Journal o f Applied Bacteriology 1990,69, 769-797

3387/05/90

A Review
Physiology and ecology of the sulphate-reducing bacteria
G . R. GIBSON Medical Research Council, Dunn Clinical Nutrition Centre, 100 Tennis Court Road, Cambridge CB2 lQL, U K Accepted 23 July 1990
1. Introduction, 769 2. Physiology, growth and classification, 770 2.1 The sulphate-reducing bacteria, 770 2.2 Growth requirements, 775 2.3 Isolation and identification, 777 2.4 Metabolism of organic carbon, 778 2.5 Products of dissimilatory sulphate reduction, 78 1 2.6 Taxonomy and genetics, 781 3. Ecology, 783 3.1 Habitats, 783 3.2 Interactions with other bacteria, 784 3.3 Syntrophic associations, 787 4. Concluding remarks, 787 5. References, 788

1. Introduction
All plants, animals and bacteria require sulphur for the synthesis of proteins. The biological transformation of sulphur in natural environments is a nutrient cycling process comprising both aerobic and anaerobic components (Postgate 1984). In its highest oxidation state, sulphur exists as the sulphate ion (SO:-) which is reduced to sulphide (Sz-) by most bacteria, fungi and plants before incorporation into amino acids. This process is termed assimilatory sulphate reduction and is purely a biosynthetic process. However, any sulphur compound with an oxidation state above that of sulphide ( - 2) can potentially function as an electron acceptor for the oxidation of carbon substrates by biological processes (Goldhaber & Kaplan 1974). For example, during dissimilatory sulphate reduction, the sulphate ion is utilized as an oxidant for the degradation of organic material. An equivalent amount of sulphide is formed per mole of sulphate reduced (Berner 1974): 2CH,O

+ SO:-

-+

H,S

+ 2HCO;

Virtually all the sulphate reduced is released as sulphide and may be converted to H,S or HS-. Dissimilatory sulphate reduction is carried out by a specialized group of anaerobes : the sulphatereducing bacteria. These micro-organisms can be defined as a mixed group of morphologically and nutritionally diverse, strictly anaerobic bacteria which utilize sulphate (or other oxidized sulphur compounds), as an electron acceptor for the dissimilation of organic compounds (Widdel 8c Pfennig 1984). Sulphatereducing bacteria are environmentally important micro-organisms. Their influences can be divided into ecological and economic effects (Postgate 1982). Probably the most significant aspect of their metabolism is the production of H,S, which, being a very strong reducing agent, is able to inhibit the growth of certain aerobic micro-organisms, Conversely, H,S plays an important role in the natural environment in that it functions as an electron donor for the growth of some sulphur bacteria. By comparing the flow of organic substrates through aerobic processes and dissimilatory sulphate

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G. R . Gibson

reduction, it has been estimated that SRB are able to metabolize over 50% of organic detritus input to coastal marine sediments (Jsrgensen 1977a, 1982a; Parkes & Buckingham 1986). Potentially therefore, the turnover of organic compounds in such an ecosystem may exceed that of the carbon cycle, with sulphate-reducing bacteria having an emphatic biological influence. The processes in which sulphate-reducing bacteria are able to impinge on mans economy have been reviewed on many occasions (Postgate 1960, 1965, 1982, 1984; LeGall & Postgate 1973). These involve the areas of water, soil and sand pollution; waste purification; mineral deposit formation; gas contamination; paper technology; food spoilage; animal nutrition; and various other biotechnological effects. The influence of sulphate-reducing bacteria upon oil technology has received much interest, mainly because of the production of corrosive H,S. The gas adversely affects pumping equipment, storage tanks and pipelines (Hamilton 1983). Contamination and souring of stored oil products and fouling of equipment due to the overproduction of hydrogen may also occur. As a result of their ubiquitous nature and economic effects, sulphate-reducing bacteria are currently the subject of considerable scientific attention. Consequently, significant progress has been made in the last decade towards the understanding of their biology.

2. Physiology, growth and classification

2.1
T H E S U L P H A T E - R E D U C IN G B A C T E R I A

Laboratory growth of sulphate-reducing bacteria was first reported by Beijerinck (1895). However, the studies of Baars were probably the first to investigate the physiological properties of these bacteria in any detail. Baars (1930) isolated the sulphate-reducing bacterium Desulfovibrio rubentschickii which was capable of oxidizing acetate directly to CO, but subsequent attempts to isolate species able to utilize acetate failed, leading Selwyn & Postgate (1959) to the conclusion that the original culture was part of a mixed commensal population. Research involving sulphate-reducing bacteria then concentrated on strains which utilized lactate as a carbon and energy source. These bacteria were classified into two groups: spore-forming straight or curved rods belonging to the genus Desulfotornaculurn (Campbell & Postgate 1965) and the non-sporing genus Desulfovibrio (Postgate & Campbell 1966) containing motile vibrios or rods. The preferred carbon sources for bacteria belonging to each of these genera are organic acids such as lactate, pyruvate and malate, and alcohols such as ethanol, propanol and butanol. The oxidation of these substrates is incomplete, however (apart from Desulfovibrio baarsii and Desulfotomuculum acetoxiduns which were isolated at a later stage), with acetate being formed as an end product. The restricted metabolic potential of these sulphatereducing bacteria was found to be in conflict with field data obtained by Jsrgensen (1977a, 1982a) and Jsrgensen & Fenchel (1974) which emphasized the ecological significance of the bacteria in coastal marine sediments. Jsrgensen & Fenchel (1974) postulated that less than a third of the organic matter present in a marine system could be degraded if the anaerobic oxidation of acetate did not occur. Moreover, Jsrgensen (1977a, 1982a) showed that over 50% of the organic material degraded in a marine sediment could be directly attributed to the activity of sulphate-reducing bacteria. These data suggested that hitherto undetected strains of sulphate-reducers that oxidized lower fatty acids, particularly acetate, were present in such sediments. The first indication that such bacteria existed was the isolation of Desulfotomaculum acetoxiduns by Widdel & Pfennig (1977, 1981a). This bacterium was assigned to the genus Desulfotomaculum on the basis of Gram reaction, morphology, spore formation, the reduction of sulphate, cytochrome profile and the absence of desulfoviridin. However, Dsm. acetoxiduns is able to completely oxidize acetate to CO, . The optimum growth temperature of this bacterium was 37C and it was isolated from animal manure and dung contaminated habitats, suggesting that it was of intestinal origin. Subsequently, a range of new genera of sulphate-reducing bacteria capable of growth upon higher and lower fatty acids were identified (Widdel 1980). These new types of sulphate reducers were both morphologically and nutritionally distinct from those classical strains belonging to the genera Desulfouibrio and Desulfotomaculum and their main characteristics are shown in Table 1.

