M E D D E L A N D E N

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S T O C K H O L M S U N I V E R S I T E T S I N S T I T U T I O N

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G E O L O G I S K A V E T E N S K A P E R

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Microbe-mineral interactions in soil Investigation of biogenic chelators, microenvironments and weathering processes

Engy Ahmed

Stockholm, May 2015

Department of Geological Sciences Stockholm University SE-106 91 Stockholm

Sweden

©Engy Ahmed, Stockholm University 2015

ISBN 978-91-7649-135-5

Printed in Sweden by Holmbergs, Malmö 2015

Distributor: Department of Geological Sciences

Cover: forest picture by Cajsa Lithell.

To my parents and brother

Thanks for your endless love

A dissertation for the Degree of Doctor of philosophy in Natural Science

Engy Ahmed Department of Geological Sciences Stockholm University, SE-106 91, Stockholm, Sweden

Abstract

The interplay between geology and biology has shaped the Earth during billions of years. Microbe-

mineral interactions are prime examples of this interplay and underscore the importance of micro-

organisms in making Earth a suitable environment for all forms of life. The present thesis takes an

interdisciplinary approach to obtain an integrated understanding of microbe-mineral interactions.

More specifically it addresses how the composition and distribution of biogenic chelators (sidero-

phores) differ with regard to soil horizon and mineral type in situ, what siderophore type soil mi-

croorganisms produce under laboratory conditions, what role microbial surface attachment plays

in mineral weathering reactions and what central roles and applications siderophores have in the

environment.

Podzol, the third most abundant soil in Europe and the most abundant soil in Scandinavia, was cho-

sen for a field experiment, where three mineral types (apatite, biotite and oligoclase) were inserted

in the organic, eluvial and upper illuvial soil horizons. The study started with an investigation of the

siderophore composition in the bulk soil profile and on the mineral surfaces (Paper I), which was

followed by a study of the siderophore producing capabilities of microorganisms isolated from the

soil profile under laboratory conditions (Paper II). Subsequently, a study was done on the impact of

microbial surface attachment on biotite dissolution (Paper III). Finally, the roles of siderophores in

nature and their potential applications were reviewed (Paper IV).

The major findings were that the concentration of hydroxamate siderophores in the soil attached to

the mineral surfaces was greater than those in the surrounding bulk soil, indicating that the

minerals stimulate the microbial communities attached to their surfaces to produce more

siderophores than the microorganisms in the bulk soil. Each mineral had a unique assemblage of

hydroxamate siderophores that makes the mineral type one of the main factors affecting

siderophore composition in the natural environment. Siderophore production varied between the

microbial species originating from different soil horizons, suggesting that the metabolic properties

of microbes in deep soil horizons function differently from those at upper soil horizons. Microbial

surface attachment enhanced the biotite dissolution, showing that attached microbes have a

greater influence than free living populations on weathering reactions in soil. In conclusion, our

findings reflected that the complicated relationship between microorganisms and mineral surfaces

reinforces the central theme of biogeochemistry that the mineral controls the biological activity in

natural environments. However, the importance of these relationships to the overall

biogeochemical systems requires further investigation.

A dissertation for the Degree of Doctor of philosophy in Natural Science

Engy Ahmed Department of Geological Sciences Stockholm University, SE-106 91, Stockholm, Sweden

Sammanfattning

Samspelet mellan geologi och biologi har format jorden under miljarder år. Mikrob-mineral

interaktioner är ett exempel på detta samspel och framhåller betydelsen av mikroorganismers roll i

att göra jorden till en beboelig miljö för olika former av liv. Denna avhandling tar ett

tvärvetenskaplig angreppssätt för att få en samordnad förståelse av mikrob-mineral interaktioner.

Mer specifikt avhandlas hur sammansättningen och fördelningen biogena kelatorer, ”sideroforer”,

som främjar vittring, skiljer sig beroende på jordhorisont och typ av mineral in situ, vilka typer av

sideroforer som marklevande mikroorganismer producerar under laboratorieförhållanden, vilken

betydelse kontakten mellan mineralytor och mikrober har i vittringen av mineraler. Dessutom

beskrivs potentiella applikationer av sideroforer i tillämpat miljöarbete.

Podsol som är den tredje vanligast förekommande jordtypen i Europa och den vanligast

förekommande i Skandinavien valdes för ett fältexperiment där tre typer av mineraler (apatit,

biotit och oligoklas) placerades i den organiska, i den eluviala och i den övre illuviala

jordhorisonten. Studien inleddes med en undersökning av sammansättningen av sideroforer i jord

adsorberad till mineralytorna och i den omgivande jorden (Artikel I). Därpå följde en undersökning

av förmågan hos marklevande mikroorganismer som isolerats från jordprofilen att producera

sideroforer under laboratorieförhållanden (Artikel II). Därefter gjordes en studie av betydelsen av

kontakten mellan mineralyta och mikroorganismer i vittring av biotit (Artikel III). Slutligen

granskades betydelsen av sideroforer i naturen och dess potentiella tillämpningar (Artikel IV).

De viktigaste resultaten var att koncentrationen av hydroxamat-sideroforer i jord som var

adsorberad till mineralytorna var högre än koncentrationen i den omgivande jorden, vilket pekar

på att mineralerna stimulerar de mikrobiella samhällena som lever i nära kontakt med mineraler

att producera mer sideroforer än mikroorganismer i den omgivande jorden. Varje typ av mineral

hade en unik uppsättning av hydroxamat-sideroforer, vilket indikerar att mineraltypen är en av de

viktigaste faktorerna som påverkar sammansättningen av sideroforer i naturliga miljöer.

Sideroforproduktionen hos isolaten varierade beroende på vilken jordhorisont mikroorganismerna

härstammade från, vilket tyder på att dessa adapterats för att fungera optimalt i de geokemiska

förhållanden som råder i deras hemma-horisont. En nära kontakt mellan mikrober och mineral

förbättrade upplösningen av biotit, vilket visar att mikroorganismer i direkt kontakt med

mineralen har en större påverkan på vittrings reaktioner än ”fritt” levande populationer.

Sammanfattningsvis återspeglar våra resultat att det komplicerade förhållandet mellan

mikroorganismer och mineralytor förstärker det centrala temat i biogeokemi att mineraler har stor

inverkan på den biologiska aktiviteten i naturliga miljöer. Betydelsen av dessa samband i de

biogeokemiska systemen kräver dock ytterligare utredning.

Microbe-mineral interactions in soil Investigation of biogenic chelators, microenvironments and weathering processes

Engy Ahmed

Department of Geological Sciences, Stockholm University, SE-106 91, Stockholm, Sweden

This doctoral thesis consists of a summary and four published articles. Paper I: Ahmed E., and Holmström S.J.M. (2014). The effect of soil horizon and mineral type on the distribu-tion of siderophores in soil. Geochim. Cosmochim. Acta 131, 184-195. Doi:10.1016/j.gca.2014.01.031. Paper II: Ahmed E., and Holmström S.J.M. (2015). Siderophore production by microorganisms isolated from a podzol soil profile. Geomicrobiology Journal, in press. Doi:10.1080/01490451.2014.925011. Paper III: Ahmed E., and Holmström S.J.M. (2015). Microbe-mineral interactions: the impact of surface at-tachment on mineral weathering and element selectivity by microorganisms. Chemical Geology 403, 13–23. Doi:10.1016/j.chemgeo.2015.03.009. Paper IV: Ahmed E., and Holmström S.J.M. (2014). Siderophores in environmental research: roles and appli-cations. Microbial Biotechnology 7, 196-208. Doi:10.1111/1751-7915.12117. The following papers are not included as a part of this thesis: Schütze E., Ahmed E., Voit A., Klose M., Greyer M., Svatoš A., Merten D., Roth M., Holmström S.J.M., Kothe E. (2015). Siderophore production by streptomycetes - Stability and alteration of ferrihy-droxamates in heavy metal contaminated soil. Environmental Science and Pollution Research, in press. Doi:10.1007/s11356-014-3842-3. Esposito A., Ahmed E., Ciccazzo S., Sikorski J., Overman J., Holmström S.J.M., Brusetti L. (2015). Comparison of rock varnish bacterial communities with surrounding non-varnished rock surfaces: Taxon-specific analysis and morphological description. Microbial Ecology, in review.

