Anaerobic Enrichment Cultures Research Articles

Anaerobic degradation of 1,2-dichloropropane in batch and continuous culture

Microbial degradation of 1,2-dichloropropane (DCP) was studied in a laboratory scale continuous flow fluidized bed reactor using polyurethane foam cubes as a carrier for the mixed culture. The anaerobic enrichment culture derived from Saale River sediment and was immobilized prior to utilization in the reactor. The DCP degradation performance was monitored by direct gas chromatographic analysis. A variety of different co-substrates were investigated for their ability to support DCP dechlorination during reactor operation. Continuous DCP removal efficiencies over 90% were achieved with a model water at substrate loading rates of up to 700 μmol/(L · d) with methanol and sodium acetate as co-substrates at 24 h hydraulic retention time in the reactor. In batch experiments the degradation potential of the culture for other chlorinated organics, like 1, 2, 3-trichloropropane and dichlorodiisopropyl ether was investigated.

Anaerobic degradation of fluorinated aromatic compounds.

Anaerobic enrichment cultures with sediment from an intertidal strait as inoculum were established under denitrifying, sulfate-reducing, iron-reducing and methanogenic conditions to examine the biodegradation of mono-fluorophenol and mono-fluorobenzoate isomers. Both phenol and benzoate were utilized within 2-6 weeks under all electron-accepting conditions. However, no degradation of the fluorophenols was observed within 1 year under any of the anaerobic conditions tested. Under denitrifying conditions, 2-fluorobenzoate and 4-fluorobenzoate were depleted within 84 days and 28 days, respectively. No loss of 3-fluorobenzoate was observed. All three fluorobenzoate isomers were recalcitrant under sulfate-reducing, iron-reducing, and methanogenic conditions. The degradation of the fluorobenzoate isomers under denitrifying conditions was examined in more detail using soils and sediments from different geographic regions around the world. Stable enrichment cultures were obtained on 2-fluorobenzoate or 4-fluorobenzoate with inoculum from most sites. Fluoride was released stoichiometrically, and nitrate reduction corresponded to the values predicted for oxidation of fluorobenzoate to CO2 coupled to denitrification. The 2-fluorobenzoate-utilizing and 4-fluorobenzoate-utilizing cultures were specific for fluorobenzoates and did not utilize other halogenated (chloro-, bromo-, iodo-) benzoic acids. Two denitrifying strains were isolated that utilized 2-fluorobenzoate and 4-fluorobenzoate as growth substrates. Preliminary characterization indicated that the strains were closely related to Pseudomonas stutzeri.

Relationship between electron donor and microorganism on the dechlorination of carbon tetrachloride by an anaerobic enrichment culture

An investigation involving the supplement of different concentrations of substrates and microorganisms was carried out under anaerobic condition to assess the feasibility of bioremediation of carbon tetrachloride (CCl 4) with the amendment of low concentrations of auxiliary substrate and microorganisms. The concentrations of substrate and microorganisms ranged from 10 to 100 mg/l and from 3.7 × 10 4 to 3.7 × 10 6 cell/ml, respectively. The biotransformation rate of CCl 4 increased progressively with the increase in the concentrations of the substrate and microorganisms. In the low biomass-amended system (3.7 × 10 4cells/ml), 28–71% and 57–96% of CCl 4 removals were exhibited when 10–100 mg/l of acetate or glucose was supplemented, respectively, whereas nearly complete degradation of CCl 4 was observed in the heavily inoculated systems (3.7 × 10 6 cells/ml). An addition of electron donor in the low microbial activity batches enhanced greater efficiency in dechlorination than in the high microbial activity batches. The second-order rate constants ranged from 0.0059 to 0.0092 l/mg/day in high biomass input system, while a two- to four-fold increase in rate constant was obtained in the low microbial activity system. This study indicates that biomass was the more important environmental parameter than substrate affecting the fate of CCl 4. The addition of auxiliary substrates was effective only in low biomass-amended batches (0.56 mg-VSS/l) and diminished inversely with the increase of microbial concentration.

