Methanogenic Consortium Research Articles

Fatty Acids Profile during Anaerobic Digestion of Night Soil-Effect of temperature, Calcium Carbonate and Selectively-enriched Inoculum

Anaerobic biodegradation of night soil was carried out at 5-30 degree centigrade with 1.8-10.62 per cent volatile solids (VS). Biogas production increased with the temperature and VS up to 6.2 per cent. Further increase in VS caused higher Volatile fatty acids (VFA) accumulation resulting in decreased gas production. Acetate and propionate accounted for 62-83 per cent of total VFA. Butyrate to isobutyrate ratio increased with VS. Calcium Carbonate promoted VS degradation, biogas production and VFA degradation. The increased methanogenic and decreased sulphate-reducing bacteria caused proportional changes in CH4 and H2S gases. Enrichment with H2 oxidising methanogenic consortium is beneficial by enhancing VFA utilisation by two to three fold.

Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP → 2,3,5,6-tetrachlorophenol → 2,3,5-trichlorophenol → 3,5-dichlorophenol → 3-chlorophenol → phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 μmol (1 reactor volume)−1 day−1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.

Study of the reductive dechlorination of pentachlorophenol by a methanogenic consortium

Pentachlorophenol (PCP) dechlorination by a methanogenic consortium was observed when glucose, formate, lactate, or yeast extract was present in the mineral medium as a secondary carbon source. Acetate was not a good substrate to sustain dechlorination. The consortium was able to dechlorinate the different monochlorophenols, although the chlorine in position ortho and meta was removed more readily than in para position. Dechlorination was most efficient at 37 degrees C. At 45 degrees C, the first PCP dechlorination steps were very rapid, but 3,5-dichlorophenol (3,5-DCP) was not further dechlorinated. At 15 and 4 degrees C, dechlorination was very slow. The dechlorination of PCP to 3-chlorophenol (3-CP) was still observed after the consortium had been subjected to heat treatment (80 degrees C, 60 min), suggesting that spore-forming bacteria were responsible. The dechlorinating activity of the consortium was significantly reduced by the presence of hydrogen, 2-bromoethanosulfonic acid (BESA), or sulfate but not of nitrate. The dechlorination of 3-CP was completely inhibited by heat treatment or the presence of BESA, suggesting that a syntrophic microorganism would be involved. Vigorous agitation of the consortium stopped the dechlorination, but the presence of DEAE-Sephacel acting as a support was very efficient in restoring the activity, suggesting that association between certain members of the consortium was important.

Microbial aldicarb transformation in aquifer, lake, and salt marsh sediments

The microbial transformation of [N-methyl-(sup14)C]aldicarb, a carbamate pesticide, occurred in aquifer, lake, and salt marsh sediments. Microbial degradation of aldicarb took place within 21 days in aquifer sediments from sites previously exposed to aldicarb (Jamesport, Long Island, N.Y.) but did not occur in sediments which were not previously exposed (Connetquot State Park, Long Island, N.Y.). At the Jamesport sites, higher aldicarb transformation rates occurred in deep, anoxic sediments than in shallow, oxic sediments. There was a significant negative relationship (P < 0.05) between transformation rates and ambient dissolved O(inf2) levels. Aldicarb hydrolysis rates in Jamesport sediments were 10- to 1,000-fold lower than rates previously reported for soils. In addition, aldicarb degradation rates were not significantly correlated with measurements of bacterial activity and density previously determined in the same sediments. Substantially higher aldicarb degradation rates were found in anoxic lake and salt marsh than in aquifer sediments. Furthermore, we investigated the anaerobic microbial processes involved in aldicarb transformation by adding organic substrates (acetate, glucose), an alternative electron acceptor (nitrate), and microbial inhibitors (molybdate, 2-bromoethanesulfonic acid) to anoxic aquifer, lake, and salt marsh sediments. The results suggest that a methanogenic consortium was important in aldicarb transformation or in the use of aldicarb-derived products such as methylamine. In addition, microbial aldicarb transformation proceeded via different pathways under oxic and anoxic conditions. In the presence of O(inf2), aldicarb transformation was mainly via an oxidation pathway, while in the absence of O(inf2), degradation took place through a hydrolytic pathway (including the formation of methylamine precursors). Under anoxic conditions, therefore, aldicarb can be transformed by microbial consortia to yield products which can be of direct benefit to natural populations of methanogens present in sediments.

