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
2Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
3Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
4Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
5Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
6Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
7Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
8Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
9Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
9Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
9Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
10Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
11Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
12Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
13Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
13Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
13Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
14Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
15Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
16Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
17Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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-)
18Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
19Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
(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-
20Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
21Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
22Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
23Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
24Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
Figure 1.8. Phylogenetic tree based on nearly complete16S ribosomal RNA (rRNA) sequences ofdescribed sulfate-reducing bacterialspecies.
(Adopted from Muyzer and Stam, 2008)
25Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
26Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
27Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
28Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
29Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
30Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
30Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
30Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
31Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
32Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
33Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
34Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
35Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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)
36Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
37Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
38Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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-
39Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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
40Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
40Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
40Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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).
41Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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.
42Jagarlapudi Sri Sasi Jyothsna, Ph.D. Thesis Introduction & Literature Survey
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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