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.     

Cresol metabolism by the sulfate-reducing bacterium Desulfotomaculum sp. strain Groll.

The metabolism of cresols under sulfate-reducing conditions was investigated in Desulfotomaculum sp. strain Groll. This strain grows on a variety of aromatic compounds, including para- and meta- but not ortho-cresol. Degradation of p-cresol proceeded by oxidation reactions of the methyl group to yield p-hydroxybenzoate, which was then dehydroxylated to benzoate. The aromatic intermediates expected for this pathway, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoate, and benzoate, were readily metabolized by strain Groll. Utilization of these intermediates generally preceded and inhibited the degradation of p-cresol. p-Hydroxybenzoate and benzoate were detected in culture fluid as metabolites of p-cresol. p-Hydroxybenzaldehyde and p-hydroxybenzoate were detected in cultures degrading p-hydroxybenzyl alcohol. Enzyme activities responsible for utilization of p- and m-cresol, induced by growth on the respective cresol, were detected in cell-free extracts of strain Groll. The compounds detected in culture fluids and the enzyme activities detected in cell-free extracts indicate that the pathways for the degradation of p- and m-cresol converge on benzoate, followed by metabolism to benzoyl-coenzyme A (CoA). Strain Groll can utilize both cresol isomers under sulfate-reducing conditions by similar reactions, but the enzyme activities catalyzing these transformations of the two isomers appear distinct.     

Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Metabolism of phenol and cresols by Bacillus stearothermophilus.

An obligate thermophilic strain of Bacillus stearothermophilus, strain PH24, isolated from industrial sediment by elective culture, grew readily at 55 C on phenol or on one of the isomers of cresol as the major carbon source. Intact cells grown in the presence of phenol, o-cresol, m-cresol, or p-cresol were induced to oxidize, without lag, these substrates together with catechol, 3-methylcatechol, and 4-methylcatechol. Cell extracts prepared from B. stearothermophilus PH24 after growth in the presence of phenol converted phenol to catechol with a concomitant uptake of 1 mol of oxygen per mol of substrate in reaction mixtures supplemented with reduced nicotinamide adenine dinucleotide. These preparations also catalyzed the oxidation of o-cresol to 3-methylcatechol and of m-cresol and p-cresol to 4-methylcatechol. Enzyme activity was inhibited by 1 mM p-chloromercuribenzoate and by 0.1 mM 0-phenanthroline. Catechol and the corresponding methylcatechol intermediates were further dissimilated by cell extracts of phenol-grown cells via the meta-cleavage route to yield 2-hydroxymuconic semialdehyde and the respective methylated derivatives.     

Effects of some alkyl phenols on methanogenic degradation of phenol.

The effects of six phenolic compounds (o-, m-, and p-cresol and 2-, 3-, and 4-ethylphenol) on the anaerobic biodegradation of phenol was examined in batch methanogenic cultures. Results showed that ethylphenols were more inhibitory of phenol degradation than were cresols. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

Effects of some alkyl phenols on methanogenic degradation of phenol

The effects of six phenolic compounds (o-, m-, and p-cresol and 2-, 3-, and 4-ethylphenol) on the anaerobic biodegradation of phenol was examined in batch methanogenic cultures. Results showed that ethylphenols were more inhibitory of phenol degradation than were cresols. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

The metabolism of the ground water contaminant 2-hydroxybiphenyl under sulfate-reducing conditions

The metabolic fate of 2-hydroxybiphenyl under different anaerobic conditions was tested with sediment slurries and enrichment cultures obtained from a shallow anoxic aquifer. 2-Hydroxybiphenyl was depleted in aquifer slurries over the course of incubation, but substrate loss in methanogenic slurries was not significantly different from either filter-sterilized or autoclaved controls. In contrast, the rate of substrate removal was significantly higher in non-sterile, sulfate-reducing aquifer slurries relative to abiotic control incubations. A 2-hydroxybiphenyl-degrading enrichment was established that was inhibited by molybdate but not by bromoethane-sulfonic acid. For every mole of substrate consumed by the bacterial consortium, 6.1±0.2 moles of sulfate were depleted from the enrichment medium. This represents about 87% of the theoretical amount of sulfate consumed and suggests that the 2-hydroxybiphenyl was largely mineralized. Oxygen, nitrate, or carbon dioxide could not replace sulfate as a terminal electron acceptor for the enrichment. Other hydroxybiphenyl isomers were not metabolized by these cultures. This study shows that aromatic substrates with multiple ring systems can undergo biotransformation by anaerobic microorganisms under some ecological conditions.     