GC-FID Determination of Chlorobenzene Isomers in Methanogenic Batch-Cultures from River Sediments

Abstract The investigation of the biological reductive dechlorination of chlorobenzenes by methanogenic consortia in river sediments is dependent on reliable analytical procedures. Two different extraction methods for the determination of chlorobenzenes from sediment samples were developed. Lower chlorinated benzenes up to the trichloro isomers were extracted with satisfactory recoveries by thoroughly mixing the sediment with an equal volume of acetone. After centrifugation, the supernatant was subjected to solid phase extraction (SPE) using RP-C18 adsorbent. The tetra-, penta- and hexachlorobenzene isomers could only be satisfactorily recovered by liquid-liquid extraction with ethyl acetate. With both methods, 2,4-dichlorotoluene was added as surrogate standard. Determination was carried out by gas chromatography using a thick film megabore column coated with methyl silicone containing 35% phenyl groups and flame ionization detection. Applying a temperature program, all chlorobenzene isomers including be...

Spore-forming bacteria that carboxylate phenol to benzoic acid under anaerobic conditions

A methanogenic consortium transforming phenol to benzoic acid was submitted to different treatments to characterize the carboxylating microorganisms and eventually to facilitate their isolation. Under aerobic conditions, phenol was not transformed by the consortium and no growth was observed on solid medium. The consortium from an inoculum that was treated with heat, or heat and ethanol, retained the ability to carboxylate phenol under strictly anaerobic conditions. Electron microscopic observations of the consortium from an inoculum that was heated for 15 min at 80 °C revealed only Gram-positive bacilli. In this culture, methane production was not detected and benzoic acid accumulated. Five colonies with distinct morphologies were isolated from this culture on solid medium. Four of these strains were identified as Clostridium spp. In contrast to the untreated culture, none of the strains isolated were able to carboxylate phenol in pure culture or in coculture, nor could they decarboxylate or dehydroxylate 4-hydroxybenzoic acid, or oxidize 2-hydroxybenzyl alcohol, or O-demethylate anisole or 2-methoxyphenol. Also, the consortium from a treated inoculum retained its ability to decarboxylate and dehydroxylate 4-hydroxybenzoic acid forming phenol and benzoic acid, respectively, but could not accomplish the other reactions. These results suggest that spore-forming microorganisms are involved in the carboxylation of phenol and in the decarboxylation and dehydroxylation of 4-hydroxybenzoic acid.Key words: spore-forming bacteria, phenol, benzoic acid, methanogenic conditions, carboxylation.

Biomethanation of tobacco waste

Tobacco is an important cash crop produced in considerable quantities in India. During cigarette manufacturing large quantities of tobacco waste are produced annually and its disposal is a serious ecological problem. In the present study a process for the biodegradation of tobacco waste, under methanogenic conditions, was developed so that environmental pollution can be reduced while producing biogas as a useful energy source. The methanogenic bacterial consortium developed for the anaerobic degradation of tobacco waste without any chemical pretreatment in a single stage digester at 15 days HRT (hydraulic retention time) has been successfully scaled-up to install a 10 m 3 biogas plant. The gas yields (1 kg −1 TS fed day −1) varied from 169 to 282 depending on the ambient temperatures. The methane content in the biogas produced was 60%. The pH and VFA content of the digested slurry were 6·8 ± 0·1 and 599 mg litre −1, respectively. Nicotine, COD and BOD reductions were 75, 60 and 80%, respectively.

Population dynamics of methanogenic biofilm consortium during a start-up period of anaerobic fluidized bed reactor

The changes in physical properties and microbial activities were investigated during initial biofilm formation in lab-scale anaerobic fluidized bed reactors. Four different upflow velocities, i.e., 4, 7, 14 and 25 m·hr−1 were applied to four respective reactors of an equal size. The upflow velocities caused a prominent difference in the pattern of initial biofilm formation. The biofilm thickness attained eventually approximately 100 μm after 100 days of operation, independent of upflow velocity. On the contrary the biofilm density varied from 4.4 to 24.1 mg-VSS·cm−3 with an increase in the upflow velocity imposed. The activity of acetoclastic methane production increased remarkably 15 to 30 fold of seed sludge, regardless of upflow velocity. Microbial activities with respect to acetate production, H2-utilizing methanogenesis and acetate-utilizing methanogenesis increased finally up to 3-4 times as large as those of suspended grown sludge in a chemostat.

