Methanogenic Enrichment Culture Research Articles

A culture apparatus for maintaining H 2 at sub-nanomolar concentrations

We devised a microbial culture apparatus capable of maintaining sub-nanomolar H 2 concentrations. This apparatus provides a method for study of interspecies hydrogen transfer by externally fulfilling the thermodynamic requirement for low H 2 concentrations, thereby obviating the need for use of cocultures to study some forms of metabolism. The culture vessel is constructed of glass and operates by sparging a liquid culture with purified gases, thereby removing H 2 as it is produced. We used the culture apparatus to decouple a syntrophic association in an ethanol-consuming, methanogenic enrichment culture, allowing ethanol oxidation to dominate methane production. We also used the culture apparatus to grow pure cultures of the ethanol-oxidizing, proton-reducing Pelobacter acetylenicus (WoAcy 1), and to study the bioenergetics of growth.

Anaerobic Formation of the Aromatic Hydrocarbon p-Cymene from Monoterpenes by Methanogenic Enrichment Cultures

The fate of monoterpenes in methanogenic habitats was investigated in enrichment cultures using defined mineral media inoculated with activated sludge and R-(-) -alpha-phellandrene, (+)-2-carene, (-)-alpha-pinene, or (+)-sabinene. The anaerobic biodegradation of alpha-pinene and 2-carene supported methanogenesis. In contrast, consumption of alpha-phellandrene and sabinene was accompanied by formation of the aromatic hydrocarbon p-cymene. This aromatization reaction did not occur in pasteurized or autoclaved cultures or in the absence of microorganisms. The monoterpene gamma-terpinene accumulated transiently. We conclude that anaerobic microorganisms exist that are able to aromatize monoterpenes with a cyclohexadiene structure. Similar biological reactions may contribute to the formation of aromatic biomarker molecules, e.g., monoaromatic steroids and hopanoids.

Effect of enclosing rocks and aeration on methanogenesis from coals

Two coals of different rank, mined in Russia, were treated by an anaerobic methanogenic enrichment culture. The addition of alkaline enclosing rock to the lower-rank coal increased the pH of the incubation medium and methane production above that of the higher-rank coal with addition of its enclosing rock. This effect was accompanied by the leaching of cations from the incubation medium. The coal was processed without a preliminary chemical treatment in a two-stage aerobic/anaerobic bioreactor containing an anaerobic methanogenic granulated enrichment culture.

Energetics of product formation during anaerobic degradation of phthalate isomers and benzoate

Methanogenic enrichment cultures grown on phthalate, isophthalate and terephthalate were incubated with the corresponding phthalate isomer on which they were grown, and a mixture of benzoate and the phthalate isomer. All cultures were incubated with bromoethanosulfonate to inactivate the methanogens in the mixed culture. Thus, product formation during fermentation of the aromatic substrates could be studied. It was found that reduction equivalents generated during oxidation of the aromatic substrates to acetate were incorporated in benzoate under formation of carboxycyclohexane. During fermentation of the phthalate isomers, small amounts of benzoate were detected, suggesting that the initial step in the anaerobic degradation of the phthalate isomers is decarboxylation to benzoate. Gibbs free energy analyses indicated that during degradation of the phthalate isomers, benzoate, carboxycyclohexane, acetate and molecular hydrogen accumulated in such amounts that both the reduction and oxidation of benzoate yielded a constant and comparable amount of energy of approximately 30 kJ mol−1. Based on these observations it is suggested that within narrow energetic limits, oxidation and reduction of benzoate may proceed simultaneously. Whether this is controlled by the Gibbs free energy change for carboxycyclohexane oxidation remains unclear.

