Preliminary characterization of four 2-chlorobenzoate-degrading anaerobic bacterial consortia.

Dechlorination was the initial step of 2CB biodegradation in four 2-chlorobenzoate-degrading methanogenic consortia. Selected characteristics of ortho reductive dehalogenation were examined in consortia developed from the highest actively dechlorinating dilutions of the original 2CB consortia, designated consortia M34(-9), P20(-9), P21(-9) and M50(-7). In addition to 2-chlorobenzoate, all four dilution consortia dehalogenated 4 of 32 additional halogenated aromatic substrates tested, including 2-bromobenzoate; 2,6-dichlorobenzoate; 2,4-dichlorobenzoate; and 2-chloro-5-hydroxybenzoate. Dehalogenation occurred exclusively at the ortho position. Both ortho chlorines were removed from 2,6-dichlorobenzoate. Benzoate was detected from 2-bromobenzoate and 2,6-dichlorobenzoate. 4-Chlorobenzoate and 3-hydroxybenzoate were formed from 2,4-dichlorobenzoate and 2-chloro-5-hydroxybenzoate, respectively. Only benzoate was further degraded. Slightly altering the structure of the parent "benzoate molecule" resulted in observing reductive biotransformations other than dehalogenation. 2-Chlorobenzaldehyde was reduced to 2-chlorobenzyl alcohol by all four consortia. 2-chloroanisole was O-demethoxylated by three of the four consortia forming 2-chlorophenol. GC-MS analysis indicated reduction of the double bond in the propenoic side chain of 2-chlorocinnamate forming 2-chlorohydrocinnamate. None of the reduction products was dechlorinated. The following were not dehalogenated: 3- and 4-bromobenzoate; 3- and 4-chlorobenzoate; 2-, 3-, and 4-fluorobenzoate; 2-, 3-, and 4-iodobenzoate; 2-, 3-, and 4-chlorophenol; 2-chloroaniline; 2-chloro-5-methylbenzoate; 2,3-dichlorobenzoate; 2,5-dichlorobenzoate; 2,4,5-trichlorophenoxyacetic acid; and 2,4-dichlorophenoxyacetic acid. Consortia M34(-9), P20(-9), P21(-9), and M50(-7) dechlorinated 2-chlorobenzoate at < or = 4 mm. Dechlorination rates were highest for consortia P20(-9) followed by those of M50(-7) with rates declining above 2 and 3 mm 2CB, respectively. The major physiological types of microorganisms in consortia M34(-9), P20(-9), P21(-9), and M50(-7) were sulfate-reducing and hydrogen-utilizing anaerobes.

Phylogenetic and physiological diversity of sulphate-reducing bacteria isolated from a salt marsh sediment.

The phylogenetic and physiological diversity of sulphate-reducing bacteria inhabiting a salt marsh rhizosphere were investigated. Sulphate-reducing bacteria were isolated from a salt marsh rhizosphere using enrichment cultures with electron donors thought to be prevalent in the rhizosphere of Spartina alterniflora. The relationship between phylogeny and nutritional characteristics of 10 strains was investigated. None of the isolates had 16S rRNA sequences identical to other delta subclass sulphate-reducers, sharing 85.3 to 98.1% sequence similarity with 16S rRNA sequences of their respective closest relatives. Phylogenetic analysis placed two isolates, obtained with ethanol as an electron donor, within the Desulfovibrionaceae. Seven isolates, obtained with acetate, butyrate, propionate, or benzoate, were placed within the Desulfobacteriaceae. One isolate, obtained with butyrate, fell within the Desulfobulbus assemblage, which is currently considered part of the Desulfobacteriaceae family. However, due to the phylogenetic breadth and physiological traits of this group, we propose that it be considered a new family, the "Desulfobulbusaceae." The isolates utilised an array of electron donors similar to their closest relatives with a few exceptions. As a whole, the phylogenetic and physiological data indicate isolation of several sulphate-reducing bacteria which might be considered as new species and representative of new genera. Comparison of the Desulfobacteriaceae isolates' 16S rRNA sequences to environmental clones originating from the same study site revealed that none shared more than 86% sequence similarity. The results provide further insight into the diversity of sulphate-reducing bacteria inhabiting the salt marsh ecosystem, as well as supporting general trends in the phylogenetic coherence of physiological traits of delta Proteobacteria sulphate reducers.

Persistence of polycyclic aromatic hydrocarbon components of creosote under anaerobic enrichment conditions.

Anaerobic biodegradation of an artificial mixture of polycyclic aromatic hydrocarbons (PAHs), which simulates the PAH component of creosote, was examined under methanogenic, sulfidogenic, and nitrate-reducing conditions using creosote-contaminated sediment as the source of inoculum. PAH degradation, CH4 formation and ion reduction were monitored for up to one year. Despite demonstrating active methanogenic and nitrate-reducing anaerobic bacterial communities, only limited degradation of a few PAHs was observed. Under methanogenic conditions limited degradation of all bicyclic (naphthalene, 1-and 2-methylnaphthalene, biphenyl, and 2,6-dimethylnaphthalene) and one tricyclic PAH, anthraquinone, was detected. 2-Methylanthracene was apparently degraded under nitrate-reducing conditions. Anthraquinone declined in sulfate enrichments, but this decline was not dependent upon sulfate reduction. None of the 4- or 5-ring PAHs were degraded under any of the enrichment conditions. These data indicate that under the anaerobic conditions tested there is only a limited potential to degrade PAHs which must be considered when proposing bioremediation technologies for PAH-contaminated sites, especially if high-molecular-weight PAHs are present.

Reduction of 3-chlorobenzoate, 3-bromobenzoate, and benzoate to corresponding alcohols by Desulfomicrobium escambiense, isolated from a 3-chlorobenzoate-dechlorinating coculture.