Sulphate-reducing bacteria

77 1

The genus Desulfobacter contains four species (Widdel & Pfennig 1984) and has been assigned to sulphate reducers which are non-sporing, NaCl and MgC1,-requiring, rod/oval shaped bacteria which are able to utilize acetate as an electron donor. Desulfobacter postgatei (Widdel & Pfennig 1981b) grows slowly on acetate and has a requirement for biotin and 4-aminobenzoic acid. Desulfobacter latus (Widdel 1987) is similar but requires biotin and thiamine for growth. Desulfobacter hydrogenophilus and Dsb. curuatus (Widdel 1987) are able to utilize ethanol as well as acetate for growth, whilst the former is able to grow autotrophically on H,/CO, . Desulfobulbus propionicus (Widdel & Pfennig 1982) is an ellipsoidal or lemon-shaped bacterium that is able to oxidize propionate, which after acetate, is the most important fermentation end product in many natural ecosystems (Hungate 1966; Cummings 1981). Desulfococci are spherical-shaped bacteria capable of fatty acid and benzoate oxidation. One species, Desulfococcus niac,Gi, can utilize nicotinic acid as an electron donor and carbon source (Imhoff-Stuckle & Pfennig 1983). Desulfosarcina variabilis consists of packets of irregularly arranged cocci or rod shaped cells which are able to oxidize fatty acids or benzoate completely and form sediments in liquid media (Widdel & Pfennig 1984). The largest known sulphate-reducing bacteria belong to the genus Desulfonema which consists of filamentous organisms 3-8 pm in diameter. These cells show gliding motility, completely oxidize their substrates to CO, and are the only known Gram-positive sulphate reducers (Widdel et al. 1983). Desulfomonas pigra (Moore et al. 1976) is a straight rod which incompletely oxidizes pyruvate and ethanol. A more recently described genus, Desulfobacterium, includes species which are able to metabolize certain phenolic (Bak & Widdel 1986a), indolic (Bak & Widdel 1986b) and betaine (Heijthuijsen & Hansen 1989) compounds. Bacteria in the genus Desulfomicrobium are similar to desulfovibrios apart from the fact that the cells do not contain the dissimilatory sulphite reductase enzyme, desulfoviridin. For this reason, Rosanova et al. (1988) proposed that Dsu. baculatus be renamed Desulfomicrobium baculatus. The isolation of Archaeoglobus fulgidis has aroused interest as the first archaebacterial sulphate reducer (AchenbachRichter et al. 1987; Stetter et al. 1987; Stetter 1988; Zellner et al. 1989b). Apart from the discovery of new genera of sulphate-reducing bacteria, many additions have recently been made to the classical Desulfouibrio and Desulfotornaculurn groups. These include the isolation of Dsu. baarsii, which is able to oxidize acetate (Widdel 1980), Dsu. sapovorans (Widdel 1980), Dsv. sulfodismutans (Bak & Pfennig 1987), Dsu. carbinolicus (Nanninga & Gottschal 1987), Dsu. simplex (Zellner et al. 1989a), Dsu. furfuralis (Folkerts et al. 1989), Dsm. sapomandens (Cord-Ruwisch & Garcia 1985) and Dsm. kuznetsouii, which is able to oxidize acetate (Nazina et al. 1989). Other newer strains not included in Table 1 are Dsu.fructosouorans (Qatibi et al. 1990a) and Dsu. alcoholouorans (Qatibi et al. 1990b). It is apparent that, individually and as a group, sulphate-reducing bacteria potentially have the ability to oxidize completely the principal organic end products of bacterial metabolism in anaerobic environments. The major metabolic reactions carried out by dissimilatory sulphate-reducing bacteria are shown in Table 2. All of these bacteria share a common ability to dissimilate sulphate for energy gain. The initial step in the biochemical sulphate reduction pathway is the transport of exogenous sulphate across the bacterial membrane into the cell (Cypionka 1987, 1989). This process may be inhibited by the structural analogue of sulphate, selenate (Newport & Nedwell 1988). Once inside the cell, sulphate dissimilation then proceeds by the action of ATP sulphurylase which combines sulphate with ATP to produce the highly activated molecule adenosine phosphosulphate (APS), as well as pyrophosphate which may be subsequently cleaved (Wilson & Bandurski 1958), to yield inorganic phosphate. APS is then rapidly converted to sulphite (SO;) by the cytoplasmic enzyme APS reductase (Peck 1962; Stille & Triiper 1984). A number of different sulphite reductases have been reported in sulphate-reducing bacteria (LeGall & Postgate 1973). However, the most commonly recognized types, particularly among the genus Desulfouibrio, are the bisulphite reductases, desulfoviridin (Lee & Peck 1971) and desulforubidin (Lee et al. 1973). Kinetically, these enzymes are similar, however the liberation of a red fluorescent s'iroporphyrin from the denaturation of desulfoviridin forms the basis of a much used diagnostic test for desulfovibrios (Postgate 1951, 1984). Sulphite may then be reduced via a variety of intermediates to form the sulphide ion (Postgate 1984). Intermediates in this pathway are thought to include

Table 1. Classification of sulphate-reducing bacteria

4
h )

Bacterium Reference 55 61 Ferments pyruvate and choline Obligate sulphate requirement

Morphology

Major electron donors

Yo G
Spores Motility Desulfoviridin Remarks

+C

Desulfovibrio desulfuricans

Vibrio

uulgaris

Vibrio

gigas africanus

Spiral Vibrio

60 61
46 Requires NaCl 57 53 66 ND

salexigens

Vibrio

baculatus

Rod

sapovorans baarsii

Vibrio Vibrio

Lactate, pyruvate, ethanol Lactate, pyruvate, ethanol, H, Lactate, pyruvate Lactate, pyruvate, malate Lactate, pyruvate, malate Lactate, pyruvate, malate Lactate, pyruvate Acetate, formate

Postgate & Campbell 1966 Postgate & Campbell 1966 LeGall 1963 Campbell et al. 1966 Postgate & Campbell 1966 Rozanova & Nazina 1976 Widdel 1980 Widdel 1980

9
Rozanova & Khudyakova 1974 Bak & Pfennig 1987

thermophilus

Rod

Lactate, pyruvate

sulfodismutans

Vibrio

Lactate, ethanol, propanol

64
65 48 61

+ + + + + + + + + +

5 e 3

carbinolicus

Vibrio

H, , alcohols,

simplex

Vibrio

Ferments pyruvate Higher fatty acids (up to C18) utilized Optimum growth temp. is 65C SO,, S,O, or S,O, disproportionated to SO, and S 2 Ferments pyruvate, fumarate & malate Oxidizes benzaldehyde derivatives

furfuralis

Vibrio

lactate Lactate, pyruvate glucose Furfural, lactate, ethanol 42 46 45 ND 37

+ + +

Nanninga & Gottschal 1987 Zellner et al. 1989a Folkerts et al. 1989

Desulfotomaculum orientis

Large vibrio

Lactate, pyruvate

ruminis

Rod

Lactate, pyruvate

Ferments pyruvate Ferments pyruvate

nigrificans

Rod

Lactate, pyruvate

antarticum

Rod

Lactate, glucose

acetoxidans

Rod

Acetate, butyrate

+ + + + +

Campbell & Postgate 1965 Campbell & Postgate 1965 Campbell & Postgate 1965 Iizuka et al. 1969 Widdel & Pfennig 1977, 1981a

sapomandens
49 52

Rod

48

+
Sensitive to H,S Cord-Ruwisch & Garcia 1985 Nazina er al. 1989 Gogiova & Vainstein 1983 Isolated from human faeces Requires NaCl Requires NaCl Requires NaCl Requires NaCl Moore et a!. 1976 ND
-

+ +
Ferments pyruvate and fumarate

kuznetsovii

Rod

guttoideum

Rod

Ethanol, higher fatty acids and alcohols Acetate, lactate, ethanol Lactate, H,

+
-

+
+

Desulfomonizs pigra
67

Rod

Ethanol, pyruvate

Desulfobactm postgatei

hydrogenophilus curvatus lotus

coccobacillus Oval Vibrio Oval

Acetate

, Acetate, H Acetate, ethanol Acetate

Widdel & Pfennig 1981b Widdel 1987 Widdel 1987 Widdel 1987

Desulfobulbus propionicus

Lemon

elongatus

Propionate, lactate, pyruvate Propionate, lactate,

Curved rod

H ,
-

Ferments lactate and pyruvate Ferments lactate and pyruvate

Widdel & Pfennig 1982 Samain et al. 1984 Ferments pyruvate Widdel 1980

Desulfococcus multivorans

Round

+
-

niacini

Oval/ round
51
-

Acetate, lactate, pyruvate, benzoate Nicotinic acid, acetate, pyruvate

Requires NaCl

Imhoff-Stuckle Pfennig 1983 Ferments pyruvate Widdel 1980

Desulfosarcina variabilis

Random packets
42
35

Acetate, ethanol, H , , pyruvate

Desulfonema

limicola

Large filaments Large filaments

Acetate, malate, benzoate Acetate, benzoate, pyruvate

+ +
(continued)

Obligate sulphate requirement Requires NaCl

Widdel et al. 1983 Widdel et al. 1983

Table 1 (continued)
%G

Bacterium Spores Motility Desulfoviridin Remarks Requires NaCl Requires NaCl Utilizes higher fatty acids 41 48
-

Morphology

Major electron donors

+C
~

Reference

Desuljobacterium phenolicum

Vibrio

Phenol, acetate

indolicum 48 58
-

autotrophicum

Oval/ rod Oval

Bak & Widdel 1986a Bak & Widdel 1986b Brysch et al. 1987

macestii 47

Oval/

Indole, acetate, formate Ethanol, formate, H z, betaine Lactate, ethanol,

+ + + +
-

9
Gogiova & Vainstein 1989 Widdel 1988

H Z

vacuolatum

cat echolicum

Lactate, H, ,, formate Catechol, formate

52

5 r
Szewzyk & Pfennig 1987 Ferments pyruvate and fumarate Rozanova et a2. 1988
0

rod Oval/ rod coccobacillus 52

Desuljomicrohium apsheronum

Rod

Lactate, pyruvate, ethanol 34

-

+
-

Thermodesuljobacterium commune

Rod

Lactate, pyruvate, H, 45
-

Optimum growth temp. is 70C ND Optimum growth temp. is 75-80C

Zeikus et al. 1983 Zellner et al. 1989b

Archaeoglobus fulgidis

Round

Lactate, pyruvate

Sulphate-reducing bacteria
Table 2. Metabolic reactions involving sulphate-reducing bacteria
References