Energetics of product formation during anaerobic degradation of phthalate isomers and benzoate

Methanogenic enrichment cultures grown on phthalate, isophthalate and terephthalate were incubated with the corresponding phthalate isomer on which they were grown, and a mixture of benzoate and the phthalate isomer. All cultures were incubated with bromoethanosulfonate to inactivate the methanogens in the mixed culture. Thus, product formation during fermentation of the aromatic substrates could be studied. It was found that reduction equivalents generated during oxidation of the aromatic substrates to acetate were incorporated in benzoate under formation of carboxycyclohexane. During fermentation of the phthalate isomers, small amounts of benzoate were detected, suggesting that the initial step in the anaerobic degradation of the phthalate isomers is decarboxylation to benzoate. Gibbs free energy analyses indicated that during degradation of the phthalate isomers, benzoate, carboxycyclohexane, acetate and molecular hydrogen accumulated in such amounts that both the reduction and oxidation of benzoate yielded a constant and comparable amount of energy of approximately 30 kJ mol −1. Based on these observations it is suggested that within narrow energetic limits, oxidation and reduction of benzoate may proceed simultaneously. Whether this is controlled by the Gibbs free energy change for carboxycyclohexane oxidation remains unclear.

Biotransformation of mixtures of chlorinated aliphatic hydrocarbons by an acetate-grown methanogenic enrichment culture

Biotransformation of chlorinated aliphatic hydrocarbons under anaerobic conditions has received considerable attention due to the prevalence of these compounds as groundwater contaminants. However, information concerning the impact of mixtures of chlorinated compounds on their own transformation is limited. The focus of this research was to investigate the effect of combinations of chlorinated aliphatics on transformation rates and the role of toxicity to a methanogenic enrichment culture. Batch studies using combinations of carbon tetrachloride (CT), perchloroethene (PCE) and 1,1,1-trichloroethane (1,1,1-TCA) were performed with acetate as the electron donor. CT was transformed most rapidly, followed by 1,1,1-TCA and PCE. Rate coefficients for the transformation of 1,1,1-TCA and CT were significantly lower when the concentration of either compound was increased. PCE transformation was limited, and its presence did not significantly impact degradation of CT or 1,1,1-TCA. In once-fed batch reactors used to assess the potential for toxicity, a transformation limit was observed for both PCE (1.91±0.21 μg of PCE per mg of cells) and 1,1,1-TCA (7.84±0.34 μg of 1,1,1-TCA per mg of cells) but not CT. The low observed limit of transformation for PCE, combined with its lack of effect on the transformation of the other two compounds, suggests that the number of PCE-degrading organisms in the mixed culture is low. Transformation of repeated CT spikes has been maintained for over 400 days without supplementation with acetate, indicating that inhibition, rather than potential inactivation, was responsible for the negative impact observed when CT is present in mixtures.

Anaerobic degradation of phthalate isomers by methanogenic consortia.

Three methanogenic enrichment cultures, grown on ortho-phthalate, iso-phthalate, or terephthalate were obtained from digested sewage sludge or methanogenic granular sludge. Cultures grown on one of the phthalate isomers were not capable of degrading the other phthalate isomers. All three cultures had the ability to degrade benzoate. Maximum specific growth rates (microseconds max) and biomass yields (YXtotS) of the mixed cultures were determined by using both the phthalate isomers and benzoate as substrates. Comparable values for these parameters were found for all three cultures. Values for microseconds max and YXtotS were higher for growth on benzoate compared to the phthalate isomers. Based on measured and estimated values for the microbial yield of the methanogens in the mixed culture, specific yields for the phthalate and benzoate fermenting organisms were calculated. A kinetic model, involving three microbial species, was developed to predict intermediate acetate and hydrogen accumulation and the final production of methane. Values for the ratio of the concentrations of methanogenic organisms, versus the phthalate isomer and benzoate fermenting organisms, and apparent half-saturation constants (KS) for the methanogens were calculated. By using this combination of measured and estimated parameter values, a reasonable description of intermediate accumulation and methane formation was obtained, with the initial concentration of phthalate fermenting organisms being the only variable. The energetic efficiency for growth of the fermenting organisms on the phthalate isomers was calculated to be significantly smaller than for growth on benzoate.

Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems

Isoprenoic compounds play a major part in the global carbon cycle. Biosynthesis and mineralization by aerobic bacteria have been intensively studied. This review describes our knowledge on the anaerobic metabolism of isoprenoids, mainly by denitrifying and fermentative bacteria. Nitrate-reducing β-Proteobacteria were isolated on monoterpenes as sole carbon source and electron donor. Thauera spp. were obtained on the oxygen-containing monoterpenes linalool, menthol, and eucalyptol. Several strains of Alcaligenes defragrans were isolated on unsaturated monoterpenes as growth substrates. A novel denitrifying β-Proteobacterium, strain 72Chol, mineralizes cholesterol completely to carbon dioxide. Physiological studies showed the presence of several oxidative pathways in these microorganisms. Investigations by organic geochemists indicate possible contributions of anaerobes to early diagenetic processes. One example, the formation of p-cymene from monoterpenes, could indeed be detected in methanogenic enrichment cultures. In man, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver from cholesterol. During their enterohepatic circulation, bile acids are biotransformed by the intestinal microflora into a variety of metabolites. Known bacterial biotransformations of conjugated bile acids include: deconjugation, oxidation of hydroxy groups at C-3, C-7 and C-12 with formation of oxo bile acids and reduction of these oxo groups to either α- or β-configuration. Quantitatively, the most important bacterial biotransformation is the 7α-dehydroxylation of CA and CDCA yielding deoxycholic acid and lithocholic acid, respectively. The 7α-dehydroxylation of CA occurs via a novel six-step biochemical pathway. The genes encoding several enzymes that either transport bile acids or catalyze various reactions in the 7α-dehydroxylation pathway of Eubacterium sp. strain VPI 12708 have been cloned, expressed in Escherichia coli, purified, and characterized.

Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems.

Isoprenoic compounds play a major part in the global carbon cycle. Biosynthesis and mineralization by aerobic bacteria have been intensively studied. This review describes our knowledge on the anaerobic metabolism of isoprenoids, mainly by denitrifying and fermentative bacteria. Nitrate-reducing beta-Proteobacteria were isolated on monoterpenes as sole carbon source and electron donor. Thauera spp. were obtained on the oxygen-containing monoterpenes linalool, menthol, and eucalyptol. Several strains of Alcaligenes defragrans were isolated on unsaturated monoterpenes as growth substrates. A novel denitrifying beta-Proteobacterium, strain 72Chol, mineralizes cholesterol completely to carbon dioxide. Physiological studies showed the presence of several oxidative pathways in these microorganisms. Investigations by organic geochemists indicate possible contributions of anaerobes to early diagenetic processes. One example, the formation of p-cymene from monoterpenes, could indeed be detected in methanogenic enrichment cultures. In man, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver from cholesterol. During their enterohepatic circulation, bile acids are biotransformed by the intestinal microflora into a variety of metabolites. Known bacterial biotranformations of conjugated bile acids include: deconjugation, oxidation of hydroxy groups at C-3, C-7 and C-12 with formation of oxo bile acids and reduction of these oxo groups to either alpha- or beta-configuration. Quantitatively, the most important bacterial biotransformation is the 7 alpha-dehydroxylation of CA and CDCA yielding deoxycholic acid and lithocholic acid, respectively. The 7 alpha-dehydroxylation of CA occurs via a novel six-step biochemical pathway. The genes encoding several enzymes that either transport bile acids or catalyze various reactions in the 7 alpha-dehydroxylation pathway of Eubacterium sp. strain VPI 12708 have been cloned, expressed in Escherichia coli, purified, and characterized.

