Diversity and Activity of Sulfate-reducing bacteria in Sulfidogenic Wastewater Treatment Reactors

Diversity and Activity of

Sulfate-reducing bacteria in Sulfidogenic

Diversity and Activity of

Sulfate-reducing bacteria in Sulfidogenic

Wastewater Treatment Reactors

Proefschrift

ter verkrijging van de graad van doctor

aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus Prof. dr. ir. J. T. Fokkema,

voorzitter van het College voor Promoties,

in het openbaar te verdedigen op vrijdag 19 oktober 2007 om

10.00 uur

door

Shabir Ahmad DAR

Master in Science of Bioprocess Technology, Asian

Institute of Technology (AIT), Thailand

geboren te Srinagar, J&K, India

Toegevoegd promotor

Dr. G. Muyzer

Samenstelling promotie commissie:

Rector Magnificus Voorzitter

Prof. dr. J.G. Kuenen Delft University of Technology, Promotor

Dr. G. Muyzer Delft University of Technology,

Toegevoegd promotor

Prof. dr. F. Widdel Max-Planck-Institute for Marine

Microbiology, Bremen, Germany

Prof. dr. ir. M.C.M. van Loosdrecht Delft University of Technology

Prof. dr. H.J. Laanbroek Utrecht University

Prof. dr. ir.A.J.M. Stams Wageningen University

Prof. dr. ir. P.N.L. Lens Wageningen University

This study was carried out in the Environmental Biotechnology group of the Department of Biotechnology at Delft University of Technology, Delft, the Netherlands.

This research was financially supported by The Netherlands Organization for Scientific Research – (NWO Earth and Life Sciences).

ISBN 978-90-9022271-4

Contents

Chapter 1 General Introduction 1

Chapter 2 Nested PCR-Denaturing gradient gel

electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities

37

Chapter 3 Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and

dsrAB genes as molecular markers

55

Chapter 4 Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single electron donor

85

Chapter 5 Competition and coexistence of sulfate

reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

107

Chapter 6 General Discussion 131

CHAPTER 1

1 Biological sulfur cycle

Sulfur is an essential element for the growth of many life forms (microorganisms, plants, animals), and their sulfur content typically varies between 0.1 and 1.5% of dry weight. The element sulfur occurs in a large variety of oxidation states (Table 1), oxidation state -2 (completely reduced) to oxidation state +6 (completely oxidized) (148). However, only three oxidation states are abundantly present in nature, i.e., -2 (sulfhydryl, R-SH and sulfide, HS-_{), 0 (elemental sulfur, S}0_{), and}

+6 (sulfate, SO42-). Most of the sulfur is found in sediments and rocks in

the form of sulfate (primarily gypsum, CaSO4) and sulfide minerals

(primarily pyrite, FeS2), although the oceans constitute the most

significant reservoir of sulfur for the biosphere (in the form of dissolved inorganic sulfate). Another large part is incorporated into the biomass as sulfur-containing compounds, such as cysteine and methionine.

Table 1 Oxidation states of sulphur in common compounds (166) Oxidation

state Compounds

-2 Dihydrogen sulfide H2S, hydrogen sulfide ion HS-_, sulfide ion S2-_{, as in FeS; thiocyanate SCN}

--1 Disulfane H2S2, disulfide S22- _{as in pyrite FeS2;} thiosulfate sulfane S1-_{; polysulfaides} –_S(S)nS

-0 Elemental sulfur Sn; organic polysulfanes R-Sn-R; polythionates -_O3S(S)nSO3

-+1 Dichlorodisulfane Cl-S-S-Cl

+2 Sulfur dichloride SCl2; sulfoxylate SO2 2-+3 Dithionate S2O4

2-+4 Sulfur dioxide SO2; sulfite SO32-_{; bisulfite HSO3}

-+5 Dithionate S2O62-_{; sulfonate RSO3}-_{; thiosulfate sulfone} SO3

-+6 Sulfur trioxide SO3; sulfate SO42-_{; peroxosulfate SO5}

2-These compounds are continuously converted into each other by a combination of biological, chemical and geochemical processes. The conversions of the inorganic sulfur compounds and to a lesser extent also those of the organic sulfur compounds are dominated by microbiological transformations. The biochemical oxidations and reductions of sulfur compounds constitute the biological sulfur cycle, which is schematically shown in Fig. 1.

Introduction

sulfur or sulfate. These conversions involve the metabolism of several different specific groups of bacteria and archaea.

SO

2-S

S

2-Assimilatory

sulfate reduction mineralization processes dissimilatory sulfate reduction biological oxidation with O2 or NO3 -biological oxidation with O2 anaerobic oxidation by phototrophic bacteria dissimilatory sulfur reduction biological oxidation with O2 or NO3- anaerobic oxidation by phototrophic bacteria chemical oxidation Organic sulphur compounds Sulfur deposits Sulfidic minerals (e.g. pyrites) Sulfate reserves (seawater)

Fig. 1 The sulphur cycle (142)

1.1 Dissimilatory oxidation of reduced sulfur compounds

Reduced sulfur compounds are used by many bacteria and some archaea to carry out dissimilatory sulfur oxidation. Winogradsky in 1887 suggested the name “Schwefelbacterien” (sulfur bacteria) for these bacteria. They include phototrophic green (Chlorobiaceae), and purple sulfur bacteria (Chromatiaceae and Ectothiorhodospiraceae) (54) and non-phototrophic colorless sulfur bacteria that belong either to the Proteobacteria (e.g. the genera Beggiatoa, Thiobacillus, Thiomicrospira,

Thioploca, Thiospira, Thiothrix and Thiovulum) or to the archaea

(Sulfolobus and Acidianus) (96, 97).