Degradation of lactate by an anaerobic mixed culture in a fluidized-bed reactor

In order to obtain syntrophic lactate degradation in a low-sulfate medium, first a methanogenic consortium consisting of Methanobrevibacter arboriphilus, Methanoculleus sp. and Methanothrix sp. was established in a fluidized-bed reactor with porous glass beads as a substratum and H 2/CO 2/acetate as energy and carbon sources. Then the reactor was inoculated with a Desulfovibrio sp. strain L1 which had been isolated from a bioreactor degrading organic acids including lactate. Racemic lactate was fed to the reactor and in the absence of H 2 the substrate was rapidly oxidized to acetate, CO 2 and hydrogen. After 1 day of operation suddenly propionate was produced by a very fast growing contaminating Clostridium sp., which was isolated thereafter. Growing on lactate the Clostridium sp. has higher μ max and K s values than Desulfovibrio sp. strain L1. Based on these data it is concluded that under non-sterile conditions of full-scale anaerobic bioreactors, lactate degradation takes place via propionate as long as the concentration of lactate is high, while at low lactate concentrations and in the presence of methanogens the oxidative decarboxylation of lactate to acetate is the preferred pathway.

Anaerobic degradation of toluene and o-xylene by a methanogenic consortium

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.

Mathematical modeling of an anaerobic butyrate degrading consortia: predicting their response to organic overloads

The steady-state and dynamic response of a butyrate using methanogenic consortia has been modeled as three sequential bacterial growth reactions. The model simulates the concentration of fermentation intermediates (acetate and hydrogen), incorporating their inhibition of butyrate oxidation and stimulation of methanogenesis. The hydrogen-producing acetogenic conversion of butyrate is modeled as a product-inhibited or reversible, modified Monod reaction, while hydrogen-using and acetoclastic methogenesis are simple Monod reactions. The growth yield coefficient for each growth reaction is variable and dependent on the Gibbs free energy of the energy reaction, which in turn depends on the time-varying concentrations of reactants and products

Fluorophenols and 3-fluorobenzoate in phenol-degrading methanogenic cultures

3-Fluorobenzoate and all three isomers of fluorophenol were used as analogues and inhibitors of phenol degradation in a methanogenic consortium. 3-Fluorobenzoate was not transformed by phenol-degrading cultures, but it facilitated the detection of the formation of 4-hydroxybenzoate and benzoate from phenol. The effects of the fluorophenols depended on their concentration in the cultures. When added at 0.90 mM, all fluorophenols prevented phenol transformation. At concentrations of 0.45 to 1.8 mM, 2-fluorophenol was transformed to 3-fluoro-4-hydroxybenzoate which accumulated in the medium. When both 2-fluorophenol and phenol were added to cultures at concentrations of 1 mM each, 3-fluoro-4-hydroxybenzoate, 4-hydroxybenzoate, 3-fluorobenzoate and benzoate were detected. 4-Fluorophenol was never transformed, and when it was present at ≦0.22 mM, it had no effect on phenol degradation. At concentrations ≦0.09 mM, 2-fluorophenol was mineralized by the phenol-degrading cultures to methane, carbon dioxide, and fluoride. The release of fluoride was also observed from 3-fluorophenol when it was initially present at ≦0.09 mM. These results support the proposed pathway for phenol degradation involving an initial para-carboxylation to 4-hydroxybenzoate followed by dehydroxylation to benzoate and further metabolism to carbon dioxide and methane. They also demonstrate defluorination of 2- and 3-fluorophenols under methanogenic conditions.

Anaerobic degradation of 2-methylbenzoic acid by a methanogenic consortium

2-Methylbenzoic has been detected as a metabolite in anaerobic consortia that degrade m-cresol and it was found to accumulate in cultures incubated under methanogenic conditions. This study was initiated to determine whether 2-methylbenzoic acid could be degraded under methanogenic conditions. Near stoichiometric amounts of methane were obtained from 2-methylbenzoic acid in enrichment cultures inoculated with 25% (v/v) domestic anaerobic sewage sludge. However, lag times of 12–36 weeks typically were required before 2-methylbenzoic acid degradation began, and it was difficult to maintain the activity of the cultures. A transient metabolite was detected but not identified. It appeared to be the product of 2-methylbenzoic acid hydroxylation because its trimethylsilyl derivative had a molecular weight of 296, but it was not 4-hydroxy-2-methylbenzoic acid, the intermediate of m-cresol metabolism from which 2-methylbenzoic acid likely originates.Key words: anaerobic, methane, 2-methylbenzoic acid.