Effect of Specific Inhibitors on the Anaerobic Reductive Dechlorination of 2,4,6-Trichlorophenol by a Stable Methanogenic Consortium

The transformation of 2,4,6-trichlorophenol (TCP) into 4-chlorophenol (4CP) was studied using a stable methanogenic enrichment culture derived from an anaerobic fixed bed reactor. Using acetate as a growth substrate, different inhibitors of methanogenesis exhibited distinct effects on TCP dechlorination. Whereas reductive dechlorination activity was not affected by 2% ethylene in the gas phase, 25 mM bromoethanesulfonic acid (BESA) had a direct inhibitory effect on this process. The choice of BESA as a specific inhibitor for identifying the subpopulations involved in reductive dechlorination of chloroaromatics is thus questionable. Inhibitors of sulfate reduction such as molybdate (20 mM) and selenate (20mM) had a direct inhibitory effect on reductive dechlorination independently of the presence of sulfate in the medium supplemented with acetate as growth substrate. Consequently much more care must also be taken with these inhibitors to prove that reductive chlorination is coupled to sulfate reduction.

Enhanced Dechlorination of Carbon Tetrachloride and Chloroform in the Presence of Elemental Iron and Methanosarcina barkeri, Methanosarcina thermophila, or Methanosaeta concillii

Previous experiments in our laboratory have demonstrated that the rate and extent of carbon tetrachloride (CT) and chloroform (CF) dechlorination were enhanced when a methanogenic enrichment culture and iron (Fe0) were incubated together. Batch experiments with three pure cultures of methanogens, Methanosarcina barkeri, Methanosarcina thermophila, and Methanosaeta concillii were performed to determine how this enhanced transformation occurred. When hydrogen (H2) was added as an electron donor, degradation of CT for all organisms and CF for M. thermophila was more rapid. H2 was produced from the oxidation of iron, which therefore served as an H2 source for the organisms, enhancing the transformation of CT and CF. Experiments with M. thermophila and M. concillii, which could not grow on H2−CO2 under the conditions tested, showed that H2 could serve as an electron donor for dechlorination of CT and CF with these organisms as well. Experiments with supernatants from M. thermophila grown with and without iron indicated the presence of an excreted biomolecule active in the enhanced transforma tion of CT and CF.

Evidence for methanogenicArchaeaand homoacetogenicBacteriain deep granitic rock aquifers

The presence and diversity of methanogens and homoacetogens in deep granitic rock groundwater from Äspö Hard Rock Laboratory were studied. Concentrations of hydrogen and methane in Äspö groundwater were 45 nM–100 μM and 19–1000 μM, respectively. Groundwater-based media were used to count viable cells in the groundwaters from the subsurface. Different physiological groups of methanogens (autotrophic H2/CO2 consuming and heterotrophic acetate, methanol and trimethylamine consuming) were found at depths ranging from 68 to 446 m below sea level in numbers from 12 to 4.5×105 cells/ml. Viable numbers of different physiological groups of methanogens and autofluorescent cells were counted repeatedly during a period of 1 year in the groundwater. The counts were reproducible in the same boreholes at different sampling times. Methane production was observed in the groundwater with added methanogenic substrates, at anaerobic conditions, at 17°C, after 20–50 days of adaptation. The most active methane production took place in those groundwaters that were sources of methanogenic enrichment and pure cultures. Methanosarcina-like organisms were observed in groundwater from depths of 45–68 m and active enrichment cultures. 16S rRNA gene sequencing of enrichment cultures indicated the presence of a psychrophilic Methanohalophilus related organism at a depth of 414 m. A new species of alkaliphilic Methanobacterium was isolated from depths of 68, 409 and 420 m and studied. Homoacetogenic Bacteria were found in the granitic groundwater as viable cells from 10 to 3.6×104 cells/ml and produced acetate autotrophically. It appears that deep granitic groundwater is inhabited by autotrophic methanogens and acetogens, which may produce methane and acetate at the expense of subterranean hydrogen and bicarbonate.

