Anaerobic Degradation of Biphenyl by the Facultative Anaerobic Strain Citrobacter freundii BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundiiwas isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in the culture medium equaled 150 mg/ml, the culture with a titer of 10⁹ cells/ml degraded up to 26–28% of biphenyl in 3 days (28°C). At 250 mg/ml, the culture with a titer of 10⁷ cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria

Screening studies with strict and facultative anaerobic bacteria showed that Clostridium app. and several other representatives of Bacillaceae and Enterobacteriaceae actively degraded -hexachlorocyclohexane (-HCH) under anaerobic conditions. Representatives of Lactobacillaceae and Propronibacterium were inactive. With ³⁶Cl-labelled -HCH a nearly complete dechlorination was shown to occur in 4–6 days by Clostridium butyricum, C. pasteurianum and Citrobacter freundii, while other facultative anaerobic species were less active.Aerobically grown facultative anaerobes also dechlorinated actively -HCH during subsequent anaerobic incubation with glucose, pyruvate or formate as substrates. The -, - and -HCH isomers were also, but more slowly, dechlorinated (>>-HCH). All species active in anaerobic degradation of -HCH formed -tetrachlorocyclohexene (TCH) as the main intermediate metabolite and no -pentachlorocyclohexene (PCH) or other isomers of TCH or PCH have been found. Small amounts of tri- and tetrachlorinated benzenes have been found too. The mechanism of dechlorination is discussed.Non-Common Abbreviations Used -HCH -hexachlorocyclohexane - -TCH -2,3,4,5-tetrachlorocyclohexene - -PCH -1,2,3,4,5-pentachlorocyclohexene - GLC gas liquid chromatography     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Catabolism of biphenyl by Pseudomonas putida BS 893 strain containing the biodegradation plasmid pBS241

The isolation and identification of biphenyl catabolism products in Pseudomonas putida BS 893 (pBS241) showed the presence of benzoic, m-hydroxybenzoic and cinnamic acids. The two latter compounds were not found in biphenyl degradation by other bacterial strains. P. putida BS 893 (pBS241) differed from other biphenyl-positive Pseudomonas strains in the enzyme activity. These differences may stem from peculiarities in the pathway of biphenyl catabolism controlled by plasmid pBS241.     

Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2

Methylocystis strain SB2, a facultative methanotroph capable of growth on multi-carbon compounds, was screened for its ability to degrade the priority pollutants 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA), and 1,1-dichloroethylene (1,1-DCE), as well as cis-dichloroethylene (cis-DCE) when grown on methane or ethanol. Methylocystis strain SB2 degraded 1,2-DCA and 1,1,2-TCA when grown on either substrate and cis-DCE when grown on methane. Growth of Methylocystis strain SB2 on methane was inhibited in the presence of all compounds, while only 1,1-DCE and cis-DCE inhibited growth on ethanol. No degradation of any chlorinated hydrocarbon was observed in ethanol-grown cultures when particulate methane monooxygenase (pMMO) activity was inhibited with the addition of acetylene, indicating that competition for binding to the pMMO between the chlorinated hydrocarbons and methane limited both methanotrophic growth and pollutant degradation when this strain was grown on methane. Characterization of Methylocystis strain SB2 found no evidence of a high-affinity form of pMMO for methane, nor could this strain utilize 1,2-DCA or its putative oxidative products 2-chloroethanol or chloroactetic acid as sole growth substrates, suggesting that this strain lacks appropriate dehydrogenases for the conversion of 1,2-DCA to glyoxylate. As ethanol: (1) can be used as an alternative growth substrate for promoting pollutant degradation by Methylocystis strain SB2 as the pMMO is not required for its growth on ethanol and (2) has been used to enhance the mobility of chlorinated hydrocarbons in situ, it is proposed that ethanol can be used to enhance both pollutant transport and biodegradation by Methylocystis strain SB2.     

