Chapter-1 Introduction & Literature...

Chapter-1

Introduction &Literature survey

1Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey

SULFUR

Sulfur is one of the most common and dominant elements present

on the Earth crust and plays an important role in essential life

processes. However, its involvement in biological processes is limited to a

series of highly specific compounds. All living organisms contain around

1% of the dry weight composed of sulfur. Sulfur is a major constituent of

proteins (which contain the S-containing amino acids like cysteine and

methionine). It is also an important constituent of coenzymes (e.g.,

coenzyme A, biotin, thiamine), metalloproteins, and also in bridging

ligands (molecules that bind to proteins, for example, in cytochrome c

oxidase) (Sievert, et. al., 2007). Sulfur is the 10th most abundant element

on the Earth, which is present in nature, mainly as pyrite (FeS2) or

gypsum (CaSO4) in rocks and sediments and as sulfate in seawater.

Sulfur exists in various oxidation states, from –2 (completely reduced) to

+6 (completely oxidized), and can be transformed both chemically and

biologically and hence the sulfur cycle (Fig. 1.1) is complex (Muyzer and

Stams, 2008).

In the sulfur cycle, plants, animals and other microorganisms

absorb or take up sulfur in the form of sulfate. The sulfur is again

released into the atmosphere as hydrogen sulfide due to the

decomposition of dead organisms in the absence of oxygen. Sulfur

dioxide is released in the environment through the combustion of fossil


fuels and emission of volcanic fumes, which when reacts with water,

forms sulfuric acid that results in acid rain. Microorganisms, especially

the sulfate reducing bacteria play an important role in the recycling of

these sulfur compounds.

1.1. Role of microorganisms in sulfur transformations:

Microorganisms play an important part in sulfur transformations

(Fig. 1.2). Sulfate is taken up as a nutrient and reduced to sulfide. The

sulfide so produced is utilized in the synthesis of amino acids like

methionine and cystine and also in the synthesis of sulfur containing

Figure 1.1. The sulfur cycle(Adopted from Muyzer and Stam, 2008)


enzymes. In this process of sulfur transformations, chemolithotropic

sulfur bacteria and sulfate reducing bacteria become important

participants in the oxidation and reduction reactions for the generation

of metabolic energy through sulfide oxidation and dissimilatory sulfate

reduction (Muyzer and Stams, 2008). SRB in particular have a key role in

this S-cycle. They use sulfate, the highly oxidized form of sulfur (SO42–)

as the terminal electron acceptor during the catabolism of organic matter

as a result of which, hydrogen sulfide (H2S) is produced.

Chemolithotrophic sulfur-oxidizing bacteria are capable of oxidizing the

so formed sulfide, either aerobically (Thiobacillus or Beggiatoa spp.) or

anaerobically (Chlorobium spp.) to elemental sulfur (S°) and SO42. Many

different groups of bacteria take part in other transformations of sulfur

like sulfur reduction (Desulfuromonas spp.) and sulfur disproportionation

(Desulfovibrio sulfodismutans). Several other groups of microorganisms

are capable of reducing compounds like dimethylsulfoxide (DMSO) to

dimethylsulfide (DMS) and vice versa (Sievert et. al., 2007).


1.2. PATHWAYS OF SULFATE REDUCTION AND BIOLOGICALPRODUCTION OF HYDROGEN SULFIDE (H2S):Sulfur is an essential nutrient for all life forms. The metabolism of

organic sulfur compounds is a key component of the sulfur cycle. Plants,

fungi, and many bacteria reduce inorganic sulfate to sulfide to cover

their need for the element. Before the sulfur can be assimilated into

biosynthetic pathways it needs to be reduced to hydrogen sulfide. Sulfur

compounds act as either electron acceptors in processes known as

sulfate/sulfur reduction or as electron donors in processes known as

and sulfur oxidation. The process of sulfur/sulfate oxidation can occur

Figure 1.2. Sulfur transformations(Adopted from Muyzer and Stam, 2008)


aerobically as well as anaerobically. The molecules of oxygen or nitrate

act as electron acceptors in aerobic oxidation and anaerobic oxidation

occurs in anoxygenic anaerobic photosynthesis. Unlike this, the sulfate

reduction occurs only under strictly anaerobic conditions. These

reactions play an important role in anoxic and sulfidic water columns

and also in the formation of microbial mats (Koblizek et. al., 2006).

Hydrogen sulfide, a product of sulfate reduction, is an extremely

active chemical. This gas produces an offensive odor, like that of rotten

egg and also foul taste if present in water (Dunnette et. al., 1985). The

nuisance associated with H2S includes its corrosive nature towards

metals like iron, copper, brass and steel. H2S is a flammable and

poisonous gas and is usually not a health risk unless the concentrations

are very high, which rarely occur in natural environments (Treleaven,

1980). There are two biological routes of production of H2S in significant

quantities in aquatic or terrestrial environments. 1) The microbial

reduction through sulfate reducing bacteria (SRB) mediated by the

reduction of higher oxidation state inorganic sulfur, i.e., sulfate to

sulfide which is evolved as H2S. Here, in the anaerobic utilization of

organic compounds under respiratory conditions, the sulfate serves as

the terminal electron acceptor. 2) However, even in anaerobic

breakdown of proteinaceous matter which is termed as putrefaction the

H2S is also produced as a product of microbially-mediated reactions

(Dunette et. al., 1985).