775

Oxidative reactions 4H2+SO:- +HS- + O H - + 3 H 2 0 4 Formate+H++SO:-+4HCO; +HSAcetate+SO:- +HS- + 2HCO; 4 Propionate+3SO:-+3HS- + H + +4HCO; + 4 Acetate 2 Butyrate+3SO:-+3HS-+H++4HCO; +2 Acetate 2 Lactate+ SO:- + H + + 2 Acetate+2CO2 + 2H,O + HS2 Malate + SO, + H -2 Acetate + 2C0, + 2HCO; + HS4 Succinate+3SO:- + 3H ++4 Acetate+4C02+4HCO; +3HS4 Pyruvate+SO:- + H + + 4 Acetate+4CO2+HS2 Fumarate + 2 H 2 0+ SO:- + H + + 2 Acetate + 2C0, + 2HCO; + HSGlucose+ SO:- + H + + 2 Acetate+ 2C0, + HS- +4H,O 4 Benzoate+ 15SO:- + 16H2O-+28HCO; + 15HS- +9H+ 2 Ethanol+SO:-+2 Acetate+HS-+H++2H2O 4 Propanol+5SO:--+4 Acetate+5HS-+3H++4H2O+4HCO; 2 Phenol+7SO:-+6H2O+12HCO; +5H++7HS2 Indole+9SO:- + 12,H20+16HCO; +2NH: +5H+ +9HS4 Nicotinic acid+ 1lSO:- +20H2O-+24HCO; + 11HS- +4NH: +5H+ 2 Furfural+SO:-+4H20+4 Acetate+2C02+HS- + H + 4 Betaine + 3SO:- + 4 N,N-dirnethylglycine+ 4C0, + 3HS- + H + + 4H,O
+

Postgate 1984 Postgate 1984 Widdel & Pfennig 1977 Widdel & Pfennig 1982 Ssrensen et al. 1981 Thauer et al. 1977 Postgate 1984 Postgate 1984 Postgate 1984 Postgate 1984 Zellner et al. 1989a Widdel et al. 1983 Kremer et al. 1988 Laanbroek et al. 1982 Bak & Widdel 1986a Bak & Widdel 1986b Imhoff-Stuckle & Pfennig 1983 Folkerts et al. 1989 Heijthuisjen & Hansen 1989 Laanbroek ef al. 1982 Magee et al. 1978 Thauer et al. 1977 Magee et al. 1978 Hayward & Stadtman 1960 Laanbroek et al. 1982 Laanbroek et al. 1982 Laanbroek et al. 1982

Fermentative reactions 3 Lactate+Acetate+2 Propionate+HCO; + H + Pyruvate+ 2H,O+Acetate+ HCO; + H, + H + Malate+ 3H20+Acetate+2HCO; + H + +2H2 Fumarate + H,-Succinate 2 Choline + H , 0 + 2 Trimethylamine + Acetate+ Ethanol Ethanol+HCO; +H,+Propionate 3 Ethanol+ 2HCO; +Acetate+ 2 Propionate+ H + + 3H,O Acetate+ HCO; + H + 3H,+Propionate + 3 H 2 0
+

metabisulphite (S20g-), dithionite (SzOi-), trithionate (S,Oi-) and thiosulphate (SzOi-). However, there are conflicting opinions as to the involvement of these compounds. For example, Chambers & Trudinger (1975) used lactate-grown Desulfovibrio strains to conclude that trithionate and thiosulphate were not free intermediates. The cyclic mechanism outlined by Postgate (1984) proposed that sulphate was dehydrated to form metabisulphite which was then reduced via dithionite to trithionate. The trithionate was then thought to be split into thiosulphate as well as regenerating sulphite. The thiosulphate thus formed may then be degraded to produce sulphide and sulphite (Fig. 1). The majority of work on sulphur metabolism has involved the use of Desulfouibrio or Desulfotomaculum. The isolation of the key enzyme APS reductase in Desulfobulbus, Desulfobacter, Desulfococcus and Desulfosarcina strains by Stille & Triiper (1984), however, suggests that the mechanism in other sulphate reducers is essentially similar. A number of the reductive enzymes involved in this metabolism have been purified and characterized and the review by Peck & LeGall(l982) summarizes some of their biological properties. 2.2
GROWTH REQUIREMENTS

The nutritional requirements of sulphate-reducing bacteria are relatively simple. Firstly, an inorganic electron acceptor is required by most strains, and this is usually provided by the sulphate ion. Certain species have been shown to utilize other oxidized sulphur compounds. For example, Desulfouibrio desulfuricans can use thiosulphate, tetrathionate and sulphite (Postgate 1951; Cypionka 1987), whilst Biebl & Pfennig (1977) grew some strains with elemental sulphur as a terminal electron acceptor. In the absence of an external electron acceptor, some sulphate-reducing bacteria can ferment substrates

776

membrane Sulphate t ATP

in

Pyrop hosphat e

+ Adenosine phosphosuIphate

2P

1
AMP

1
Sulphtte Thiosulphate

+ suiphite
I I

Metabisulphite

Tri t hionate

Dithionite

e-

- Cytochrome c3
Fig. 1. The sulphite regeneration pathway of dissimilatory sulphate reduction (e= electron).

such as pyruvate, fumarate, malate and lactate (Table 1). A number of studies have also demonstrated the ability of Desulfooibrio spp. and Desulfobulbus spp. to reduce nitrate and nitrite, with energy yields as great or higher than those recorded with sulphate (Liu et al. 1980; Steenkamp & Peck 1981; Widdel & Pfennig 1982; Keith & Herbert 1983; McCready et al. 1983; Mitchell et al. 1986; Seitz & Cypionka 1986). The type of carbon source utilized for the reduction of an electron acceptor varies according to genus. Major electron donors are shown in Table 1. Essentially, these consist of volatile fatty acids (VFA), e.g. acetate, propionate, butyrate; C 3 and C 4 fatty acids, e.g. lactate, pyruvate, malate; alcohols, e.g. ethanol, propanol; H,/CO,, and occasionally sugars and longer chain fatty acids. A number of sulphate reducers are also thought to metabolize certain hydrocarbons, including methane, although the bacteria and processes involved have not been clearly resolved (Postgate 1984). Ammonium ions serve as a nitrogen source for the growth of most sulphate-reducing bacteria, although the dissimilatory reduction of nitrate and nitrite may also provide a nitrogen source. Furthermore, some sulphate reducers are able to fix nitrogen (Rieder-Henderson & Wilson 1970; Widdel & Pfennig 1984; Postgate & Kent 1985; Postgate et al. 1985). Stams et al. (1985, 1986) and Stams & Hansen (1986) showed that several marine strains of Desulfovibrio could utilize a number of amino acids as carbon and nitrogen sources. Other nutrients important for the growth of sulphate reducers are phosphorus and iron (Postgate 1984). A low E, medium is often needed to initiate the growth of sulphate-reducing bacteria (Grossman & Postgate 1953). This effect may be achieved by the use of E, poising agents such as Na,S, sodium dithionite (Pfennig et al. 1981), sodium ascorbate or sodium thioglycollate (Postgate 1966). However, sodium thioglycollate, particularly at high concentrations, has been shown to inhibit the growth of many strains of sulphate-reducing bacteria (Khosrovi & Miller 1975; Pfennig et al. 1981). Yeast

Sulphate-reducing bacteria

777

extract, vitamins, NaCl and MgCl, have also been used to supplement growth media (Pfennig et al. 1981; Postgate 1984; Widdel & Pfennig 1984).