Contents

1. Introduction.…………………………………………………………………………………… 8

1.1 Podzol soil………………………………………………………………………… 8

1.2 Mineral weathering by soil microorganisms…………………………………… 9

1.3 Microbial siderophores in soil ecosystem…………………………………………… 10

2. Scope of the thesis……………………………………………………………………………… 13 3. Sampling site and field experiment set up……………………………………… 14 4. Summary of materials and methods………………………………………………… 15

4.1 Extraction of siderophores from soil and microbial isolates………………. 15

4.2 Isolation and molecular identification of siderophore producing microorganisms from soil 15

4.3 Quantification of siderophores using HPLC-ESI-MS 15

4.4 Biotite weathering by attached and unattached soil microorganisms 16

4.5 Data analysis and statistics 16 5. Summary and discussion of major findings…………………………………… 18

5.1 What is the impact of soil horizon and mineral type on siderophore composition in soil? 18.................................................................

5.2 What siderophore types are produced by soil microorganisms?............. 20

5.3 What is the role of microbial surface attachment in mineral weathering reactions? 22

6. Overview of the potential roles and applications of siderophores…… 26 7. Conclusion and future prospective…………………………………………………… 27 Acknowledgements…………………………………………………………………………… … 29 References……………………………………………………………………………… 30

8

1. Introduction

Soil is a heterogeneous system that consists of a variety of microhabitats with different physico-

chemical properties and discontinuous environmental conditions (Havlicek, 2012). Mineral nutri-

ents are cycled between living and dead organic components of soil, and mineral weathering is a

primary source of most of the essential elements for microorganisms and plants (Gadd, 2013). Soil

microorganisms influence weathering through physical and chemical processes (Banfield et al.,

1999). One of the main chemical processes used by most soil microorganisms is the production of

siderophores as metal chelating agents (Schwyn and Neilands, 1987).

Up to the present, most studies have focused on investigating mineral weathering under laboratory

conditions, and only a few studies have reported the biogenic chelator concentrations in natural

soil. The present thesis takes an interdisciplinary approach to obtain a comprehensive understand-

ing of microbe-mineral interactions in the podzol forest soil. Field experiments have been conduct-

ed to explore the composition and distribution of siderophores in a soil profile and on different

mineral substrates. In addition, laboratory investigations have been conducted linking siderophore

production capabilities and microbial surface attachment efficiency of soil microorganisms to min-

eral weathering reactions. The main hypothesis is that the mineral type controls the activity of soil

microbial communities. Furthermore the nature of the microbe-mineral interaction affects the

weathering capacity and element selectivity of the microorganisms.

1.1 Podzol soil

Podzol soils cover approximately 485 million hectares worldwide and are located mainly in the

temperate and boreal regions of the Northern Hemisphere (Lundström et al., 2000). Podzol is the

third most abundant soil in Europe, covering more than 0.5 million km2 or 14% of its total area

(Figure 1). This reference soil group is present in 22 member states of the EU and is only absent in

Hungary, Slovenia, Bulgaria, Malta and Cyprus. Vast areas of podzols are found in the Scandinavian

countries; for example they cover approximately 14 M ha or 60% of the forest land area of Sweden

(Figure 1).

Figure 1. Distribution of podzols in European Union and Sweden (FAO, 1988).

9

A typical podzol profile consists of a litter layer (O), a leached ash gray eluvial mineral layer (E), an

accumulation (illuvial) layer of organic matter in combination with Fe and Al (B) and the parent

material (C) (Figure 2). Podzol soils are typically acidic throughout the profile with the lowest pH

values in the O-horizon and an increasing pH with depth (Fritze et al., 2000). The O-horizon con-

tains various organic acids like fulvic acids, humic acids, low molecular mass organic acids (LMMO-

As) and other chelating agents which are produced mainly by soil biota and decaying plant litters

(Lundström et al., 2000; Holmström et al., 2004; van Scholl et al., 2006; Winkelmann, 2007). These

organic acids and chelating agents are leached downwards into the underlying E-horizon to pro-

mote the weathering of primary minerals by ion-exchange, dissolution and formation of metal-

organic complexes (Jongmans et al., 1997; van Breemen et al., 2000; Eriksson et al., 2005). When

the primary minerals dissolve, their surface color turns gray, which is the main reason for the char-

acteristic pale ash color of the E-horizon (Sauer et al., 2007). The weathering products are trans-

ported downward with the percolating soil solution, making the E-horizon very poor in nutrients

(Eriksson et al., 2005). As the soil solution percolates farther down in the soil profile, Al and Fe in

complex with organic compounds are precipitated and accumulated in the B-horizon (Lundström et

al., 2000). There are three main reasons for the precipitation and accumulation in the B-horizon

(Buurman and Jongmans, 2005): a) weathering of silicate minerals that leads to the formation and

precipitation of allophanic material, b) migration of metal-organic complexes down the soil profile,

and c) decomposition of the metal-organic complexes and release of Fe and Al and their subsequent

precipitation as sesquioxides which may be responsible for the reddish brown color of the B-

horizon. Compared with the E-horizon, the B-horizon contains higher levels of both nutrients and

organic matter (Buurman and Jongmans, 2005). Finally, the C-horizon is overlies the bedrock,

which is commonly granite and gneiss (FAO, 1990).

Figure 2. Description of podzol soil profile of the study area (Bispgården Forest Research Park).

1.2 Mineral weathering by soil microorganisms

Mineral weathering is important for understanding a variety of environmental issues such as nutri-

ent cycling, neutralization of acidic rain and long-term sequestration of atmospheric CO2 (Drever

and Hurcomb, 1986; Berner and Berner, 1997; Huntington et al., 2000). Soil microorganisms play

an essential role in releasing the key nutrients from primary minerals that are required for their

own and plants’ nutrition (Uroz et al., 2009). Microorganisms can directly or indirectly enhance

mineral dissolution, disaggregation and secondary mineral formation (Finlay et al., 2009). Microbi-

al attachment to mineral surfaces leads directly to the formation of a low pH microenvironment

O-horizon, organic layer

E-horizon (weathered) Fe and Al in soluble organic complexes

B-horizon (Fe and Al have precipitated)

C-horizon, parent material

10

that contains extracellular polymers. This increases the water retention time on the mineral surface

and the diffusion of ions away from the mineral; therefore it increases mineral dissolution and en-

hances secondary mineral production (Banfield et al., 1999; Liermann et al., 2000; Ojeda et al.,

2006). In these microenvironments, mineral nutrients can be chelated directly from the soil miner-

als or shared amongst the surrounding microorganisms (Rogers and Bennett, 2004). The surface

attached microorganisms contribute to mineral dissolution by using different mechanisms such as

lowering pH, complexing with surface ions, catalyzing redox reactions and by physical forcing

(Brown et al., 1999; Rogers et al., 1998; Rogers et al., 2001; Bennett et al., 2001). The unattached

microorganisms that live as individuals or in aggregates in the soil solution, on the other hand, ob-

tain their element nutrition mainly by complexing with ions and oxido-reduction reactions (Madi-

gan et al., 2000).

The production of biogenic chelators is considered to be one of the most important weathering

mechanisms by all soil microorganisms. The chelators can either complex with ions on the mineral

surface and weaken the metal–oxygen bonds or alternatively complex with ions in solution, thereby

decreasing solution saturation state (Banfield et al., 1999; Finlay et al., 2009). The most common

biogenic chelators produced by microorganisms are siderophores and LMMOAs. Siderophores form

1:1 complexes with Fe(III), with stability constants ranging between K=1022 and K=1052 (Jalal and

van der Helm, 1991; Matzanke, 1991), while the stability constants of LMMOAs like oxalic and citric

acids with Fe(III) are only K=107.6 and 1012.3, respectively (Perrin, 1979). Thus, the impact of sider-

ophores on soil mineral weathering is more effective than LMMOAs. However, when both LMMOAs

and siderophores are present they may function synergistically, which results in a higher mineral

dissolution rate compared to that of the siderophore only (Reichard et al., 2007).

There are several mechanisms for siderophore promoted Fe-bearing mineral dissolution in soil

(e.g., Holmén and Casey, 1996; 1998). The general mechanism is that a Fe-siderophore complex is

formed at the mineral surface and is subesquently transferred into the surrounding soil solution,

thereby becoming available for uptake by the cell membrane of microorganisms or plants (Kali-

nowski et al., 2000a; Liermann et al., 2000; Kraemer, 2004). Siderophores are either recycled or

destroyed upon Fe reduction. The reduced iron Fe(II) that is not used by the cell can act as an elec-

tron donor in electron transport chains (Kalinowski et al., 2000b). On the other hand, LMMOAs

could promote the dissolution of minerals directly through three mechanisms: by altering the dis-

solution rate far from equilibrium through decreasing solution pH, by forming complexes with cati-

ons at mineral surface, and by affecting the saturation state and speciation of ions in solution

(Drever and Stillings, 1997).