1.2.1. Production of H2S by putrefying bacteria throughAssimilatory Sulfate Reduction (ASR):Most organisms utilize sulfate as a sole sulfur source. They

reduce it to H2S intra-cellularly to incorporate sulfur in to the S-

containing amino acids like cysteine which then gets combined in to

proteins (Killham, 1994, Brüser et. al., 2000). The reduction of sulfate to sulfide

becomes necessary in the absence of external reduced sulfur compounds that can

be readily incorporated or assimilated. Certain bacteria may lack sulfate reduction

as they thrive in habitats rich in reduced sulfur compounds and hence sulfide for

cellular assimilation of sulfur is not done by these eg. Chlorobiaceae (Brüser et. al.,

2000). The pathway of assimilatory sulfate reduction is a pathway which plays an

important role in the synthesis of organic sulfur compounds by contributing the

necessary sulfur to the cell. It is regulated in such a way that under normal

conditions, release of sulfide from the cells does not take place (Zehnder and Zinder,

1980). The sulphate assimilation pathway was first resolved in the

enteric bacteria Escherichia coli and Salmonella typhimurium using

mutants auxotrophic for different sulphur compounds (Jones-Mortimer,

1968 and Kredich, 1971). Since then, a number of bacterial strains have

been identified to produce H2S in process of assimilation of various

oxidized form of sulfur to sulfide (Table 1.1)


1.2.1.1. Assimilatory Sulfate Reduction (ASR) pathway:

Assimilatory sulfate reduction is a pathway in which inorganic

sulfate is converted to sulfide. This pathway finds its place in many fungi

and also in plants. The sulfide is incorporated into the vital cellular

components, the proteins containing amino acids like cysteine or

homocysteine (Brunold, 1993). The first step in this pathway is the

Names of bacterial genera producing H2S Reference

Actinobacillus, Actinomyces, Actinoplanes, Agrobacterium,Aeromonas, Alysiella, Arachnia, Bacteroides, Brucella,Butyrivibrio, Campylobacter, Cardiobacterium, Citrobacter,Clostridium, Edwardsiella, Erwinia, Erysipelothrix,Eubacterium, Flavobacterium, Francisella, Flexibacteria,Francisella, Fusobacterium, Halobacterium, Halococcus,Helicobacter, Hypomicrobium, Klebsiella, Megasphaera,Mycoplasma, Neisseria, Pasteurella, Peptococcus,Peptostreptococcus, Planobispora, Proteus, Rhodospirillum,Rothia, Salmonella, Selenomonas, Simonsiella, Spirillum,Staphylococcus, Streptobacillus, streptoverticillium,Treponema, Thermomonospora, Veillonella, Yersinia,Xanthomonas, Zymomonas

Sobsey and Pfaender,2008

Azotobacter, Bacillus, Bacterionema, Budvicia, Kingella,Lactobacillus, Pseudomonas, Rhizobium

Barrett and Clark,1987

Escherichia coli, Shigella Treleaven et. al., 1980

Prevotella, Streptococcus Washio et. al., 2005

Haemophilus Sottnek et. al., 1980

Leminorella Hickman-Brenner et.al., 1985

Streptomycetes Kluster and Williams,1964

Leptospira Fiehn, 1986Enterobacter Closs, 1971

Corynebacterium, Propionibacterium Moss, et. al.,, 1967

Table: 1.1. Bacterial genera producing H2S duringassimilatory sulfate reduction


activation of sulfate to adenosine 5’-phosphosulfate (APS). This reaction is

catalyzed by the enzyme sulfate adenylyltransferase or ATP sulfyrylase. In

higher plants, APS is directly reduced to sulfite by the enzyme APS

reductase (APR). The APS so formed is subsequently reduced to sulfide by

sulfite reductase which is then converted by the enzyme O-acetylserine

(thiol)lyase into O-acetylserine (Fig.1. 3.) (Neumann et. al., 2000 and

Kopriva et. al., 2002). For the next step to occur in this pathway, i.e.,

reduction of PAPS by PAPS reductase, the APS needs to be

phosphorylated, which is catalyzed by the enzyme APS kinase to form o

phosphoadenosine-5'-phosphosulfate (PAPS). This step is generally

observed in fungi and some enteric bacteria, like Salmonella typhimurium

and Escherichia coli (Suter et. al., 2000). In contrast to this step, in

certain other bacteria such as Allochromatium vinosum (Neumann et. al.,

2000) and in plants, without phosphorylation to form an intermediate

PAPS, sulfite is directly formed through reduction of APS.


These facultative anaerobic bacteria which are involved in

nitrification, putrefaction etc. are quite common in sewage, sludge and

other anaerobic environments that are rich in organic matter. They also

constitute a part of human and other animal colon. The common

putrefying bacteria involved in such environments include Coliforms,

Streptococci, Clostridia, Lactobacilli, Micrococci, Proteus and Pseudomonas.

Figure: 1.3. Pathway of Assimilatory sulfatereduction in Escherichia coli(Adopted from Kopriva et. al., 2002)


















Most of these bacteria are the causative agents of diseases like gastritis,

typhoid, cholera and food poisoning in humans (Bhawsar, 2011).

1.2.2. Production of H2S by Sulfate Reducing Bacteria throughDissimilatory Sulfate Reduction (DSR):Sulfur is used by many Archaeal and bacterial members in the

most important energy yielding reactions through a metabolic pathway

called dissimilatory sulfur metabolism. These processes are essential for

the cycling of sulfur on our planet. In short, pathways of sulfate

reduction for the purpose of energy production are called pathways of

dissimilatory sulfate reduction. These dissimilatory processes occur in

strictly anaerobic environments where the sulfur or sulfate acts as the

terminal electron acceptor in the electron transport system where H2S is

released in large amounts as the end product.

2CH2O + SO42-+ H+ 2CO2+ HS-+ 2H2O

Dissimilatory sulfate-reducing prokaryotes are a heterogenous

group of Bacteria which include members of the phyla Proteobacteria

(Beeder et. al., 1995), Nitrospirae (Henry et. al., 1994), Firmicutes (Daumas

et. al., 1988), and Archaea consisting the phylum Archaeoglobi (Dahl and

Truper, 2001) all members can use sulfate as a terminal electron

acceptor. All Sulfate Reducing Prokaryotes are characterized by their use

of sulfate as a terminal electron acceptor during anaerobic respiration.


1.2.2.1. Dissimilatory Sulfate Reduction (DSR) Pathway:

Sulfate is unique among microbial electron acceptors in that

it must be activated before it can be reduced. The enzyme adenosine

triphosphate (ATP) sulfurylase activates sulfate. The attachment of the

sulfate to a phosphate of ATP is catalyzed by the enzyme ATP sulfurylase,

leading to the formation of adenosine 5'-phosphosulfate (APS). Although

ATP is hydrolyzed, the reaction is energy requiring, and must be pulled

to completion by the removal of the end products. Pyrophosphatase

catalyzes the reaction where Pyrophosphate (PPi) is hydrolyzed to

phosphate (Sekowska, 2000) (Fig. 1.4).