2.3

ISOLATION AND IDENTIFICATION

Cultures of sulphate-reducing bacteria may be enriched from mixed inocula such as sediment or faecal samples by conventional batch (Widdel 1980; Postgate 1984) or chemostat methods (Keith et al. 1982; Gibson & Macfarlane 1988). An appropriate choice of electron donor and media conditions is critical. As batch culture enrichments are closed systems where all nutrients are present initially in excess and no additional input of substrate or removal of metabolic end product occurs, they are simple to perform. Micro-organisms are selected upon the basis of their maximum specific growth rate, i.e. those bacteria which grow fastest under the imposed conditions will predominate. Unless a selective medium is employed, this method will not be particularly useful for the isolation of sulphatereducing bacteria, which generally grow relatively slowly and may therefore be unable to compete in the enrichment system. The use of continuous culture systems, where bacteria can be selected which have a high substrate affinity and low growth rate are therefore ideally suited for the specific enrichment of sulphate reducers from mixed cultures. Another procedure that has been employed for the enrichment of sulphate-reducing bacteria is the use of gel stabilized model ecosystems (Macfarlane et al. 1984). Culture purity may be determined by microscopic examination coupled with plating methods to determine the absence of culture contamination. Different genera of sulphate-reducing bacteria can be identified by a number of parameters. The most obvious feature of the group is the reduction of sulphate to produce sulphide and the subsequent blackening of media containing ferrous ions (due to a production of FeS). The oxidation of specific carbon sources can also be used for diagnostic purposes (Pfennig et al. 1981). Other criteria useful for the characterization of different genera include spore formation, motility, Gram reaction, cell size, morphology, % mol GC, cytochrome profiles, growth requirements for NaCl and MgCl, , vitamins, serology, DNA-rRNA homology, temperature requirements, flagella type and antibiotic resistance (Postgate 1984). The desulfoviridin test has also been used as a rapid method to distinguish some sulphate-reducing bacterial strains (Postgate 1951; Sharma & Hobson 1987), as has the use of immunofluorescent assays (Abdollahi & Nedwell 1980; Smith 1982; Beeder et al. 1990; Brakstad 8~Hamouda 1990). With the recent and ongoing isolation of new types of sulphate-reducing bacteria, alternative identification methods are also being considered, particularly as some stains can show overlapping nutritional and morphological characteristics. One such approach is the analysis of specific fatty acid components in the cell membrane. Phospholipids constitute a specific and relatively constant proportion of the cell membrane of all micro-organisms (White et a/. 1979). Differences in the lipid membrane profile of individual bacteria have previously been applied as a useful taxonomic determinant (Shaw 1974; Federle et a/. 1983). The method has also been used to identify different populations of sulphate reducers. Bacteria belonging to the genus Desulfouibrio contain high levels of a branched C17 fatty acid in their membranes which appears to be specific to the group (Boon et al. 1977). Taylor & Parkes (1983) also analysed the cellular fatty acids of Desulfobacter spp. and Desulfobulbus spp. and compared the profiles obtained with those of Dsu. desulfiricans. They found that the three genera could be distinguished by their characteristic membrane fatty acids. This work was then extended by determining the presence of specific lipid biomarkers in a marine sediment slurry in which sulphate reduction had been stimulated by the addition of appropriate electron donors (Taylor & Parkes 1985).Thus, the analysis of specific fatty acid biomarkers can potentially be applied to the study of sulphate-reducing bacteria in complex biological systems. With the exception of Desulfotornaculurn acetoxidans (Dowling et al. 1986), lipid profiles of other sulphate-reducing types have not as yet been determined. However, if each of the different genera could be distinguished by the presence of a specific biomarker, this approach could be an important tool for the study of sulphate-reducing bacteria in mixed culture, assuming in situ stability of the lipids and the possibility that inter-site or genetic variation does not occur.

778

G. R . Gibson

The potential use of the lipid analysis method has been demonstrated (G.R. Gibson, R.J. Parkes & R.A. Herbert, unpublished results). The membrane fatty acids of nine apparently pure cultures of sulphate-reducing bacteria were extracted and compared with those of previously published genera. Bacteria were isolated by chemostat enrichment with either acetate, lactate or propionate as the carbon source and sediment from sampling sites situated at Lochs Etive and Eil, West Scotland and the Tay Estuary, East Scotland (Parkes et al. 1989). Culture purity was determined by standard aerobic and anaerobic plating methods (Postgate 1984) and microscopic examination. Sulphatereducing bacteria tentatively identified as Desulfobacter spp. from Lochs Etive and Eil, Desulfouibrio spp. from Loch Etive, Desulfobulbus spp. from Loch Eil and Desulfotomaculum acetoxidans from the Tay Estuary had membrane profiles consistent with authentic cultures of these bacteria (Taylor & Parkes 1983; Dowling et al. 1986). These results indicated that the bacteria did belong to these genera and that they were present in pure culture. In contrast, however, sulphate reducers isolated from Loch Eil and the Tay Estuary and assigned to the genus Desulfouibrio as well as a strain resembling Desulfobulbus from Loch Etive contained a higher than expected number of even chained C12-Cl8 fatty acids. This would indicate some contamination of the cultures by acetate-oxidizing sulphatereducing bacteria, e.g. Desulfobacter spp. The anaerobic plating methods described by Postgate (1984) do not distinguish between different genera of sulphate reducers. Moreover, bacteria which belong to the groups Desulfobacter and Desulfobulbus are not easily differentiated microscopically (although the bacteria have different nutritional requirements) and in impure lactate grown cultures, the opportunistic Desulfouibrio spp. would become numerically predominant leading to the incorrect assumption that no other bacteria were present. Laanbroek et al. (1984) demonstrated the enhanced capability of desulfovibrios to utilize sulphate in comparison with either Desulfobacter spp. or Desulfobulbus spp. Acetate is a metabolic end product of the oxidation of lactate and propionate by sulphate-reducing bacteria (Table 2) and it is possible for desulfobacters to cross feed and grow in cultures utilizing these substrates. These findings confirm the difficulty of obtaining pure cultures of sulphate-reducing bacteria as well as a need for rigorous tests of culture purity and identification. This becomes important in the performance of subsequent physiological tests such as the extraction and analysis of membrane fatty acids. Alternatively, the determination of bacterial lipids could itself be used as a test of culture purity. 2.4
M E T A B O L I S M OF O R G A N I C C A R B O N

The major electron donors utilized by sulphate-reducing bacteria are shown in Table 1. Although these are predominantly volatile fatty acids, it is now agreed that sulphate reducers have the ability to oxidize completely the principal end products of fermentation in situ, provided sufficient electron acceptor is available (Jsrgensen 1977a, 1982a). A number of studies have therefore been conducted to assess the major carbon sources for sulphate-reducing bacteria in different ecosystems. Principally, these have involved the incubation of samples from marine and estuarine sediments. Enumeration of viable populations present in natural environments results in information concerning the relative distribution of species, but not the importance of individual substrates. An alternative approach is the addition of 14C-labelledsubstrates to in uitro systems (in combination with specific metabolic inhibitors of processes of interest) and the subsequent elucidation of pathways of degradation and turnover rates of individual compounds (Cappenberg & Prins 1974; Winfrey & Zeikus 1979; Mountford et al. 1980; Balba & Nedwell 1982; Christensen & Blackburn 1982; Banat & Nedwell 1983; Parkes et al. 1984). With this approach, acetate and hydrogen have been cited as substrates quantitatively important to sulphate-reducing bacteria. A potential difficulty with this technique however, is the fact that varying substrate availability occurs in situ (Christensen & Blackburn 1982; Parkes et al. 1984; Gibson et nl. 1989), which may cause a considerable overestimate of the carbon tracer turnover rates. As an alternative, structural inhibitors of carbon compounds thought to be important oxidative substrates have been added to sediment systems. For example, fluorolactate and fluoroacetate have been used for lactate and acetate dependent reactions respectively (Kun 1969; Cappenberg 1974; Cappenberg & Prins 1974; Banat et al. 1981). The use of specific metabolic inhibitors of sulphate reduction such as molybdate (Peck 1959)and selenate (Postgate 1949) has also been investigated. The

