Methanogenesis from furfural by defined mixed cultures

Methanogenesis from furfural by defined mixed cultures was studied. Under sulfate-reducing conditions, a Desulfovibrio strain was used as the furfural-degrading species producing acetic acid. This sulfate-reducing bacterium (SRB) Desulfovibrio strain B is an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. Introduction of acetate-utilizing methanogenic archaeon Methanosarcina barkeri 227 converted acetic acid to methane. This well-defined mixed consortium used furfural as its sole source of carbon and converted it to methane and CO₂. In the mixed culture, when a methanogen inhibitor was used in the culture medium, furfural was converted to acetic acid by the Desulfovibrio strain B, but acetic acid did not undergo further metabolism. On the other hand, when the growth of Desulfovibrio in the consortium was suppressed with a specific SRB inhibitor, namely molybdate, furfural was not degraded. Thus, the metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural. Received: 15 August 2001 / Accepted: 20 September 2001     

Inhibition Experiments on Anaerobic Methane Oxidation

Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria.     

Anaerobic degradation of volatile fatty acids at different sulphate concentrations

The effect of sulfate on the anaerobic breakdown of mixtures of acetate, propionate and butyrate at three different sulfate to fatty acid ratios was studied in upflow anaerobic sludge blanket reactors. Sludge characteristics were followed with time by means of sludge activity tests and by enumeration of the different physiological bacterial groups. At each sulfate concentration acetate was completely converted into methane and CO₂, and acetotrophic sulfate-reducing bacteria were not detected. Hydrogenotrophic methanogenic bacteria and hydrogenotrophic sulfate-reducing bacteria were present in high numbers in the sludge of all reactors. However, a complete conversion of H₂ by sulfate reducers was found in the reactor operated with excess sulfate. At higher sulfate concentrations, oxidation of propionate by sulfate-reducing bacteria became more important. Only under sulfate-limiting conditions did syntrophic propionate oxidizers out-compete propionate-degrading sulfate reducers. Remarkably, syntrophic butyrate oxidizers were well able to compete with sulfate reducers for the available butyrate, even with an excess of sulfate. Correspondence to: A. Visser     

Inhibition of hydrogen sulfide, methane, and total gas production and sulfate-reducing bacteria in in vitro swine manure by tannins, with focus on condensed quebracho tannins

Management practices from large-scale swine production facilities have resulted in the increased collection and storage of manure for off-season fertilization use. Odor and emissions produced during storage have increased the tension among rural neighbors and among urban and rural residents. Production of these compounds from stored manure is the result of microbial activity of the anaerobic bacteria populations during storage. In the current study, the inhibitory effects of condensed quebracho tannins on in vitro swine manure for reduction of microbial activity and reduced production of gaseous emissions, including the toxic odorant hydrogen sulfide produced by sulfate-reducing bacteria (SRB), was examined. Swine manure was collected from a local swine facility, diluted in anaerobic buffer, and mixed with 1 %?w/v fresh feces. This slurry was combined with quebracho tannins, and total gas and hydrogen sulfide production was monitored over time. Aliquots were removed periodically for isolation of DNA to measure the SRB populations using quantitative PCR. Addition of tannins reduced overall gas, hydrogen sulfide, and methane production by greater than 90 % after 7 days of treatment and continued to at least 28 days. SRB population was also significantly decreased by tannin addition. qRT-PCR of 16S rDNA bacteria genes showed that the total bacterial population was also decreased in these incubations. These results indicate that the tannins elicited a collective effect on the bacterial population and also suggest a reduction in the population of methanogenic microorganisms as demonstrated by reduced methane production in these experiments. Such a generalized effect could be extrapolated to a reduction in other odor-associated emissions during manure storage.     