In dissimilatory sulfate reduction, the sulfate moiety of APS is

reduced directly to sulfite (SO32-) by the enzyme APS reductase with the

release of adenosine monophosphate (AMP). In assimilatory reduction,

Figure: 1.4. Pathways of dissimilatory sulfate reduction(Adopted from Fauque, 1991 and Widdel, 1992).


another phosphate group is added to APS to form phosphoadenosine 5'-

phosphosulfate (PAPS), and only then is the sulfate moiety reduced to

sulfite with the release of phosphoadenosine 5'-phosphate (PAP). The

sulfite is reduced to H2S through a pathway that has not been resolved

to date (Postgate, 1984). The reduction may occur directly to sulfide or

via intermediates, trithionate and thiosulfate. In the dissimilatory

sulfate reduction, H2S is excreted into the environment whereas in the

assimilative reduction, the H2S formed is immediately converted into

organic sulfur compounds, such as amino acids (Hansen, 1994).

1.3. SULFATE REDUCING BACTERIA (SRB)

Sulfate Reducing Bacteria (SRB) is a large heterogeneous group of

prokaryotic microorganisms, both Bacteria and Archaea that use

sulfate as the terminal electron acceptor, in the energy metabolism

(Mori et. al., 2003), i.e., these are capable of dissimilatory sulfate

reduction as mentioned above. The end product of dissimilatory sulfate

reduction is hydrogen sulfide (hereafter referred to as sulfide), which

unlike the inert sulfate molecule, is very reactive, foul smelling, and

generally highly toxic to especially eukaryotic and also prokaryotic

organisms. The sulfide so formed reacts in presence of iron minerals

forming black precipitates of ferrous sulphide, which makes the

recognition of SRB habitats easy (Rabus et al., 2006).


1.3.1. Distribution of SRB in various habitats:

Sulfate-reducing Bacteria (SRB) are more generally preferred to

be called as Sulfate-Reducing Prokaryotes (SRP) (Fauque, 1995)

because of Archeal members being involved in this physiologically

grouped microorganisms. In fact, SRP are widespread in nature and

are mainly found in sulphate-rich anoxic habitats such as soil, marine

and fresh waters and sediments as well as in the oxic-anoxic interfaces

of all these biotopes and in the gut of many animals, including

humans. The members of SRP have been successfully isolated from

extreme barophilic, thermophilic, psychrophilic and halophilic

environments (Odom and Singleton, 1993). They have been identified

as responsible for the bio-geo-chemical nutrient cycles, biocorrossion,

food spoilage etc (Fig. 1.5).

Figure 1.5. Interactions of sulfate reducing bacteria(Adopted from Barton et. al., 2007)
































1.3.2. Brief history of Sulfate reducing bacteria:

The first person to observe and report SRB was the Dutch

microbiologist, Martinus Beijerinck in 1895. He described the first SRB

as Spirillum desulfuricans based on its spiral shape and its property to

reduce sulfur. He isolated from a Dutch City canal and described it as

the cause of contamination of the city sewage in the summer due to the

production of hydrogen sulfide. He also observed that Spirillum

desulfuricans was difficult to grow in absence of aerobic bacteria, that

consumed the oxygen in the culture medium and hence the bacterium

was classified as strict anaerobe (Voordouw, 1995 & Muyzer, 2008). In

1930, Baars studied that Spirillum desulfuricans was capable to oxidize

lactate and ethanol to acetate, when the name Vibrio was used to

represent this strain. Finally, it was renamed as Desulfovibrio

desulfuricans by Kluyver and Van Neil (1936). Desulfovibrio was the only

recognised Gram-negative SRP genus until the 1970s (Postgate, 1984).

1.3.3. Physiology of Sulfate reducing bacteria:

The physiology of SRB is influenced by various aspects like the

physic-chemical conditions, chemotaxis, dissimilation of organic and

inorganic compounds and assimilatory pathways. The three key

physiological aspects of SRB are reduction of sulfate, reduction of

nitrate, nitrite and metabolism of electron donors and the pigments

involved in dissimilatory sulfate reduction.


1.3.4. Reduction of Sulfate:

As discussed in the previous section, all SRB gain energy

through the dissimilatory sulfate reduction process where sulfate is

reduced to sulfide, coupled with the oxidation of H2 or other organic

substrates. Apart from sulfate, many other electron acceptors like:

sulfite, thiosulfate, sulfur, nitrate, elemental Fe (III), CO2 and fumerate

are also used by several other SRB. Sulfate is transported to the

cytoplasm initially by an ion-gradient. The uptake may be either

simultaneous along with protons (Cypionka, 1989) or with sodium ions

(Kreke and Cypionka, 1993). The reduction of sulfate occurs in the

cytoplasm near the inner side of the cytoplasmic membrane by an

overall reaction that involves eight electrons and requires ATP (Rudolf,

2007):

SO42- + ATP + 8H+ + 8e- HS- + AMP + PPi

The activation of inert sulfate, the enzymes involved in the dissimilatory

sulfate reduction occur as discussed in section (1.2.2) SRB are the only

organisms known to carry out inorganic sulfur fermentation and all

reactions of the sulfur cycle a few of which are shown in Table. 1.2.


1.3.5. Reduction of nitrate and nitrite:

The nitrate or nitrite reduction by SRB was first demonstrated in

washed cells or cell extracts of different Desulfovibrio species from a

lactate-sulfate culture (Barton et. al., 1983). Later nitrate has been

reported to be reduced to ammonia (with nitrite as intermediate) by a few

Desulfovibrio species. Eg. D. desulfuricans, D. furfuralis, D. profundus, D.

termitidis, D. gigas. (Dalsgaard and Bak, 1994). The dissimilatory nitrate

or nitrite reduction also called as ammonification can function as sole

energy-conserving process in some SRB (Fauque and Ollivier, 2004).

Nitrate is reduced to ammonia in a dissimilatory pathway that involves

the nitrate reductase and nitrite reductase enzymes. The first step is the

Table 1.2. Various oxidation reduction reactions of sulfurcarried out by sulfate reducing bacteria.


conversion of nitrate to nitrite, performed by the enzyme nitrate

reductase (Dias et. al., 1999).

1.3.6. Metabolism of electron donors:

The range of electron donors used by SRB is broad. The electron

donors which are used by most of the SRB are compounds like fatty acid,

alcohols and H2, i.e., products of fermentation of organic compounds

(Widdel, 1988). SRB have been differentiated into two groups based on

their oxidative metabolism. Those SRB which are not capable of oxidizing

acetyl-coenzyme A and produce incompletely oxidized product of organic

compound i.e., acetate are termed “incomplete oxidizers” (Rabus et. al.,

2000). These include species belonging to Desulfovibrio,

Desulfomicrobium, Desulfobotulous, Desulfofustis, Desulfotomaculum,

Desulfomanile, Desulfobacula, Desulfobulbus, Desulforhopalus,

Archaeoglobus and Thermodesulfobactereium (Madigan, 2006). On the

other hand some SRB oxidize organic compounds completely to produce

CO2 as they possess various types of enzymes catalyzing pathways of

complete oxidation of Acetyl coenzyme-A. These are called “complete

oxidizers” (Rabus et. al., 2000). Species of Desulfobacter,

Desulfobacterium, Desulfococcus, Desulfonema, Desulfosarcina,

Desulfoarculus, Desulfacinum, Desulforhabdus and

Thermodesulforhabdus are complete oxidizers. (Madigan, 2006).

The list of compounds oxidized by SRB is ever increasing with the

recent studies in this regard. SRB capable of oxidizing Phosphite (HPO32-)


to phosphate (Desulfotignum phosphitoxidans) (Schink, 2002), metallic

iron (FeO) to ferrous iron (Desulfobacterium corrodens) (Dinh et. al., 2004)

have been reported. Some strains of SRB have been reported to utilize

glucose and fructose (Goorissen et. al., 2003 and Rabus, et. al., 2000),

Archaeoglobus fulgidis strain 7324 was demonstrated to grow on starch

(Trinkler et. al., 1990). Many sulfate reducers have been described over

the past two decades that have the ability to grow on various different

substrates, including sugars (Ollivier et. al., 1988 and Sass et al., 2002),

amino acids (Baena et. al., 1998 and Stams et. al., 1985) and one-carbon

compounds, such as methanol (Nanninga et. al., 1987 and Nazina et. al.,

1987), carbon monoxide (Parshina et. al., 2005 and Henstra et. al., 2007)

and methanethiol (Tanimoto and Bak, 1994). Apart from benzoate and

phenol, other aromatic hydrocarbons (for example, toluene and

thylbenzene) are also degraded by a number of SRB (Harms et. al., 1999

and Morasch et. al., 2004). Recently, SRB that can grow on long- chain

alkanes (Cravo-Laureau et. al., 2004), alkenes (Grossi et. al., 2007) and

short-chain lkanes (Kniemeyer et. al., 2007) have also been described.

1.3.7. Pigments involved in dissimilatory sulfate reduction:

The reduction of sulfite to sulfide is an intermediate step of

sulfate reduction in the sulfate assimilatory and dissimilatory pathways.

Both the assimilatory and dissimilatory sulfite reductase catalyses the

six-electron reduction of sulfite to sulfide (Dzierzewicz et. al., 1993). A

variety of sulfite reductases have been purified from SRB: Desulfoviridin


(Postgate, 1956; Lee and Peck, 1971), Desulforubidin (Lee et. al., 1973),

P-582 (Trudinger, 1970) and Desulfofuscidin (Zeikus, 1983).

These sulfite reductases are called dissimilatory sulfite

reductases. These are identical with respect to tetrahydroporphyrin

structure, but they differ with respect to the presence of iron atoms in

porphyrin groups. The chromophore of desulforubidin, desulfofuscidin

and P-582 contains an iron atom siroheme [SIR(Fe)], whereas a metal-

free siroheme called sirohydrochlorin [SIR] is the chromophore of

desulfoviridin (Dzierzewicz et. al., 1993).

The desulfoviridin pigment was isolated from the extracts of

Desulfovibrio vulgaris by Postgate (1956). This pigment decomposes

under alkaline conditions to yield red fluorescent chromophore group

when exposed to ultraviolet light at 365 nm. This characteristic property

of fluorescing under ultraviolet light is used as a diagnostic test for

identification of Desulfovibrio species as all species of Desulfovibrio

possess this pigment desulfoviridin except the mutant, Desulfovibrio

desulfuricans Norway 4, isolated by Miller and Saleh (1964).

1.3.8. Fate of SRB: From ecological nuisance organisms tobiotechnologically potential bacteria:SRB have been recognized to be associated with microbial

corrosion which occurs in many industrial systems as most of the

industrial processes use sulfuric acid. The early work on SRB focused

on the role of SRB as nuisance organisms as these have been reported

to cause deterioration of steel. They are undesirable especially in petro-


chemical industries, agro-industrial waste water systems, drinking

water distribution systems and gas-industries. (Muyzer and Stams,

2008).

In recent research, SRB have also been applied in beneficial

aspets. SRB find their application in removal of heavy metals from waste

waters and ground waters. This is based on the property of various metal

sulfates (Cd, Co, Zn, Fe, Ni, Cu) which are highly soluble but their

corresponding metal sulfides are less soluble. The H2S produced through

dissimilatory sulfate reduction by SRB reacts with metal sulfates to form

insoluble sulfides and thus precipitated (Kaufmann et. al., 1996 and

Koschorreck et. al., 2007).

SO42– + 8 [H] + H+ → HS– + 4 H2O

HS– + Me2+ → MeS↓ + OH–

HS– + 0.5 O2 → S° ↓ + OH–

SRB are also applied for the removal and reuse of sulfur

compounds from off gases and waste water. This is based on the

principle that under oxygen limiting conditions, SRB produce elemental

sulfur instead of sulfate through oxidizing sulfide (Buisman et. al., 1990).

Recent findings have shown that the highly explosive nitrocellulose when

treated with enrichment cultures of SRB (Freedman et. al., 2002) and

with Desulfovibrio desulfuricans strain 1388 (Tarasova et. al., 2004) had

undergone transformation in the structure making the explosive

nitrocellulose less reactive. Bioremediation of metals or metalloids is an


application for which SRB are particularly suitable. Bio remediation of

metal processing wastes, acid mine-drainage waters are the most

successfully applied aspect of SRB. (Hockin and Gadd, 2007).

1.4. PHYLOGENETIC DIVERSITY OF SRB:

Various techniques have been used to detect and study the

diversity of SRB. The initial methods included cultivation based

techniques but as only a small percentage of bacteria (less than 1%) can

be cultured, methods based on other physiological or genetic markers

were searched for. In this regard, analysis of phospholipid fatty acids

was used over a period of time (Parkers, 1987).

1.4.1. Phylogeny of SRB based on 16S rRNA gene:

The most commonly used marker gene used for the identification

of SRB was the gene that codes for the 16S ribosomal RNA. Based on the

analysis of 16S rRNA gene, SRB have can be grouped into seven

phylogenetic lineages with five bacterial and two archaeal lineages (Fig

1.8.). The lineages include (Thauer et. al., 2007 and Muyzer and Stams,

2008):

1) Delta proteobacteria with 23 genera covering maximum

species of SRB (Mori et. al., 2003).

2) Gram positive SRB under Clostridia (Desulfotomaculum,

Desulfosporosinus and Desulfosporomusa) (Mori et. al.,

2003).

3) Nitrospirae with SRB under the genus

Thermodesulfovibrio (Mori et. al., 2003).


4) Thermodesulfobacteriaceae with SRB under the genus

Thermodesulfobacterium (Mori et. al., 2003).

5) Thermodesulfobiaceae with SRB under the genus

Thermodesulfobium (Castro et. al., 2000).

6) SRB under archaeal lineage Archaeoglobus in

Euryarchaeota (Castro et. al., 2000).

7) SRB under the genera Thermocladium and Caldivira in

Crenarchaeota (Castro et. al., 2000).

Untill July 2011, there are 200 validly published names of SRB

belonging to both archeal and bacterial phyla. This information is based

on the data collected from the website: List of Prokaryotic names with

standing in Nomenclature (http://www.bacterio.cict.fr).

Taxa Validly published names of SRBunder different phyla

Archea Bacteria Total

Phyla 2 4 6

Class 2 4 6

Order 2 9 11

Family 2 15 17

Genus 2 52 54

Species 11 179 200

Table 1.3. Number of validly published names ofsulfate reducing bacterial taxa

(Data source (http://www.bacterio.cict.fr) till July 2011)


Figure 1.6. Distribution of sulfate reducing bacteria underdifferent families and orders of the DomainBacteria

Figure 1.7. Distribution of sulfate reducing bacteria underdifferent families and orders of the DomainArchaea


Figure 1.8. Phylogenetic tree based on nearly complete16S ribosomal RNA (rRNA) sequences ofdescribed sulfate-reducing bacterialspecies.

(Adopted from Muyzer and Stam, 2008)


1.4.2. Phylogeny of SRB based on dsrAB gene:

Since the first report of SRB by Beijerinck in 1895 as Spirillum

desulfuricans which was later renamed as Desulfovibrio desulfuricans,

there are only about 188 validly reported names of SRB contributing to

the cultured diversity, which shows that a vast unexplored diversity of

SRB could inhabit the ecologically diverse habitats. The difficulty in

isolation and culturing of SRB limit their description through culturable

methods. Hence recent research is more focused on understanding the

natural populations and native diversity of SRB using molecular

approaches through the identification of marker functional genes specific

for SRB, like the dsrAB or ApsA (Stahl et. al., 2007). Moreover, the

culture-based methods recover merely a fraction of the natural

population. Additionally, for the SRP, the isolation of axenic cultures

from environmental samples is not straight forward and requires a lot of

time and effort. To overcome such difficulties, molecular based culture-

independent methods were introduced through which the native SRB

populations present in a wide range of ecosystems were studied, which

helped in understanding many different and diverse types of SRB. Hence,

this shortcoming has forced microbial ecologists to search for alternative

phylogenetic marker genes that are more specific for SRP. Consequently,

subsequent research focused on genes encoding enzymes which are part

of the unique pathway for dissimilatory reduction of sulfate and are thus

common and more specific to all SRP (Barton and Hamilton, 2007).


FISH (fluorescence in situ hybridization) method targeting

the 16S rRNA oligonucleotide SRB specific probes were

used to detect SRB in a variety of environments

(Ravenschlag, 2000).

Genes encoding important enzymes in the sulfur cycle

have also been used to detect sulfate-reducing bacteria in

different environments (Wawer and Muyzer, 1995).

Phylogenetic sub groups of SRB were detected using

specially designed, highly specific PCR primers for the

16S rRNA gene (Daly, 2000).

DNA microarray suitable for SRB diversity analysis has

been developed and applied to detect SRB in complex

environmental samples (Loy, et. al., 2001).

Presence and distribution of different groups of SRB was

also detected using the molecular method of DGGE,

(Denaturing gradient gel electrophoresis) of PCR amplified

DNA fragments (Dar, 2005).

In all recognized SRP, the dissimilatory sulfate reduction process is

mediated by three key enzymes. The chemically inert sulfate is activated

to adenosine-5’-phosphosulfate (APS) by ATP sulfurylase (Sat), which is

then converted to AMP and sulfite by the enzyme APS reductase (Apr).

The sulfite so formed is finally reduced to Sulfide by the enzyme

dissimilatory sulfite reductase (DSR) (Meyer and Kuever, 2001). The DSR


enzyme is coded by the dsrAB genes. Due to its crucial functional role,

ubiquitous presence in all SRP, a high degree of conservation and

appropriate matching phylogenetic topology with 16S rRNA, dsrAB gene

sequences have been used as suitable molecular markers for SRB

(Zverlov et. al., 2005). The dsrA and dsrB genes are only known to be

located adjacent to each other and present in one copy in genomes of

SRP and can therefore be PCR amplified with a single primer pair. The

generally used dsrAB specific PCR primer pair (DSR1F and DSR4R, Table

2) was designed by Wagner et. al., (1998) and amplifies an approximately

1900 bp fragment of the dsrAB genes. However, as more dsr operon

sequences recently have become available it is now evident that it is not

practically possible to design single primers specific for the dsrAB genes

of all SRP (Zverlov et. al., 2005). Therefore a mix of different DSR1F and

DSR4R primer variants must be used for the PCR amplification of dsrAB

gene fragments to ensure maximum coverage (Table 1.4.).