Anaerobic malonate decarboxylation by Citrobacter diversus

Citrobacter diversus ATCC 27156 was able to grow by decarboxylation of malonate to acetate under strictly anaerobic conditions, in the presence of yeast extract. The growth yield, corrected for growth on yeast extract, was 2.03 g cell dry mass per mol malonate. The addition of malonate to ATP-depleted cell suspensions (less than 0.2 nmol ATP/mg cell protein) resulted in a rapid increase in cellular ATP levels to between 4.5 and 6.0 nmol/mg cell protein. Intact cells decarboxylated malonate at rates of up to 1.5 mumol/min.mg protein. Enzyme assays on malonate-grown cells indicated activation of malonate by an ATP-dependent ligase reaction and by CoA transfer from acetyl-CoA, followed by decarboxylation of malonyl-CoA to acetyl-CoA with subsequent recovery of the invested ATP by substrate level phosphorylation through the activity of acetate kinase. Net ATP synthesis is postulated to be mediated by gradient formation coupled to the decarboxylation of malonyl-CoA. The protonophore CCCP and H(+)-ATPase inhibitor DCCD significantly reduced cellular ATP levels, suggesting a role for proton gradients in the energy metabolism of this strain when growing an malonate. Inhibitors of sodium metabolism or ommission of sodium had no effect on ATP levels or malonate decarboxylation.     

Multiple and flexible roles of facultative anaerobic bacteria in microaerophilic oleate degradation

Anaerobic degradation of long-chain fatty acids (LCFA) involves syntrophic bacteria and methanogens, but facultative anaerobic bacteria (FAB) might have a relevant role as well. Here we investigated oleate degradation by a syntrophic synthetic co-culture of Syntrophomonas zehnderi (Sz) and Methanobacterium formicicum (Mf) and FAB (two oleate-degrading Pseudomonas spp. I1 + I2). Sz + Mf were first cultivated in a continuous bioreactor under strict anaerobic conditions. Thereafter, I1 + I2 were inoculated and microaerophilic conditions were provided. Methane and acetate were the main degradation products by Sz + Mf in anaerobiosis and by Sz + Mf + I1 + I2 in microaerophilic conditions. However, acetate production from oleate was higher in microaerophilic conditions (5% O₂) with the four microorganisms together (0.41 ± 0.07 mmol day⁻¹) than in anaerobiosis with Sz + Mf (0.23 ± 0.05 mmol day⁻¹). Oleate degradation in batch assays was faster by Sz + Mf + I1 + I2 (under microaerophilic conditions) than by Sz + Mf alone (under strict anaerobic conditions). I1 + I2 were able to grow with oleate and with intermediates of oleate degradation (hydrogen, acetate and formate). This work highlights the importance of FAB, particularly Pseudomonas sp., in anaerobic reactors treating oleate-based wastewater, because they accelerate oleate conversion to methane, by protecting strict anaerobes from oxygen toxicity and also by acting as alternative hydrogen/formate and acetate scavengers for LCFA-degrading anaerobes.     

Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G Isolated from an anaerobic bacterial consortium

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gram-negative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to beta-Asp-Arg and beta-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the beta-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.     

Use of molecular sulfur as an agent oxidizing H2 by a facultative anaerobic Pseudomonas strain

Pseudomonas Z-731, subspecies P. mendocina, was grown under anaerobic conditions in the atmosphere of hydrogen. Apart from hydrogen nitrate reduction and Fe3+ reduction, the organism was able to reduce S0, S2O3 and SO3 with the formation of H2S. The reduction of S0 with hydrogen was shown to be typical of certain facultative anaerobic microorganisms.     

Biodegradation of estrogens by facultative anaerobic iron-reducing bacteria

Natural estrogens such as estrone, 17β-estradiol, estriol, and the synthetic component of contraceptive pills, 17α-ethinylestradiol, enter the municipal wastewater treatment plant via human excretions. A significant portion of these substances is found to remain in reject water produced after anaerobic digestion of activated sludge. In this study, the effect of the oxidant, Fe(III), and facultative anaerobic strain of iron-reducing bacteria on the anaerobic degradation of estrogens in reject water was investigated. Synthetic 17α-ethinylestradiol remained resistant to anaerobic biodegradation by iron-reducing bacteria, while natural estrogens such as 17β-estradiol, estriol, and estrone were removed by 92%, 60% and 27%, respectively, after 15 days of batch cultivation of iron-reducing bacteria in reject water with the addition of all estrogens to concentrations 100 μg l⁻¹ each. The ability of facultative anaerobic iron-reducing bacteria to degrade estrogens can be used for the anaerobic removal of trace organics from reject water in municipal wastewater treatment plant.     

