30-50% bacterial biomass

continuous increase of pH


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The rumen ecosystem

Enlargement of the oesophagus

Fermentation chamber (large volume) cow app. 100-250 l

sheep app. 6 l

residence time 9-12 h

Physico-chemical conditionspH 5,5 - 6,9 (mean: 6,4)temperature 37-42Cdry mass 10-18 %redox potential -350 to -400 mVgas phase 65 % CO2, 27 % CH4, 7 % N2, 0,6 % O2, 0,2 % H2dissolved fatty acids 68 mM acetate, 20 mM propionate, 10 mM butyrate, 2 mM FA > C 4ammonium 2-12 mM

Biologyprokaryontes 1010 - 1011 g-1 (more than 200 species)ciliates 104 - 106 g-1

fungy 102 - 104 g-1 (zoospores)

Mouth: food is roughly hackled, swallowed, mixed with spittle(bicarbonate buffered)

Rumen: mass is mixed thoroughly (muscle movement of rumen wall)

Reticulum: fibrous compounds are sieved, densified to chunks, refluxed andruminated

Omasum : water removal

Abdomasum: normal digestion

How does the cow eat?

duodenum

reticulum

oesophagus

omasumabdomasum

Fig.: Campbell und Reece 2003 (mod.)

rumen


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starch cellulose pectine hemicelluloses

glucose fructose

pyruvate

CH4 acetate CO2 butyrate (lactate) propionate

What happens in the rumen?

Fermentation of plant material100 Glucose 113 acetate + 35 propionate + 26 butyrate+ 104 CO2 + 61 CH4 + 43 H2O

What is the benefit for the cow?

fermentation products (acetate, propionate and butyrate)

bacterial biomass, gets into abdomasum after reflux

N2 fixation in the rumen by anaerobic microorganisms


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What groups of microorganisms are found in the rumen?

Cellulose degrader Ruminococcus albus, Butyrivibrio fibrisolvens,Fibrobacter succinogenes, Clostridium locheadii

Hemicellulose degrader Ruminococcus albus, Butyrivibrio fibrisolvens,Fibrobacter succinogenes, Lachnospira multiparus

Sarch and sugar degrader Selenomonas ruminantium, Succinomonas amylolytica,Bacteroides ruminicola, Streptococcus bovis

Lactate utiliser Selenomonas lactilytica, Megasphaera elsdenii,Lac Prop + Ac Veillonella sp.

Succinate utiliser Selenomonas ruminantium, Veillonella parvulaSucc Prop + CO2

Methanogens Methanobrevibacter ruminantium,CO2 + H2 CH4 Methanomicrobium mobile

Fungi and ciliates play a minor role: degradation of polymeric substancesCiliates feed on bacteria: important for a stable microbial community

Wood feeding termites (e.g. Reticulitermes flavipes, app. 3 mm long)

have an enlarged hindgut as a fermentation chamber.

The termite gut


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Measurement of physico chemical parameter within the gut

embedding of gut in agarose (the tip of the microelectrode is marked)

Oxigen profiles

within the hindgut of

Reticulitermes flavipes

polysaccharides from wood

disolved disaccharidesand oligosaccharides

homoacetogenicbacteria

CO2, H2, acetate, propionate, butyrate,

lactate, formiate

fermenters

protozoa

absorption by termite

CH4

homoacetogenicbacteria

methanogens

CO2, H2acetate

What happens in the termite gut?

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