As unfortunately, dsrAB sequence information is only available for

a few closely related species which hampers the interpretation of novelty

of species based on the comparative phylogenetic distances between the

16S rRNA and dsrAB gene sequences, using a 90% sequence similarity

threshold value for grouping dsrAB sequences into OTU probably would

be a choice value if wanting to discriminate at the species level (Zverlov

et. al., 2005).


1.4.3. Genetic diversity analysis of Sulfate reducing bacteria usingPyrosequencing:The research on microbial diversity from environmentally

important niches is difficult to estimate and culture based methods have

not been successful in identifying these hidden populations of bacteria.

Recent estimates of existence of around 40 million bacterial species in

one gram of soil have dragged much attention in developing methods

that reveal a maximum percentage of this uncultured diversity. Such

Organism dsrA forward primerbinding site (5´- 3´)

Perfectmatchprimera

dsrB reverse primerbinding site (5´- 3´)

Perfectmatchprimerb

Desulfovibrio desulfuricans(AJ249777)

ACCCATTGGAAACACG DSR1Fa TGCGGAAACTGCTACAC DSR4Rc

Desulfovibrio vulgaris(AE017285)

ACCCACTGGAAGCACG DSR1F TGCGGTAACTGCTACAC DSR4R

Bilophila wadsworthia(AF269147)

ACGCACTGGAAGCACG DSR1F TGCGGTAACTGCTACAC DSR4R

Desulfobactervibrioformis(AJ250472)

ACCCACTGGAAACACG DSR1Fa TGCGGTAACTGTTACAC DSR4Rb

Desulfobacula toluolica(AJ457136)

ACCCATTGGAAACATG DSR1Fc GTGGTAACTGCTACAC DSR4Rd

Desulfobulbusrhabdoformis (AJ250473)

ACCCATTGGAAACACG DSR1Fa TGCGGTAACTGCTACAC DSR4R

Desulfotaleapsychrophila(NC006138)

ACTCACTGGAAGCACG _ TGTGGTAACTGTTACAC _

Thermodesulforhabdusnorvegica (AJ277293)

GCCACTGGAAGCACG DSR1Fb TGCGGAAACTGCTACAC DSR4Rc

Desulfotomaculumthermocisternum(AF074396)

ACCCACTGGAAACACG DSR1Fa TGCGGCAACTGCTACAC DSR4Rc

Archaeoglobusfulgidus(NC000917)

ACGCACTGGAAGCACG DSR1F TGCGGTAACTGCTACAC DSR4R

Archaeoglobus profundus(AF071499)

ACGCACTGGAAGCACG DSR1F GTGGAAACTGTTACAC DSR4Ra

Table 1.4. dsrAB gene primer binding sites in completely sequenced dsrABgenes (and dsrAB gene targeting primers).(Adopted from Wanger et. al., 1998 and Zverlov et. al., 2005).


estimates in ecologically important habitats are of great interest as these

samples, like soils, sediments etc. are considered to harbor the most

diverse populations of bacteria than any other environment on earth

(Roesch et. al., 2007). Molecular methodologies developed over the past

decade now enable researchers to examine the diversity of SRB

independent of cultural methods. The use of rapid sequencing

technologies combined with molecular methods is becoming a gold

standard for evaluating the microbiomes of ecologically important niches

(Liu et. al., 2007 and Wolcott, 2008).

The proprietary technology, Pyrosequencing, for automated DNA

sequence-based analysis is based on ‘sequence-by-synthesis’, a method

which originates from breakthrough research undertaken at the Royal

Institute of Technology (Stockholm, Sweden). The generation of light

plays a key role in how the technology works. Four enzymes; DNA

polymerase, ATP sulfurylase, luciferase and apyrase play the key role in

‘sequence-by-synthesis’ based pyrosequencing. The initial sample

consists of single stranded DNA of unknown sequence with a short

annealed primer. The enzymes are automatically dispensed into the

sample well and the 4 nucleotides are added in a defined order, for

example A,G,T,C. These 4 enzymes are formulated in such a way that

light is produced whenever an added nucleotide (A, T, G or C) forms a

base pair and is incorporated into the growing DNA strand. When a base

pair is formed, the polymerase incorporates the nucleotide into the


strand which releases pyrophosphate (PPi). The PPi so released is

immediately converted to energy source ATP by the enzyme ATP

sulfurylase. The energy so produced is used by the enzyme luciferase to

generate light, which is detected by a camera under the sample well. If

any of the nucleotides is not incorporated in to the growing strand, it is

immediately lysed by the enzyme apyrase. By repeatedly adding A,G,T

and C and recording which produce light, the correct sequence of bases

in the DNA template is built up (Winge, 2000).

Figure 1.9. The Principle, steps and enzymes involved in theprocess of pyrosequencing.






















The most recent research on studying the native populations of

bacteria independent of cultivation techniques have used a combination

of methodologies like the above discussed pyrosequencing with novel tag

bacterial diversity amplification methods. The first culture-independent

estimation of bacterial species present in 1 gram of soil revealed the

presence of around 10000 species which were identified based on the

DNA:DNA hybridization of all bacterial genomes extracted from the

sample (Torsvik et. al., 1990). Later, by employing 454 pyrosequencing,

amplification of the hypervariable V9 region of highly conserved 16S

rRNA gene was achieved which could detect 25000 16S rRNA gene

fragment sequences from each of 4 soil samples tested (Roesch et. al.,

2007). A little later, the diversity of bacteria present in the cattle feces

was estimated by using 16S rDNA bacterial tag-encoded FLX amplicons

pyrosequencing (bTEFAP), where special fusion primer are tagged to the

initial primers like, Linker A-Tags-530F and Linker B-Tags-1100R and

9.6 million DNA capture beads are added to capture maximum number

of DNAs present the sample. By using this method, several thousand

sequences per sample were analyzed (Dowd et. al., 2008).