Anaerobic degradation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by a marine Desulfobacterium strain

Dimethylsulfoniopropionate, an osmolyte of marine algae, is thought to be the major precursor of dimethyl sulfide, which plays a dominant role in biogenic sulfur emission. The marine sulfate-reducing bacterium Desulfobacterium strain PM4 was found to degrade dimethylsulfoniopropionate to 3-S-methylmercaptopropionate. The oxidation of one of the methyl groups of dimethylsulfoniopropionate was coupled to the reduction of sulfate; this process is similar to the degradation betaine to dimethylglycine which was described earlier for the same strain. Desulfobacterium PM4 is the first example of an anaerobic marine bacterium that is able to demethylate dimethylsulfoniopropionate.Abbreviations DMSP dimethylsulfoniopropionate - DMS dimethyl sulfide - MMPA 3-S-methylmercaptopropionate     

Quantification of denitrification by strain T1 during anaerobic degradation of toluene

Summary Strain T1 is a denitrifying bacterium that is capable of toluene degradation under anaerobic conditions. During anaerobic growth on toluene, the specific growth rate of strain T1 was 0.14 h^–1. Nitrite accumulated in the medium stoichiometrically with the depletion of nitrate. When nitrate was nearly depleted from the medium nitrite reduction and dinitrogen formation began. A non-kinetic model was formulated that was based on a hypothesis of non-simultaneous nitrate and nitrite reduction, independent of the concentrations of nitrate and nitrite. The model was verified experimentally over a wide range of conditions that included nitrate and nitrite limitation, toluene limitation, and various ratios of nitrate to nitrite. The model and its experimental verification demonstrated that strain T1 reduces nitrate and nitrite non-simultaneously, even if nitrite is initially present in the medium in addition to nitrate. Offprint requests to: L. Y. Young     

Anaerobic degradation of isobutyrate by methanogenic enrichment cultures and by a Desulfococcus multivorans strain

Methanogenic enrichment cultures with isobutyrate as sole source of carbon and energy were inoculated with sediment and sludge samples from freshwater and marine origin. Over more than 20 transfers, these cultures fermented 2 mol isobutyrate with 1 mol CO₂ via an intermediate formation of n-butyrate to 4 mol acetate and 1 mol CH₄. The primary isobutyrate-fermenting bacteria could not be purified. From one of the marine enrichment cultures, a sulfate-reducing bacterium was isolated which oxidized isobutyrate with sulfate completely to CO₂. Based on its physiological and morphological properties, this strain was assigned to the known species Desulfococcus multivorans. It also oxidized many other fatty acids without significant release of short-chain intermedeates. The enzymes involved in isobutyrate degradation by this bacterium were assayed in cell-free extracts. The results indicate that isobutyrate is activated to its CoA derivative and oxidized via methylmalonate semialdehyde to propionyl-CoA. Propionyl-CoA is further converted via the methylmalonyl-CoA pathway to acetyl-CoA which is finally cleaved by the CO-dehydrogenase system. It is evident that this is not the pathway used by the fermenting bacteria prevailing in the methanogenic enrichment cultures. There results are discussed on the basis of energetical considerations.     

Genome sequence of the facultative anaerobic arsenite-oxidizing and nitrate-reducing bacterium Acidovorax sp. strain NO1

Acidovorax sp. strain NO1, isolated from gold mine soil, was shown to be a facultative anaerobic arsenite-oxidizing and nitrate-reducing bacterium. The reported draft genome predicts the presence of genes involved in arsenic metabolism, nitrate reduction, phosphate transport, and multiple metal resistances and indicates putative horizontal gene transfer events.     

