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Quinones and humus are ubiquitous in the biosphere and play an important role in the anaerobic biodegradation and biotransformation of organic acids, poisonous compounds as well as inorganic compounds. The impact of humic model compound, anthraquinone-2, 6-disulfonate (AQDS) on anaerobic phenol and p-cresol degradation were studied. Four methanogenic AQDS-free phenol and p-cresol enrichments and two phenol-AQDS enrichments were obtained using two sludges with potential biodegradability of phenol and cresol isomers as inoculum. 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that syntrophic aromatic compound degrading bacterium Syntrophorhabdus aromaticivorans was dominant in four AQDS-free enrichments, whereas phenol degrading Cryptanaerobacter phenolicus was dominant in two phenol-AQDS enrichments. Neither co-culture of S. aromaticivorans with Methanospirillum hungatei nor two phenol-AQDS enrichments could metabolize phenol using AQDS as the terminal electron acceptor. Further degradation experiments suggested that C. phenolicus related microbes in two phenol-AQDS enrichments were responsible for the conversion of phenol to benzoate, and benzoate was further degraded by benzoate degraders of Syntrophus aciditrophicus or Sporotomaculum syntrophicum to acetate. HIGHLIGHTS Anaerobic degradation of phenol and p-cresol were faster than o- and m-cresols.; 2 mM AQDS mitigated phenol and completely inhibited p-cresol degradation.; The dominant bacteria in AQDS-free and phenol-AQDS enrichments were different.; Neither S. aromaticivorans nor C. phenolicus affinities could use AQDS as TEA.; The characteristic of key phenol degraders in phenol-AQDS enrichments was described.

Five different inocula from cow manure, goat manure, pig manure, and rumen fluid (RU) and microbial sludge from anaerobic digestion were screened to select anaerobic lignocellulolytic microbial consortium (ALMC) of high efficacy in degradation of filter paper (FP), microcrystalline cellulose (MCC), and rice straw (RS) to produce methane. RU from Bubalus bubalis demonstrated the highest growth rate RU inoculum (10.45 mgVS/day); fastest degradation rate of FP, MCC, and RS (125.00, 50.07, and 25.43 mgVS/day); and highest methane production rate (6.23 ml/day). To obtain the enriched and stabilized ALMC from RU, consecutive batch subculturing was used and reached at 25 passages, namely, E25. The efficacies of E25 in RS removal and methane production were 78%VS and 293 ml/gVSadded compared with initial inoculum (E0) at 46% VS and 138 ml/gVSadded, respectively, within 14 days of incubation time. The efficacies of E25 in RS removal and methane production were 1.7- and 2.12-folds higher than of E0, respectively. E25 showed high ability to utilize various carbon sources (68%) from Biolog EcoPlate assay. It was confirmed that E25 could degrade derivatives of cellulose, hemicellulose, and lignin. Metagenomic analysis by 16 s and shotgun sequencing of E25 found dominant species of hydrolytic/fermentative bacteria (Proteiniphilum acetatigenes, Pyramidobacter piscolens, Syntrophus aciditrophicus, Mesotoga prima, and Candidatus Cloacimonas acidaminovorans) and methanogens (Methanoculleus bourgensis, Methanoculleus marisnigri, Methanofollis liminatans, Methanosarcina mazei, and Methanosaeta harundinacea). This stabilized E25 with enrichment of lignocellulolytic and fermentative bacteria as well as hydrogenotrophic and acetoclastic methanogens shows its efficacy in accelerating RS degradation and methane production.

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron transfer because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Heterologous expression of the putative S. aciditrophicus type IV pilin gene in Geobacter sulfurreducens yielded conductive pili of the same diameter (4 nm) and conductance of the native S. aciditrophicus pili and enabled long-range electron transport in G. sulfurreducens. S. aciditrophicus lacked abundant c-type cytochromes often associated with DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.Access opt

A non-bifurcating NADH-dependent, dimeric [FeFe]-hydrogenase (HydAB) from Syntrophus aciditrophicus was heterologously produced in Escherichia coli, purified and characterized. Purified recombinant HydAB catalyzed NAD+ reduction coupled to hydrogen oxidation and produced hydrogen from NADH without the involvement of ferredoxin. Hydrogen partial pressures (2.2–40.2 Pa) produced by the purified recombinant HydAB at NADH to NAD+ ratios of 1–5 were similar to the hydrogen partial pressures generated by pure and cocultures of S. aciditrophicus (5.9–36.6 Pa). Thus, the hydrogen partial pressures observed in metabolizing cultures and cocultures of S. aciditrophicus can be generated by HydAB if S. aciditrophicus maintains NADH to NAD+ ratios greater than one. The flavin-containing beta subunits from S. aciditrophicus HydAB and the non-bifurcating NADH-dependent S. wolfei Hyd1ABC share a number of conserved residues with the flavin-containing beta subunits from non-bifurcating NADH-dependent enzymes such as NADH:quinone oxidoreductases and formate dehydrogenases. A number of differences were observed between sequences of these non-bifurcating NADH-dependent enzymes and [FeFe]-hydrogenases and formate dehydrogenases known to catalyze electron bifurcation including differences in the number of [Fe-S] centers and in conserved residues near predicted cofactor binding sites. These differences can be used to distinguish members of these two groups of enzymes and may be relevant to the differences in ferredoxin-dependence and ability to mediate electron-bifurcation. These results show that two phylogenetically distinct syntrophic fatty acid-oxidizing bacteria, Syntrophomonas wolfei a member of the phylum Firmicutes, and S. aciditrophicus, a member of the class Deltaproteobacteria, possess functionally similar [FeFe]-hydrogenases that produce hydrogen from NADH during syntrophic fatty acid oxidation without the involvement of reduced ferredoxin. The reliance on a non-bifurcating NADH-dependent [FeFe]-hydrogenases may explain the obligate requirement that many syntrophic metabolizers have for a hydrogen-using partner microorganism when grown on fatty, aromatic and alicyclic acids.

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

Short-chain fatty acids such as acetic, propionic, butyric or lactic acids are typical primary fermentation products in the anaerobic feeding chain. Fifteen years ago, a novel fermentation type was discovered in the obligately anaerobic Deltaproteobacterium Syntrophus aciditrophicus. During fermentative growth with crotonate and/or benzoate, acetate is formed in the oxidative branch and cyclohexane carboxylate in the reductive branch. In both cases cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) is a central intermediate that is either formed by a class II benzoyl-CoA reductase (fermentation of benzoate) or by reverse reactions of the benzoyl-CoA degradation pathway (fermentation of crotonate). Here, we summarize the current knowledge of the enzymology involved in fermentations yielding cyclohexane carboxylate as an excreted product. The characteristic enzymes involved are two acyl-CoA dehydrogenases specifically acting on Ch1,5CoA and cyclohex-1-ene-1-carboxyl-CoA. Both enzymes are also employed during the syntrophic growth of S. aciditrophicus with cyclohexane carboxylate as the carbon source in coculture with a methanogen. An investigation of anabolic pathways in S. aciditrophicus revealed a rather unusual pathway for glutamate synthesis involving a Re-citrate synthase. Future work has to address the unresolved question concerning which components are involved in reoxidation of the NADH formed in the oxidative branch of the unique cyclohexane carboxylate fermentation pathway in S. aciditrophicus.

The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase., IMPORTANCE Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.

In the denitrifying bacterium Thauera aromatica , the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of β-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA ( K m , 80 and 35 μM; V max , 350 and 550 μmol min −1 mg −1 , respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 ± 0.24 mol of acetate and 0.16 ± 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U- 13 C]crotonate was metabolized to [1,2- 13 C]acetate and [1,2,3,4,5,7- 13 C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [ 13 C]sodium bicarbonate formed [6- 13 C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U- 13 C]crotonate, the m/z -15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z -15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [ 13 C]sodium bicarbonate and unlabeled crotonate, the m/z -15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2- 13 C]acetate and unlabeled crotonate, the m/z -15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z -15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO 2 addition, rather than the use of the intact four-carbon skeleton of crotonate.

Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Summary We used a combination of genomic, transcriptional and enzymatic analyses to determine the mechanism of interspecies electron transfer by two model syntrophic microorganisms, Syntrophomonas wolfei and Syntrophus aciditrophicus. Both organisms contain multiple hydrogenase and formate dehydrogenase genes, but lack genes for outer membrane cytochromes and nanowire formation. Syntrophically grown cells and cell-free extracts of S. aciditrophicus and S. wolfei had both hydrogenase and formate dehydrogenase activities. Butyrate metabolism and CH4 production by washed cell suspensions of S. wolfei and Methanospirillum hungatei were inhibited by hydrogenase inhibitors (cyanide and carbon monoxide), but not by a formate dehydrogenase inhibitor (hypophosphite). Syntrophic benzoate oxidation and CH4 production by washed cell suspensions of S. aciditrophicus and M. hungatei were inhibited by hypophosphite, but not cyanide and carbon monoxide. All three inhibitors halted syntrophic cyclohexane-1-carboxylate metabolism. Two hydrogenase genes, hydA1 and hydA2, were more highly expressed when S. wolfei was grown syntrophically. S. aciditrophicus expressed multiple hydrogenase and formate dehydrogenase genes during syntrophic benzoate and cyclohexane-1-carboxylate growth, one of which (fdhA2) was highly differentially expressed during syntrophic benzoate growth. Thus, these syntrophic microorganisms have flexible metabolisms that allow them to use either H2 or formate transfer depending on the substrate involved.

Transformations of 2-hydroxybenzoate and fluorobenzoate isomers were investigated in the strictly anaerobic Syntrophus aciditrophicus to gain insight into the initial steps of the metabolism of aromatic acids. 2-Hydroxybenzoate was metabolized to methane and acetate by S. aciditrophicus and Methanospirillum hungatei cocultures and reduced to cyclohexane carboxylate by pure cultures of S. aciditrophicus when grown in the presence of crotonate. Under both conditions, transient accumulation of benzoate but not phenol was observed, indicating that dehydroxylation occurred prior to ring reduction. Pure cultures of S. aciditrophicus reductively dehalogenated 3-fluorobenzoate with the stoichiometric accumulation of benzoate and fluorine. 3-Fluorobenzoate-degrading cultures produced a metabolite that had a fragmentation pattern almost identical to that of the trimethylsilyl (TMS) derivative of 3-fluorobenzoate but with a mass increase of 2 units. When cells were incubated with deuterated water, this metabolite had a mass increase of 3 or 4 units relative to the TMS derivative of 3-fluorobenzoate. 19 F nuclear magnetic resonance spectroscopy ( 19 F NMR) detected a metabolite in fluorobenzoate-degrading cultures with two double bonds, either 1-carboxyl-3-fluoro-2,6-cyclohexadiene or 1-carboxyl-3-fluoro-3,6-cyclohexadiene. The mass spectral and NMR data are consistent with the addition of two hydrogen or deuterium atoms to 3-fluorobenzoate, forming a 3-fluorocyclohexadiene metabolite. The production of a diene metabolite provides evidence that S. aciditrophicus contains dearomatizing reductase that uses two electrons to dearomatize the aromatic ring.

In methanogenic environments, the main fate of benzoate is its oxidization to acetate, H(2) and CO(2) by syntrophic associations of hydrogen-producing benzoate degraders and hydrogen-using methanogens. Here, we report the use of benzoate as an electron acceptor. Pure cultures of S. aciditrophicus simultaneously degraded crotonate and benzoate when both substrates were present. The growth rate was 0.007 h(-1) with crotonate and benzoate present compared with 0.025 h(-1) with crotonate alone. After 8 days of incubation, 4.12 +/- 0.50 mM of cyclohexane carboxylate and 8.40 +/- 0.61 mM of acetate were formed and 4.0 +/- 0.04 mM of benzoate and 4.8 +/- 0.5 mM of crotonate were consumed. The molar growth yield was 22.7 +/- 2.1 g (dry wt) of cells per mol of crotonate compared with about 14.0 +/- 0.1 g (dry wt) of cells per mol of crotonate when S. aciditrophicus was grown with crotonate alone. Cultures grown with [ring-(13)C]-benzoate and unlabelled crotonate initially formed [ring-(13)C]-labelled cyclohexane carboxylate. No (13)C-labelled acetate was detected. In addition to cyclohexane carboxylate, (13)C-labelled cyclohex-1-ene carboxylate was detected as an intermediate. Once almost all of the benzoate was gone, carbon isotopic analyses showed that cyclohexane carboxylate was formed from both labelled and non-labelled metabolites. Glutarate and pimelate were also detected at this time and carbon isotopic analyses showed that each was made from a mixture labelled and non-labelled metabolites. The increase in molar growth yield with crotonate and benzoate and the formation of [ring-(13)C]-cyclohexane carboxylate from [ring-(13)C]-benzoate in the presence of crotonate are consistent with benzoate serving as an electron acceptor.

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S . aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (Δ G ′) of −9.2 kJ/mol was reached (−4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO 2 , and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than −20 kJ/mol, the postulated minimum free energy value for substrate metabolism.

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by “ Syntrophus aciditrophicus ” in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. 13 C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ ring - 13 C 6 ]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 μM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of “ S. aciditrophicus ” and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of “ S. aciditrophicus ” grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of “ S. aciditrophicus ”- Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of “ S. aciditrophicus ”. These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris .

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.

Anaerobic bacteria, particularly the species Syntrophus aciditrophicus, that dwell in oil reservoirs are key to converting crude oil to methane, according to a consortium of researchers from Canada, the United Kingdom, and Norway. If those bacterial processes could be accelerated while also harnessed, it could lead to environmentally friendly means for converting heavy oil deposits into extractable and more readily transported gases for use as cleaner fuels, the researchers suggest in their report in the December 12, 2007 online edition of Nature.

The sensitivity of the requirement of Methanobacterium ruminantium strain M1 to a new coenzyme, 2-mercaptoethanesulfonic acid (HS-CoM) was examined by use of new techniques that were developed for rapid and efficient handling of large numbers of cultures of methanogenic bacteria. The system uses sealed tubes that contain a gas mixture of 80% hydrogen and 20% carbon dioxide under a pressure of 2 to 3 atm. This modification of the Hungate technique reduces variability among replicate cultures and simplifies the dispensing, sterilization, and storage of liquid media as well as the transfer and maintenance of methanogenic bacteria. Results indicate a limit of sensitivity of the assay at 5 nM HS-CoM, with half-maximal growth at 25 nM HS-CoM. Coenzyme activity could be replaced by 2,2'-dithiodiethanesulfonic acid at a half-molar equivalent of the HS-CoM concentration, or by 2-(methylthio)ethanesulfonic acid on an equimolar basis. These data reveal a very sensitive and precise requirement for HS-CoM in the nutrition of this fastidious anaerobe.