Potential for carboxylation-dehydroxylation of phenolic compounds by a methanogenic consortium.

An anaerobic consortium that carboxylated and dehydroxylated phenol to benzoate, and 2-cresol to 3-methylbenzoic acid, under methanogenic conditions was studied. Phenol induced this transformation activity. Addition of 4-hydroxypyridine or an increase in the concentration of proteose peptone to 0.5% (w/v) delayed the transformation. Phenol enhanced the rate of transformation of 2-cresol whereas 2-cresol delayed the transformation of phenol. Phenols with ortho-substitutions (chloro-, fluoro-, bromo-, hydroxyl-, amino-, or carboxyl-) were transformed to meta-substituted benzoic acids. However, meta- and para-substituted phenols (cresols, fluorophenols, and chlorophenols) were not transformed. Phenol was most rapidly metabolized, followed by catechol, 2-cresol, 2-fluorophenol, 2-aminophenol, 2-chlorophenol, 2-hydroxybenzoic acid, and 2-bromophenol. The consortium O-demethylated anisole to phenol and 2-methoxyphenol to catechol, and oxidized 2-hydroxybenzyl alcohol to 2-hydroxybenzoic acid. Aniline, 2-ethylphenol, 2-hydroxypyridine, 2-acetamidophenol, 2,6-dimethylphenol, 2-phenylphenol, and 1-naphthol were not metabolized.

Use of Fluorinated Compounds To Detect Aromatic Metabolites from m -Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol --> 4-hydroxy-2-methylbenzoic acid --> 4-hydroxybenzoic acid --> benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol --> 5-fluoro-4-hydroxy-2-methylbenzoic acid --> 3-fluoro-4-hydroxybenzoic acid --> 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-C]cresol was recovered predominantly as [C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols.

Toxicity of a mixture of polychlorinated organic compounds towards an unacclimated methanogenic consortium

The toxicity of a mixture of polychlorinated aliphatic compounds towards an unacclimated methanogenic consortium was assayed in carrier-free, continuously-stirred batch experiments. Results obtained indicate that a 50 % inhibition of methanogenesis occurs at 3.3 mg toxic mixture per litre mixed liquor and that at 100 mg toxic mixture per litre mixed liquor, the inhibition of methanogenesis is complete.

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.

Inhibitory effects of kraft bleachery effluents on methanogenic consortia

A systematic investigation of the inhibition exerted by kraft pulp mill bleach plant effluents on a methanogenic consortium was completed using anaerobic serum bottle techniques. The inhibition exerted on unacclimated biomass was found to be correlated to the wastewater AOX. A significant portion of the inhibition was determined to be present in the permeate of a 1000 Da ultrafiltration membrane. Treatment of the wastewater with powdered activated carbon prior to biomass exposure removed the inhibitory effects of the wastewater on the biomass. A methanogenic consortium was found to acclimate to a wastewater volumetric fraction of 80% v/v. The wastewater volumetric fraction did not affect the overall time required for acclimation to occur.

Reductive dechlorination of chlorophenols by a pentachlorophenol- acclimated methanogenic consortium.

Anaerobic digester sludge fed 5,300 mg of acetate per liter, 3.4 microM pentachlorophenol, and nutrients for 10 days biotransformed pentachlorophenol by sequential ortho dechlorinations to produce 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol. Upon acclimation to 3.4 microM pentachlorophenol for 6 months, the methanogenic consortium removed chlorines from the ortho, meta, and para positions of pentachlorophenol and its reductive dechlorination products. Pentachlorophenol was degraded to produce 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and 2,3,5,6-tetrachlorophenol. Dechlorination of 2,3,4,5-tetrachlorophenol produced 3,4,5-trichlorophenol, which was subsequently degraded to produce 3,4-dichlorophenol and 3,5-dichlorophenol. 2,3,4,6-Tetrachlorophenol was dechlorinated at the ortho and meta positions to produce 2,4,6-trichlorophenol and 2,4,5-trichlorophenol. 2,3,5,6-Tetrachlorophenol yielded 2,3,5-trichlorophenol, followed by production of 3,5-dichlorophenol. 2,4,6-Trichlorophenol was degraded to form 2,4-dichlorophenol, and 2,4,5-trichlorophenol was dechlorinated at two positions to form 2,4-dichlorophenol and 3,4-dichlorophenol. Of the three dichlorophenols produced (2,4-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol), only 2,4-dichlorophenol was degraded significantly within 3 weeks, to produce 4-chlorophenol.