Hydroxylation and carboxylation--two crucial steps of anaerobic benzene degradation by Dechloromonas strain RCB

Benzene is a highly toxic industrial compound that is essential to the production of various chemicals, drugs, and fuel oils. Due to its toxicity and carcinogenicity, much recent attention has been focused on benzene biodegradation, especially in the absence of molecular oxygen. However, the mechanism by which anaerobic benzene biodegradation occurs is still unclear. This is because until the recent isolation of Dechloromonas strains JJ and RCB no organism that anaerobically degraded benzene was available with which to elucidate the pathway. Although many microorganisms use an initial fumarate addition reaction for hydrocarbon biodegradation, the large activation energy required argues against this mechanism for benzene. Other possible mechanisms include hydroxylation, carboxylation, biomethylation, or reduction of the benzene ring, but previous studies performed with undefined benzene-degrading cultures were unable to clearly distinguish which, if any, of these alternatives is used. Here we demonstrate that anaerobic nitrate-dependent benzene degradation by Dechloromonas strain RCB involves an initial hydroxylation, subsequent carboxylation, and loss of the hydroxyl group to form benzoate. These studies provide the first pure-culture evidence of the pathway of anaerobic benzene degradation. The outcome of these studies also suggests that all anaerobic benzene-degrading microorganisms, regardless of their terminal electron acceptor, may use this pathway.     

Pollutant degradation by a Methylocystis strain SB2 grown on ethanol: bioremediation via facultative methanotrophy

A facultative methanotroph, Methylocystis strain SB2, was examined for its ability to degrade chlorinated hydrocarbons when grown on methane or ethanol. Strain SB2 grown on methane degraded vinyl chloride (VC), trans-dichloroethylene (t-DCE), trichloroethylene (TCE), 1,1,1-trichloroethane (1,1,1-TCA), and chloroform (CF), but not dichloromethane (DCM). Growth on methane was reduced in the presence of any chlorinated hydrocarbon. Strain SB2 grown on ethanol degraded VC, t-DCE, and TCE, and 1,1,1-TCA, but not DCM or CF. With the exception of 1,1,1-TCA, the growth of strain SB2 on ethanol was not affected by any individual chlorinated hydrocarbon. No degradation of any chlorinated hydrocarbon was observed when acetylene was added to ethanol-grown cultures, indicating that this degradation was due to particulate methane monooxygenase (pMMO) activity. When mixtures of chlorinated alkanes or alkenes were added to cultures growing on methane or ethanol, chlorinated alkene degradation occurred, but chlorinated alkanes were not, and growth was reduced on both methane and ethanol. Collectively, these data indicate that competitive inhibition of pMMO activity limits methanotrophic growth and pollutant degradation. Facultative methanotrophy may thus be useful to extend the utility of methanotrophs for bioremediation as the use of alternative growth substrates allows for pMMO activity to be focused on pollutant degradation.     

Anaerobic degradation of protocatechuate (3,4-dihydroxybenzoate) by Thauera aromatica strain AR-1

The denitrifying bacterium Thauera aromatica strain AR-1 grows anaerobically with protocatechuate (3,4-dihydroxybenzoate (DHB)) as sole energy and carbon source. This bacterium harbors two distinct pathways for degradation of aromatic compounds, the benzoyl-coenzyme A (CoA) pathway for benzoate degradation and the hydroxyhydroquinone (HHQ) pathway for degradation of 3,5-DHB. In order to elucidate whether protocatechuate is degraded via the benzoyl-CoA or the HHQ pathway, induction experiments were carried out. Dense suspensions of cells grown on protocatechuate or benzoate readily degraded benzoate and protocatechuate but not 3,5-DHB. Dense suspensions of 3,5-DHB-grown cells degraded 3,4- and 3,5-DHB at similar rates, but benzoate was not degraded. 3,5-DHB hydroxylating activity was found only in cells grown with this substrate. HHQ dehydrogenase activity was found in extracts of cells grown with 3,5-DHB and at a low rate also in protocatechuate-grown cells, but not in extracts of cells grown with benzoate. Activities of protocatechuyl-CoA synthetase and protocatechuyl-CoA reductase leading to 3-hydroxybenzoyl-CoA were found in extracts of cells grown with protocatechuate. There was no repression of the HHQ pathway by the presence of protocatechuate, unlike by degradation of benzoate. We conclude that protocatechuate is not degraded via the HHQ pathway because there was no evidence of a hydroxylation reaction involved in this process. Instead, our results strongly suggest that protocatechuate is degraded via a pathway which connects to the benzoyl-CoA route of degradation.     

Anaerobic benzene degradation by bacteria

Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.