Sulphate-reducing bacteria

779

addition of sodium molybdate to natural ecosystems has been extensively used and it is this procedure which has resulted in the majority of information concerning in situ substrates for sulphatereducing bacteria (Peck 1962; Huisingh et al. 1974; Oremland & Taylor 1978; Nedwell & Banat 1981; Smith & Klug 1981; Ssrensen et al. 1981; Banat et al. 1983; Winfrey & Ward 1983; Christensen 1984; Parkes et al. 1989). Molybdate is a structural isomer of sulphate which competitively inhibits the enzyme adenosine triphosphate sulphurylase (Peck 1959; Taylor & Oremland 1979). Newport & Nedwell(l988) reported that molybdate has more than one site of inhibition, but that its primary effect was upon sulphate transport. Thus, because the inhibition is specific for processes involving sulphate, molybdate has been added to various in uitro systems. Any compound which accumulates in the presence of molybdate is therefore a potential substrate for sulphate reduction. If the accumulation is linear, rates of sulphate reduction (in the absence of inhibitor) and relative reaction stoichiometries can be taken into account, and the importance of individual electron donors estimated. From such studies, it is now accepted that acetate is the predominant carbon source for sulphate reduction in marine and estuarine sediments (Ssrensen et al. 1981; Winfrey & Ward 1983; Christensen 1984). However, other substrates are also recognized as being significant in sustaining the growth of sulphate-reducing bacteria in the field. These include lactate, hydrogen, propionate, butyrate, 2-methylbutyrate, valerate, glutamate, serine, alanine, arginine, aspartate, leucine, isoleucine, phenylalanine and lysine (Parkes et al. 1989). Differences in the relative importance of substrates in a particular environment occur and this is largely dependent upon the input and nature of organic material to the site, as well as the sulphate-reducing activity therein. Elevated amounts of electron donors that are important for sulphate reduction have also been added to model ecosystems in an attempt to investigate rates of carbon turnover (Laanbroek & Pfennig 1981; Taylor & Parkes 1985). The habitats studied are normally carbon-limited ecosystems where sulphate is present in excess, thus, the activities of sulphate-reducing bacteria become stimulated. Time course measurements of changes in sulphate, sulphide and electron donor concentrations as well as viable sulphate reducer numbers reveals how the metabolism of individual substrates can be potentially related to sulphate reduction. Such experiments have demonstrated that not only do differences in the type of substrate utilized occur, but that the pathway of carbon source degradation also shows inter-site variability. This appears to be largely due to the interactions of sulphatereducing bacteria with other fermentative and H, producing organisms in the environment, and emphasizes subtle differences between laboratory and in situ growth of these bacteria. The majority of bacterial species belonging to the most studied sulphate reducing genus, Desulfovibrio have a restricted catabolic capacity. The favoured substrate for their growth is lactate which is oxidized via pyruvate to form acetate and CO, . Similarly, a number of other compounds including TCA cycle acids, pyruvate and alcohols are incompletely oxidized. Many of these reactions involve the use of an 0,-labile NADPH dehydrogenase (Stams & Hansen 1982; Kremer et al. 1988, 1989). However, as acetate is now considered to be quantitatively the principal sulphate-reducing substrate in natural ecosystems (Laanbroek & Pfennig 1981; Serensen et al. 1981), it is the pathway of utilization of this carbon source which has received considerable attention in recent years. A functional citric acid cycle which converts one acetate into two CO, molecules, has been demonstrated in cell extracts of Desulfobacter postgatei (Brandis et al. 1983; Gebhart et al. 1983). The major enzymes involved are citrate synthase, aconitase, NADP-dependent isocitrate dehydrogenase, 2-oxoglutarate ferredoxin oxidoreductase, membrane bound succinate dehydrogenase (which couples with a menadione), fumarase and membrane bound malate dehydrogenase (which couples with a quinone). Acetate was activated by CoA transfer from succinyl CoA with succinate thiokinase being absent. Further studies by Moller et al. (1987) demonstrated that the enzyme mediating citrate formation was an ATP-citrate lyase rather than a citrate synthase. Other sulphate-reducing bacteria such as Desulfotomaculum acetoxidans employ a different method for acetate metabolism. The non-involvement of a TCA cycle was demonstrated by the absence of 2-oxoglutarate dehydrogenase and citrate synthase (Schauder et al. 1986). The bacterium seemingly catalyses an exchange between CO, and the carboxyl group of acetate, that is to say, a reverse acetogenic pathway (Fuchs 1986) operates and involves the use of acetyl CoA rather than acetate or acetyl phosphate as substrate for carbon4arbon cleavage (Schauder et al. 1986; Spormann & Thauer

780
Desulfovibrio vulgaris

G. R. Gibson
Pyruvote

Acetyl COP.

t
k

Amino acids

Cellular proteins

(Assimilation)

Acetate

I
I Pyruvote
HCO; PEP
Acetyl CoA

7
(Oxidation)

Oxoloacetote i/

yTc'trat\
co
\ Acetyl CoA

CH3X

lsoci trate
2

Malote

2-Oxoglutorote

Fumarote

\Succiny:A

LOA

\ Succinote

3 (
Acetate
Desulfotomaculum ocetoxidans, Desulfovtbrio boorsii, Desulfobacter autotrophicum

Desulfobacter postgatet

Fig. 2. Possible pathways of acetate utilization carried out by sulphate-reducing bacteria

1988; 1989). Desulfooibrio baarsii (Jansen et al. 1985) and Desulfobacter hydrogenophilus (Schauder et al. 1987; Lange et al. 1989) oxidize acetate by an almost identical pathway to Desulfotomaculum acetoxidans. Thus, acetate-oxidizing sulphate-reducing bacteria can be separated into two groups depending upon whether metabolism by the TCA cycle or acetyl CoA pathway predominates (Fig. 2). The demonstration of an acetyl CoA pathway in Desulfiuibrio baarsii is particularly pertinent. Strains of Desulfioibrio have been shown to degrade H,/CO,. However, the effect has been attributed to mixotrophic rather than strictly autotrophic growth, as the culture medium requires the addition of acetate before bacterial growth occurs. Less than 30% of cell carbon is derived directly from CO, assimilation (Badziong et al. 1978; 1979; Brandis & Thauer 1981). Although Desulfouibrio baarsii does not utilize hydrogen, it is able to grow with CO, , formate and sulphate as sources of carbon and energy. By the use of the acetyl CoA pathway, the organism is able to derive over 60% of its cell carbon from CO, (the remainder being provided by formate) and is therefore a truly autotrophic bacterium (Jansen et al. 1984; 1985) and can be distinguished from other Desulfouibrio spp. which require acetate as well as CO, for growth. Ferredoxins, flavodoxins, menaquinones, rubredoxins and cytochromes b and c act as hydrogen and electron carriers in sulphate-reducing bacteria (Peck & LeGall 1982; Postgate 1984). For example, in Desulfouibrio desulfiricans, cytochrome c3 acts as an electron carrier for hydrogenase; ferredoxins and flavodoxin for pyruvate dehydrogenase and sulphite reductase; cytochrome c553 for

Sulphate-reducing bacteria

78 1

lactate and formate dehydrogenase; whilst high molecular weight cytochrome c3 and rubredoxin are involved in lactate metabolism (Yagi & Ogata 1990). The interaction between cytochrome c3 and the reversible dehydrogenase of Desulfouibrio spp. is perhaps most interesting. It has been suggested that a proton motive force can be established in this genus (Odom & Peck 1981, 1984). Lactate is fermented to acetate and CO, in the cytoplasm. Electrons thus produced are converted to hydrogen by an accompanying hydrogenase. The hydrogen is then able to diffuse across the membrane and be oxidized by a periplasmic hydrogenase. This hydrogen oxidation produces protons and electrons. The protons are utilized for ATP synthesis, whilst the electrons are transferred back across the cytoplasmic membrane (cytochrome c3 is reduced) which causes endogenous or exogenous electron acceptor reduction in an energy yielding reaction. 2.5
P R O D U C T S OF D I S S I M I L A T O R Y S U L P H A T E R E D U C T I O N

Although any sulphur compound with a redox potential below that of the sulphate ion can be excreted by dissimilatory sulphate-reducing bacteria, their primary metabolic end product is sulphide, which can be further converted to H,S in the presence of external H + ions. Strong sulphate reducing activiy is therefore easily detected by the characteristic odour of H,S. Sulphide is extremely corrosive and binds rapidly to metals. Hydrogen sulphide also has marked effects upon the external conditions by virtue of being a potent reducing agent (EHat pH 7.0 is approximately -320 mV; Postgate 1984) and thus is able to suppress the growth of some aerobic organisms. Furthermore, H,S is directly toxic to a large range of bacteria and higher organisms, including man (Karrer 1960). These factors make sulphate-reducing bacteria extremely important micro-organisms, especially in the oil industry where their growth and corrosive nature can cause great economic problems (see earlier). In a natural ecosystem such as a marine coastal sediment, only about 10% of the free sulphide produced by sulphate reducers is precipitated by metal ions (Jsrgensen 1977b). The remainder is potentially available for oxidation by chemical or biological processes. Sulphide can be re-oxidized by phototrophic anaerobic bacteria which also require light and a supply of organic carbon for growth. These micro-organisms are grouped as purple (e.g. Chromatium, Thiocapsa, Thiopedia spp.) or green (e.g. Chlorobium, Pelodictyon, Prosthecochlarius) sulphur bacteria (Jsrgensen 1982b; Kelly 1982; Triiper & Fischer 1982). They are able to produce purple or green colorations on beaches and commonly form elemental sulphur as an end product (van Gemereden et al. 1989a, b). Cyanobacteria (e.g. Oscillatoria, Microcoleus spp.) or colourless (e.g. Beggiatoa, Thiothrix, Thiouulum spp.) sulphur bacteria can act similarly (Jsrgensen & Des Marais 1986, Jsrgensen et al. 1979, 1986). If sulphide diffuses to aerobic regions, bacteria such as the chemolithotrophic thiobacilli (Kuenen & Beudeker 1982) are able to oxidize sulphide or sulphur to sulphate, again stimulating the activity of sulphatereducing bacteria. Thus, a complete sulphur cycle can develop which has been termed a sulfuretum (Goldhaber & Kaplan 1974). Other metabolic end products of sulphate reduction are oxidized carbon componds. For example, most desulfovibrios have an incomplete TCA cycle and therefore excrete acetate from the oxidation of higher fatty acids such as lactate. Similarly, propionate and butyrate may be incompletely oxidized to produce acetate. The acetate thus formed may then be further utilized by other sulphate-reducing bacteria such as Desulfobacter spp. This leads to considerable difficulties in attempts to isolate pure cultures, as the end products of the metabolic activities of one group may frequently serve as carbon sources for another. 2.6
TAXONOMY A N D GENETICS