1.3 Microbial siderophores in soil ecosystem

Fe is an important element for the growth of almost all living microorganisms since it acts as a cata-

lyst in various enzymatic processes, oxygen metabolism, electron transfer, and DNA and RNA syn-

thesis (Touati, 2000; Verkhovtseva et al., 2001). Thus, microorganisms have developed specific Fe

uptake strategies like siderophores. Siderophores (Greek: “iron carriers”) are defined as low mo-

lecular mass chelating agents produced by bacteria and fungi growing under Fe-limiting condition

(Neilands, 1995). The main role of siderophores is to scavenge Fe from the environment and make

it available to the microbial cell (Kraemer, 2004). Microorganisms can produce both extracellular

and intracellular siderophores. Extracellular siderophores contribute in the solubilization and

transportation of Fe from environments to microbial cells, while intracellular siderophores are

responsible for storage of Fe inside the cells (Wilhelm and Trick, 1994). Bacteria produce four

groups of siderophores (catecholates, carboxylates, hydroxamates “ferrioxamines only” and mixed

11

siderophores) (Matzanke, 1991), while fungi produce mainly hydroxamates (i.e. ferrichromes,

coprogens and fusarinines) (Winkelmann, 2007).

Hydroxamates are the most common group of siderophores in natural environments. They form

1:1 complexes with Fe(III) and the stability constants are in the range of K=1022 to K=1033 (Robert

and Chenu, 1992). Fe-hydroxamate complexes are stable against hydrolysis and enzymatic degra-

dation in the soil (Winkelmann, 2007). The main types of hydroxamate siderophores are ferrioxam-

ines, ferrichromes, coprogens and fusarinines (Figure 3). Ferrioxamines have both linear and cyclic

compounds containing 1-amino-5-hydroxyaminopentane (Dhungana et al., 2001). Cyclic ferriox-

amine structures like (Ferrioxamine E and D) have a higher stability constant with Fe than the line-

ar structures (Ferrioxamine B and G) (Boukhalfa and Crumbliss, 2002). They are commonly pro-

duced by many soil bacteria, such as Erwinia, Nocardia, Streptomyces, Arthrobacter, Chromobacte-

rium and Pseudomonas species (Berner et al., 1988; Berner and Winkelmann, 1990; Gunter et al.,

1993; Meyer and Abdallah, 1980; Muller and Raymond, 1984; Wei et al., 2007). Ferrichromes are

the predominant fungal siderophores and are based on a cyclic hexapeptide structure (Leong and

Nielands, 1982; Deml et al., 1984). Ferrichromes are further divided into five groups depending on

the side chain of the hydroxamate functional group: acetyl (ferrichrome, ferrichrome C, ferricrocin,

and ferrichrysin), malonyl (malonichrome), trans-b-methylglutaconyl (ferrichrome A), trans-

anhydromevalonyl (ferrirubin) and cis-anhydromevalonyl (ferrirhodin) (Renshaw et al., 2002;

Winkelmann, 2007). Common soil fungi that produce ferrichromes are the following: Ustilago

sphaerogena for ferrichrome (Emery, 1971), Aspergillus fumigatus for ferricrocin (Wallner et al.,

2009), Neurospora crassa for tetraglycylferrichrome (Winkelmann, 2007) and Cenococcum geophi-

lum and Hebeloma crustuliniforme for ferrichrysin (Martino and Perotto, 2010). Coprogens consist

of a cyclic diketopiperazine ring that is formed by two N5-acyl-N5-hydroxy-L-Orn units (Budzikie-

wicz, 2010). Coprogens are commonly produced by fungal species such as Trichoderma spp. and

Neurospora crassa (Zähner et al., 1963). The ester bonds in coprogen can be split by esterase en-

zyme produced by microorganisms and plants in soil and yield one dihydroxamate siderophore

(dimerum acid) and one monohydroxamate siderophore (fusigen) that can support the plants with

the required Fe nutrition (Winkelmann, 2007). Fusarinines consist of acyl unit (5- hydroxy-3-

methyl-pent-2-enoic acid) bound to N5-hydroxy L-ornithine that could form liner or cyclic struc-

tures (Barry and Challis, 2009). Fusarinines, especially fusigen, are commonly produced by Fusari-

um spp. (Diekmann and Zahner, 1967; Sayer and Emery, 1968; Neilands, 1973).

Siderophores in soil are present in two different forms, dissolved in soil solution and adsorbed on

soil particles (Cervini-Silva, 2008). It has been suggested that adsorption controls the concentration

of siderophores in bulk soil solution and protects siderophores against hydrolysis and enzymatic

degradation (Powell et al., 1982; Haselwandter et al., 2011). The presence of adsorbed sidero-

phores is dependent on the following: a) the physico-chemical properties of their functional groups,

b) the different chemical structure of the specific types of siderophores and c) the different electri-

cal charge of the respective siderophores that will affect their mobility in the soil profile (Winkel-

mann, 2007). The main factor influencing the siderophore adsorption capacity is the effect of soil

solution pH on the siderophore charge, rather than the effect of the charge of the mineral and soil

particles. On the other hand, the adsorption of siderophores in the soil ecosystem is strongly influ-

enced by their interactions with solid phases, like clay minerals, that constitute a major part of the

surface area in soils (Siebner-Freibach et al., 2004).

12

Ferrichrome: R1 = R2 = H, R3 = CH3

Ferrichrome A: R1 = R2 = H, R3 = A

Ferricrocin: R1 = H, R2 = CH2OH, R3 = CH3

Ferrichrysin: R1 = R2 = CH2OH, R3 = CH3

Ferrirubin: R1 = R2 = CH2OH, R3 = B

Ferrirhodin: R1 = R2 = CH2OH, R3 = C

Figure 3. Chemical structure of some hydroxamate siderophores. Bacterial hydroxamate sidero-

phores include ferrioxamine (B, E, D and G). Fungal hydroxamate siderophores are ferrichrome, ferri-

crocin, ferrichrome, ferrichrysin, ferrirubin, ferrirhodin, ferrichrome A, fusigen (linear) and copro-

gens. All the chemical structures were drawn using ChemDraw Standard 13.0 software.

Coprogen: R1 = R2 = A, R3 = COCH3 Neocoprogen I: R1 = H, R2 = COCH3, R3 = A Neocoprogen II: R1 = H, R2 = COCH3, R3 = CH3

Ferrioxamine E

Fusigen

Ferrioxamine B: R1 = H, R2 = CH3, n = 5 Ferrioxamine D: R1 = COCH3, R2 = CH3, n = 5 Ferrioxamine G: R1 = H, R2 = CH2CH2COOH, n = 5

(A)

(B)

(C) (A)

13

2. Scope of the thesis

Earlier research focusing on microbe-mineral interactions considered mostly the mineral dissolu-

tion rates by microbial species in lab conditions and concluded that different microbial species

have different abilities in mineral weathering. The present thesis examines in situ the responses of

soil microbial activity to different mineral substrates in comparison to those in bulk soil. Further-

more, laboratory investigations have been conducted to explore the siderophore production capa-

bilities of soil microorganisms and to gain a better understanding of the weathering performance of

the mineral attached and free living microorganisms in soil environment.

The central hypothesis is that the siderophore production changes in response to different mineral

substrates and soil horizons. In addition, the weathering capacity and element selectivity of micro-

organisms are believed to be dependent on the type of interaction they have with minerals. Field

and laboratory studies have been conducted to answer the following questions: What is the impact

of soil horizons and different mineral types on siderophore composition in soil? What siderophores

are produced by soil microorganisms? What is the role of microbial surface attachment in mineral

weathering reactions? In addition, the main roles and potential biotechnological applications of

siderophores were reviewed. The specific objectives of the four papers are formulated as shown in

figure 4.

Figure 4. Diagram of the four papers included in the thesis and their objectives.

In situ investigations Lab investigations Insights

14

3. Sampling site and field experiment setup

Soils were sampled from central Sweden in the vicinity of the village Bispgården (63°07′N,

16°70′E) in September 2011 (Figure 5). The site is located on a slope (angle 2°) at an altitude of

258 m above sea level and is forested with 80-yr-old Norway spruce (Picea abies) and Scots pine

(Pinus sylvestris). The annual average precipitation is 700 mm, and is not acidic. The annual average

temperature is +2 °C. The bedrock in the area is granite and gneiss. The soil is a typical haplic pod-

zol (FAO, 1990). The soil horizons in the studied soil profile have the following depths: 12 cm for O

(organic horizon), 10 cm for E (elluvial horizon), 19 cm for B (upper illuvial horizon) and finally C

(parent material). Three polished minerals were used: biotite (purchased from Wards, Canada),

apatite and oligoclase (purchased from Krantz, Germany) with dimensions of 3 cm X 4 cm X 3 mm.