嫌気性集積培養菌を用いたテトラクロロエチレン汚染土壌の修復

The solvent tetrachloroethylene (PCE), a common soil contaminant, can be completely dechlorinated to ethylene and ethane by microorganisms under anaerobic conditions. Ethylene and ethane are harmless and environmentally acceptable products. This study was conducted to investigate factors influencing on an anaerobic bioremediation of PCE-contaminated soil.An anaerobic enrichment culture, which is capable of dechlorinating PCE completely, sustained its transformation ability in the presence of soil, although the activity was lowered. Addition of the enrichment culture was indispensable for progressing complete dechlorination of PCE in actual contaminated soils. PCE contained in soil was first leached to liquid-phase and then dechlorinated. Therefore, desorbing rate of PCE from soil was an important factor in the bioremediation. PCE was also dechlorinated to ethylene and ethane under the condition of low liquid/solid (L/S) ratio of 0.2l · kg-1 by adding the enrichment culture.

Establishment of a polychlorinated biphenyl-dechlorinating microbial consortium, specific for doubly flanked chlorines, in a defined, sediment-free medium.

Estuarine sediment from Charleston Harbor, South Carolina, was used as inoculum for the development of an anaerobic enrichment culture that specifically dechlorinates doubly flanked chlorines (i.e., chlorines bound to carbon that are flanked on both sides by other chlorine-carbon bonds) of polychlorinated biphenyls (PCBs). Dechlorination was restricted to the para chlorine in cultures enriched with 10 mM fumarate, 50 ppm (173 microM) 2,3,4, 5-tetrachlorobiphenyl, and no sediment. Initially the rate of dechlorination decreased upon the removal of sediment from the medium. However, the dechlorinating activity was sustainable, and following sequential transfer in a defined, sediment-free estuarine medium, the activity increased to levels near that observed with sediment. The culture was nonmethanogenic, and molybdate, ampicillin, chloramphenicol, neomycin, and streptomycin inhibited dechlorination activity; bromoethanesulfonate and vancomycin did not. Addition of 17 PCB congeners indicated that the culture specifically removes double flanked chlorines, preferably in the para position, and does not attack ortho chlorines. This is the first microbial consortium shown to para or meta dechlorinate a PCB congener in a defined sediment-free medium. It is the second PCB-dechlorinating enrichment culture to be sustained in the absence of sediment, but its dechlorinating capabilities are entirely different from those of the other sediment-free PCB-dechlorinating culture, an ortho-dechlorinating consortium, and do not match any previously published Aroclor-dechlorinating patterns.

Reduction of halogenated derivatives of benzoic acid to the corresponding alcohols by Desulfovibrio vulgaris PY1

AbstractDesulfovibrio vulgaris strain PY1 was isolated from a 3‐chlorobenzoic acid (3CBA) degrading anaerobic enrichment culture, using anaerobic Percoll density centrifugation. When grown on pyruvate (20 mM), in the absence of sulphate and under strict anaerobic conditions, this organism converted not only the co‐substrates benzoate (BA), 3‐amino‐BA and 3CBA to the corresponding alcohols but also ten other different halogenated benzoic acids, viz., 4‐Cl‐, 3‐Br‐, 4‐Br‐, 3‐I‐, 3‐F‐, 4‐F‐, 2,4‐di‐Cl‐, 2,5‐di‐Cl‐, 3,4‐di‐Cl‐ and 3,5‐di‐Cl‐BA. This was verfied with HPLC and GC/MS spectrometric analyses. The yields of the co‐substrate converted after 30 days of growth were between 20% and 88%, depending on the compounds which had been added at initial concentrations of 500 μM. Sulphate, sulphite, thiosulphate and disulphite inhibited the formation of 3‐Cl‐benzyl alcohol (3CBOH), i.e. a 97 to 99% inhibition, and nitrate and sulphur had no effect (a 7–10% inhibition). In cell‐free extracts, the reduction of 3CBA to 3CBOH required strict anaerobic conditions, pyruvate or H2 as electron donors and the addition of methylviologen (MV), FAD, FMN or ferredoxin as electron carriers. The specific activity of the reduction of 3CBA to 3CBOH in crude extract was 5.3 nmol/(mg protein min). The reaction was not inhibited by additions of sulphate or sulphite (5 mM), but was completely inhibited at concentrations of 10 mM 3CBA or 50 mM BA. A carboxylic acid reductase (aldehyde dehydrogenase), which acted on non‐activated 3CBA and was responsible for the reduction of 3CBA to 3‐Cl‐benzaldehyde, was found in the solube fraction (94% of the total activity). These results demonstrate that strain PY1 was able to effectively reduce a wide range of halogenated benzoic acids to the corresponding alcohols.