Growth and Activities of Sulfate-Reducing and Methanogenic Bacteria in Human Oral Cavity

Viable counts and activities of sulfate-reducing bacteria (SRB) and methanogenic bacteria were determined in the oral cavities of eight volunteers. Of these, seven harbored viable SRB populations, and six harbored viable methanogenic bacterial populations. Two volunteers classified as type III periodontal patients had both SRB and methanogenic bacteria. Six separate sites were sampled: posterior tongue, anterior tongue, mid-buccal mucosa, vestibular mucosa, supragingival plaque, and subgingival plaque. The SRB was found in all areas in one volunteer, and it was mostly present in posterior tongue, anterior tongue, supragingival, and subgingival plaques in many volunteers. The methanogenic bacteria were mostly found in supragingival and subgingival plaques. The activities of sulfate reduction and methane production were determined in randomly selected isolates. Received: 27 July 2002 / Accepted: 27 August 2002     

Toxicity of nitrite toward mesophilic and thermophilic sulphate-reducing, methanogenic and syntrophic populations in anaerobic sludge

The various problems associated with treating sulphate-containing wastewaters stem inherently from successful competitive interactions between sulphate reducing bacteria (SRB) and other bacteria involved in the process, resulting in the formation of H₂S. Prevention of in-reactor sulphide generation by use of specific SRB inhibitors presents a potential solution. Nitrite has been reported to be a specific inhibitor of SRB but its possible toxicity to syntrophic and methanogenic members of the anaerobic consortium has not been investigated. In batch activity and toxicity tests, under both mesophilic and thermophilic conditions, nitrite, at concentrations of up to 150 mg L^–1, was found to be ineffective as a specific inhibitor of SRB, and was also shown to have an inhibitory effect on the activity of syntrophic and methane-producing bacteria in mesophilic and thermophilic digester sludge samples.     

Metabolism of low molecular weight organic compounds by sulfate-reducing bacteria in a Delaware salt marsh

Oxidation of acetate, lactate, pyruvate, and ethanol to CO₂ in anaerobic salt marsh sediments was rapid, with the oxidation rate being significantly inhibited (60–90% decrease) in the presence of 2 mM sodium molybdate, an inhibitor of sulfate-reducing bacteria (SRB). 2-Bromoethanesulfonic acid (BES), an inhibitor of methanogenic bacteria, generally had no effect on the oxidation rate. Acetate was the only intermediate product detected in the oxidation of lactate and ethanol. Competition studies with lactate, acetate, and ethanol indicated that the preferred order of substrate utilization was lactate, then acetate, then ethanol. The turnover times of these three compounds in salt marsh sediments via the combined CO₂ plus acetate pool was rapid (10–13 hours) with a two- to threefold increase in the turnover time in the presence of molybdate. These results strongly suggest that SRB play a major role in the terminal metabolism of low molecular weight organic compounds in anaerobic salt marsh sediment.     

Isolation and characterization of a furfural degrading sulfate-reducing bacterium from an anaerobic digester

A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO₂. This SRB isolate could use furfural, furfuryl alcohol, and 2-furoic acid as sole source of carbon and energy in a defined mineral sulfate medium. Acetic acid was the major end product of furfural degradation. This organism also used wide varieties of other carbon sources, including ethanol, pyruvate, lactate, succinate, propanol, formate, and malate. The SRB isolate contained the electron carrier desulfoviridin. It used SO₄, NO₃, and thiosulfate as electron acceptors. This isolate used ammonium chloride, nitrate and glutamate as nitrogen source. The characteristics of the SRB isolate were closely similar toDesulfovibrio sp.     

Anaerobic degradation of m-cresol in anoxic aquifer slurries: carboxylation reactions in a sulfate-reducing bacterial enrichment

The anaerobic biodegradation of m-cresol was observed in anoxic aquifer slurries kept under both sulfate-reducing and nitrate-reducing but not methanogenic conditions. More than 85% of the parent substrate (300 microM) was consumed in less than 6 days in slurries kept under the former two conditions. No appreciable loss of the compound from the corresponding autoclaved controls was measurable. A bacterial consortium was enriched from the slurries for its ability to metabolize m-cresol under sulfate-reducing conditions. Metabolism in this enrichment culture was inhibited in the presence of oxygen or molybdate (500 microM) and in the absence of sulfate but was unaffected by bromoethanesulfonic acid. The consortium consumed 3.63 mol of sulfate per mol of m-cresol degraded. This stoichiometry is about 87% of that theoretically expected and suggests that m-cresol was largely mineralized. Resting-cell experiments demonstrated that the degradation of m-cresol proceeded only in the presence of bicarbonate. 4-Hydroxy-2-methylbenzoic acid and acetate were detected as transient intermediates. Thus, the parent substrate was initially carboxylated as the primary degradative event. The sulfate-reducing consortium could also decarboxylate p- but not m-hydroxybenzoate to near stoichiometric amounts of phenol, but this reaction was not sulfate dependent. The presence of p-hydroxybenzoate in the medium temporarily inhibited m-cresol metabolism such that the former compound was metabolized prior to the latter and phenol was degraded in a sequential manner. These findings help clarify the fate of a common groundwater contaminant under sulfate-reducing conditions.     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.     

Anaerobic degradation of m-cresol in anoxic aquifer slurries: carboxylation reactions in a sulfate-reducing bacterial enrichment.

The anaerobic biodegradation of m-cresol was observed in anoxic aquifer slurries kept under both sulfate-reducing and nitrate-reducing but not methanogenic conditions. More than 85% of the parent substrate (300 microM) was consumed in less than 6 days in slurries kept under the former two conditions. No appreciable loss of the compound from the corresponding autoclaved controls was measurable. A bacterial consortium was enriched from the slurries for its ability to metabolize m-cresol under sulfate-reducing conditions. Metabolism in this enrichment culture was inhibited in the presence of oxygen or molybdate (500 microM) and in the absence of sulfate but was unaffected by bromoethanesulfonic acid. The consortium consumed 3.63 mol of sulfate per mol of m-cresol degraded. This stoichiometry is about 87% of that theoretically expected and suggests that m-cresol was largely mineralized. Resting-cell experiments demonstrated that the degradation of m-cresol proceeded only in the presence of bicarbonate. 4-Hydroxy-2-methylbenzoic acid and acetate were detected as transient intermediates. Thus, the parent substrate was initially carboxylated as the primary degradative event. The sulfate-reducing consortium could also decarboxylate p- but not m-hydroxybenzoate to near stoichiometric amounts of phenol, but this reaction was not sulfate dependent. The presence of p-hydroxybenzoate in the medium temporarily inhibited m-cresol metabolism such that the former compound was metabolized prior to the latter and phenol was degraded in a sequential manner. These findings help clarify the fate of a common groundwater contaminant under sulfate-reducing conditions.     

Characterization of Anaerobic Dechlorinating Consortia Derived from Aquatic Sediments

Four methanogenic consortia which degraded 2-chlorophenol, 3-chlorophenol, 2-chlorobenzoate, and 3-chlorobenzoate, respectively, and one nitrate-reducing consortium which degraded 3-chlorobenzoate were characterized. Degradative activity in these consortia was maintained by laboratory transfer for over 2 years. In the methanogenic consortia, the aromatic ring was dechlorinated before mineralization to methane and carbon dioxide. After dechlorination, the chlorophenol consortia converted phenol to benzoate before mineralization. All methanogenic consortia degraded both phenol and benzoate. The 3-chlorophenol and 3-chlorobenzoate consortia also degraded 2-chlorophenol. No other cross-acclimation to monochlorophenols or monochlorobenzoates was detected in the methanogenic consortia. The consortium which required nitrate for the degradation of 3-chlorobenzoate degraded benzoate and 4-chlorobenzoate anaerobically in the presence of KNO₃, but not in its absence. This consortium also degraded benzoate, but not 3-chlorobenzoate, aerobically.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Formation of Aniline as a Transient Metabolite During the Metabolism of Tetryl by a Sulfate-Reducing Bacterial Consortium

A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenylmethylnitramine) can be degraded by an anaerobic process. The results indicated that the metabolic conversion of tetryl to aniline is possible by a sulfate-reducing bacterial (SRB) consortium. This SRB consortium metabolized tetryl by co-metabolism with pyruvate as a growth substrate. For every mole of tetryl metabolized, 1 mole of aniline was produced, and the aniline was further metabolized. This metabolic conversion of tetryl is likely to be of value in the anaerobic treatment of tetryl-contaminated soil and ground water, such as found at many military ammunition sites. Received: 18 August 1999 / Accepted: 15 September 1999     

Methanogenic and Other Strictly Anaerobic Bacteria in Desert Soil and Other Oxic Soils

Strictly anaerobic bacteria such as methanogenic, sulfate-reducing, and homoacetogenic bacteria could be enriched from all five oxic soils tested. The number of cells was lower than that in typical anoxic habitats. Spores did not always dominate the population of sulfate-reducing bacteria. In all soils, the methanogenic population displayed a long lag phase after anoxic conditions were imposed before methane production began.     