The minerals were polished with 6, 3 and 1 μm diamond paste to obtain a fresh unweathered sur-

face. The three minerals were buried in the O-, E- and B-horizons at depths of 7, 14 and 28 cm, re-

spectively, from the top of the soil at June 2009. The soil samples for this study were taken from the

bulk soil of the whole profile and mineral surfaces and kept cold (+4 °C) until further analysis.

Figure 5. Sampling site and field experiment set up. a) Map of the sampling area in Bispgården, Sweden. b) The podzol soil profile in the sampling site. c) Diagram of the field experiment set up. Apatite (A), biotite (B) and oligoclase (O) were inserted in O-, E- and B-horizons. Soil samples were collected from the mineral surfaces and bulk soil (BS).

15

4. Summary of materials and methods

The following series of methods were used to cover all the objectives of the thesis (Figure 6).

4.1 Extraction of siderophores from soil and microbial isolates

In order to determine the siderophore content in soil samples, we extracted the siderophores by

two methods, water extraction (dissolved siderophores) and methanol extraction (adsorbed sider-

ophores) as described in paper I. Siderophores were also extracted from the soil microbial isolates

as described in paper II. All isolated cultures were inoculated into 10 ml broth medium and were

incubated on a rotary shaker (200 rpm) for 7 days. A low concentration (1%) of FeCl3 was added to

the culture filtrates during stirring to obtain ferri-siderophores. Subsequently the brown culture

filtrates were filtrated through 0.45 μm filters. The extracts from both soil and microbial isolates

were pre-concentrated by freeze-drying. When all water in the sample had been evaporated a yel-

low-white solid dust remained that was dissolved in 1 ml of MilliQ water. To remove the high mo-

lecular mass compounds (>3000 Da), centrifugal ultrafiltration (3000 Da cutoff) filters (Nanosep

3K Omega, Pall, Mexico) were used. The pre-concentration and purification method was developed

by Holmström et al. (2004).

4.2 Isolation and molecular identification of siderophore producing microorganisms from

soil

Siderophore producing microorganisms were isolated from soil solution and identified as de-

scribed in paper II. Soil solutions were obtained by shaking 5 g of soil in 15 ml of MilliQ water with

glass pearls for 4 h and then were serially diluted. 0.1 ml of the 10-3 and the 10-4 dilutions were

plated using spread plate technique onto two replicates of the appropriate media containing dilut-

ed chrome azurol S (CAS) indicator. Fungi were isolated using modified ½MMN (Melin Norkans

agar) and bacteria were isolated using soybroth agar. The color change from blue to orange was an

indicator for the production of siderophores (Schwyn and Neilands, 1987). The most effective si-

derophore producing isolates were selected by choosing those that formed the largest orange halos

around the cultures on the plates. Microbial genomic DNA was extracted from the isolated microor-

ganisms using Genomic DNA Miniprep Kit (Axygen Biosciences, USA) according to the manufactur-

er’s instructions. Molecular identification was carried out by amplification using universal primers.

Primers 518F 5'-CCA GCA GCC GCG GTA ATA CG-3' and 800R 5'-TAC CAG GGT ATC TAA TCC-3' tar-

geting a subset of the 16S rRNA gene were used for bacteria, while ITS1F '5-CTT GGT CAT TTA GAG

GAA GTA A-3' and ITS4 5'-TCC TCC GCT TAT TGA TAT GC-3' primers targeting ITS were used for

fungi. DNA sequencing was done by the sequencing service Macrogen inc.

(http://www.macrogen.com). The obtained sequences were compared to sequences in the NCBI

GenBank database using online BLASTN (Altschul et al., 1990). Phylogenetic trees were generated

using a neighbor-joining tree-building algorithm in the MEGA 5 Software. All sequences were sub-

mitted to GeneBank (accession numbers: KJ081748- KJ081778).

4.3 Quantification of siderophores using HPLC-ESI-MS

The extracted siderophores from both the soil samples and the isolated microorganisms were ana-

lyzed using a method modified from Duckworth et al. (2009) as described in paper I. The four main

groups of hydroxamate siderophores (ferrichromes, ferrioxamines, coprogens and fusigen) were

quantified by high performance liquid chromatography coupled to electrospray ionization mass

spectrometry (HPLC-ESI-MS) (Ultimate 3000 RS, Thermo Scientific, USA). The HPLC system con-

16

sisted of two pumps with flow rate of 0.030 ml/min for the low pressure gradient pump and 0.15

ml/min for the high pressure gradient pump and a column compartment set at 10°C. The switch

time between the pre-column and analytical column was 10.35 min. The column types and eluents

used in this method were described in more detail in paper I. The HPLC system was connected to a

mass spectrometer in which the hydroxamate siderophores were detected in mass range (550-

1055 m/z) and scan rate (8 sec). The ferric complex of each individual hydroxamate siderophore

was detected by selected ion monitoring (SIM) of the proton adducts [M + H]+: i.e. m/z 797.3 for

tetraglycyl ferrichrome, 771.3 for ferricrocin, 741.2 for ferrichrome, 801.2 for ferrichrysin, 1011.3

for ferrirubin, 1011.2 for ferrirhodin, 1052.2 for ferrichrome A, 614.2 for ferrioxamine B, 672.2 for

ferrioxamine G, 656.3 for ferrioxamine D, 654.3 for ferrioxamine E, 682.5 for neocoprogen II, 793.2

for fusigen (lin.), 752.3 for neocoprogen I, and 821.2 for coprogen.

4.4 Biotite weathering by attached and unattached soil microorganisms

Sterilized microplate devices (VWR International, Sweden) were filled with biotite (>2mm) fol-

lowed by 15.5 ml of an iron limited liquid medium. The microplate wells were inoculated separate-

ly with six microbial species isolated from podzol soil Erwinia amylovora, Pseudomonas stutzeri,

Pseudomonas mendocina, Neurospora crassa, Penicillium melinii and Streptomyces pilosus. The ex-

periment was designed in two modes, 1) microbial attachment, in which the microorganisms inocu-

lated directly to the biotite surface, 2) microbial unattachment, in which 0.4 µm PET track-etched

devices (VWR International, Sweden) were used to separate the microbial cells from the biotite

surface. The following analyses were done in interval times (4, 7, 10, 13, 16 days of incubation): pH

determination; quantification of dissolved elements using inductively coupled plasma atomic emis-

sion spectroscopy (ICP-OES) (Varian Vista AX, SPS-5, USA) and quantification of siderophores using

HPLC–ESI-MS. Uninoculated biotite-amended medium was used as a control. More details with re-

gard to the experiment setup and the dissolution analysis are described in paper III.

4.5 Data analysis and statistics

The sum of tetraglycyl ferrichrome, ferricrocin, ferrichrome, ferrichrysin, ferrirubin, ferrirhodin

and ferrichrome A was calculated and denoted as the total concentration of ferrichromes. The sum

of ferrioxamine B, ferrioxamine G, ferrioxamine D and ferrioxamine E corresponds to the total con-

centration of ferrioxamines. The sum of neocoprogen II, neocoprogen I and coprogen was denoted

as the total concentration of coprogens. Fusigen (linear) represents the fusigen. The sum of all the

ferrichromes, ferrioxamines, coprogens and fusigen was denoted as the total concentration of hy-

droxamate siderophores. The data were normalized and one/two-way ANOVA for multiple compar-

isons were used to compare the averages for Fe, Al and Si released from the biotite by the attached

and unattached microbial species. The ternary plots were carried out using XLSTAT

(http://www.xlstat.com/en/).

17

Fig

ure

6. S

eq

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s of th

e m

eth

od

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s use

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pre

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18

5. Summary and discussion of major findings

5.1 What is the impact of soil horizon and mineral type on siderophore composition in soil?

Soil horizon strongly influenced the composition of siderophores (Paper I). The average concentra-

tion of total dissolved ferrioxamines was 2-7 pmol/g dry soil, ferrichromes 1-4 pmol/g dry soil,

fusigen 0-39 pmol/g dry soil and coprogens 2-16 pmol/g dry soil (Figure 7A). On the other hand,

the average concentration of total adsorbed ferrioxamines was 0-2 pmol/g dry soil, ferrichromes

0.3-2 pmol/g dry soil, fusigen 0-32 pmol/g dry soil and coprogens 8-18 pmol/g dry soil (Figure 7B).