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.     

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.     

Anaerobic biotransformation of carbon tetrachloride under various electron acceptor conditions

The biotransformation of carbon tetrachloride (CT) under various electron acceptor conditions was investigated using enrichment cultures developed from the anaerobic digester sludge of Thibodaux sewage treatment plant. The results indicated that CT was biotransformed under sulfate-reducing, methanogenic, nitrate-reducing, iron-reducing, fermenting, and mixed electron acceptor conditions. However, the rates of CT removal varied among the conditions studied. The fastest removal of CT (100% removal in 12 days) was observed under mixed electron acceptor conditions followed in order by sulfate-reducing, methanogenic, fermenting, iron-reducing, and nitrate-reducing conditions. Under mixed electron acceptor conditions, the CT was converted to methyl chlorides, which was further metabolized. Under sulfate, iron, nitrate-reducing, and methanogenic conditions, the major metabolite produced from CT metabolism was chloroform (CF). Under fermenting conditions, methylene chloride was produced from CT metabolism. This study showed evidence for CT metabolism in a mixed microbial population system similar to many contaminated field sites where a heterogeneous microbial population exists.     

Enhanced Biotransformation of Trichloroethylene Under Mixed Electron Acceptor Conditions

The biotransformation of trichloroethylene (TCE) under various electron acceptor conditions was investigated by using enrichment cultures developed from the anaerobic digester sludge of Thibodaux sewage treatment plant. The results indicated that TCE was biotransformed under sulfate reducing, methanogenic, nitrate reducing, iron reducing, and fermenting conditions. However, the rates of TCE removal varied among the conditions studied. The fastest removal of TCE (100% removal in 9 days) was observed under mixed electron acceptor conditions, followed in order by methanogenic, fermenting, iron reducing, sulfate reducing, and nitrate reducing conditions. Under mixed electron acceptor conditions, the TCE was converted to ethene, which was further metabolized. Under sulfate and nitrate reducing conditions, the major metabolites produced from TCE metabolism were cis and trans dichloroethylene (DCE). Under methanogenic, iron reducing, and fermenting conditions, cis and trans DCE and ethene were produced from TCE metabolism. This study showed evidence for TCE metabolism in a mixed microbial population system similar to any contaminated field sites, where heterogeneous microbial population exists. Received: 21 July 2000 / Accepted: 5 September 2000     

Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids.

Nitrate, sulfate, and carbonate were used as electron acceptors to examine the anaerobic biodegradability of chlorinated aromatic compounds in estuarine and freshwater sediments. The respective denitrifying, sulfidogenic, and methanogenic enrichment cultures were established on each of the monochlorinated phenol and monochlorinated benzoic acid isomers, using sediment from the upper (freshwater) and lower (estuarine) Hudson River and the East River (estuarine) as source materials. Utilization of each chlorophenol and chlorobenzoate isomer was observed under at least one reducing condition; however, no single reducing condition permitted the metabolism of all six compounds tested. The anaerobic biodegradation of the chlorophenols and chlorobenzoates depended on the electron acceptor available and on the position of the chlorine substituent. In general, similar activities were observed under the different reducing conditions in both the freshwater and estuarine sediments. Under denitrifying conditions, degradation of 3- and 4-chlorobenzoate was accompanied by nitrate loss corresponding reasonably to the stoichiometric values expected for complete oxidation of the chlorobenzoate to CO2. Under sulfidogenic conditions, 3- and 4-chlorobenzoate, but not 2-chlorobenzoate, and all three monochlorophenol isomers were utilized, while under methanogenic conditions all compounds except 4-chlorobenzoate were metabolized. Given that the pattern of activity appears different for these chlorinated compounds under each reducing condition, their biodegradability appears to be more a function of the presence of competent microbial populations than one of inherent molecular structure.     

P-cresol and 3,5-xylenol methylhydroxylases in Pseudomonas putida N.C.I.B. 9896.

Pseudomonas putida N.C.I.B. 9869, when grown on 3,5-xylenol, hydroxylates the methyl groups on 3,5-xylenol and on p-cresol by two different enzymes. 3,5-Xylenol methylhydroxylase, studied only in relatively crude extracts, requires NADH, is not active with p-cresol and is inhibited by cyanide, but not by CO. The p-cresol methylhydroxylase requires an electron acceptor and will act under anaerobic conditions. It was purified and is a flavocytochrome c of mol.wt. approx. 114,000 consisting of two subunits of equal size. The enzyme catalyses the hydroxylation of p-cresol (Km 16 micron) and the further oxidation of product, p-hydroxybenzyl alcohol (Km 27 micron) to p-hydroxybenzaldehyde. A different p-cresol methylhydroxylase of the flavocytochrome c type is induced by growth on p-cresol. It too was purified and has mol.wt. approx. 100,000, and again consisted of two equal-size subunits. The Km for p=cresol 3.6 micron and for p=hydroxybenzyl alcohol, 15 micron.     

Degradation of high concentrations of cresols by Pseudomonas sp. CP4

Pseudomonas sp. CP₄, a potent phenol-degrading laboratory isolate could mineralize all three isomers of cresol. This strain readily utilized up to 1.4, 1.1 and 2.2 g/l of o- m- and p-cresol, respectively as the sole sources of carbon and energy. These are the highest concentrations of cresols reported to be degraded by a bacterial strain. The rates of degradation of the three isomers were in the order: o- > p- > m-cresol. All the isomers of cresol were catabolized through a meta-cleavage pathway. Fairly high catechol 2,3-dioxygenase (C230) activity against catechol was observed in the cell-free extracts of the culture grown on these compounds and were in the order: m- > o- > p-cresol.     

Anaerobic biodegradation ofPara-cresol under three reducing conditions

The anaerobic degradation ofp-cresol was studied with one sediment source under three reducing conditions—denitrifying, sulfidogenic, and methanogenic. Loss ofp-cresol (1 mM) in all the anaerobic systems took initially 3 to 4 weeks. In acclimated culturesp-cresol was degraded in less than a week.p-Cresol was completely metabolized under denitrifying, sulfidogenic, and methanogenic conditions, with formation of nitrogen gas, loss of sulfate, and formation of methane and carbon dioxide, respectively.p-Cresol metabolism proceeded throughp-hydroxybenzal-dehyde andp-hydroxybenzoate under denitrifying and methanogenic conditions. These compounds were rapidly degraded in cultures acclimated top-cresol under all three reducing conditions. These results suggest that the initial pathway ofp-cresol degradation is the same under denitryfying, sulfidogenic, and methanogenic conditions and proceeds via oxidation of the methyl substituent top-hydroxybenzaldehyde andp-hydroxybenzoate. The initial rate ofp-hydroxybenzaldehyde degradation was high in both the unacclimated cultures and in the cultures acclimated top-cresol, suggesting that this step is nonspecific. Benzoate was additionally detected as a metabolite followingp-hydroxybenzoate in the methanogenic cultures, but not in the denitrifying or sulfidogenic cultures. The degradation pathway therefore may diverge afterp-hydroxybenzoate formation depending on which electron acceptor is available.     

Methanogenic Conversion of 3-S-Methylmercaptopropionate to 3-Mercaptopropionate

Anaerobic metabolism of dimethylsulfoniopropionate, an osmolyte of marine algae, in anoxic intertidal sediments involves either cleavage to dimethylsulfide or demethylation to 3-S-methylmercaptopropionate (MMPA) and subsequently to 3-mercaptopropionate. The methanogenic archaea Methanosarcina sp. strain MTP4 (DSM 6636), Methanosarcina acetivorans DSM 2834, and Methanosarcina (Methanolobus) siciliae DSM 3028 were found to use MMPA as a growth substrate and to convert it stoichiometrically to 3-mercaptopropionate. Approximately 0.75 mol of methane was formed per mol of MMPA degraded; methanethiol was not detected as an intermediate. Eight other methanogenic strains did not carry out this conversion. We also studied the conversion of MMPA in anoxic marine sediment slurries. Addition of MMPA (500 (mu)M) resulted in the production of methanethiol which was subsequently converted to methane (417 (mu)M). In the presence of the antibiotics ampicillin, vancomycin, and kanamycin (20 (mu)g/ml each), 275 (mu)M methane was formed from 380 (mu)M MMPA; no methanethiol was formed during these incubations. Only methanethiol was formed from MMPA when 2-bromoethanesulfonate (25 mM) was added to a sediment suspension. These results indicate that in natural environments MMPA could be directly or indirectly a substrate for methanogenic archaea.     

The effect of concentration of phenols on their batch methanogenesis

The biodegradability of phenol and six other phenolic compounds (o-, m-, and p-cresol, 2-, 3-, and 4-ethylphenol) was examined in batch methanogenic cultures. The effect of concentration of these alkyl phenols on the anaerobic biodegradation of phenol was also evaluated. The inoculum used in this study was cultivated in a continuous flow laboratory fermenter with phenol as the primary substrate. Phenol, at initial concentrations as high to 1400 mg/L was completely degraded to methane and carbondioxide after 350 hours incubation. Complete degradation of m- and p-cresol was also observed while the ethylphenols and o-cresol were not significantly degraded.At initial concentrations exceeding 600 mg/L, phenol inhibited the phenol-degrading microorganisms but not the methanogens. At about 600 mg/L, cresols reduced the rate of phenol degradation to 50% of that observed in a control culture containing only 200 mg/L phenol. Ethylphenols were more inhibitory than cresols. Phenol degrading microorganisms were more susceptible to inhibition by cresols and ethylphenols than were the methanogens. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

Dehalogenation and biodegradation of brominated phenols and benzoic acids under iron-reducing, sulfidogenic, and methanogenic conditions.

The anaerobic biodegradation of monobrominated phenols and benzoic acids by microorganisms enriched from marine and estuarine sediments was determined in the presence of different electron acceptors [i.e., Fe(III), SO4(2-), or HCO3-]. Under all conditions tested, the bromophenol isomers were utilized without a lengthy lag period whereas the bromobenzoate isomers were utilized only after a lag period of 23 to 64 days. 2-Bromophenol was debrominated to phenol, with the subsequent utilization of phenol under all three reducing conditions. Debromination of 3-bromophenol and 4-bromophenol was also observed under sulfidogenic and methanogenic conditions but not under iron-reducing conditions. In the bromobenzoate-degrading cultures, no intermediates were observed under any of the conditions tested. Debromination rates were higher under methanogenic conditions than under sulfate-reducing or iron-reducing conditions. The stoichiometric reduction of sulfate or Fe(III) and the utilization of bromophenols and phenol indicated that biodegradation was coupled to sulfate or iron reduction, respectively. The production of phenol as a transient intermediate demonstrates that reductive dehalogenation is the initial step in the biodegradation of bromophenols under iron- and sulfate-reducing conditions.     

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.     

Potential for anaerobic bioremediation of BTEX in petroleum-contaminated aquifers

Laboratory incubations of aquifer material or enrichments derived from aquifer material as well as geochemical data have suggested that, under the appropriate conditions, BTEX components of petroleum (benzene, toluene, ethylbenzene and xylene) can be degraded in the absence of molecular oxygen with either Fe(III), sulfate, or nitrate serving as the electron acceptor. BTEX degradation under methanogenic conditions has also been observed. However, especially for benzene, the BTEX contaminant of greatest concern, anaerobic degradation is often difficult to establish and maintain in laboratory incubations. Although studies to date have suggested that naturally occurring anaerobic BTEX degradation has the potential to remove significant quantities of BTEX from petroleum-contaminated aquifers, and mechanisms for stimulating anaerobic BTEX degradation in laboratory incubations have been developed, further study of the organisms involved in this metabolism and the factors controlling their distribution and activity are required before it will be possible to design rational strategies for accelerating anaerobic BTEX degradation in contaminated aquifers. Received 21 November 1995/ Accepted in revised form 20 February 1996