The extraction of DNA from various strains of sulphate-reducing bacteria and subsequent analysis of base pair ratios has been extensively used as an aid to their classification (Sigal et al. 1963; Saunders et al. 1964; Skyring & Jones 1972; Skyring et al. 1977; Hardy 1981). Table 1 shows the % mol G C of different sulphate reducers. As a large number of sulphate-reducing bacteria are recognized, % GC ratios in different genera do show a certain degree of overlap. It seems therefore that phenotypic variation is a potentially more satisfactory basis upon which to classify sulphate reducers. Also, the

782

G. R . Gibson

method of % GC analysis may give varying results. For example, Skyring et al. (1977) employed the buoyant density method to conclude that Desulfouibrio species with low GC ratios (4659%) were more prevalent in saline environments than those with high (61-64%) ratios, whilst Hardy (1981) found contrasting results in North Sea waters by the thermal denaturation method (inter-site variation should not be excluded, however). Alternative methods of genetic analysis have also been considered to study the taxonomy of sulphate-reducing bacteria however. One such method is the analysis of 16s rRNA sequences. Ribosomal RNA (rRNA) is the predominant transcript product in prokaryotic cells, comprising 80-90% of the total cellular RNA (Lewin 1987). rRNA in bacteria is represented by multiple genes with a large amount of base pairing, and the comparison of rRNA sequences in related organisms has been used to analyse secondary structure. In micro-organisms, 16s rRNA is highly dispersed but is also genetically related. In bacteria therefore, 16s rRNA may be isolated, purified and oligonucleotide sequences compared. Similarly coefficients (SAB values) are then determined and phylogenetic relationships assessed (Fox et al. 1977). With this method a phylogenetic relationship between Desulfouibrio desulfuricans and purple bacteria was thought to exist (Fox et al. 1980; Stackebrandt & Woese 1981). However, it is now believed that desulfovibrios are genetically related to bdellovibrios (Hespell et al. 1984). In a comprehensive study of seven sulphate reducing genera, Fowler et al. (1986) attempted to correlate 16s rRNA interrelationships with different physiological and morphological categories of sulphate reducers. The genetic status of sulphate-reducing bacteria with respect to other bacteria was also assessed. The authors concluded that S,, values were different in the seven groups tested (Desulfouibrio, Desulfotomaculum, Desulfobulbus, Desulfobacter, Desulfococcus, Desulfonema and Desulfosarcina). Generally, 16s rRNA analyses agreed with the classification of genera by other physiological criteria. The sporeforming genus Desulfotomaculum was found to be phylogenetically related to clostridia, whilst other sulphate reducers formed a distinctive relationship with aerobic myxobacteria and Bdellouibrio spp. Although the estimation of 16s rRNA similarity coefficients alone should not be used to classify bacteria, the method taken together with % GC ratios does have potential, from the genetic viewpoint, as a taxonomic aid. For example, Folkerts et al. (1989) have used oligonucleotide cataloguing to group a furfural-degrading anaerobic bacterium in the genus Desulfouibrio. Furthermore, Devereux et al. (1989) developed hybridization probes based upon 16s rRNA specificities of bacteria belonging to the genera Desulfouibrio, Desulfobacter, Desulfobacterium, Desulfobulbus, Desulfococcus, and Desulfosarcina, which seem to be useful for the detection of these micro-organisms in mixed samples, without a need for prior cultivation (Deveruex et al. 1990). Little information is available upon other aspects of the genetics of suiphate-reducing bacteria, mainly because of their variable growth on plates. However, Postgate et al. (1984) used a double restriction digest method to analyse the genomes of Desulfouibrio gigas and Desulfooibrio vulgaris. The bacterial genomes were characterized and sized and the authors concluded that cryptic plasmids were present in some strains of Dsu. vulgaris, Dso. gigas and Dsu. desulfuricans, although the plasmids seemed to be inconsistent with regard to size or incidence in the different bacteria. The work of Postgate et al. (1984) stimulated further research into genetic aspects of sulphate-reducing bacteria. This involved the cloning, sequencing and possible expression of certain proteins important to sulphate reducer metabolism. The hydrogenase enzyme in particular has received attention, because of its central role in energy generation in Desulfouibrio spp. Three types of hydrogenase exist in desulfovibrios (Fauque et al. 1988):

(1) Iron sulphur-containing; (2) Nickel-iron sulphur-containing ; (3) Nickel-selenium-iron sulphur-containing.

Each of the hydrogenases have two subunits of various sizes, but differ in their N-terminal amino acid sequences and biological characteristics. Genes encoding the Dsu. vulgaris (Hildenborough) iron hydrogenase have been cloned (into Escherichia coli) and characterized by Voordouw & Brenner (1985) and Voordouw et al. (1985). Although both subunits characteristic of the hydrogenase were expressed in E. coli, the gene product was not formed in the recombinant (Voordouw et al. 1987a). An

Sulphate-reducing bacteria

783

essential iron sulphur cluster in the catalytic domain of the enzyme was missing. Furthermore, van Dongen et al. (1988) observed that whilst the Dsu. uulgaris hydrogenase was periplasmically located, the enzyme in Escherichiu coli was largely confined to the cytoplasm (larger subunit) or membrane (smaller subunit). Nevertheless, Voordouw et al. (1987b) used cloned hydrogenase genes of Dsu. uulgaris to probe the genome of other desulfovibrios and concluded that the enzymes were chromosomal in origin. More recently, another gene from Dsu. uulgaris (hydy) was cloned into E . coli by Stokkermans et al. (1989). Although the protein encoded by hydy was similar in terms of amino acid sequence to the hydrogenase enzyme, the authors were unable to determine its functional role. Genes encoding the periplasmic nickel-iron hydrogenase of Dsu. gigas (Li et al. 1987) and the nickel-selenium-iron hydrogenase of Dsu. baculatus (Menon et al. 1987) have both been cloned into E. coli. Successful cloning of the hydrogenase genes of sulphate-reducing bacteria has increased knowledge of its enzymic physiological and biochemical properties. These are summarized by Fauque et al. (1988) and Voordouw (1988; 1990). The genes which encode flavodoxin (Curley & Voordouw 1988) and cytochrome c3 (Voordouw & Brenner 1986; Voordouw et al. 1987b) have also been cloned and sequenced. The above observations along with the complementation of pyr F (Li et al. 1986) and pro (Fons et al. 1987) mutations in E. coli using DNA from desulfovibrios, suggest that cloning systems in the facultatively anaerobic organism are extremely useful for further studies of the molecuiar biology of sulphate-reducing bacteria. Another important area of genetic research involving these bacteria has been the exploitation of the diazotrophic potential of desulfovibrios. Postgate et al. (1986) showed that the genomic DNA of 13 Desulfouibrio strains was homologous to nitrogen fixation (nif) DNA from Klebsiella pneumoniue. DNA from three non-diazotrophic sulphate reducers were non-homologous. In three Dsu. uulgaris strains, the nif DNA was plasmid derived. Postgate et al. (1988) summarizes plasmids in species of Desulfouibrio which carry nif DNA. The recent study of Powell et al. (1989) demonstrated that a broad host range antibiotic resistance plasmid could be transferred into two strains of Desulfouibrio. Similarly, Rapp & Wall (1989) attained conjugal transfer of Q incompatibility group plasmids from E. coli to Dsu. desulfuricans with an acquired resistance to neomycin or streptomycin occurring. Although genetic research of sulphatereducing bacteria has only begun to proliferate in recent years, a number of important studies have shown that recombinant DNA technology may be applied to these micro-organisms. Future research should result in a rapid acquisition of new information regarding the metabolism of sulphate reducers.