In addition, the soil horizon affected the distribution of individual dissolved and adsorbed hydrox-

amate siderophores differently. For dissolved hydroxamate siderophores, all the individual ferriox-

amines were correlated to the E-horizon, most of the ferrichromes and coprogens were correlated

to the O-horizon, and fusigen was correlated to the C-horizon, whereas for adsorbed hydroxamate

siderophores, most of the ferrichromes and ferrioxamines were correlated to the E-horizon, and

fusigen and most of the coprogens were correlated to the O-horizon (Paper I).

Figure 7. The concentration (pmol/g dry soil) of (A) dissolved and (B) adsorbed hydroxamate sidero-

phores (ferrioxamines, ferrichromes, fusigen and coprogens) per each soil horizon.

Three possible reasons could explain the variety of hydroxamate siderophore concentration and

distribution within the soil horizons: a) chemical and mineralogical properties, b) organic matter

content and c) the pH of the soil. The chemical and mineralogical properties of podzol soil change

with depth, which creates a number of different habitats for microorganisms throughout the soil

profile (Fierer et al., 2003; LaMontagne et al., 2003; Rosling et al., 2003). The maximum sidero-

phore metabolic diversity was usually found in the O- and E-horizons as compared to B- and C-

horizons. Furthermore, soil organic matter content could have an impact on hydroxamate sidero-

phore concentration, in which the low clay soils yielded almost twice as many hydroxamate sidero-

phores as did high clay soils suggesting that adsorption might be an important determinant of hy-

droxamate siderophore concentration in soil solution (Powell et al., 1982). That could explain the

highly adsorbed hydroxamate siderophores found in the O- and E-horizons with regard to the high

organic matter found in those horizons. However, the pH of the soil solution in our study was in the

range of 4.4-5.4 and the majority of iron was soluble; therefore, the microorganisms that produce

siderophores at these pH values have a distinct advantage over non-siderophore producers. That is

because of the extreme acid stability of the hydroxamate siderophore molecules that give them the

ability to scavenge the needed iron from competing microorganisms and also protect themselves

from the stress of Fe overdose (Matsumoto et al., 2001; Wittenwiler, 2007).

E

O

19

The mineral types (apatite, biotite and oligoclase) had a greater impact on the composition of si-

derophores than soil horizon (Paper I). High variable concentrations of hydroxamate siderophores

in the soil attached to the different polished mineral surfaces were observed. The average concen-

tration of total dissolved hydroxamate siderophores found on apatite, biotite and oligoclase was

20-83, 42-107 and 18-36 pmol/g dry soil, respectively (Paper I). Biotite had the maximum concen-

tration of dissolved ferrichromes (66 pmol/g dry soil) and fusigen (60 pmol/g dry soil) in O-

horizon, apatite had the maximum concentration of dissolved coprogens (67 pmol/g dry soil) in B-

horizon, whereas oligoclase had the minimum concentration of most of dissolved hydroxamate

siderophores (Figure 8A). The average concentration of total adsorbed hydroxamate siderophores

was 35-64 pmol/g dry soil for apatite, 6-55 pmol/g dry soil for biotite and 0.09-21 pmol/g dry soil

for oligoclase (Paper I). The adsorbed fusigen and coprogens were found in higher concentrations

than ferrichromes and ferrioxamines for all mineral surfaces and they were completely absent on

oligoclase in the O-horizon (Figure 8B).

Figure 8. The concentration (pmol/g dry soil) of (A) dissolved and (B) adsorbed hydroxamate sidero-

phores (ferrioxamines, ferrichromes, fusigen and coprogens) of the soil attached to mineral surfaces

per each soil horizon.

There are some theories that could explain our findings. The higher hydroxamate siderophore con-

centration that was detected on the mineral surfaces in comparison to the bulk soil could be due to

the microbial formation of microenvironments, in which mineral nutrients can be chelated directly

by certain microorganisms or shared among the surrounding microorganisms (Bennett et al., 2001;

Rogers and Bennett, 2004). Thus the conditions in the microenvironment induce the microorgan-

isms to produce siderophores to obtain their elemental nutrients from the mineral surfaces. On the

other hand, the variability in hydroxamate siderophore concentrations between mineral types

could indicate that the composition of the mineral affects the physico-chemical conditions in the

soil attached to its surface. This leads to different sources of elemental nutrients and physical sup-

port for selective microbial colonization (Hutchens, 2009; Uroz et al., 2012). In the present study,

biotite (mica) contains high concentration of different elements (4.5% Fe, 4% K, 8% Mg and 5% Al).

Apatite (Fe-phosphate) contains high content of P (12%) and Ca (18.5%), and low content of Fe

A B

20

(0.24%). Oligoclase (feldspar) contains a very low content of Fe (0.1%), K (0.2%) and Ca (1.5%),

and a high content of Al (8%). The high concentration of the major elements required by the micro-

organisms in both biotite and apatite could enhance the production of siderophores. However, the

high content of Al in oligoclase may inhibit the growth of microorganisms and the siderophore pro-

duction in the attached soil, especially if the Al present in its free toxic form (Al3+) and the essential

nutrients were very low (Roberts, 2004). Our new finding that fusigen and coprogens were the

predominant hydroxamate siderophore types in both the bulk soil and soil attached to minerals

could be related to the nature of production of the different siderophore types. Coprogens and fusi-

gen are extracellular siderophores produced by microorganisms for Fe chelation, while ferri-

chromes and ferrioxamines are mostly intracellular siderophores that participate in Fe storage and

sporulation processes (Winkelmann, 2007; Wallner et al., 2009).

A variation in the concentration of the two siderophore phases (dissolved and adsorbed) was also

detected with respect to the soil horizons and different mineral types. The concentration of dis-

solved hydroxamate siderophores was higher than the concentration of adsorbed hydroxamate

siderophores. These findings could be due to their strong cyclic hexapeptide structure that make

them highly resistant to the environmental degradation by some enzymes produced by plants (i.e.

hydrolases and proteases), which affect the lifetime of the siderophores (e.g. Hider and Kong,

2010). In addition, the ferrichromes and ferrioxamines were found in higher concentrations in the

dissolved phase than in the adsorbed phase, while the coprogens were more abundant in the ad-

sorbed phase in most of soil horizons and minerals. These findings could be related to the different

chemical structures of the specific hydroxamate functional groups. The fusigen, ferrichromes, ferri-

oxamines and coprogens differ with regard to the characteristic bonds within the molecule that

could form strong or weak bonds with the soil particles. For instance, three ester bonds are present

in fusigen, six peptide bonds in ferrichromes, five peptide bonds in ferrioxamines, and one ester

plus two peptide bonds in the coprogens (Winkelmann, 2007).

5.2 What siderophore types are produced by soil microorganisms?

Among the different siderophore types, hydroxamate siderophores have received much attention

since they are highly resistant to environmental degradation due to their cyclic hexapeptide struc-

ture (Winkelmann, 2007). Our findings showed that the isolated bacteria and fungi from podzol soil

horizons produced different types of hydroxamate siderophores, and each individual microorgan-

ism could produce a set of siderophore types covering a wide range of physico-chemical properties

(Paper II). The isolated siderophore producing bacteria showed different abilities in the production

of ferrioxamines (E, B, G and D) (Table 1). All the bacterial isolates could produce ferrioxamine D,

except Micrococcus luteus (C-horizon). The maximum concentration of ferrioxamine D, 18.6 nM,

was produced by Streptomyces olivaceus (O-horizon). Ferrioxamine G was the second most com-

mon hydroxamate siderophore produced by most of the isolated bacteria. The highest concentra-

tion of ferrioxamine G, 24.3 nM, was produced by Streptomyces nitrosporeus (B-horizon). Ferriox-

amine B was produced in low concentration by most of the isolated bacterial species and was com-

pletely absent for all the isolates from the C-horizon. In comparison, not all the isolated bacteria

had the ability to produce ferrioxamine E, but Pseudomonas mendocina isolated from the B-horizon

was found to produce the maximum concentration (11.4 nM) of ferrioxamine E. The isolated sider-

ophore producing fungal species showed a high variety in their ability to produce hydroxamate

siderophores, i.e. ferrichromes, coprogens and fusarinines (Table 1). Most of the fungal isolates

produced high concentrations of coprogen that ranged between 101.3 nM (Trichothecium roseum,

O-horizon) and 1649.3 nM (Ustilago maydis, E-horizon). Neocoprogen II was also produced by

many of the isolated fungi, especially those that were isolated from the E-horizon. Fe-dimerum acid

21

Fusarinines (nM)

FOX B FOX G FOX D FOX E FCH FC Tetra-FCH FCHR FRU FCH A Fe-DA Neo-COP II Neo-COP I COP FUS (lin.)

HORIZON ^O^

Fungi

Penicillium glabrum - - - - - - 1.8 ±0.04 0.01 ±0.003 - - 0.9 ±0.004 - - 304.7 ±5.9 -

Thielavia terrestris - - - - - 0.2 ±0.002 4.4 ±0.21 0.05 ±0.001 0.7 ±0.002 - - - - - -

Trichothecium roseum - - - - 0.1 ± 0.001 - - - 0.3 ±0.0001 - - 4.8 ±0.41 - 101.3 ±8.2 -

Bacteria/Actinobacteria

Streptomyces olivaceus 14.1 ±0.22 21.7 ±5.1 18.6 ±3.2 - - - - - - - - - - - -

Pseudomonas stutzeri 9.4 ±1.77 1.6 ±0.03 1.3 ±0.007 1.2 ±0.001 - - - - - - - - - - -

Nocardia tenerifensis 8.1 ±0.06 14.1 ±1.7 16.6 ±1.9 - - - - - - - - - - - -

Klebsiella pneumoniae 14.7 ±0.17 18.2 ±0.07 8.4 ±0.18 - - - - - - - - - - - -

Rhodococcus sp. - 17.5 ±0.8 16.0 ±0.65 3.7 ±0.05 - - - - - - - - - - -

HORIZON ^E

Fungi

Penicillium melinii - - - - 0.6 ±0.02 - - - - - - - - 539.7 ±7.8 -

Ustilago maydis - - - - - - - - - - - 6.0 ±0.11 - 1649.3 ±9.6 -

Neurospora crassa - - - - - - - - - - 0.81 ±0.002 5.9 ±0.27 - 892.7 ±10.5 -

Fusarium sporotrichioides - - - - - - 1.0 ±0.003 - - - - 3.1 ±0.04 81.1 ±6.5 462.4 ±3.7 14.8 ±0.16

Bacteria/Actinobacteria

Enterobacteriaceae bacterium 11.8 ±0.22 21.3 ±0.27 0.8 ±0.001 0.7 ±0.02 - - - - - - - - - - -

Bacillus thuringiensis 13.7 ±0.09 14.4 ±0.13 9.6 ±0.1 1.1 ±0.001 - - - - - - - - - - -

Pseudomonas sp 6.7 ±0.78 12.7 ±0.59 13.5 ±0.59 3.9 ±0.24 - - - - - - - - - - -

Streptomyces sp. 19.3 ±0.87 15.5 ±0.16 11.1 ±0.15 - - - - - - - - - - - -

Pseudoalteromonas sp. 7.9 ±0.26 13.2 ±0.16 13.8 ±1.4 4.3 ±0.31 - - - - - - - - - - -

Actinosynnema mirum 8.9 ±0.26 - 8.1 ±0.01 - - - - - - - - - - - -

Streptomyces pilosus 15.3 ±0.35 19.7 ±1.06 10.8 ±0.15 - - - - - - - - - - - -

Nocardia farcinica 9.6 ±0.45 6.7 ±0.94 14.5 ±0.31 - - - - - - - - - - - -

Erwinia amylovora 7.7 ±0.24 11.7 ±0.37 7.3 ±0.13 1.6 ±0.002 - - - - - - - - - - -

Gamma proteobacterium - 16.2 ±1.8 16.3 ±0.71 4.1 ±0.16 - - - - - - - - - - -

HORIZON ^B^

Fungi

Aspergillus fumigatus - - - - - - 19.3 ±0.16 0.04 ±0.0001 4.1 ±0.003 - 8.9 ±0.28 - - - -

Bacteria/Actinobacteria

Micrococcus luteus 5.0 ±0.14 2.8 ±0.03 7.2 ±0.27 1.5 ±0.002 - - - - - - - - - - -

Pseudomonas mendocina - 2.8 ±0.01 14.6 ±0.15 11.4 ±0.16 - - - - - - - - - - -

Streptomyces nitrosporeus 17.5 ±0.18 24.3 ±1.06 10.8 ±0.15 - - - - - - - - - - - -

Uncultured Streptomyces sp. 11.4 ±0.53 13.2 ±0.18 16.3 ±0.21 - - - - - - - - - - - -

HORIZON ^C^

Fungi

Epicoccum nigrum - - - - - - - - - 10.7 ±0.17 - 4.5 ±0.02 - 514.4 ±9.1 -

Bacteria/Actinobacteria

Uncultured gamma proteobacterium - - 0.9 ±0.01 2.9 ±0.01 - - - - - - - - - - -

Micrococcus luteus - - - 5.0 ±0.02 - - - - - - - - - - -

Uncultured bacterium - 18.4 ±0.54 6.9 ±0.09 - - - - - - - - - - - -

Horizon/MicroorganismsFerrioxamines (nM) Ferrichromes (nM) Coprogens (nM)

was sparsely detected. In line with this, neocoprogen I (81.1 nM) and fusigen (lin.) (14.8 nM) were

restricted to Fusarium sporotrichioides (E-horizon). Ferrichromes were found in much lower con-

centration compared to the coprogen. The fungal species isolated from the O- and B-horizons had

the ability to produce most of ferrichromes, except ferrichrome A. Ferrichrome A was restricted to

Epicoccum nigrum (C-horizon), whereas ferricrocin was only produced by Thielavia terrestris (O-

horizon).

Our findings raise the question, why do soil microorganisms produce a variety of different sidero-

phores? An explanation could be that the diversity in hydroxamate siderophore production helps

soil microorganisms to overcome the limits in Fe bioavailability and allow them to compete against

other microorganisms for key nutrients (Winkelmann, 2007; Haselwandter et al., 2013). Therefore,

microorganisms that produce various types of siderophores may have an ecological advantage,

since the successful competition for Fe is based on the concentration of siderophores produced by

the microorganisms, the chemical properties of the siderophore with regards to its stability con-

stant in binding with Fe and the resistance of siderophore types to the environmental degradation.

In addition, the different affinities of hydroxamate siderophores for Fe not only enable the micro-

organisms to chelate it, but may also allow the microorganisms to mobilize Fe from weaker Fe che-

lating complexes (like Fe-LMMOAs) produced by other microorganisms (Desai and Archana, 2011).

Table 1. The concentration of hydroxamate siderophores produced by isolated microorganisms from each soil horizon. Ferrioxamine B (FOX B), Ferrioxamine G (FOX G), Ferrioxamine D (FOX D), Ferriox-amine E (FOX E), Ferrichrome (FCH), Ferricrocin (FC), Tetraglycyl ferrichrome (Tetra-FCH), Ferrri-chrysin (FCHR), Ferrirubin (FRU), Ferrichrome A (FCH A), Fe-dimerum acid (Fe-DA), Neocoprogen II (Neo-COP II), Neocoprogen I (Neo-COP I), Coprogen (COP), Fusigen (linear) (FUS (lin.).

22

Thus we could assume from our results that the bacterial and fungal isolates that produce a wide

range of hydroxamate siderophores with high concentration had a greater chance to gain the ele-

ment nutrition than the other soil microbes. Another explanation could be that under Fe deficiency

conditions several genes involved in the biosynthesis of the siderophores and their receptors are

expressed (Barona-Gòmez et al., 2006). In addition, the receptors can identify siderophores that

have common properties. As a consequence, several siderophores can be produced by individual

microorganisms to obtain Fe from different sources (Pattus and Abdallah, 2000).

Even though the cultured microorganisms do not exceed 1-5% of the total soil microorganisms

(Janssen et al., 2002), we still found relatively positive correlation between the hydroxamate sider-

ophores detected in E-horizon (Paper I) and the siderophore producing cultures isolated from the

same horizon (Paper II). E-horizon comprised high concentrations of dissolved and adsorbed

coprogens and fusigen. Dissolved coprogens and fusigen were 13 pmol/g dry soil and 39 pmol/g

dry soil, respectively, while adsorbed coprogens and fusigen were 14 pmol/g dry soil and 32

pmol/g dry soil, respectively. When we correlated between those findings and the abundance of

siderophore producing microorganisms and their ability to produce hydroxamate siderophores

under laboratory conditions, we found that all the fungal species Ustilago maydis, Neurospora cras-

sa, Fusarium sporotrichioides and Penicillium melinii isolated from E-horizon could produce very

high concentrations of coprogen (up to 1649.3 nM). In addition, Fusarium sporotrichioides pro-

duced 14.8 nM of fusigen. Thus, we found a positive correlation between the high concentrations of

coprogen and fusigen in E-horizon and those that produced by isolated microorganisms from the

same horizon. But still a question should be addressed: why was the concentration of the coprogen

produced by microorganisms higher than fusigen in laboratory conditions and the opposite was

found in the soil? The answer could be that the fusigen in soil was coming from coprogen’s origin.