Evidence for methanogenic Archaea and homoacetogenic Bacteria in deep granitic rock aquifers

The presence and diversity of methanogens and homoacetogens in deep granitic rock groundwater from Äspö Hard Rock Laboratory were studied. Concentrations of hydrogen and methane in Äspö groundwater were 45 nM–100 μM and 19–1000 μM, respectively. Groundwater-based media were used to count viable cells in the groundwaters from the subsurface. Different physiological groups of methanogens (autotrophic H 2/CO 2 consuming and heterotrophic acetate, methanol and trimethylamine consuming) were found at depths ranging from 68 to 446 m below sea level in numbers from 12 to 4.5×10 5 cells/ml. Viable numbers of different physiological groups of methanogens and autofluorescent cells were counted repeatedly during a period of 1 year in the groundwater. The counts were reproducible in the same boreholes at different sampling times. Methane production was observed in the groundwater with added methanogenic substrates, at anaerobic conditions, at 17°C, after 20–50 days of adaptation. The most active methane production took place in those groundwaters that were sources of methanogenic enrichment and pure cultures. Methanosarcina-like organisms were observed in groundwater from depths of 45–68 m and active enrichment cultures. 16S rRNA gene sequencing of enrichment cultures indicated the presence of a psychrophilic Methanohalophilus related organism at a depth of 414 m. A new species of alkaliphilic Methanobacterium was isolated from depths of 68, 409 and 420 m and studied. Homoacetogenic Bacteria were found in the granitic groundwater as viable cells from 10 to 3.6×10 4 cells/ml and produced acetate autotrophically. It appears that deep granitic groundwater is inhabited by autotrophic methanogens and acetogens, which may produce methane and acetate at the expense of subterranean hydrogen and bicarbonate.

Fate and toxic effects of nitrophenols on anaerobic treatment systems

Modern industrial societies discharge vast quantities of chemicals into the environment. Removal of priority pollutants is a vital task of increasing importance. A literature search shows that more quantitative information about the effects and fates of these pollutants during anaerobic treatment is needed. The objective of this research was to study the fate and toxic effects of nitrophenols (2-nitrophenol, 3-nitrophenol, 4-nitrophenol and 2,4-dinitrophenol) on methanogenic bacteria. Both batch and continuous flow experiments were performed. Batch anaerobic toxicity assay (ATA) were performed in methanogenic enrichment cultures. The effects of 4-nitrophenol were also studied using chemostats at 15-day solids retention time. Stock acetate enrichment cultures were developed and used in this research. An inhibition coefficient model was used to quantify the effects of 4-nitrophenol. The competitive inhibition coefficient model can adequately describe the fate of the systems exposed to 4-nitrophenol. The results of this research showed that about 81% of 4-nitrophenol can be removed by biodegradation. Among the nitrophenols studied, 4-nitrophenol and 2,4-dinitrophenol were most toxic.

Biotransformation of nitrocellulose under methanogenic conditions

Treatment of wastewater containing nitrocellulose (NC) fines is a significant hazardous waste problem currently facing manufacturers of energetic compounds. Previous studies have ruled out the use of biological treatment, since NC has appeared to be resistant to aerobic and anaerobic biodegradation. The objective of this study was to examine NC biotransformation in a mixed methanogenic enrichment culture. A modified cold-acid digestion technique was used to measure the percentage of oxidized nitrogen (N) remaining on the NC. After 11 days of incubation in cultures amended with NC (10 g/L) and methanol (9.9 mM), the % N (w/w) on the NC decreased from 13.3% to 10.1%. The presence of NC also caused a 16% reduction in methane output. Assuming the nitrate ester on NC was reduced to N2, the decrease in CH4 represented almost exactly the amount of reducing equivalents needed for the observed decrease in oxidized N. An increase in the heat of combustion of the transformed NC correlated with the decrease in % N. There was no statistically significant decrease in % N when only NC was added to the culture, or in controls that contained only the sulfide-reduced basal medium. The biotransformed NC has a % N comparable to nonexplosive nitrated celluloses, suggesting that anaerobic treatment may be a technically feasible process for rendering NC nonhazardous.