Growth of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a High-Pressure Membrane Capsule Bioreactor

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-μm-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.     

Sulfate reduction with methanol by a thermophilic consortium obtained from a methanogenic reactor

 An enrichment culture obtained from anaerobic granular sludge of a bench-scale anaerobic reactor degraded methanol at 65°C via sulfate reduction and acetogenesis. Sulfate reduction was the dominant process (S^2-/acetate=2.5). No methane formation was observed. Approximately 30% of the methanol was converted by acetogenic bacteria to acetate, while the remainder was degraded by these bacteria to H₂ and CO₂ in syntrophy with hydrogen-consuming sulfate-reducing bacteria. Pure cultures of sulfate-reducing and acetogenic bacteria were isolated and characterized. Received: 4 December 1995 / Received revision: 15 April 1996 / Accepted: 22 April 1996     

Effects of Sulfuroxy Anions on Degradation of Pentachlorophenol by a Methanogenic Enrichment Culture

We studied the degradation of pentachlorophenol (PCP) under methanogenic and sulfate-reducing conditions with an anaerobic mixed culture derived from sewage sludge. The consortium degraded PCP via 2,3,4,5-tetrachlorophenol, 3,4,5-trichlorophenol, and 3,5-dichlorophenol and eventually accumulated 3-chlorophenol. Dechlorination of PCP and metabolites was inhibited in the presence of sulfate, thiosulfate, and sulfite. A decrease in the rate of PCP transformation was noted when the endogenous dissolved H₂ was depleted below 0.11 μM in sulfate-reducing cultures. The effect on dechlorination observed with sulfate could be relieved by addition of molybdate, a competitive inhibitor of sulfate reduction. Addition of H₂ reduced the inhibition observed with sulfuroxy anions. The inhibitory effect of sulfuroxy anions may be due to a competition for H₂ between sulfate reduction and dechlorination. When cultured under methanogenic conditions, the consortium degraded several chlorinated and brominated phenols.     

Transformation of Phenol into Phenylalanine by a Methanogenic Consortium

The conversion by a methanogenic consortium of phenol into phenylalanine, with benzoic and phenylpropionic acid as intermediates, was investigated. When (sup14)C-labelled phenol was fed to the consortium, the radioactivity was mostly transferred into methane and CO(inf2), but 4% of the radioactivity was found in the water fraction after extraction of the culture medium with an organic solvent. Utilization of labelled compounds and analysis by gas chromatography coupled with mass spectrometry revealed that a fraction of the benzoic acid produced was transformed into 3-phenylpropionic acid. When fully (sup13)C-labelled acetic acid was fed to the consortium, the labels were incorporated at the 1 and 2 positions of 3-phenylpropionic acid. When deuterium-labelled 3-phenylpropionic acid was fed to the consortium, part of the phenylalanine of the biomass was labelled. These metabolic transformations are reversible, since deuterium-labelled phenylalanine generated labelled 3-phenylpropionic acid. Cinnamic acid was also transformed into 3-phenylpropionic acid.     

Anaerobic degradation of benzoate to methane by a microbial consortium

A stabilized consortium of microbes which anaerobically degraded benzoate and produced CH₄ was established by inoculation of a benzoate-mineral salts medium with sewage sludge; the consortium was routinely subcultured anaerobically in this medium for 3 years. Acetate, formate, H₂ and CO₂ were identified as intermediates in the overall conversion of benzoate to CH₄ by the culture. Radioactivity was equally divided between the CH₄ and CO₂ from the degradation of uniformly ring-labeled [¹⁴C]benzoate. The methyl group of acetate was stoichiometrically converted to CH₄. Acetate, cyclohexanecarboxylate, 2-hydroxycyclohexanecarboxylate, o-hydroxybenzoic acid and pimelic acid were converted to CH₄ without a lag suggesting that benzoate was degraded by a reductive pathway. Addition of o-chlorobenzoate inhibited benzoate degradation but not acetate degradation or methane formation. Two methanogenic organisms were isolated from the mixed culture, neither organism was able to degrade benzoate, showing that the methanogenic bacteria served as terminal organisms of a metabolic food chain composed of several organisms. Removal of intermediates by the methanogenic bacteria provided thermodynamically favorable conditions for benzoate degradation.