That is because the coprogen is a trihydroxamate siderophore which contains ester bonds that can

be split by esterase enzyme produced by microorganisms and plants in soil. This splitting results in

one dihydroxamate siderophore (dimerum acid) and one monohydroxamate siderophore (fusigen)

that can support the plants with the required Fe due to their low Fe reduction potential (Winkel-

mann, 2007).

5.3 What is the role of microbial surface attachment in mineral weathering reactions?

Mineral attached microorganisms had a greater ability in biotite dissolution than unattached mi-

croorganisms that is evidenced by the higher mobilization of the elements (Fe, Al and Si), lower

solution pH and relatively higher production of siderophores (Paper III). It was also found that the

interaction of both attached and unattached microorganisms with the mineral enhanced their ele-

ment selectivity. Both the microbial forms dissolved very high concentration of Fe and Si in com-

parison to Al. The attached microorganisms dissolved up to 25-54 µmol/l of Fe, 21-98 µmol/l of Si

and 2.6-11 µmol/l of Al, while the unattached microorganisms dissolved up to 18-35 µmol/l of Fe,

13-74 µmol/l of Si and 2.2-8.5 µmol/l of Al (Figure 9). The dissolution concentrations of all the

studied elements were significantly (P < 0.05) different among the different microbial species. Fun-

gal species Neurospora crassa was the most efficient strain that was able to mobilize Fe, Al and Si to

the greatest extent in both the attached and unattached form (Figure 9). However, the attached

Neurospora crassa dissolved a higher concentration of Fe (up to 54 µmol/l), Al (up to 11 µmol/l)

and Si (up to 98 µmol/l) than the unattached form that dissolved Fe (up to 35 µmol/l), Al (up to 8.5

µmol/l) and Si (up to 74 µmol/l) after 16 days. In contrast, bacterial species Erwinia amylovora had

the lowest efficiency in element mobilization (Figure 9). The attached Erwinia amylovora dissolved

Fe (up to 25 µmol/l), Al (up to 2.6 µmol/l) and Si (up to 21 µmol/l) whereas the unattached form

dissolved Fe (up to 18 µmol/l), Al (up to 2.2 µmol/l) and Si (up to 13 µmol/l) after 16 days.

23

Fig

ure

9. T

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of F

e, A

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i from

bio

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y (A

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24

Thus a key question should be addressed: why does the microbial surface attachment play an im-

portant role in mineral weathering? The reason could be that the attachment of the microbial cells

on mineral surfaces forms a chemically complex and reactive microenvironment that promotes the

microbial ability of mineral dissolution (Roberts, 2004; Roberts et al., 2006; Hutchens, 2009; Dong,

2010). That is because the microbial cells in the microenvironment produce extracellular polymeric

substances (EPS) and other biogenic molecules including chelating agents (i.e. siderophores) that

can directly influence the surface reactivity of the mineral, and may also further strongly influence

the porosity and the permeability characteristics of the colonized mineral (Liermann et al., 2000;

Fowle et al., 2004; Buss et al., 2007; Uroz et al., 2009). However, the unattached microorganisms

could also produce chelating agents, but there is a high possibility that these compounds dilute in

the solution and as a consequence their mineral leaching ability decreases in comparison to mineral

surface attached microorganisms (Madigan et al., 2000). Another explanation could be that the di-

rect attachment between the microbes and minerals could stimulate the genes that are involved in

the biosynthesis of the chelating agents and enzymes that promote high and fast dissolution of the

mineral. Regarding that point of view, we found that most of the attached microbial species pro-

duced a relatively higher siderophore concentration than the unattached microorganisms.

A variation in dissolution processes was found to be used by the attached microorganisms in com-

parison to unattached microorganisms (Figure 10) (Paper III). After 4 days, the Fe and Al dissolu-

tion was due mostly to the complexation process by most of the attached microbial species. After

10 days, some attached microbial species were mostly dependent on the complexation process and

some others were mostly dependent on the acidification process. After 16 days, the Fe and Al disso-

lution was due to the acidification process by most of the attached microorganisms. In contrast,

after 4 and 10 days, all the unattached microbial species depended on the complexation process.

After 16 days, three Fe and Al dissolution patterns for the unattached microbial species were dis-

tinguished: 1) some species depended on the acidification process, 2) some species depended on

the complexation process and 3) some species depended on both the acidification and complexa-

tion processes.

Some explanations for these findings are possible. The dependence of some microorganisms on the

acidification process could be related to the production of high amount of LMMOAs and/or respira-

tory CO2. It was previously known that both the respiratory CO2 and LMMOAs are the main sources

of acid pH during mineral weathering processes (Landeweert et al., 2001; Hoffland et al., 2004; Wu

et al., 2008). It was also suggested that there were some differences between the fungi and bacteria

with regards to their abilities in solution acidity. The acidity in the beginning of the weathering

process by fungal species could be relevant to LMMOAs, followed by respiration of the organic ani-

on to CO2 (Hoffland et al., 2004). However, the main source of acidity for bacteria could be relevant

to the production of LMMOAs rather than the respiratory CO2 (Wu et al., 2008). The dependence of

some microorganisms on the complexation process could be explained by the high production of

siderophores that would have a great impact on mineral dissolution. The impact of siderophores on

mineral weathering can be more effective compared to that of LMMOAs since siderophores form

1:1 complexes with Fe(III), with stability constants ranging between K=1022 and K=1052 (Jalal and

van der Helm, 1991; Matzanke, 1991), while the stability constants of some LMMOAs like oxalic and

citric acids with Fe(III) are K=107.6 and 1012.3, respectively (Perrin, 1979). On the other hand, when

some microorganisms depended on both the acidification and complexation in the biotite dissolu-

tion, we could assume that both LMMOAs/protons and siderophores were present in a relatively

similar quantity and they might function synergistically (Reichard et al., 2007; Cheah et al., 2003).

25

Fig

ure

10

. Te

rna

ry p

lots re

pre

sen

ting

the

rela

tive

con

tribu

tion

of co

mp

lex

atio

n a

nd

acid

ificatio

n p

roce

sses in

Fe

an

d A

l disso

lutio

n b

y

atta

che

d (A

) an

d u

na

ttach

ed

(B) m

icrob

ial sp

ecie

s du

ring

the

ex

pe

rime

nt (4

, 10

an

d 1

6 d

ay

s). Micro

bia

l spe

cies: E

rwin

ia a

my

lov

ora

(EA

), Pseu

do

mo

na

s stutze

ri (PS

), Pseu

do

mo

na

s men

do

cina

(PM

C), S

trep

tom

yces p

ilosu

s (SP

), Neu

rosp

ora

crassa

(NC

) an

d P

enicilliu

m

melin

ii (PM

).

26

It was also observed that most of the microorganisms depended on the complexation when the pH

was near to neutral but depended on the acidification process when the pH was acidic. This finding

could be explained by the proton promoted dissolution predominates at acidic pH and ligand pro-

moted dissolution predominates at weak acidic/neutral pH (Welch and Ullman, 1999; Balland et al.,

2010). Our finding was in good agreement with Duckworth et al. (2014), which confirmed the same

conclusion by conducting a series of dissolution experiments in the presence of siderophores over a

range of pH values. They found that at pH < 5 the dissolution rates were predominantly by a proton

promoted mechanism and that at pH > 5 the dissolution was predominantly by a ligand (sidero-

phore) promoted mechanism.

6. Overview of the potential roles and applications of siderophores

Investigating the roles and applications of siderophores in nature can lead to new ways of resolving

critical environmental questions (Paper IV). Siderophores play important roles in both terrestrial

and aquatic ecosystems. They are incorporated in Fe dissolution especially in soils that are en-

riched with insoluble Fe oxides (Hersman et al., 1995; Shirvani and Nourbakhsh, 2010). They are

also involved in the biogeochemical cycling of Fe in the ocean by enhancing the competition be-

tween marine bacteria and phytoplankton for Fe that affect the Fe abundance and solubility

(Hutchins and Bruland, 1998; Granger and Price, 1999; Amin et al., 2012).