In the present study, the bTEFAP method was outsourced from the

Research and testing laboratories, Lubbock, TX. The bacterial diversity

and species richness harboring a few randomly selected samples was

analyzed from the results. The diversity of sulfate reducing and other H2S

producing bacterial species was calculated.


1.5. SULFATE REDUCING BACTERIA IN POLLUTION CONTROL:

Sulfur conversions involve the metabolism of several different

specific groups of bacteria, like, SRB, phototrophic sulfur bacteria and

thiobacilli specialized to use these sulfur compounds in various redox

states (Lens and Kueven, 2001). As many of these microorganisms

possess many unique metabolic and ecophysiological features with

respect to microbial conversions of sulfur cycle, they can be successfully

implemented in pollution control technologies (Lens et. al., 2007).

Technological utilization of SRB was thought to be controversial

for several years after their discovery, as sulfate reduction has been

considered an unwanted process in industrial and water treatment.

From 1990 onwards, interest has grown in applying SRB for treatment of

specific waste streams like inorganic sulfate-rich waste waters, acid mine

drainage, metal polluted ground water and flue-gas scrubbing waters.

Now-a-days, sulfur cycle based technologies are applied for pollution

prevention, metal or water recovery and re-use (Lens et. al., 2007).

In recent years, many genera of SRB have been recognized, which

are nutritionally far more diverse than previously perceived. Electron

donors participating in sulfate reduction include hydrogen, a variety of

alcohols, fatty acids, mono- and dicarboxylic acids, amino acids, sugars,

phenyl substituted acids, homocyclic aromatic compounds, long chain

saturated alkanes (Widdel and Bak, 1992). Many other compounds

which are involved in anaerobic metabolism can be linked with the


consumption of sulfate as terminal electron acceptors are also

metabolized by the SRB.

The almost ubiquitous distribution of SRB and the vast

information on their wide applicability in anaerobic transformations

emphasizes their use in treatment of environmental pollutants. As many

environments are anaerobic or become anaerobic rapidly due to

contamination with pollutants, and as they will also harbor sulfate or

other sulfur rich compounds, these environments become ideal niches

for the growth and activity of SRB (Ensley and Suflita, 1995).

1.5.1. Aromatic sulfur compounds as environmentalcontaminants:Aromatic compounds form the second largest group of

organic compounds in nature after carbohydrates (Shinoda et. al.,

2005). Hazardous aromatic compounds get into the environment in the

form of diverse detergents with oil spills, sewage from petroleum

refineries and chemical plants, and with municipal waste waters.

Aerobic processes in waste treatments plants may not completely

remove aromatic compounds from waste waters, turning researchers'

interest to the study of the anaerobic metabolism of these compounds

(Harwood et. al., 1999). Various kinds of compounds like lignin

monomers, quinones, amino acids, flavonoids which are released into

the environment serve as good substrates for many microorganisms. As

a major part of natural environment has little or no access to


atmospheric oxygen, anaerobic microbes hold a major role in the

processing of the nutrient cycles in nature.

Table: 1.5. Chemical structure and industrial use of aromatic sulfurcompounds para-toluene sulfonic acid (PTSA), Sulfanilic acid(SFA) and Thiophene-2-acetic acid (TPA)


1.5.2. Para-toluene sulfonic acid (PTSA), sulfanilic acid (SFA) andthiophene-2-acetic acid (TPA) as environmental pollutants:Several aliphatic, glycol, aryl sulfonates and aromatic sulfur

compounds occur naturally and their appearance in rivers and water

bodies indicates a need for their biodegradation (Cain, 1981). Synthetic

detergents, sulfonated dye stuffs, fabric brighteners and their precursors

are generally released from textile and dye industries that contribute to

the pollution of water bodies with aromatic sulfur compounds through

S. No Compound Acute effects Chronic effects

1 Ρ-toluenesulfonic acid

Severe skin, nose andeye irritation andburns. Can irritate thethroat causingburning, dryness andcoughing.

Irritate the lungs.Repeated exposuremay cause bronchitisto develop with cough,phlegm, and/orshortness of breath.

2 Sulfanilic acid Skin irritant , eyeirritant, inflammatory

Toxic to blood, cancause target organdamage on prolongexposure

3 Thiophene Very hazardous in caseof ingestion.Hazardous in case ofskin contact (irritant,permeator), of eyecontact (irritant), ofinhalation.

Toxic to blood,kidneys, the nervoussystem, liver, mucousmembranes. Repeatedor prolonged exposureto the substance canproduce target organsdamage.

Table 1.6. Health hazards caused to human beings by the aromatic sulfurpollutants para-toluene sulfonic acid (PTSA), Sulfanilic acid

(SFA) and Thiophene-2-acetic acid (TPA)


the effluents from these industries (Meyer, 1981 and Bretscher, 1978).

Degradative product of azo dyes are sulphonated aromatic amines, which

can be divided into two main groups, linear alkyl benzene sulphonic acid

(Jimenez et. al., 1991; Mumpel et. al., 1998) and the sulphonated

aromatic compounds. The latter group comprises 2-aminobenzene

sulphonic acid, 3-aminobenzene sulphonic acid, 4-aminobenzene

sulphonic acid (4-ABS or sulphanilate) and 4-toluene sulphonic acid

(Tan & Field, 2000)Also the, anthropogenic sources such as petroleum

spillage or refuse disposal sites account for contamination of soil and

water by compounds like benzene, toluene, ethylbenzene and xylene

(U.S. Public Health Service, 1989).

p-Toluenesulphonate (PTSA), which is mainly used as a

hydrotropic agent in detergent formulations, has served as a model

compound in studies of the biodegradation of alkylated

benzenesulphonates and was found to be degraded via 4-methylcatechol

(Focht & Williams, 1970). The aerobic degradation pathway of toluene

was demonstrated in Pseudomonas testosterone (Locher et. al., 1989) way

back in 1989 (Fig. 1.10), while there is no clear pathway elucidated for

the degradation of the same compound under anaerobic conditions till

today. The degradation of toluene with sulfate as electron acceptor was

demonstrated in enrichment cultures of river sediment (Beller et. al.,

1992).


Desulfobacula tolulica strain Tol2 was the first reported SRB which

could degrade toluene under strict anaerobic conditions. The complete

anaerobic oxidation, i.e., degradation of toluene to CO2 was

demonstrated in this strain by testing the hypothetical intermediates of

toluene degradation in growth experiments (Rabus et. al., 1993).

Sulfanilic acid (p-amino-benzenesulfonate) is an important

representative compound of aromatic sulfonated amines. It is widely

used as an intermediate in production of azo-dyes, plant protectives and

pharmaceuticals (Magony, 2004). Other important applications of

Figure: 1.10. The degradative pathway of Para toluene sulfonic acid inPseudomonas testosteroni.

(Adopted from Locher et. al.,., 1989). The numbers represent: (1), p-toluenesulphonate (2), p-sulphobenzyl alcohol (3), p-sulphobenzaldehyde (4), p-sulphobenzoate (5), theoretical intermediate (6), protocatechuate (7), 4-carboxy-2-hydroxymuconate semialdehyde and (8), meta-cleavage pathway.


sulfanilic acid are discussed in table 1.5. The effluents from textile and

dyeing and printing industries release this compound in larger quantities

which poses the problem of the compound being recalcitrant and

mutagen. The ill effects of this compound on human beings when they

become pollutants in water or soil are listed in table 1.6. A 40%

degradation of sulfanilic acid under aerobic conditions by fungal strains

Phanerocheate chrysosporium (Paszezynski et. al., 1992) and Aspergillus

niger RH19 (Faryal, 2006) were demonstrated. Sphingomonas subartica

strain was demonstrated to utilize sulfanilic acid as sole carbon, nitrogen

and sulfur source indicating its degradation by the strain (Perei et. al.,

2001). There are no reports on the anaerobic degradation of the same

compound.

The organic sulfur in coal exists as both aliphatic and aromatic or

heterocyclic forms, which can be classified into four groups (Klein et. al.,

1994). 1) aliphatic or aromatic thiols (mercaptans, thiophenols); 2)

aliphatic, aromatic, or mixed sulfides (thioethers); 3) aliphatic, aromatic,

or mixed disulfides (dithioethers); and 4) heterocyclic compounds or the

thiophene type (dibenzothiophenes). Depending on its origin, products of

petroleum and coal may contain high quantities of organic sulfur

compounds. When the organically bound sulfur is not sufficiently

removed, SO2 will be formed during combustion, which poses a serious

concern in the environment (Marcelis et. al., 2003). Similarly these

compounds enter into environment through the effluents from petro-


chemical industries, petroleum spillages, refuse disposal sites

contributing to the contamination of water and soil by products like

thiophene, benzene, xylene etc. Other important uses of thiophene and

its ill effects on human beings as a contaminant have been listed in

tables 1.5 & 1.6. Aerobic microbiological conversion of thiophenes has

been studied extensively (Kobayashi et. al., 2001; Hirasawa et. al.,

2001). However, only limited data are available in the literature

concerning the sulfur specific anaerobic conversion of thiophenes. The

pathway of desulfurization of dibenzothiophene (DBT) was elucidated for

the first time in Rhodococcus erythrophilus IGTS8 through a sulfur

specific pathway which was called the 4S pathway proposed by Kilbane,

(1990). In this pathway, DBT is sequentially metabolized to DBT-

sulfoxide, DBT-sulfonate, DBT-sulfinate, hydroxybiphenyl (HBP) and

sulfite as shown in (fig 1.11). In this pathway, oxidation of sulfur atom

in DBT occurs without cleavage of C-C bonds.

Very little information is available on the anaerobic biodegradation

of sulfur heterocyclic compounds under sulfate- reducing conditions.

Furthermore, clear evidence for significant anaerobic desulphurisation is

scarce. It is proposed that thiophene molecules can be used as

alternative electron acceptor leading to the formation of the remaining

hydrocarbon molecule and H2S (Kim et. al., 1990). The degradation of

DBT under anaerobic nitrogen flushed condition was identified initially

in two SRB strains, Desulfomicrobium escambium and Desulfovibrio


longreachii where more than 10% of DBT present initially was degraded

(Yamada et. al., 2000). Kuhn and Suflita (1989) found no vidence for the

microbial removal of thiophene, thiophenecarboxylate, 0r 2- or 3-

methylthiophene following a 3 month incubation period in sulfate

reducing aquifer sediment incubations.

Figure: 1.11. Pathway of biodegradation ofDibenzothiophene(Bressler et. al., 1998).
















Origin of Present study:

The diversity studies of Anoxygenic Phototophic Bacteria (APB) are

carried out as major research studies in the laboratory in which the

present thesis work is been carried out. As a part of this, different kinds

of samples obtained from various habitats of India were collected and

enriched in the medium specific for APB. In this context, many samples

of soil, sludge and water of various habitats were observed to be black in

colour and with strong sulfide smell resembling those with rich sulfate

reduction activity. Also, the attempts to enrich APB were dominated by

the growth of Sulfate Reducing Bacteria in a few samples. This drew our

interest in understanding the diversity of SRB from samples of different

habitats India, as only a few taxa of SRB reported from India. The

diversity of SRB based solely on pure culture techniques was difficult

and more time consuming and generated very little data in

understanding the diversity of SRB. Hence, attempts were also made to

understand the phylogenetic diversity of SRB based on a single

universal, SRB marker gene, dsrAB.

The growth of SRB in enrichment cultures was always

accompanied with other chemotrophic bacteria which were not sulfate

reducing but produced H2S like SRB in SRB specific medium, giving a

wrong interpretation on diversity of SRB. Hence the studies also focused

on understanding the diversity of these non-sulfate reducing, but H2S

producing chemotrophs.


Finally, an attempt was made to use the isolated SRB in

bioremediation studies with a few selected aromatic sulfur compounds

that pose environmental threat as pollutants so that they can be applied

in natural environments like soil and sludge, which are natural and

popular niches of SRB.

Objectives

1. To study the cultured and phylogenetic diversity of H2S

producing bacteria of diverse habitats of India, with a special

focus on the sulfate reducing bacteria.

2. To describe novel taxa, if any, based on polyphasic taxonomic

analysis.

3. To study capability of the novel sulfate reducing bacterium

Desulfovibrio psychrotolerans, JS1T in biodegradation of a few

aromatic sulfur compounds.

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