Fate and toxic effects of nitrophenols on anaerobic treatment systems

Modem industrial societies discharge vast quantities of chemicals into the environment. Removal of priority pollutants is a vital task of increasing importance. A literature search shows that more quantitative information about the effects and fates of these pollutants during anaerobic treatment is needed. The objective of this research was to study the fate and toxic effects of nitrophenols (2-nitrophenol, 3-nitrophenol, 4-nitrophenol and 2,4-dinitrophenol) on methanogenic bacteria.Both batch and continuous flow experiments were performed. Batch anaerobic toxicitys assay (ATA) were performed in methanogenic enrichment cultures. The effects of 4-nitrophenol were also studied using chemostats at 15-day solids retention time. Stock acetate enrichment cultures were developed and used in this research.An inhibition coefficient model was used to quantify me effects of 4-nitrophenol. The competitive inhibition coefficient model can adequately describe the fate of the systems exposed to 4-nitrophenol. The results of this research showed that about 81% of 4-nitrophenol can be removed by biodegradation. Among the nitrophenols studied, 4-nitrophenol and 2,4-dinitrophenol were most toxic.

Biotransformation of nitrocellulose under methanogenic conditions

Treatment of wastewater containing nitrocellulose (NC) fines is a significant hazardous waste problem currently facing manufacturers of energetic compounds. Previous studies have ruled out the use of biological treatment, since NC has appeared to be resistant to aerobic and anaerobic biodegradation. The objective of this study was to examine NC biotransformation in a mixed methanogenic enrichment culture. A modified cold-acid digestion technique was used to measure the percentage of oxidized nitrogen (N) remaining on the NC. After 11 days of incubation in cultures amended with NC (10 g/L) and methanol (9.9 mM), the % N (w/w) on the NC decreased from 13.3% to 10.1%. The presence of NC also caused a 16% reduction in methane output Assuming the nitrate ester on NC was reduced to N2, the decrease in CH4 represented almost exactly the amount of reducing equivalents needed for the observed decrease in oxidized N. An increase in the heat of combustion of the transformed NC correlated with the decrease in % N. There was no statistically significant decrease in % N when only NC was added to the culture, or in controls that contained only the sulfide-reduced basal medium. The biotransformed NC has a % N comparable to nonexplosive nitrated celluloses suggesting that anaerobic treatment may be a technically feasible process for rendering NC nonhazard'ous.

Effect of Cobalt on Methanogenesis

Anaerobic toxicity assays were performed with 150 ml serum bottles using acetate-utilizing methanogenic enrichment cultures spiked with cobalt at 35–1500 mg-l−1. Free cobalt concentrations in the bottles were measured by a combination of dialysis and ion exchange at the end of the experiments. Free cobalt and total soluble cobalt of 70 and 280 mg-l−1, respectively, caused complete inhibition of methanogenesis. Free cobalt/VSS and total soluble cobalt/VSS ratios of and exceeding 0.05 and 0.16, respectively, caused complete failure of methanogenesis.

Use of Cyanocobalamin To Enhance Anaerobic Biodegradation of Chloroform

Biodegradation of chloroform (CF) was examined in a methanogenic enrichment culture grown on dichloromethane (DCM) as the sole organic carbon and energy source, with and without the addition of supplemental cyanocobalamin. In the absence of cyanocobalamin, the principal products of P4C1 CF biodegradation were 14C02 and V4C1DCM. The extent of CF reduction to DCM increased significantly when CF was biodegraded in the presence of a large amount of DCM. The addition of cyanocobalamin enhanced CF biodegradation in two ways. First, the rate of CF biodegradation increased approximately 10-fold. Second, the metallocofactor increased the extent of CF oxidation to C02 and virtually eliminated the accumulation of DCM. These effects were not observed in autoclaved cultures supplemented with cyanocobalamin. When cyanocobalamin was added to viable cultures, as much as 10 % of the [l4C1CF transformed accumulated as 14C-labeled carbon monoxide. This suggested that the oxidation of CF to C02 proceeds via net hydrolysis to CO. CF levels as high as 2.2 mM were readily transformed, without accumulation of DCM, at cyanocobalamin to CF molar ratios of 3-596. Although the organism or consortium responsible for CF biodegradation was not identified, prior work with DCM suggests that acetogenic bacteria are involved.

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures