The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation.

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Dehalogenation: a novel pathway for the anaerobic biodegradation of haloaromatic compounds.

Microorganisms of lake sediment and sewage sludge anaerobically metabolized halobenzoates by a novel pathway. The primary degradative event was loss of the aryl halide without the alteration of the aromatic ring. Dehalogenation required strict anaerobic conditions and depended on the halogen and position, but not the number of halogen substituents. A stable methanogenic bacterial consortium was enriched from sludge and found capable of dehalogenating and often mineralizing a variety of halobenzoates to CH(4) and CO(2). The results suggest that reductive dehalogenation of aromatics could be important in removal of some chlorinated xenobiotics from the environment.

Ecology and evolution of microbial populations for bioremediation.

Bioremediation exploits the genetic diversity and metabolic versatility of microorganisms for the transformation of contaminants into less-harmful end-products, which are then integrated into natural biogeochemical cycles. Understanding the ecology, physiology and evolution of degradative microorganisms is critical for the successful consideration and implementation of bioremediation. This article focuses on the common ecological and evolutionary constraints that influence bioremediation processes.

Microbial ecology of a shallow unconfined ground water aquifer polluted by municipal landfill leachate.

The microflora of a shallow anoxic aquifer underlying a municipal landfill in Oklahoma was characterized by direct light microscopy, most probable number determinations of sulfate reducers and methanogens, and measurements of methanogenesis in aquifer samples containing either endogenous or exogenous electron donors and various sulfate concentrations. Acridine orange direct counts of bacteria did not vary significantly with time or between 2 major sampling areas (1.70±0.16×10(7) to 11.2±2.1×10(7) cells/gdw). One site (B) was high in organic matter and low in sulfate, and methanogens generally outnumbered sulfate-reducers at most times of the year, whereas the opposite was true for another site (A). Greater than 75% of the theoretical amount of methane was detected within 7 weeks in both site A and B aquifer slurries amended with noncompetitive electron donors like methanol and trimethylamine. However, only site B slurries efficiently converted competitive donors like acetate, H2, and formate to the expected amount of methane. A mapping of sulfate and methane levels indicated that site A is relatively localized. These results suggest that the predominant flow of carbon and energy is through methanogenesis at aquifer site B whereas sulfate reduction predominated at site A. However, both methanogens and sulfate reducers could be isolated from either site.

Biodegradation of cresol isomers in anoxic aquifers.

The biodegradation of o-, m-, and p-cresol was examined in material obtained from a shallow anaerobic alluvial sand aquifer. The cresol isomers were preferentially metabolized, with p-cresol being the most easily degraded. m-Cresol was more persistent than the para-isomer, and o-cresol persisted for over 90 days. Biodegradation of cresol isomers was favored under sulfate-reducing conditions (SRC) compared with that under methanogenic conditions (MC). Slurries that were acclimated to p-cresol metabolism transformed this substrate at 18 and 330 nmol/h per g (dry weight) for MC and SRC, respectively. Inhibition of electron flow to sulfate reduction with 2.0 mM molybdate reduced p-cresol metabolism in incubations containing sulfate. When methanogenesis was blocked with 5 mM bromoethanesulfonic acid in incubations lacking sulfate, p-cresol catabolism was retarded. Under SRC 3.4 mol of sulfate was consumed per mol of p-cresol metabolized. The addition of sulfate to methanogenic incubations stimulated p-cresol degradation. Simultaneous adaptation studies in combination with spectrophotometric and chromatographic analysis of metabolites indicated that p-cresol was oxidized under SRC to p-hydroxybenzoate via the corresponding alcohol and aldehyde. This series of reactions was inhibited under sulfate-limited or aerobic conditions. Therefore, the primary catabolic event for p-cresol decomposition under SRC appears to involve the hydroxylation of the aryl methyl group.

Anaerobic aquifer transformations of 2,4-dinitrophenol under different terminal electron accepting conditions.

We evaluated the susceptibility of 2,4-dinitrophenol (2,4-DNP) and 2,4-diaminophenol to anaerobic biodegradation in aquifer slurries. Aquifer microorganisms depleted 2,4-DNP at rates of 25, 9 and 0.4 microM/day under methanogenic, sulfate-reducing and nitrate-reducing conditions, respectively. Rates of abiotic, 2,4-DNP loss in autoclaved control incubations were 7.2, 6.2 and 0.95 microM/day respectively. Abiotic, 2,4-DNP reduction was especially important as the first step in its transformation. 2-Amino-4-nitrophenol was produced by this process, but this compound was further metabolized in methanogenic and sulfate-reducing aquifer slurries. This partially reduced compound persisted in autoclaved controls and in the nitrate-reducing aquifer slurries. Aquifer slurries incubated with either 2,4-DNP or 2,4-diaminophenol produced methane when incubated with no other electron acceptor suggesting that mineralization had occurred under these conditions. In parallel experiments, aquifer slurries amended with 2,6-dinitrophenol or picric acid did not produce methane at levels above the substrate unamended controls.

Role of electron-donating cosubstrates in the anaerobic biotransformation of chlorophenoxyacetates to chlorophenols by a bacterial consortium enriched on phenoxyacetate.

A bacterial consortium that anaerobically mineralized phenoxyacetate, with transient production of phenol as an intermediate, was obtained from a methanogenic aquifer site near the Norman, OK municipal landfill. This consortium was able to convert the eight halogenated chlorophenoxyacetates tested to the corresponding chlorophenols. The chlorophenols were not subsequently metabolized. The addition of reduced substrates increased the rate of degradation of all chlorophenoxyacetates, with 78% of mono- and di-chlorinated substrates being transformed to chlorophenols in butyrate-amended cultures, compared to less than 37% transformed in unsupplemented cultures. Butyrate increased the transformation of 2,4,5-trichlorophenoxyacetate from 10% to 20%. An experiment evaluating the effects of several compounds on the side-chain cleavage reaction of 3-chlorophenoxyacetate showed that addition of compounds which readily act as hydrogen donors (butyrate, crotonate, ethanol, propionate, and hydrogen) resulted in 2 to 5 times the amount of 3-chlorophenoxyacetate transformed compared to controls with no amendment, formate had a slight stimulatory effect, and acetate and methanol had no effect. Butyrate addition also increased the rate of phenoxyacetate degradation, resulting in transient phenol accumulation not observed in butyrate-unamended controls. These results support the hypothesis that the side-chain cleavage of phenoxyacetate is a reductive process that is stimulated by the oxidation of reduced cosubstrates.

Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation.

We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates.

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

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

Environmental factors influencing methanogenesis in a shallow anoxic aquifer: a field and laboratory study.

The environmental factors influencing methanogenesis in a shallow anoxic aquifer were probed in a combined field and laboratory study. Field data collected over a year revealed that 'in situ' rates of methane production were depressed in winter and elevated in summer. Over the same period, ground water pH values ranged from 6.0 to 7.8 while temperatures varied from 7-22 degrees C. 'In situ' methanogenesis was severely inhibited at temperatures less than 13 degrees C or by pH values less than 7. The influence of these factors on microbial methane formation from both endogenous and exogenous substrates were tested in aquifer slurries adjusted to pH 5-9 and incubated at temperatures ranging from 5-45 degrees C. Temperature optima for methane production from endogenous substrates varied as a function of pH, but the pH optimum was 8 at all temperatures. Optimal conditions for acetoclastic methanogenesis were found at pH 8 and 35 degrees C. An analysis of variance revealed that pH, temperature, and a pH-temperature interaction are all significant variables influencing aquifer methanogenesis. In addition transient sulfate accumulations were also found to limit methane production in some areas. A comparison of field and laboratory methane production patterns suggest that pH, temperature, and sulfate accumulations are important, but not the only environmental variables influencing the mineralization of organic matter in shallow aquifers.

Reductive dehalogenation of a nitrogen heterocyclic herbicide in anoxic aquifer slurries.

We studied the metabolic fate of bromacil in anaerobic aquifer slurries held under denitrifying, sulfate-reducing, or methanogenic conditions. Liquid chromatograhy-mass spectrometry of the slurries confirmed that bromacil was debrominated under methanogenic conditions but was not degraded under the other incubation conditions. This finding extends the range of aryl reductive dehalogenation reactions to include nitrogen heterocyclic compounds.

Anaerobic biodegradation of 2,4,5-trichlorophenoxyacetic Acid in samples from a methanogenic aquifer: stimulation by short-chain organic acids and alcohols.

The herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was dehalogenated in samples from a methanogenic aquifer to form 2,4- and 2,5-dichlorophenoxyacetic acids as the first detected intermediates. Further incubation of the aquifer slurries resulted in the formation of several intermediates including monochlorophenoxyacetic acids, di- and monochlorophenols, as well as phenol. No transformation of the parent substrate or production of intermediates was detected in autoclaved controls. The pattern of intermediate formation suggested that the anaerobic degradation of 2,4,5-T proceeded by a series of sequential dehalogenation steps with side-chain cleavage reactions occurring at some point before ring cleavage. The addition of short-chain organic acids or alcohols stimulated the onset and rate of 2,4,5-T dehalogenation and decreased the amount of parent substrate still detectable as halogenated intermediates at the end of the experiment. Sulfate addition had the opposite effect on dehalogenation regardless of whether supplemental carbon was added to the aquifer slurries. The inhibitory effect of sulfate on dehalogenation could sometimes be relieved with molybdate, although this effect seemed to be related to the supplemental carbon compound that was used.

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei".

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.

Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei.

The inhibition of aryl reductive dehalogenation reactions by sulfur oxyanions has been demonstrated in environmental samples, dehalogenating enrichments, and the sulfate-reducing bacterium Desulfomonile tiedjei; however, this phenomenon is not well understood. We examined the effects of sulfate, sulfite, and thiosulfate on reductive dehalogenation in the model microorganism D. tiedjei and found separate mechanisms of inhibition due to these oxyanions under growth versus nongrowth conditions. Dehalogenation activity was greatly reduced in extracts of cells grown in the presence of both 3-chlorobenzoate, the substrate or inducer for the aryl dehalogenation activity, and either sulfate, sulfite, or thiosulfate, indicating that sulfur oxyanions repress the requisite enzymes. In extracts of fully induced cells, thiosulfate and sulfite, but not sulfate, were potent inhibitors of aryl dehalogenation activity even in membrane fractions lacking the cytoplasmically located sulfur oxyanion reductase. These results suggest that under growth conditions, sulfur oxyanions serve as preferred electron acceptors and negatively influence dehalogenation activity in D. tiedjei by regulating the amount of active aryl dehalogenase in cells. Additionally, in vitro inhibition by sulfur oxyanions is due to the interaction of the reactive species with enzymes involved in dehalogenation and need not involve competition between two respiratory processes for reducing equivalents. Sulfur oxyanions also inhibited tetrachloroethylene dehalogenation by the same mechanisms, further indicating that chloroethylenes are fortuitously dehalogenated by the aryl dehalogenase. The commonly observed inhibition of reductive dehalogenation reactions under sulfate-reducing conditions may be due to similar regulation mechanisms in other dehalogenating microorganisms that contain multiple respiratory activities.

Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds.

The reductive biodegradation of a variety of haloaromatic substrates was monitored in samples from two sites within a shallow anoxic aquifer and was compared with freshwater sediment and sewage sludge. The metabolic capacity existing in methane-producing aquifer material was very similar to that in sediment in that three of four chlorobenzoates, five of seven chlorophenols, and one of two chlorophenoxyacetate herbicides were reductively dehalogenated in both types of incubations. The 2,4-dichlorophenoxyacetate was first converted to a dichlorophenol before dehalogenation occurred. Sewage sludge microorganisms dehalogenated four of seven chlorophenols tested and degraded both phenoxyacetate herbicides by first converting them to the corresponding chlorophenols, but the microorganisms did not transform the chlorobenzoates. In general, the same suite of initial metabolites were produced from a test substrate in all types of samples, as confirmed by cochromatography of the intermediates with authentic material. Aquifer microbiota from a sulfate-reducing site was unable to significantly degrade any of the haloaromatic substrates tested. Biological removal of the sulfate in samples from this site permitted dehalogenation of a model substrate, while stimulation of methanogenesis without removal of sulfate did not. These results demonstrate that dehalogenating microorganisms were present at this site but that their activity was at least partially inhibited by the high sulfate levels.

Anaerobic biodegradation of methyl esters by Acetobacterium woodii and Eubacterium limosum.

The ability of Acetobacterium woodii and Eubacterium limosum to degrade methyl esters of acetate, propionate, butyrate, and isobutyrate was examined under growing and resting-cell conditions. Both bacteria hydrolyzed the esters to the corresponding carboxylates and methanol under either condition. Methanol was further oxidized to formate under growing but not resting conditions. Unlike the metabolism of phenylmethylethers, no H2 requirement was evident for ester biotransformation. The hydrolysis of methyl carboxylates is thermodynamically favorable under standard conditions and the mixotrophic metabolism of ester/CO2 allowed for bacterial growth. These results suggest that the degradation of methyl carboxylates may be a heretofore unrecognized nutritional option for acetogenic bacteria.

H(2)-CO(2)-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium.

The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N(2)-CO(2) atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H(2)-CO(2) but not a N(2)-CO(2) or N(2) atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H(2) and CO(2) in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C(7)H(3)O(3)(OCH(3))(n) + nHCO(3) + nH(2) --> C(7)H(3)O(3)(OH)(n) + nCH(3)COO + nH(2)O.

Alternative nonlinear model for estimating second-order rate coefficients for biodegradation.

A modification of the second-order model for biodegradation was derived, applied to an example data set, and shown to be superior for describing the anaerobic biodegradation of p-cresol by an enriched bacterial consortium. The modified model circumvents the no-growth assumption implicit in the use of the second-order rate equation, but still requires the assumption of first-order kinetics over the course of substrate depletion. Violation of the no-growth assumption is particularly important since overestimates of the pseudo-first-order rate coefficient lead to underestimates of the time required for the removal of a xenobiotic chemical from a contaminated environment. Our calculations show that the errors introduced into the pseudo-first-order rate coefficient (and the resulting estimates of the second-order rate coefficient) approach 100% if one doubling occurs in activity over the course of substrate depletion. For an exemplary data set, use of a first-order model resulted in a 100% overestimate of the first-order decay coefficient, which would in turn lead to a corresponding overestimate of the second-order rate coefficient. The modified model we describe is a potential alternative to the pseudo-first-order model for the routine estimation of second-order rate coefficients.

Characterization of the acclimation period before anaerobic dehalogenation of halobenzoates.

The acclimation periods prior to detectable dehalogenation of halogenated benzoates in anaerobic lake sediments ranged from 3 weeks to 6 months. These acclimation periods were reproducible over time and among sampling sites and were characteristic of the chemical tested. The lengthy acclimation period appears to represent an induction phase in which little or no aryl dehalogenation is observed, followed by an exponential increase in activity typical of an enrichment response. Continuous growth from the time of the first exposure to the chemical is inconsistent with the extremely low per-cell activities estimated for the early days of the acclimation period and the fact that the dehalogenation yields no carbon to support microbial growth. The finding of a characteristic acclimation time for each chemical argues against nutritional deficiency, inhibition, or predation as an explanation for this phase of metabolism, while the reproducibility of the findings with time and space and among replicates argues against genetic changes as the explanation. The acclimation times did correlate with the eventual dehalogenation rates. This may reflect the general energy limitations in the anaerobic communities and suggests that those chemicals with faster dehalogenation rates provide more energy for the induction and growth phases of the active population.

A rapid and simple method for estimating sulfate reduction activity and quantifying inorganic sulfides.

A simplified passive extraction procedure for quantifying reduced inorganic sulfur compounds from sediments and water is presented. This method may also be used for the estimation of sulfate reduction rates. Efficient extraction of FeS, FeS(inf2), and S(sup2-) was obtained with this procedure; however, the efficiency for S(sup0) depended on the form that was tested. Passive extraction can be used with samples containing up to 20 mg of reduced sulfur. We demonstrated the utility of this technique in a determination of both sulfate reduction rates and reduced inorganic sulfur pools in marine and freshwater sediments. A side-by-side comparison of the passive extraction method with the established single-step distillation technique yielded comparable results with a fraction of the effort.