Sulfidogenesis from 2-aminoethanesulfonate (taurine) fermentation by a morphologically unusual sulfate-reducing bacterium, Desulforhopalus singaporensis sp. nov.

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The name Desulforhopalus singaporensis is proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-beta-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genus Desulforhopalus which has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297-301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation. D. singaporensis thus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.

Dehalogenation of lindane (γ-hexachlorocyclohexane) by anaerobic bacteria from marine sediments and by sulfate-reducing bacteria

Marine sediments from around burrows of Saccoglossus kowalevskii, a tribromopyrrole-producing marine hemichordate, were used to develop anaerobic enrichment cultures supplemented with lindane and a mixture of short chain fatty acids. These enrichments consumed lindane and both monochlorobenzene and benzene were detected as transformation products. Cultures transferred to sediment-free media containing citrate, lactate, yeast extract and sulfate also dehalogenated lindane. Lindane transformation was inhibited by the addition of molybdate. Lindane loss was not observed when the enrichment was cultured on lactate and citrate in the absence of sulfate, suggesting that sulfate-reducing bacteria transform lindane. Monochlorobenzene and benzene were identified as two transformation products in the lactate, citrate and sulfate enrichment culture. Pure cultures were used in order to confirm the ability of sulfate-reducing bacteria to dehalogenate lindane. Cell suspensions of Desulfovibrio gigas ATCC 19364, Desulfovibrio africanus ATCC 19997 and Desulfococcus multivorans ATCC 33890 were also able to dehalogenate lindane to benzene and monochlorobenzene. Actively growing and autoclaved cell suspensions of Desulfovibrio gigas were capable of lindane transformation while filter-sterilized cultures were not.

Dehalogenation of lindane (γ-hexachlorocyclohexane) by anaerobic bacteria from marine sediments and by sulfate-reducing bacteria

Marine sediments from around burrows of Saccoglossus kowalevskii, a tribromopyrrole-producing marine hemichordate, were used to develop anaerobic enrichment cultures supplemented with lindane and a mixture of short chain fatty acids. These enrichments consumed lindane and both monochlorobenzene and benzene were detected as transformation products. Cultures transferred to sediment-free media containing citrate, lactate, yeast extract and sulfate also dehalogenated lindane. Lindane transformation was inhibited by the addition of molybdate. Lindane loss was not observed when the enrichment was cultured on lactate and citrate in the absence of sulfate, suggesting that sulfate-reducing bacteria transform lindane. Monochlorobenzene and benzene were identified as two transformation products in the lactate, citrate and sulfate enrichment culture. Pure cultures were used in order to confirm the ability of sulfate-reducing bacteria to dehalogenate lindane. Cell suspensions of Desulfovibrio gigas ATCC 19364, Desulfovibrio africanus ATCC 19997 and Desulfococcus multivorans ATCC 33890 were also able to dehalogenate lindane to benzene and monochlorobenzene. Actively growing and autoclaved cell suspensions of Desulfovibrio gigas were capable of lindane transformation while filter-sterilized cultures were not.

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.

包括固定化した嫌気性菌と好気性菌によるテトラクロロエチレン・トリクロロエチレンの回分処理

Degradation of tetrachloroethene (PCE) and trichloroethene (TCE) by combining an anaerobic consortium with an aerobic bacterium was investigated. The anaerobic enrichment culture that dechlorinated high concentration of PCE to cis-1, 2-dichloroethene (cDCE) without methane production and sulfate reduction, was obtained from TCE contaminated soil. The aerobic phenol-oxidizing bacterium that degraded TCE and cDCE was isolated from leachate of a waste landfill site. Each culture retained chloroethene degrading activities after immobilization in κ-carrageenan gel beads. In a batch bioreactor containing the immobilized cells, PCE and TCE were converted to cDCE and chloride ion under anaerobic condition. After addition of phenol and oxygen (or hydrogen peroxide) to the reactor, cDCE was degraded. PCE and TCE (40-50mg·l-1) were degraded in 3 days with the bioreactor. The immobilized cells retained the degrading activities in repeated use.

Methanogenic and perchloroethylene-dechlorinating activity of anaerobic granular sludge.

The biodegradation and toxicity of tetrachlorethylene (C2Cl4) and trichloroethylene (C2HCl3) were studied with different anaerobic enrichment cultures using the following electron donors: acetate, propionate, butyrate, methanol, formate and hydrogen. All of them sustained dechlorination except propionate, for which C2Cl4 biodegradation rates were not significant. The best results were obtained with butyrate. Hydrogen appeared to be a relevant electron donor for dechlorination with the present cultures. In the presence of specific inhibitors such as bromoethanesulphonate or molybdate, a slight inhibition of dechlorination was observed. According to dechlorination kinetics, Monod-type behaviour was observed up to 120 microM C2Cl4 or 200 microM C2HCl3 with Ks values around 7 microM for both compounds. Dechlorination was partially inhibited at higher concentrations. In contrast, methanogens, or at least methane production, were more sensitive to the presence of chlorinated ethylenes and inhibitions of methanogenesis was observed to different extents over all the C2Cl4/C2HCl3 concentration range tested, even at the lowest concentrations.

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria.

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30 degrees C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30 degrees C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments.

Enrichment and characterization of an anaerobic methyl ethyl ketoxime degrading culture from an anoxic/anaerobic/aerobic activated sludge sequencing batch reactor

An anaerobic enrichment culture was developed from an anoxic/anaerobic/aerobic activated sludge sequencing batch reactor using methyl ethyl ketoxime (MEKO), a potent nitrification inhibitor, as the sole carbon and energy source in the absence of molecular oxygen and nitrate. The enrichment culture was gradually fed decreasing amounts of biogenic organic compounds and increasing concentrations of MEKO over 23 days until the cultures metabolized the oxime as the sole carbon source; the cultures were maintained for an additional 41 days on MEKO alone. Turbidity stabilized at approximately 100 mg/l total suspended solids. Growth on selective media plates confirmed that the microorganisms were utilizing the MEKO as the sole carbon and energy source. The time frame required for growth indicated that the kinetics for MEKO degradation are slow. A batch test indicated that dissolved organic carbon decreased at a rate comparable to MEKO consumption, while sulfate was not consumed. The nature of the electron acceptor in anaerobic MEKO metabolism is unclear, but it is hypothesized that the MEKO is hydrolyzed intracellularly to form methyl ethyl ketone and hydroxylamine which serve as electron donor and electron acceptor, respectively.

Enrichment and characterization of an anaerobic methyl ethyl ketoxime degrading culture from an anoxic/anaerobic/aerobic activated sludge sequencing batch reactor

An anaerobic enrichment culture was developed from an anoxic/anaerobic/aerobic activated sludge sequencing batch reactor using methyl ethyl ketoxime (MEKO), a potent nitrification inhibitor, as the sole carbon and energy source in the absence of molecular oxygen and nitrate. The enrichment culture was gradually fed decreasing amounts of biogenic organic compounds and increasing concentrations of MEKO over 23 days until the cultures metabolized the oxime as the sole carbon source; the cultures were maintained for an additional 41 days on MEKO alone. Turbidity stabilized at approximately 100 mg/l total suspended solids. Growth on selective media Plates confirmed that the microorganisms were utilizing the MEKO as the sole carbon and energy source. The time frame required for growth indicated that the kinetics for MEKO degradation are slow. A batch test indicated that dissolved organic carbon decreased at a rate comparable to MEKO consumption, while sulfate was not consumed. The nature of the electron acceptor in anaerobic MEKO metabolism is unclear, but it is hypothesized that the MEKO is hydrolyzed intracellularly to form methyl ethyl ketone and hydroxylamine which serve as electron donor and electron acceptor, respectively.

Biotransformation of explosives by anaerobic consortia in liquid culture and in soil slurry

A laboratory study was conducted to study the feasibility of removing explosives in contaminated soil under anaerobic conditions. Anaerobic enrichment cultures were prepared from soil samples under various electron-accepting conditions, namely, sulfate-reducing, methanogenic, and nitrate-reducing conditions. The sulfate-reducing condition was very effective in removing all of the explosive compounds from the soil. The sulfate-reducing consortium removed 100% of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitrobenzene (TNB) within 10–15 days of incubation and removed 75 to 95% of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), within 21 days of incubation. The consortium used explosive compounds as the nitrogen source, however, it did not use these compounds as the sole carbon source. The various metabolites obtained from TNT metabolism were 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT), 2,4-diamino-6-nitrotoluene (2,4- d-6-NT), and 2-methyl pentanoic acid. This sulfate-reducing consortium was further studied for its usefulness in removing TNT at the contaminated site. The results showed that the consortium can remove TNT under 5% and 10% soil slurry conditions. This laboratory study demonstrated that under anaerobic conditions, sulfate-reducing bacteria can be useful in the bioremediation of contaminated soil with TNT and other explosives.

Decarboxylation of 2,3-Dihydroxybenzoate to Catechol Supports Growth of Fermenting Bacteria

Anaerobic enrichment cultures with 2,3-dihydroxybenzoate as sole source of energy and organic carbon yielded slow-growing cultures of fermenting bacteria. A culture derived from a marine inoculum, strain Pe23DHB, consisted of two morphotypes: spirilloid, highly motile bacteria (the predominant type), and very few long, rod-shaped bacteria. The culture was able to grow by decarboxylation of 2,3-dihydroxybenzoate to catechol. The decarboxylating activity was localized mainly in the cytosol; addition of avidin or of sodium salts had no effect on the decarboxylating activity in cell-free extracts. These results suggest that strain Pe23DHB did not conserve energy by a membrane-bound biotin-containing decarboxylase, creating a Na+-gradient across the cytoplasmic membrane. The energy conservation mechanism remains unknown at present.

Characterization of an anaerobic soil enrichment capable of dechlorinating high concentrations of tetrachloroethylene

An anaerobic soil enrichment culture could dechlorinate high concentrations of tetrachloroethylene (PCE; 150 mg/liter nominal concentration; approximately 58 mg/liter in aqueous concentration) nearly stoichiometrically to cis-1,2-dichloroethylene (cis-DCE) via trichloroethylene (TCE) at high rates; a maximum dechlorination rate was 0.4 μmol of PCE transformed/mg volatile suspended solids per hr, using citrate as an electron and carbon source and yeast extract as a nutritional requirement. This dechlorinating activity was comparable with those of the previously-reported, efficient bacterial cultures. Some substrates such as pyruvate, succinate, formate, acetate, and acetate with H2 could replace citrate but propionate could not, and yeast extract could be replaced by a vitamin mixture. However the PCE dechlorination rate decreased more than threefold by the addition of the vitamin mixture, suggesting that the vitamin mixture could not be a complete supplement for the nutritional requirement. Optimal pH and temperature of the enrichment for PCE dechlorination were 7 and 30 °C, respectively. Dechlorination of PCE was completely inhibited by the addition of NO3− and NO2− as potential alternative electron acceptors. S2O3−2 and SO3−2 delayed PCE dechlorination but SO4−2 had no significant effect on PCE reduction. 2-bromoethanesulfonic acid (BES, an inhibitor of methanogenesis) also showed no influence on PCE dechlorination, suggesting methanogens were not concerned with PCE removal in this enrichment. Further, microbial investigations on the enrichment showed that it contains four types of bacteria; cocci, large rods, curved rods, and small rods. The small rods seemed to nutritionally support the PCE dechlorinating bacteria, presumably the curved rods.

Fe(III) as an electron acceptor for H2 oxidation in thermophilic anaerobic enrichment cultures from geothermal areas.

Six sustainable enrichment cultures of thermophilic H2-oxidizing microorganisms utilizing Fe(III) as an electron acceptor were obtained from geothermally heated environments located on two continents (America, Eurasia) and on islands in the Northern (Iceland) and Southern (Fiji) hemispheres, demonstrating the wide distribution of these microorganisms. The main products of amorphic Fe(III) oxide reduction were magnetite and siderite. The observed temperature range for Fe(III) reduction in growing cultures was from 55 degrees C to 87 degrees C, extending the known limits for growth of Fe(III)-reducing microorganisms producing extracellular magnetite to nearly 90 degrees C.

Response to Comment on “Comparative Kinetics of Hydrogen Utilization for Reductive Dechlorination of Tetrachloroethene and Methanogenesis in an Anaerobic Enrichment Culture”

Response to Comment on “Comparative Kinetics of Hydrogen Utilization for Reductive Dechlorination of Tetrachloroethene and Methanogenesis in an Anaerobic Enrichment Culture”