The anaerobic sulfur and thiosulfate oxidizers are represented by photosynthetic green and purple sulfur bacteria. These bacteria oxidize H2S by using it as source of reducing power in CO2 fixation.

Green sulfur bacteria are strict anaerobes that deposit the sulfur (So₎

they produce extracellularly. The purple sulfur bacteria, with the exception of Ectothiorhodospiraceae spp., deposit the sulfur

intracellularly (127). Some of the latter can grow as chemolithoautotrophs under microaerophilic conditions. Under H2S

limitation, they oxidize the sulfur further to sulfate.

Non-phototrophic colorless sulfur bacteria comprise aerobes as well as anaerobes. Among the bacterial aerobes, the most important groups comprise Thiobacillaceae and Beggiatoacea. These groups include obligate and facultative autotrophs as well as mixotrophs and heterotrophs. Other H2S oxidizers found in aquatic environments

include Thiovulum (autotrophic) (186), Achromatium, Thiothrix,

Thiobacterium (98) and Thiomicrospira (97). Many of these groups

produce intracellular and extracellular sulfur when oxidizing H2S. The

archaea, Sulfolobus spp. and Acidianus spp. are able to oxidize sulfur to sulfuric acid at temperatures between 55 and 85°C (17). These organisms are facultative autotrophs. Two examples of facultative, anaerobic sulfur oxidizing bacteria are Thiobacillus denitrificans (83) and Thermothrix thiopara (15). The former a mesophile and the latter a thermophile, use nitrate as terminal electron acceptor and reduce it to oxides of nitrogen and dinitrogen. It should be noted that many other bacterial groups such as Paracoccus and Hydrogenobacter species posses the capability to oxidize sulfur compounds.

1.2 Dissimilatory sulfate reduction and the key enzymes

involved.

The most oxidized form of sulfur is sulfate (SO42-). A broad range of

organisms, such as higher plants, algae, fungi and most prokaryotes can use sulfate as a sulfur source and carry out assimilatory sulfate-reduction. A variety of fermentative bacteria can use partially reduced sulfur. However, the ability to use sulfate as electron acceptor during the degradation of organic compounds is only restricted to the group of sulfate reducing prokaryotes. Even though an alternative microaerobic metabolism of some sulfate-reducing bacteria (SRB) was reported (43, 93), these bacteria can grow only under anoxic, reduced conditions (181).

Dissimilatory sulfate reduction, the focus of this thesis, is the central metabolic pathway that drives the global sulfur cycle. The SRB reduce sulfate by oxidizing hydrogen and various organic compounds and directing the electrons arising from the oxidation to the sulfate reducing system. Compounds such as sulfur or thiosulfate are used as alternative external electron acceptor, whereas sulfide is the end product. Typical substrates (electron donors and/or carbon sources) for SRB are lactate, ethanol, propionate and H2. Three enzymes are

Introduction

flavo-protein adenosine-5’-phosphosulfate (APS) reductase and the dissimilatory sulfite reductases.

The free sulfate anion (SO42-) is chemically inactive and not

easily reduced and thus the initial reaction in the reduction of sulfate is an activation step where ATP and sulfate form adenylyl sulfate (APS), and pyrophosphate (PPi). This reaction is catalyzed by ATP sulfurylase

and has been studied in several SRB belonging to the genera

Desulfovibrio and Desulfotomaculum (47). Although the equilibrium for

this reaction lies in the direction of ATP and sulfate (ΔG0’ _{= +46kJ/mol)}

(1), the reaction is pulled by the hydrolysis of pyrophosphate (PPi)

(ΔG0’ _{= -33 kJ/mol) by pyrophosphatase and the subsequent reduction}

of APS (ΔG0’ _{= -60 kJ/mol) formed.} 2 4 i

ATP SO

+

_←⎯

⎯⎯

_⎯

→

APS PP

+

ATP sulfurylase

2

PP H O

+

⎯⎯

→

P

Inorganic pyrophosphatase

2

APS H

₊

₊

e

⎯⎯

→

HSO

₊

AMP

←⎯

⎯

APS reductase

3

6

6

3

HSO

₊

H

₊

e

_⎯⎯

_→

HS

₊

H O

_{Sulfite reductase}

APS is the actual electron acceptor, which is converted to sulfite or bisulfite and AMP (Adenosine monophosphate). APS reduction is catalyzed by a reductase that has been purified and characterized from several sulfate reducers e.g. Desulfovibrio vulgaris (14), Desulfovibrio gigas (100) and Archaeoglobus fulgidus (99). All APS reductases are nonheme iron-sulfur flavoproteins composed of a large flavin subunit (70-80 kDa) and a small ferredoxin subunit (18-25 kDa). They together form heterodimeric structure (αβ). It has been proposed that the same enzyme activity catalyzes also the inverse reaction in a variety of sulfur-oxidizing bacteria (53).

The bisulfite arising from the reduction of APS is subsequently reduced to the end product, sulfide, through a pathway that is yet to be resolved fully. The reduction of bisulfite to sulfide by sulfite reductase involves the transfer of six electrons. Dissimilatory sulfite reductases have been isolated from many sulfate reducers and their classification has been based on the differences in their optical absorption spectra. Four major types are distinguished in SRB, the desulfoviridin, desulforubidin, P582 and desulfofuscidin. They generally have a α2β2

tetrameric subunit composition (32). However a third type of subunit (γ) has been observed in a desulfoviridin type of dissimilatory sulfite reductase in Desulfovibrio vulgaris (130). Desulfoviridin has been identified in virtually all Desulfovibrio species (132), most

Desulfonema species (57) and Desulfococcus multivorans (180).

Desulforubidin was identified in Desulfomicrobium species and

Desulfosarcina variabilis (6, 102)), whereas Desulfofuscidin was

purified and characterized from Thermodesulfobacterium commune (70). P582 was identified in the spore-forming Desulfotomaculum

nigrificans.

Another significant enzyme characteristic of SRB belonging to the genus Desulfovibrio, are the hydrogenases, which catalyze the oxidation of H2 coupled to the reduction of sulfate. Based on their metal

composition, three types of hydrogenases are distinguished in

Desulfovibrio species, the [Fe] hydrogenases, the [NiFe] hydrogenases

and the [NiFeSe] hydrogenases (48). All three types of hydrogenases have heterodimeric αβ-structures and are mostly located in the periplasm. Voordouw et al. (176) analyzed the distribution of the hydrogenase encoding genes in 22 different Desulfovibrio species and found that they are not uniformly distributed. While the genes for [NiFe] hydrogenase could be identified in all the tested strains, the distribution of [Fe] and [NiFeSe] hydrogenases was limited.

Genes encoding for different subunits of APS reductase, bisulfite reductases and hydrogenases have been cloned and sequenced. Since the genes encoding for these subunits have been found highly conserved across different lineages of SRB and archaea, they have been thus proposed as useful phylogenetic markers.

1.2.1 Carbon metabolism of sulfate-reducing bacteria

Previously, the SRB were thought to be of restricted metabolic and physiological diversity. This thought persisted until Widdel and Pfennig in 1977 (182) isolated Desulfotomaculum acetoxidans which oxidized acetate to CO2. Since then several studies, mostly on SRB isolated from

marine and freshwater sediments and sewage sludge, have indicated that these bacteria are more metabolically versatile than previously believed (65). A wide range of different compounds act as electron donors for dissimilatory sulfate reduction including short chain fatty acids, other carboxylic acids, alcohols, sugars, amino acids, aromatic compounds, methylated N- and S- compounds and some special inorganic electron donors other than hydrogen, such as phosphite (135, 180).

The metabolic capacities of SRB appear to fall into two categories- those species that are capable of complete oxidation of organic compounds to CO2, and those that carry out incomplete

oxidations, usually with acetate as end-product (180). This metabolic limitation of the latter group is due to the absence of a biochemical pathway for oxidation of acetyl-CoA to CO2 (65). Genera capable of

complete oxidation include Desulfobacter, Desulfobacterium,

Desulfococcus, Desulfosarcina, Desulfomonile, Desulfonema,

Introduction

Desulfomicrobium, Desulfobulbus, Desulfobotulus,

Thermodesulfobacterium and the species of the genera Desulfovibrio

and Desulfotomaculum (181).

Hydrogen and formate are an excellent energy source for the growth of many incompletely and completely oxidizing SRB (181). Besides acetate these are the major fermentation products in anaerobic environments. Thermodynamically, growth on hydrogen is more favorable than growth on acetate or other 2, 3 or 4 carbon-compounds (171). Growth of incomplete oxidizers, such as Desulfovibrio sp., on molecular hydrogen is usually much more rapid than that of complete oxidizers. Both autotrophic and heterotrophic growth on hydrogen and formate is possible. The incomplete oxidizers such as Desulfovibrio sp. require acetate as a source of carbon, whereas complete oxidizers such as Desulfobacterium sp. can use CO2 as the sole source of carbon (65,

181).

The ability of sulfate reducers to couple acetate oxidation to sulfate reduction is of particular interest, due to the importance of acetate as a major fermentation end-product during anaerobic degradation of organic matter. Organisms capable of significant growth on acetate belong to the genera Desulfobacter and Desulfotomaculum.

Desulfoarculus baarsii and the members of the genera Desulfococcus, Desulfosarcina, Desulfobacterium and Desulfonema oxidize acetate

slowly and sometimes without any substantial formation of cell mass (180). Desulfobacter species oxidize acetate via a modification of citric acid cycle. Acetate activation takes place via co-enzyme A transferred from succinyl CoA (65). In other completely oxidizing genera a non-cyclic pathway is used to oxidize acetate. The non-non-cyclic pathway involves cleavage of the two-carbon unit into a methyl and a carbon monoxide moiety, each of which is oxidized via independent pathways.

Propionate and butyrate are among the most important fermentation end-products in many ecosystems (60) and also a key intermediate in anaerobic digesters serves as electron donor and carbon source for the incompletely oxidizing Desulfobulbus species and several completely oxidizing sulfate-reducers. Propionate in Desulfobulbus is oxidized to acetate via a randomizing pathway with succinate as free intermediate (94). The oxidation of propionate to CO2 by complete

oxidizers like Desulfococcus multivorans also proceeds via the succinate pathway (167).

Lactate is one of the most widely used substrates for cultivating SRB. It is utilized by most species of almost each genus and may be oxidized completely or incompletely. Several species or genera, however are unable to grow on lactate, e.g., completely oxidizing

Desulfotomaculum and Desulfobacter species, Desulfoarculus baarsii, Desulfobacterium or Desulfonema (180). The oxidation of L- and

lactate is mediated by NAD(P)+- independent lactate dehydrogenases that are mainly membrane bound. Many SRB, including both the incompletely and completely oxidizing SRB can grow with ethanol as an energy source. Ethanol is oxidized via acetaldehyde to acetate, which may be further oxidized. Only a few strains of SRB, such as

Desulfococcus multivorans, appear to utilize secondary alcohols such as

2-propanol and 2-butanol (180). In addition, several types of SRB can grow by fermentation of some organic substrates in the absence of sulfate or other inorganic electron acceptors [for review see (31)].

1.2.2 Phylogeny of sulfate-reducing bacteria

The study of the microorganisms responsible for dissimilatory sulfate reduction was already initiated at the end of the 19th _{century with the}

pioneering work of the famous microbiologist Beijerinck. The first pure culture isolated was named Spirillum desulfuricans (10) and was described as strict anaerobe. Kluyver and van Niel (87) finally established the genera Desulfovibrio with D. desulfuricans as the type species for an organism hitherto classified as Spirillum desulfuricans by Beijerinck.

In 1925, Elion was the first to describe the thermophilic sulfate reducing bacterium Vibrio thermodesulfuricans (46). The capability of some SRB’s to form endospores was initially recognized for the thermophile Clostridium nigrificans (179) and Sporovibrio

desulfuricans (164). Later, Campbell et al. demonstrated that both

bacteria were members of the same species (21). The continuous accumulation of newly described SRB demanded thorough reclassification of all existing strains. As a result, all non-sporulating SRB were assigned to the vibrio-shaped genus Desulfovibrio (133), whereas the endospore-forming species formed the new sausage-shaped genus Desulfotomaculum (22). At that time it was thought that SRB comprise a small and nutritionally limited guild, growing preferentially on electron donors such as lactate and pyruvate that were incompletely oxidized to acetate. This view changed considerably with the description of new types of SRB capable of oxidizing acetate, higher fatty acids, or aromatic compounds (8, 9, 18, 128, 129, 180-184).

In addition, the novel genus Thermodesulfobacterium was established for thermophilic SRB which was isolated from hot aquatic habitats in the Yellowstone National Park (USA), contained unusual ether lipids, and was phylogenetically distinct from previously known SRB (101, 191). Studies on hyperthermophiles led to the description of the archaeal genus Archaeoglobus and demonstrated that the capacity for dissimilatory sulfate reduction is not restricted to the Bacteria.

Archaeoglobus fulgidus (165) and Archaeoglobus profundus (20),

Introduction

dissimilatory sulfate reducing members of the domain Archaea so far described.

Previously, phylogenetic relationships among SRB were inferred by more classical comparative techniques of phenotypic and biochemical properties such as nutrition, morphology, presence of desulfoviridin, lipid fatty acids or menaquinones. The discovery of ribosomal RNA (rRNA) as the ultimate universal molecular marker provided a basis for prokaryotic phylogeny and taxonomy (51). Together with the advent of nucleic acid sequencing, comparative 16S rRNA sequence analysis became decisive for the inference of natural relationships among prokaryotes. The application of comparative 16S rRNA sequence approach demonstrated that the delta subdivision of the phylum purple bacteria included bacteria with different phenotypes such as the SRB, sulfur-reducing bacteria, myxobacteria and bdellovibrios (124). Later all members of the phylum purple bacteria were reclassified into the new class Proteobacteria (159). A more comprehensive phylogenetic study of 20 nonspore-forming and two endospore-forming SRB based on comparison of nearly complete 16S rRNA sequences was performed by Devereux et al. (38). This study confirmed the classification of the genus Desulfotomaculum within the gram-positive bacteria as suggested previously (50).

Several reviews have summarized phylogenetic and taxonomic relationships among SRB (26, 41, 160, 181), wherein the SRB were classified mainly into four main groups: the mesophilic gram-negative, the thermophilic gram-negative, the thermophilic gram-positive SRB and the hyperthermophilic sulfate-reducing archaea. All of these groups were characterized by their use of sulfate as a terminal electron acceptor during anaerobic respiration. Assigning of individual species into appropriate groups based on 16S rRNA analysis was in agreement with those obtained by traditional taxonomy, although some exceptions did exist.

Comparative 16S rRNA sequence analyses have placed the mesophilic gram-negative species of SRB within the delta subclass of the Proteobacteria (50, 124). Two families were suggested for mesophilic Gram-negative SRB, the Desulfovibrionaceae and the

Desulfobacteriaceae (39, 181). The Desulfovibrionaceae family

includes the genera Desulfovibrio and Desulfomicrobium. The original

Desulfobacteriaceae family included all SRB within the

delta-proteobacteria that were not part of the Desulfovibrionaceae (39, 181). This rather broad definition included species of the genera

Desulfobulbus, Desulfobacter, Desulfobacterium, Desulfococcus, Desulfosarcina, Desulfomonile, Desulfonema, Desulfobotulus and Desulfoarculus. Several newly proposed genera may fall within the Desulfobacteriaceae on the basis of rRNA sequence analysis as

proposed by Castro et al. (26). With the increasing number of newly described SRB species the precise definition of the family

Desulfobacteriaceae is not possible. Especially Desulfobulbus and some

newly added genera, like Desulfocapsa and Desulfofustis, may represent a deeply branching group that constitutes a separate family.

Phylogenetically independent from the deltaproteobacterial SRB are the genera Thermodesulfobacterium (phylum

Thermodesulfobacteria) and Thermodesulfovibrio (phylum Nitrospirae)

which encompass the thermophilic gram-negative members of the SRB guild. Two most well-characterized species in this group of SRB are

Thermodesulfobacterium commune (191) and Thermodesulfovibrio yellowstonii (72). The Gram-positive group is dominated by the genus Desulfotomaculum, and is placed within low G+C Gram-positive

bacteria, which also contain Bacillus and Clostridium species (phylum Firmicutes). As mentioned earlier, the only sulfate-reducing archaea recognized to date are members of the genus Archaeoglobus (phylum

Euryarchaeota).

1.2.3 Ecology of Sulfate-reducing bacteria

The taxonomy and the picture of the physiology of SRB have undergone enormous changes in the last 20 years. Concomitant with these large changes in the understanding of taxonomy and physiology has been a proliferation of ecological studies of SRB. The increased interest in the ecological studies is firstly due to the central role played by SRB in the mineralization of organic matter in the anaerobic environments and secondly due to the concern of the role played by SRB in the pollution of various environments.

Four different trophic groups of bacteria are involved in the active decomposition of organic matter in anaerobic environments (Fig. 2). These include fermentative microorganisms that hydrolyze high molecular weight polymers and produce a variety of fermentative products i.e. alcohols, ketones and acids, the acetogenic bacteria that ferment organic acids and alcohols into acetate, H2 and CO2, the

methanogenic bacteria that utilize the end-products of all previous processes (H2, CO2, acetate, formate) to produce methane, the acetogens

that convert H2 and CO2 into acetate and the SRB which compete with

the methanogens and acetogens for available substrates. Methanogens and H2-utilizing acetogens dominate the sulfate-poor habitats such as

Introduction

for instance that sulfate reduction accounts for upto 50% of the total organic matter degradation in marine sediments (82). Because of their profound ecological importance in these systems, SRB in marine sediments were subject of many extensive studies (40, 88, 141, 150). The significance of SRB in marine environments can be understood from the variety of ecological processes that they are involved in. Anaerobic methane oxidation has been known for a long time, but it was only recently demonstrated that microbial aggregates composed of SRB and methane-oxidizing archaea catalyze this geobiologically important process (11, 36). Besides sediments stratified (salt) lakes, such as the Black Sea and the rhizosphere of marsh and sea grasses (73, 146) are important habitat for SRB’s. In addition, microhabitats in marine environments colonized by SRB include gutless marine oligochaete Olavius algarvensis (45) and the rhizosphere of marsh and sea grasses (73, 146).

Although SRB mainly inhabit anoxic zones they are also found in the oxic/anoxic interface of various environments. They were thought to be obligate anaerobes for a long time until several environmental studies showed their existence in high numbers in oxic environments (23, 92, 170). Despite a high respiration rate, it has been assumed that this process has only a protective function (33). In addition some SRB respond to oxygen exposure by simple migration to anoxic regions (156). Although all these protective mechanisms allow SRB to survive oxygen stress, substantial aerobic growth in pure culture has not yet been observed.

Sulfate reduction is generally assumed to be less important for the mineralization of organic carbon in freshwater sediments. This view needs to be changed in light of the studies that detected a highly diverse SRB population in freshwater lake sediments (81, 153). Some even suggest that dissimilatory sulfate reduction may contribute to more than 20% of the total anaerobic mineralization in freshwater environments (24, 76). Other freshwater habitats where the occurrence of SRB has been demonstrated are waterlogged paddy fields (154), ground water from aquifers (103), and wastewater treatment systems (16, 105, 140).

From an economic point of view, some SRB have the metabolic capacity to degrade environmental pollutants, such as oil (69). Such SRB can prove to be potential candidates for the use in bioremediation. In contrast, the high metabolic activity of SRB is undesirable in petroleum industry as they are the driving force for microbiologically-induced metal corrosion (144).

1.3 Anaerobic degradation of organic material

Anaerobic digestion has long been used successfully for the treatment of industrial and domestic wastewaters. It consists of a series of

microbiological processes that convert organic compounds to methane and CO2. The application of anaerobic wastewater treatment has

increased rapidly since the late 1970’s (52). This was made possible through a better understanding of the microbiology of this process and through improved reactor designs. Contrary to anaerobic wastewater treatment systems, conventional aerobic systems demand high energy inputs for aeration and large amounts of surplus sludge are produced during the purification process (158). A break-through in the application of anaerobic wastewater treatment systems came with the development of several new anaerobic reactor types with immobilized biomass, e.g. the Upflow Anaerobic Sludge Bed (UASB) reactor (106), the Fluidized Bed reactor (80) and the Anaerobic filter (190). The system with the widest application so far has been the UASB reactor. At present more than 1000 full-scale UASB reactors are in operation for the treatment of high strength industrial wastewaters (163).

Consortia of microorganisms, mostly bacteria and methanogens, are involved in the transformation of complex high-molecular weight organic compounds to methane. Although some fungi and protozoa (49) may be found in anaerobic digesters, bacteria and methanogens are undoubtedly the dominant microorganisms. Contrary to aerobic wastewater treatment, the anaerobic conversion of complex organic pollutants proceeds along a food chain (Fig. 2) in which interaction between several microorganisms are required to convert organic pollutants to the end product methane. Hydrolytic, fermentative acidogenic, acetogenic and methanogenic bacteria are involved in the transformation of complex materials into methane and CO2. These

microbial groups operate in a synergistic relationship (5). SRB compete with methanogens and acetogens for the available substrates in sulfate-rich environments.

1.3.1 Anaerobic treatment of sulfate-rich wastewaters

Introduction

alcohols etc.) Acetate H2, CO2 CH4, CO2 hydrolysis fermentation fermentation acetogenesis homoacetogenesis methanogenesis Complex Biopolymers (proteins, carbohydrates, lipids) Monomers (sugars, aminoacids) Acetate H2, CO2 HS-_{, CO} 2 hydrolysis fermentation fermentation acetogenesis homoacetogenesis sulfidogenesis Intermediary products (fatty acids, succinate,

alcohols etc.)

Fig. 2 Anaerobic degradation of organic compounds in the absence of sulfate (A) and in the presence of sulfate (B)

Hence the removal of higher concentrations of sulfate from wastewater is desirable and sometimes mandatory before it is released into the environment. The problems associated with anaerobic treatment of high sulfate wastewaters are primarily linked to the production of sulfide by the SRB. The major problems associated with anaerobic treatment of wastewaters containing high concentrations of sulfate (31) include:

• Reduced methane production due to effective competition between SRB and methanogens for the same substrates.

• Poor biogas quality due to the presence of sulfide which may also cause severe problems of corrosion. Besides hydrogen sulfide, even at concentrations of ≤ 2 ppm, is recognizable by its distinctive smell and may cause

significant malodour problems. This necessitates the need for H2S removal from the biogas.

• Potential toxicity of gaseous sulfide to many anaerobes resulting in reduced COD-removal efficiency.

• Accumulation of inert material in the sludge due to metal sulfide precipitation.

In most cases of anaerobic wastewater treatment, since carbon removal is the prime target, methane production is the preferred process. Problems caused due to the presence of sulfate ion in the wastewater had thus made sulfate reduction in conventional anaerobic bioreactors as an unwanted process. For many years, research on sulfate reduction was focused on its negative role in anaerobic wastewater treatment (143) and as such research efforts were directed towards strategies to suppress sulfate reduction (30, 169, 189) and to stimulate the competition between SRB and methanogens in the direction of methanogenesis.

However, compared to methanogens, SRB are very diverse in terms of their metabolic possibilities (181). The development of treatment processes using their capacity to degrade a wide range of organic compounds opens up new alternatives for environmental biotechnology. Sulfate reduction can be used together with sulfide removal techniques as a biotechnological method for the removal of sulfate (112, 168). The use of sulfide, generated during sulfate reduction, to form insoluble metal sulfide precipitates can be used for the treatment of heavy metal-contaminated wastewaters (67). In addition, the coupling of a sulfate reducing process to an aerobic sulfur producing process has opened the possibility to remove sulfate in the form of insoluble elemental sulfur. Therefore in the last decade, microbial sulfate reduction has drawn much attention as a remedial process of sulfate-rich wastewaters. In recent years, bioreactors have been designed specifically for sulfate removal (75). The aim in these reactors is maximal sulfate reduction coupled to a complete suppression of methanogenesis.

1.3.2 Competition of sulfate reducers with methanogens and

acetogens

Introduction

The main intermediates in the anaerobic mineralization of complex organic compounds are hydrogen, acetate, propionate and butyrate. SRB will directly compete for substrates like hydrogen and acetate with methanogens. Compounds like propionate and butyrate, which require syntrophic consortia in methanogenic environments, are degraded directly by single species of SRB in environments where sufficient sulfate is present (123). Even in the absence of sulfate certain SRB species are able to degrade propionate in syntrophic association with H2-consuming anaerobes (188). Thermodynamically (163), SRB

should be able to outcompete acetogens and methanogens (see Table 2) and thus would become a dominant process in anaerobic bioreactors treating sulfate-rich wastewaters.

Table 2 Acetogenic, methanogenic and sulfidogenic reactions involved in the degradation of organic matter in methanogenic and sulphate-reducing bioreactors (163)

Kinetic properties of sulfate reducers, methanogens and acetogens can be used to predict the outcome of the competition for these common substrates (95, 123). Although the thermodynamic and kinetic data

(163) of methanogens and sulfate reducers can partly explain the outcome of the competition, in practice many other factors, such as the substrate concentration, sulfide toxicity, pH, temperature, immobilisation of bacteria, substrate and product gradients in biofilms and granules etc may affect the competition outcome (78, 115, 142, 175).

Previous studies (68, 114) have also identified the influent chemical oxygen demand (COD)/sulfate ratio as one of the significant factors that influences the outcome of the competition between SRB and methanogens. This ratio determines which part of the organic material (based on grams COD) can be maximally degraded through sulfate reduction. For wastewaters with a COD/sulfate ratio of less than 0.67, theoretically all organic matter (COD) available can be degraded via sulfate reduction (143). For the ratios above 0.67, SRB will have to compete with methanogens and acetogens for the available substrate.

1.4 Sulfidogenic bioreactors

Since the beginning of the nineties, there has been an increasing interest in applying sulfate reduction for the treatment of specific wastewaters, generated by many industrial processes that use sulfuric acid or sulfate-rich feed stocks e.g. wastewaters from edible oil industry, paper mills, potato starch factories or the sulfate-rich wastewaters generated as a result of acid mine drainage, flue gas scrubbing or metal smelting etc. It is a cost-effective alternative for costly and complex physico-chemical sulfate removal methods (111).

Introduction

An alternative to passive treatment of sulfate and metal-rich wastewaters is an active system based on new bioreactor configurations, which offers more control over the process and its performance. In these bioreactors wastewater purification is mainly accomplished by SRB, which convert sulfate to sulfide. Numerous sulfidogenic reactor design studies have been reported including anaerobic filters (27), completely mixed reactors (110), anaerobic packed bed reactors (112), gas-lift reactors (44, 173) UASB (19) and anaerobic baffled reactors (63). For wastewater that contains no or insufficient amounts of electron donor and carbon source for a complete reduction of sulfate, addition of an appropriate electron donor is required. Several electron donors have been proposed for sulfidogenic bioreactors, including lactate, methanol, and ethanol, mixture of volatile fatty acids and hydrogen or complex organic substrates like molasses (84, 110, 172). Simple organic compounds (ethanol, methanol) or synthesis gas (a mixture of H2, CO

and CO2) are preferred to more complex organic substrates (e.g.

molasses) as they are easier to dose and can be utilized rapidly and completely. The major sulfidogenic bioreactor technologies applied at full-scale are the Biosulfide® _{and the THIOPAQ}® _{processes. The}

Biosulfide® _{technology is marketed by Bioteq (Vancouver, B.C.,}

Canada) and THIOPAQ® _{by Paques B.V. (Balk, Netherlands). The}

Biosulfide process (147) is divided into two stages: a biological stage and a chemical precipitation stage. The biological stage is a reagent-generating system that produces dissolved and gaseous sulfide and alkalinity as a consequence of sulfate reduction. In the chemical stage, which is separated from the biological stage, the reagents are used to precipitate the metal pollutants and increase the alkalinity. Hydrogen is used as electron donor in the bioreactors.

The Paques Thiopaq-process for sulfate removal consists of two biological processes, which take place in separate bioreactors. First, the sulfate is reduced to sulfide under anaerobic conditions in the sulfidogenic bioreactor; subsequently the sulfide produced is oxidized to sulfur in a second sulfur oxidizing reactor. The sulfur produced can be recycled for the production of sulfuric acid, or fertilizers. Due to increased alkalinity during the conversion of sulfide to sulfur, influent neutralization can be achieved by recirculation of this stream eliminating the need to add large amounts of alkaline chemicals. Several full-scale sulfate removal plants are currently in operation, for instance at the synthetic fiber production plant of Akzo Nobel (Emmen, The Netherlands) (75). Paques has also installed a groundwater treatment system, based on combined sulfate reduction and sulfide oxidation and metal precipitation for the Zinifex Budel Company in the Netherlands to remove sulfate, zinc and cadmium (13). The Thiopaq process for metal

and sulfate removal has also been successfully used for flue-gas desulfurization.

1.5 Characterization of microbial populations in anaerobic

bioreactors

The major microbial processes that take place in anaerobic bioreactors, such as methanogenesis, sulfidogenesis and acetogenesis, are nowadays well understood. However, our knowledge of the diversity and dynamics of the microbial communities responsible for these processes is still limited. This is because microbial communities in large-scale biotechnological processes, such as wastewater treatment facilities, are often treated as a “black box” (71). This is not due to the underestimation of the significance of the biological component but is due to the limitations of traditional identification and enumeration techniques, such as selective enrichment, pure culture isolation and most probable number estimates. It has frequently been reported that direct microscopic counts exceed viable cell counts by several orders of magnitude; the majority of microscopically visualized cells are viable but do not form viable colonies on plates (3). It has been estimated that >99% of microorganisms observable in nature typically are not cultivated by standard techniques (74). These problems are even more exacerbated in studies of anaerobes; because of their low growth rates and obligate anaerobiosis, SRB and methanogens are among the microorganisms that are most difficult to study by culture-based techniques. Fortunately, molecular techniques have provided alternative approaches to overcome the problems associated with culture dependent analysis of complex microbial communities (3). These allow the identification and quantification of the most contributing populations in the ecosystem and thus establish a link between microbial structure and function. The individual molecular methods which found widespread application in studies on natural SRB communities include membrane lipid analysis, Immunodetection, Denaturing gradient gel electrophoresis (DGGE), Fluorescence in situ hybridization, DNA microarrays etc. We have summarized a few of these methods in the next few paragraphs. The benefits and potential drawbacks of these methods have already been reviewed in detail elsewhere (3, 55, 119).

1.5.1 Denaturing gradient gel electrophoresis

Introduction

decreased electrophoretic mobility of a partially melted DNA fragment in polyacrylamide gels containing a linearly increasing gradient of DNA denaturants (119). Besides general biases that are characteristic of all molecular analytical methods based on nucleic acid extraction and PCR-amplification (12, 113, 131), a DGGE-specific drawback is that only short PCR fragments of up to 500 bp can be well separated which limits phylogenetic information retrieved after sequencing of the individual bands. Analyses of complex SRB communities by DGGE mainly used 16S rRNA genes as target molecules (85, 89, 152, 170). The DGGE of 16S rRNA gene has been successfully used in detecting SRB in diverse habitats, such as hydrocarbon-contaminated aquifers (85), hypersaline microbial mats (174), marine sediments (178, 185), deep sub-surface environment (90), anaerobic digesters (149) etc. However, in contrast to phylogenetically and functionally homogeneous bacterial groups, the polyphyletic origin of SRP does not allow the design of a single 16S rRNA-targeted primer pair that is specific for all SRP. 16S rRNA targeted primer sequences specific for SRB subgroups have been designed (35), which can be used in a PCR-DGGE protocol to specifically target different groups of SRB.

The use of functional genes that encode for enzymes has given a new direction in molecular microbial ecology that allows establishing a link between the presence of a microbial population and the specific metabolic process. Generally these genes have more sequence variation than the relatively conserved 16S rRNA encoding genes, and might, therefore, be better molecular markers to discriminate between closely related but ecologically different populations (125). The gene encoding for the large subunit of [NiFe] hydrogenase, an enzyme which plays an important role in the hydrogen metabolism of Desulfovibrio species, has been used specifically to monitor the metabolically active populations of Desulfovibrio spp. in environmental samples (177). However, the limited distribution of this gene among the SRB has limited its use to the study of Desulfovibrio species only.

Dissimilatory sulfite reductase (dsrAB) encodes α and β subunits of an enzyme that catalyses the six-electron reduction of sulfite to sulfide (161). Due to a remarkably high degree of conservation observed in

dsrAB across SRB and archaea (117), it is a potential candidate for

phylogenetic studies of these organisms. The dsrAB gene-based molecular approach has been used to discriminate among SRB in diverse environments (25, 42, 56, 121, 126). Recently, Geets and coworkers described DGGE of PCR-amplified dsrB gene fragments to specifically follow population dynamics of SRB (59). However, one downside of the dsrAB approach is that these genes appear to have been subject to several lateral gene transfer events. This is indicated by partly inconsistent phylogenetic tree topologies of SRB pure cultures when the

taxonomy is based on 16S rRNA or DsrAB sequence analysis (86), respectively. This fact hampers exact identification of environmental sequences that are not closely related to known SRB lineages.

1.5.2 Hybridization with rRNA-targeted oligonucleotide

probes

a) Fluorescence

in situ hybridization (FISH)

Use of rRNA-targeted oligonucleotide probes has become very important in microbial ecological studies including studies focusing on identification and abundance of SRB (4, 11, 109). These probes allow the direct identification and enumeration of microbial populations in complex environments (62). The suitability of fluorescently labeled rRNA-targeted oligonucleotide probes for cultivation independent identification of microorganisms was reported for the first time by DeLong et al.., (37). Since then, this technique has become the method of choice for reliable and rapid identification of microorganisms in environmental samples (2). The essence of FISH is that, using fluorescently labeled probes, it allows the specific visualization of morphologically intact organisms (hence the name whole-cell hybridization) directly in their natural environment. FISH not only provides insight into microbial community structure, but absolute and/or relative numbers of visualized cells can also be determined (34, 61). FISH has been used in combination with techniques such as micro sensors (136) or microautoradiography (79) to elucidate the ecophysiology of identified SRB.