Siderophores can function as plant growth promoters (Verma et al., 2011), biocontrol agents

(Schenk et al., 2012) and bioremediation agents (Ishimaru et al., 2012). Microbial siderophores can

provide the plants with Fe nutrition to enhance their growth when the bioavailability of Fe is low in

the soil (Crowley, 2006). Kloepper et al. (1980) were the first to show that different Pseudomonas

species improved plant growth by producing siderophores and protecting them from pathogens. In

addition, mycorrhizal fungi were recommended to be used as a biofertilizer because of their sider-

ophore production capabilities (Van Schöll et al., 2008). Siderophores have been also used as a bio-

control agent in which they worked as competitors for Fe in the soil that reduce the Fe availability

for the phytopathogens (Scher and Baker, 1982; Thomashow et al., 1990).

In addition, previous studies showed that the production of siderophores can be used in quick iden-

tification of microbes to the species level according to the siderophore types they produce and

called this application “siderotyping” (Meyer and Stintzi, 1998; Meyer et al., 2002). However, the

application of siderophores in microbial identification was limited to specific microbial species and

generally not recommended. Siderophores are extremely effective in solubilizing and increasing the

mobility of a wide range of metals such as Cd, Cu, Ni, Pb, Zn, and the actinides Th(IV), U(IV) and

Pu(IV) (Schalk et al., 2011). Thereby, siderophores become a useful tool in heavy metal bioremedia-

tion, which is a cost-effective and environment friendly technique (Rajkumar et al., 2010). Sidero-

phores could also participate in the biodegradation of petroleum hydrocarbons through an indirect

mechanism, by facilitating the Fe acquisition for the degraded microorganisms under Fe-limiting

conditions (Barbeau et al., 2002). In addition, siderophores have many other applications including

biocontrol of fish pathogens, nuclear fuel reprocessing, optical biosensor and bio-bleaching of

pulps, which were reported in details in paper (IV).

27

7. Conclusion and future prospective

A major challenge in biogeochemistry is to resolve the questions of how mineral influences micro-

bial activity in the natural environment. The present thesis focuses on resolving some key ques-

tions in a comprehensive manner. What is the impact of soil horizon and mineral type on sidero-

phore composition in soil? What siderophore types are produced by soil microorganisms? What is

the role of microbial surface attachment in mineral weathering reactions? What are the main roles

and potential applications of siderophores in nature?

Paper I

Our findings demonstrate that mineral type is one of the main factors affecting siderophore concen-

tration and distribution in soil. Each mineral type had a unique assemblage of dissolved and ad-

sorbed hydroxamate siderophores in the different soil horizons. The characteristics of soil horizons

strongly influenced the distribution of hydroxamate siderophores within the soil profile. Hydrox-

amate siderophores could be dissolved or adsorbed depending on their susceptibility to degrada-

tion. However, a wide range of hydroxamate siderophores, both fungal (ferrichromes, coprogens

and fusigen) and bacterial (ferrioxamines), were detected. To our knowledge, this the first time that

coprogens and fusigen were found to be the predominant hydroxamate siderophore types in soil.

Paper II

Our findings indicate that the composition of siderophore producing microorganisms significantly

varies within the horizons of a podzol soil profile. Siderophore production varied between the mi-

crobial species originating from different soil horizons. A wide range of fungal hydroxamate sider-

ophores (ferrichromes, coprogens, and fusigen) and bacterial hydroxamate siderophores (ferriox-

amine B, D, E and G) were produced by the isolated microorganisms and that may help them to

overcome the limits in Fe bioavailability and allow them to compete against other microorganisms

for key nutrients.

Paper III

Our findings demonstrate that the microbial surface attachment enhances the biotite weathering

reactions and both the attached and unattached microorganisms have a powerful ability in element

selectivity. Higher mobilization of elements, lower solution pH and relatively higher production of

siderophores were observed for attached microorganisms in comparison to unattached microor-

ganisms. The biotite dissolution was enhanced initially by ligand promoted dissolution processes

and later by proton promoted dissolution processes. Mineral composition and nutritional needs of

microorganisms were considered to be the main factors affecting the microbial attachment and

element selectivity by microorganisms.

Paper IV

Our literature review shows that siderophores represent central organic compounds for element

uptake by many microorganisms and plants in various environmental habitats. The important roles

and applications of siderophores such as mineral weathering, biogeochemical cycling of Fe, plant

growth promoting, biocontrol and bioremediation processes make them powerful tools in envi-

ronmental research.

28

Unresolved questions and future directions

Some questions remain open with regard to microbe-mineral interactions, in spite of current re-

search efforts. What parameters control the microbe-mineral interaction, and how can we include

those parameters in predictive biogeochemical models? What are the relative contributions of abi-

otic and biotic processes to mineral formation or dissolution in nature? How can traces of past mi-

crobe-mineral interactions be identified as biosignatures in ancient habitats? To what extent have

the functional genes of microbial species influenced the mechanisms involved in their interactions

with minerals? How can such interactions contribute to ocean chemistry and atmospheric composi-

tion?

In addition to these outstanding questions, other issues need further investigation, like the direct

and intimate relationship between the activities of microbial community, the geochemical signature

of their activity and how that signature may translate between environments. This investigation

will enhance the understanding of these current and past processes that drive our planet. As a fu-

ture study, an implementation of a model including the critical microbe-mineral interactions in

future climate assessments will help to overestimate or underestimate of the role those interac-

tions play in influencing global climate.

The present thesis shows how minerals control the microbial activity in soil. More in situ investiga-

tions are also needed for long-term microbe-mineral interactions in other terrestrial and aquatic

habitats. Such investigations will clarify new scientific perspectives that have yet to be known in a

comprehensive manner.

29

Acknowledgements

Good times go fast! I would like to take this opportunity to express my deep gratitude to my super-

visors, colleagues, family and friends who have helped and supported me during my PhD studies.

I would like to express my sincere gratitude to my supervisors, Sara Holmström, Nils Holm, Volker

Brüchert and Anders Andersson. Thank you, Sara, for offering me the opportunity to have this great

PhD journey in Stockholm, and for your encouragements and patient along the way. I will never

forget how much support and inspirations you offered me. Thank you Nils and Volker for your re-

sourceful ideas, constructive feedbacks and advices along my studies. Thank you, Anders, for giving

me the opportunity to work in SciLife lab, and providing me with valuable ideas and constructive

feedbacks.

I am very grateful for Luisa Hugerth and Jürg Brendan Logue for their helpful discussions. I also

want to thank Madelen Olofsson and Dan Bylund for taking part in the experiment setup and sam-

pling. Thanks Yue Hu for guiding me in SciLife lab. Thanks Chang Yin, Gustav Berggren and Britt-

Marie for giving me the opportunity to work in the Department of Biochemistry and Biophysics.

Many thanks to Jayne Rattray for her great assistance with HPLC-ESI-MS, Carina Johansson for her

assistance with Freeze dryer, Marianne Ahlbom for her assistant with ESEM, Clarisse Bolou-Bi for

her helpful discussions, Hildred Crill for her language editing and great advices and discussions

along the way.

I want to express my gratitude to Patrick Crill for his great support and encouragements and to the

geochemistry group for insightful discussions during the lunch seminars. Special thanks to Eve Ar-

nold for her support and nice meetings along the PhD way, to Barbara Kleine for her assistance

from my first day in Sweden, to my roommates Anna Neubeck and Kweku Yamoah for their nice

discussions, and to all former and present PhD students.

I am also grateful for the help from Monica Rosenblom, Margita Jensen, Elisabeth Däcker and Dan

Zetterberg with all of administrative work. Many thanks to all the former and present colleagues at

Department of Geological Sciences, Department of Materials and Environmental Chemistry, De-

partment of Biochemistry and Biophysics at Stockholm University, and School of Biotechnology at

the Royal Institute of Technology (KTH) for creating a warm working environment. Many thanks to

Cajsa Lithell and to all the publishers for giving us the permissions to use the forest picture in cover

image and the pictures in the methods diagram.

First and foremost, all praise is to ALLAH “GOD’’ as much as the heavens and earth and what is be-

tween or behind for all blessing and favors given me. With love and gratitude, I would like to thank

all my family and friends in Egypt and Sweden. The greatest thank you is deserved by my parents,

my lovely brother and my best friend Hekmat Morshedy for your endless love and always encour-

agements. I will never forget your great support along the way.

Funding from the Swedish Research Council for Environment, Agricultural Sciences and Spatial

Planning (FORMAS), Magnus Bergvall Foundation, Mineralogy foundation, Albert & Maria Berg-

ströms Foundation, Stiftelsen Elisabeth och Herman Rhodins Minne fund, John Söderbergs fund,

Liljevalchs travel grant, K & A Wallenberg Foundation, Geochemistry floor budget, Bert Bolin Cen-

tre for Climate Research and Stockholm University Astrobiology Centre are greatly acknowledged.

30

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