3. Ecology
3.1
HABITATS

Because of their high sulphate content, the anaerobic regions of marine and estuarine sediments as well as saline ponds are primary sites for the growth of sulphate-reducing bacteria (Triiper et al. 1969). Carbon sources for these bacteria can be derived from the activities of heterotrophic microorganisms in the water column overlying the sediments, or from a direct organic influence, especially those habitats which are contaminated by a secondary nutrient input such as sewage emuent. Many of the species isolated by Widdel (1980) employed the use of anaerobic muds derived from brackish water and marine environments as inocula. Although these bacteria are strictly anaerobic, their presence has been detected in many ostensibly aerobic regions. For example, Battersby et al. (1985) detected low sulphate reduction rates in oxic North-East Atlantic sediments. The presence of sulphate-reducing bacteria may be associated with the anaerobic reduced microniches described by Jsrgensen (1977b) but Hardy (198 1) enumerated and isolated sulphate-reducing bacteria from North Sea waters. The economic significance of this observation is that high pressure injection of seawater into exhausted oil reservoirs is used to disturb residual oil causing an increased (secondary) recovery. However, if the water contains barotolerant sulphate reducers, the oil will be soured. Stott & Herbert (1986) have discussed how the addition of biocides with enhanced activities at high temperature and pressure can alleviate this problem.

784

G. R . Gibson

7
a b

Fig. 3. Substrates metabolized by sulphate-reducing bacteria in the human large intestine (n = 17). Results show mean values +S.E. mean. a, acetate; b, lactate; c, propionate; d, butyrate; e, H,/CO,; f, succinate; g, valerate; h, ethanol; i, pyruvate;j, Glu/Ser/Ala.

A number of sulphate-reducing bacteria are able to grow in non-saline environments (saltrequiring species are shown in Table 1). Active sulphate reduction has been demonstrated in freshwater sediments (e.g. Ingvorsen et al. 1981; Smith & Klug 1981; Cappenberg & Verdouw 1982; Jones & Simon 1984; Herlihy & Mills 1985; Hordijk et al. 1985). Soils are also potential habitats (Adams & Postgate 1961). Desulfotornaculurn sapornandens (Cord-Ruwisch & Garcia 1985) was isolated from putatively aerobic gasoline-contaminated soil. Thus, these bacteria are particularly ubiquitous in both aquatic and terrestrial ecosystems. Other habitats in which sulphate-reducing bacteria have been detected include polluted environments such as sour whey digesters (Zellner et al. 1989a), spoiled foods (Campbell et al. 1957), anaerobic purification plants (Nanninga & Gottschal 1987) and sewage plants (Schoberth 1973; Ueki et al. 1988). Barross & Deming (1983) extended the work of Zobell (1958) who showed that barotolerant strains could grow in deep sea vents. Some sulphate reducers are able to grow at temperatures below 5C (Zobell 1958), whilst at the opposite extreme, spore forming thermophilic species (able to grow at 65-80C) have aso been detected in deep aquifiers (Olson et al. 1981). Sulphate-reducing bacteria are able to multiply in the intestinal contents of man and animals. The occurrence of Desulfouibro spp. in ruminal contents has long been recognized (Coleman 1960; Huisingh et al. 1974). They have also been detected in the termite gut (Traore et al. 1990) and human faeces (Moore et al. 1976; Beerens & Romond 1977). More recently, Gibson et al. (1988a) demonstrated that over 40% of individuals tested in two different human population groups harboured significant numbers of intestinal sulphate-reducing bacteria. Substrates metabolized by the human colonic bacteria are shown in Fig. 3. The principal species present were identified as belonging to the genera Desulfouibrio, Desulfobacter, Desulfobulbus, Desulfornonas and Desulfotornaculurn.
3.2
I N T E R A C T I O N SW I T H OTHER BACTERIA

In aerobic environments, heterotrophic micro-organisms are able to carry out a complete mineralization of organic carbon to CO, . In anaerobic ecosystems, however, mineralization is much more

Sulphate-reducing bacteria
Polymeric molecules (eg proteins, Carbohydrates, lipids, nucleic acids) hydrolysis

785

Low molecular weight products chain fatty acids, etc ) fermentation

Fermentation intermediates (e.g alcohols, lactate, pyruvate, succinate)

t
~

Volatile f a t t y acids (acetate, propionate, butymte)

1 i'
S O : -

[-Acetate

1
so '4-

ab

mb

srb

mb

Fig. 4. Pathway of the anaerobic degradation of organic matter, showing potential interactions of sulphatereducing bacteria. (srb = sulphate-reducing bacteria; mb = methanogenic bacteria; ab = acetogenic bacteria).

complex and requires the interaction of number of different groups of micro-organisms (Vosjan 1982). Each microbial group performs a partial oxidation of the organic carbon available to it and their metabolic end products are in turn assimilated by the next member of the food chain until complete oxidation has occurred. Classically, the process involves the hydrolysis of large molecular weight compounds such as proteins, nucleic acids, carbohydrates and lipids to lower molecular weight products such as organic acids and alcohols. These may then be fermented into volatile fatty acids (acetate, propionate and butyrate) and gases (H,, CO,). Terminal oxidative processes are then able to degrade these substrates further (Fig. 4). In environments with a plentiful supply of sulphate, it is during these terminal stages that sulphate-reducing bacteria have a major role in the metabolism of organic detritus. If low molecular weight compounds such as acetate and hydrogen are allowed to accumulate, initial oxidative processes higher in the food chain are inhibited (Bryant 1976). Until the discovery of sulphatereducing bacteria which utilize acetate, it was thought that acetate oxidation occurred by methanogenic and sulphate-reducing bacteria acting in syntrophy (Cappenberg 1974; Cappenberg & Prins 1974). Indeed, in sulphate depleted ecosystems such as freshwater sediments, methanogens are mainly responsible for hydrogen and acetate utilization. However, in the presence of sulphate, it is clear that methanogenesis is inhibited (Martens & Berner 1974; Cappenberg 1975; Winfrey & Zeikus 1977, 1979; Oremland & Taylor 1978; Mountford et al. 1980). It is now agreed that sulphate reducers are

786

G . R. Gibson

able to outcompete methanogens for the mutual growth substrates hydrogen and acetate, since they have a higher affinity for these substrates (Bryant et al. 1977; Abram & Nedwell 1978a, b ; Oremland & Taylor 1978; King & Wiebe 1980; Kristjansson et al. 1982; Oremland & Polcin 1982; Schonheit et al. 1982; Robinson & Tiedje 1984). Consequently, the in situ concentrations of these substrates are maintained at levels below those a t which methanogens can effectively compete (Lovley et al. 1982). Low rates of methanogenesis can occur even in sulphate rich habitats (Senior et a!. 1982). A possible reason for this may be the fact that the amount of available electron donor reaches a high enough level for non-competitive utilization by both methanogenic and sulphate-reducing bacteria. In this respect, it has been shown that sparging of Tay Estuary sediment slurries with H,/CO, simultaneously stimulated methanogenesis and sulphate reduction (Gibson 1986). Alternatively, methanogens are able to utilize certain substrates such as methlyamines, which are not degraded by sulphate reducers. Competitive interactions between these two groups of bacteria can be further demonstrated by considering processes which occur in the human large intestine. For a number of years it was unclear to gastroenterologists why only a certain percentage (30-50%) of the population produce methane in breath, faeces or flatus (Bond et al. 1971). Methanogenic bacteria in the colon have an obligate requirement for hydrogen (Wolin & Miller 1983; Miller & Wolin 1985) and species that utilize acetate, formate or methylamine are not present. Recent work however, has indicated that the activities of sulphate-reducing bacteria and methanogenic bacteria are mutually exclusive in the gut (Gibson et al. 1988a), that is to say, methane excretion only occurs when sulphate reduction is absent or inactive. Further studies (Gibson et al. 1988b) demonstrated that this effect could partly be explained by a direct competitive influence of hydrogen utilizing sulphate reducers upon the methanogenic population, with the concentration of sulphate available in the colon being critical to the outcome of hydrogen utilization. In the gut, sulphate may be present in host secretions or dietary in origin. The potential of endogenous substrates such as highly sulphated glycoproteins to sustain rates of sulphate reduction high enough to inhibit methanogenesis was demonstrated by adding mucin (a highly sulphated polymer secreted by goblet cells of the intestinal epithelium) to a three-stage continuous culture model of the gut which was inoculated with faeces that contained predominantly methThe activity of anogenic bacteria, but also low numbers of sulphate reducers (Gibson et al. 1988~). sulphatase-containing fermentative bacteria releasing free sulphate from the mucopolysaccharide allowed an outcompetition of methanogens. More recent studies (Christ1 et al. 1990) have also demonstrated how diet can significantly affect these processes. Six methanogenic volunteers were fed a diet enhanced with sulphate (15 mmol/d) for three weeks. During this time, sulphate reduction and methanogenesis were continually monitored and compared with values obtained during a control diet. In half of the subjects tested, the addition of sulphate caused an inhibition of the activity of methanogenic bacteria, whilst sulphate reduction was concomitantly stimulated. In the other 50% of the volunteers, little difference in methane excretion rates occurred. These studies suggested that two types of methane-producing persons exist. One groups colonic microflora contains low and relatively inactive (in terms of hydrogen uptake), populations of sulphate-reducing bacteria, which may be growing fermentatively under normal circumstances. These bacteria become active when a supply of sulphate enters the colon, thus enabling them to metabolize hydrogen and so inhibit methanogenesis. Although dietary sulphate is partially absorbed in the small intestine, it is clear from these studies that a significant proportion reaches the large gut. In the other group of methanogenic individuals, sulphate-reducing bacteria are completely absent, thus the addition of exogenous sulphate does not affect the activity of methanogens in the short term. In a further study, (G. R. Gibson & J. H. Cummings, unpublished results) a nonmethanogenic volunteer was fed a sulphate-deficient diet for four weeks. During this time, sulphatereducing activity decreased whilst hydrogen uptake rates remained unaffected even though methanogenesis did not occur. This suggests a role for other hydrogen utilizing bacteria in the colon. The presence of homoacetogenic bacteria (Zeikus 1983) has been demonstrated in the human gut (Lajoie et al. 1988). We have previously shown, however, that relatively low activities of these bacteria occur in intestinal contents under normal circumstances, and that they only become significant when conditions unfavourable for methanogenesis or sulphate reduction prevail (Gibson et al. 1990).

Sulphate-reducing bacteria
3.3
SYNTROPHIC ASSOCIATIONS

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The majority of natural anaerobic ecosystems consist of a series of integrated microbial processes. It is possible, therefore, that a number of consortia and syntrophic bacterial associations will be allowed to develop. An example of this type of interaction can be demonstrated by considering relationships which occur in the biological sulphur cycle. Here, the sulphur atom, depending upon its oxidation state, can act either as an electron acceptor for the dissimilation of organic carbon and hydrogen, or as an electron donor for some autotrophic bacteria. Thus, a complex system can develop with inorganic sulphur compounds serving as electron carriers and the bacteria involved growing in syntrophic associations (Pfennig 1978; Nedwell 1982; Pfennig & Widdel 1982). Desulfouibrio baculatus was originally isolated from the consortium Chloropseudomonas ethylica which consisted of a sulphide oxidizing Chlorobium spp. (Gray 1977). Another potentially important in situ role for sulphate-reducing bacteria in anaerobic consortia is related to their ability to utilize hydrogen, although some acetate may be required for cell carbon synthesis (Badziong & Thauer 1978, 1980; Badziong et al. 1978, 1979: Brandis & Thauer 1981; Widdel & Pfennig 1982; Widdel 1987). These bacteria are able to interact with other microorganisms (proton-reducing acetogenic bacteria) which generate H, from the oxidation of organic matter, a process known as interspecies hydrogen transfer. In these consortia, the sulphate reducer is essential for the disposal of reducing equivalents. A number of examples have been cited. Syntrophomonas wolinii was isolated in co-culture with a hydrogen utilizing Desulfouibrio spp. (Boone & Bryant 1980) and is able to metabolize propionate, whilst Syntrophornonas wolfei (McInerney et al. 1981) utilizes butyrate in a similar manner. The studies of McInerney et al. (1979), Stams & Hansen (1984), Stieb & Schink (1985, 1986), Cord-Ruwisch & Oliver (1986), Dolfing & Tiedje (1986) and Dwyer et al. (1988) report similar interactions. Hydrogen-utilizing methanogenic bacteria are also able to take part in such consortia (Wolin 1976). Desulfovibrios have, by the use of a reversible hydrogenase enzyme, the ability to produce as well as utilize hydrogen during the degradation of organic matter (Odom & Peck 1981, 1984; Fauque et al. 1988). Thus, under certain circumstances, such as low electron acceptor availability, sulphatereducing bacteria are able to fermentatively yield hydrogen from some organic substrates. The hydrogen evolved may in turn be metabolized by syntrophic methanogens (Bryant et al. 1977). For example, a co-culture of Methanosarcina barkeri and Desulfouibrio spp. was able to degrade lactate in the absence of sulphate (McInerney & Bryant 1981; Traore et al. 1983). Similar, more recent, associations have reported the metabolism of 3-trimethoxybenzoate (Kriekenbohm & Pfennig 1985), tetrachloromethane and 1,2-dichloroethane (Eglie et al. 1987), chitin (Pel et al. 1989), furfural (Schoberth et al. 1990), glycerol (Qatibi et al. 1990a) and propanediol (Qatibi et al. 1990b). Environmentally, these consortia are important with respect to the energy gained from fermentation, as more oxidized substrates can be produced from the detrital input to a particular ecosystem.

4. Concluding remarks

This review has outlined some aspects of sulphate-reducing bacterial metabolism. However, as the search for new genera and species continues, it follows that opinions regarding their physiology, ecology, biochemistry and genetics will need to be constantly revised. The economic effects of dissimilatory sulphate reduction, whether detrimental or otherwise, are fairly diverse, however the current understanding is that sulphate-reducing bacteria are not involved in any pathogenic mechanism towards man, although Postgate (1984) did cite one example of bacteriaemic Desulfouibrio strains in an unspecified clinical condition. The presence of high numbers of sulphate reducers (up to in excess of 10IO/g dry weight of faeces) in human intestinal contents has been demonstrated (Gibson et al. 1988a). Moreover, the toxicity of hydrogen sulphide is recognized (Postgate 1984) and it has been speculated that over-production of this gas in uiuo is related to the initiation of some colonic diseases, many of which are of unknown aetiology. The majority of large intestinal disorders arise in the left colon, and autopsy studies have shown that highest numbers and activities of sulphate reducers also

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occur in this region of the gut (Macfarlane et al. 1990), where the prevailing physical and chemical conditions favour their growth. Ulcerative colitis is a chronic inflammatory large bowel disease which usually occurs distally, but may spread up and throughout the colon. The disorder is characterized by sympton-free remissions between acute relapses when the patient suffers severe abdominal pain and frequently passes watery diarrhoea. Faeces contains excessive levels of mucin, blood and pus which may stain stools (Thomas 1980). The disease is of unknown aetiology, however bacterial involvement has been implicated (Onderdonk 1983). An unidentified micro-organism may have a triggering effect whereby a cascade of clinical features and subsequent immune response occurs. Animal models where guinea pigs were fed the sulphated polysaccharide carrageenan, caused a stimulation of colitis-like lesions (Marcus & Watt 1969; Watt & Marcus 1971). In a recent study (Florin et al. 1990) we measured counts and activities of sulphate-reducing bacteria in the gut contents of 24 persons with ulcerative colitis. It was found that virtually all the patients (96%) carried colonic sulphate reducers and that faecal sulphide concentrations were significantly higher than those detected in a similar control group of healthy individuals. Although there is no positive evidence to associate these bacteria with the initiation of the disease, we have found (unpublished results), that some sulphate reducers in uitro are able to survive some of the clinical symptoms of ulcerative colitis. A physical manifestation of the disease is the continuous flushing of the bowel with watery stools. This will have the dual effect of diluting substrates for sulphatereducing bacterial growth, as well as washing out bacteria from the colon. We therefore compared the ability of sulphate reducers from the healthy and colitis bowel to withstand these effects in uitro. Chemostat studies demonstrated that ulcerative colitis associated bacteria were able to grow at high dilution rates which did not allow the growth of control cultures. Moreover, Desulfouibrio spp. from diseased persons had a higher affinity for limiting amounts of sulphate compared to standard isolates. A number of questions arise from these studies:

1. Are sulphate-reducing bacteria directly involved in the aetiology of ulcerative colitis, or is the disease caused by another unidentified mechanism which is responsible for colonocyte damage, with sulphate reduction as an irrelevant consequence? 2. Do individuals who develop the disease lack the necessary absorptive bacteriological or chemical means to dispose of excess H,S? 3. Are sulphatase producing saccharolytic bacteria that ferment sulphated polysaccharides (e.g. mucins, chondroitin sulphate) able to release enough free sulphate to stimulate sulphate reducer growth, and are they prevalent in diseased patients? 4. Is the degree of sulphate availability, either from endogenous or dietary sources a contributing factor ?
It should be stressed that this research is currently at a preliminary stage, However, further studies are currently under way in our laboratory to determine whether sulphate-reducing bacteria play a role in this disease. The implication with respect to the sulphate-reducing bacteria is that the biological potential of this group of micro-organisms may not yet be fully recognized.
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