A first application of FISH for SRB community analysis identified Desulfovibrio vulgaris related bacteria in sulfidogenic biofilms established in anaerobic bioreactors (4). Since then the use of FISH for the identification of SRB in different environments has been steadily growing. Manz et al. (109) monitored the abundance and spatial organization of single SRB populations in activated sludge. FISH was used to investigate the response of SRB to oxygen stress under oligotrophic conditions in particle-free systems (7). Santegoeds et al. (151) combined the microsensor measurements for H2S and CH4 with

FISH to study the population structure and activity in anaerobic aggregates from different reactors. Other habitats include estuarine sediments (134), activated sludge (118), wastewater biofilms grown under microaerophilic conditions (122) etc.

b)

Quantitative membrane hybridization

Introduction

amount of rRNA measured by a universal probe (137, 138). Despite biases due to different rRNA operon copy numbers in different species, quantitative dot-/slot-blot hybridization has been extensively used in different sulfate reducing environments such as cyanobacterial mats (116), sediments (141) and anaerobic biofilms (139).

1.5.3 DNA microarrays

DNA microarrays are the DNA phylochips that consist of up to thousands of diagnostic nucleic acid sequences (referred to as probes) attached to a solid support (usually a glass slide) in an arrayed order. Probes can be oligonucleotides or PCR amplificates. The labeled nucleic acid sequences, usually PCR amplificates from the unknown sample (referred to as target), is revealed after hybridization to the microarray. Various studies have shown that DNA microarrays hold much promise in the determinative studies in environmental microbiology (28, 29, 64, 89, 157, 187). Recently Loy et al. (108) designed an oligonucleotide microarray for 16S rRNA gene based detection for SRP. The potential applicability of the developed microarray was applied for identification of SRB in different environmental samples (107).

1.6 Scope and outline of the thesis

Wastewater treatment is generally performed with mixed microbial populations that work synergistically for the removal of organic and inorganic pollutants. Anaerobic, aerobic or combined anaerobic-aerobic processes are applied under controlled conditions in different bioreactor designs. There are thus two aspects to the wastewater treatment; technological and microbiological. Technological aspects include the design of a proper reactor and improvement of reactor operation, whereas microbiological aspects include the physiological and ecological properties of the microbial communities present in the bioreactors. Insights into both the technological and microbiological aspects are required for stable and efficient functioning of the bioreactors. The major microbiological processes that take place in these bioreactors have been extensively studied (104, 105, 139, 143, 162, 163), which resulted in the development of improved reactors. However, little or no information on phylogenetic and functional composition of the microbial communities responsible for these processes is available.

In order to better understand the stable and efficient functioning of anaerobic reactor in general and the sulfidogenic bioreactors in particular, the research presented in this thesis focused on the microbial and ecological aspects of these reactors. The specific aims were to determine the diversity and also to identify the active populations of

SRB in the anaerobic reactors with particular focus on sulfidogenic wastewater treatment reactors. Many ecological studies in natural environments have indicated an enormous diversity of closely related coexisting SRB populations. In light of such studies, the aim was also to investigate the coexistence of metabolically related SRB in engineered environments and the significance of this coexistence on the performance of sulfate reduction process.

Various studies have indicated the significant effect of electron donor to sulfate ratios on the competition for electrons between different anaerobic communities. Although the effect on the major processes like sulfidogenesis and methanogenesis is well studied, the effect on the diversity and dynamics of the microbial communities itself has been largely ignored. Hence, this project also aimed at investigating the effect of changing substrate to sulfate ratio on the microbial population structure and function with particular focus on acetogens, SRB and methane producing archaeal populations.

The molecular detection of microorganisms by DGGE may become difficult if they are present in low numbers. The DGGE of 16S rRNA gene fragment obtained with general bacterial primers mainly detects the major constituents of the analyzed community overlooking the less abundant, but potentially important species. Chapter 2 illustrates a novel strategy of nested-PCR DGGE approach using primers targeting 16S rRNA genes of different groups of SRB, to overcome the difficulty in detecting low numbers of SRB in complex microbial communities. The use of SRB group-specific primers in a three step nested amplification strategy ensured the specific amplification of SRB, thereby increasing not only the specificity but also the sensitivity of detection of SRB in mixed microbial communities containing SRB in low number.

Most microbial ecology studies have focused on the diversity of microorganisms. However, more important for the cycling of chemical elements, such as sulfur, are the microorganisms that are active.

Chapter 3 compared the structure and function of sulfate-reducing

bacterial communities in different lab- and full-scale sulfidogenic wastewater treatment reactors by targeting both the DNA and RNA of the two different molecular markers, i.e. 16S rRNA and dsrB gene fragments.

Introduction

bioreactor treating sulfate rich wastewater using a combination of cultivation and molecular techniques.

In order to get more insight into the effect of the lactate/sulfate ratio on the structure and function of a microbial population maintained in a lab-scale anaerobic continuously stirred tank reactor (CSTR),

Chapter 5 investigated the competition and coexistence among

different trophic groups of bacteria as affected by changing substrate to sulfate ratio. The focus was laid on the interaction between dominant microbial communities of SRB, acetogens and Archaea. DGGE and FISH together with chemical analysis was used to link the microbial population dynamics with changes in the lactate/sulfate ratio.

In Chapter 6, a general discussion summarizing the results and conclusions is presented.

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