An anaerobic bacterial coculture which dechlorinated 3-chlorobenzoate (3CB) to benzoate was obtained by single-colony isolation from an anaerobic bacterial consortium which completely degraded 3CB in defined medium. Of 29 additional halogenated aromatic compounds tested, the coculture removed the meta halogen from 2,3- and 2,5-dichlorobenzoate, 3-bromobenzoate (3BB), 5-chlorovanillate (5CV), and 3-chloro-4-hydroxybenzoate. Dechlorinating activity in the coculture required the presence of pyruvate. 5CV was also O-demethoxylated. The coculture contained two cell types: a short, straight gram-negative rod and a long, thin, curved gram-positive rod. The short rod, Desulfomicrobium escambiense, was recently isolated and identified as a new sulfate-reducing bacterial species (B. R. Sharak Genthner, S. D. Friedman, and R. Devereux, Int. J. Syst. Bacteriol. 47:889-892, 1997; B. R. Sharak Genthner, G. Mundfrom, and R. Devereux, Arch. Microbiol. 161:215-219, 1994). D. escambiense did not dehalogenate any of the compounds dehalogenated by the coculture, nor dit it O-demethoxylate 5CV or vanillate. However, D. escambiense reduced 3CB, EBB, and benzoate to their respective benzyl alcohols. Reduction to alcohols required the presence of pyruvate, which was transformed to acetate, lactate, and succinate in the presence of absence of 3CB, 3BB, or benzoate. Alcohol formation did not occur in pyruvate-sulfate medium. Under these conditions, sulfate was preferentially reduced. Other electron donors that supported the growth of D. escambiense during sulfate reduction did not support benzoate reduction to benzyl alcohol.

para-hydroxybenzoate as an intermediate in the anaerobic transformation of phenol to benzoate.

Anaerobic phenol transformation was studied using a consortium which transformed phenol to benzoate without complete mineralization of benzoate. Products of monofluorophenol transformation indicated para-carboxylation. Phenol and benzoate were detected during para-hydroxybenzoate (p-OHB) degradation. p-OHB was detected in phenol-transforming cultures containing 6-hydroxynicotinic acid (6-OHNA), a structural analogue of p-OHB, or at elevated initial concentrations of phenol (greater than or equal to 5 mM), or benzoate (greater than or equal to 10 mM).

Effect of fluorinated analogues of phenol and hydroxybenzoates on the anaerobic transformation of phenol to benzoate.

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At greater than or equal to 250 microM 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of greater than or equal to 250 microM 3-fluorophenol. In contrast, 4-fluorophenol at less than or equal to 2 mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (approximately 3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2:1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para-carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.

Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline.

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10(6) ml(-1)) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10(6) ml(-1)). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10(6) ml(-1)) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10(6) ml(-1)) or tetraclycline (1.5×10(6) ml(-1)). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10(2) ml(-1)) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO4 to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.

Anaerobic transformation of phenol to benzoate via para-carboxylation: use of fluorinated analogues to elucidate the mechanism of transformation.

Isomeric fluorophenols were used as phenol analogues to investigate the transformation of phenol to benzoate by an anaerobic, phenol-degrading consortium derived from freshwater sediment. Transformation of 2-fluorophenol and 3-fluorophenol led to the accumulation of fluorobenzoic acids. 2-Fluorophenol was transformed in the presence or absence of phenol, while 3-fluorophenol transformation was only observed in the presence of phenol. Identification of the resulting fluorobenzoate products as 3-fluorobenzoate and 2-fluorobenzoate isomers, respectively, together with the nontransformation of 4-fluorophenol indicated that the carboxyl group was introduced para to the phenolic hydroxyl group.

Anaerobic Degradation of Chloroaromatic Compounds in Aquatic Sediments under a Variety of Enrichment Conditions.

Anaerobic degradation of monochlorophenols and monochlorobenzoates in a variety of aquatic sediments was compared under four enrichment conditions. A broader range of compounds was degraded in enrichments inoculated with sediment exposed to industrial effluents. Degradation of chloroaromatic compounds was observed most often in methanogenic enrichments and in enrichments amended with 1 mM bromoethane sulfonic acid. Degradation was observed least often in enrichments with added nitrate or sulfate. The presence of 10 mM bromoethane sulfonic acid prevented or inhibited degradation of most compounds tested. Primary enrichments in which KNO(3) was periodically replenished to maintain enrichment characteristics degraded chlorobenzoates, but not chlorophenols. In contrast, primary enrichments in which Na(2)SO(4) was periodically replenished failed to degrade any chloroaromatic compounds. Upon transfer to fresh medium, none of the sulfate enrichments required the presence of Na(2)SO(4) for degradation, while only two nitrate enrichments required the presence of KNO(3) for degradation. As a class of compounds, chlorophenols were degraded more readily than chlorobenzoates. However, as individual compounds 3-chlorobenzoate, 2-chlorophenol, and 3-chlorophenol degradation was observed most often and with an equal frequency. Within the chlorophenol class, the relative order of degradability was ortho > meta > para, while that of chlorobenzoates was meta > ortho > para, In laboratory transfers, 2-chlorobenzoate, 3-chlorobenzoate, and 2-chlorophenol degradation was most easily maintained, while degradation of para-chlorinated compounds was very difficult to maintain.

Characterization of anaerobic dechlorinating consortia derived from aquatic sediments.

Four methanogenic consortia which degraded 2-chlorophenol, 3-chlorophenol, 2-chlorobenzoate, and 3-chlorobenzoate, respectively, and one nitrate-reducing consortium which degraded 3-chlorobenzoate were characterized. Degradative activity in these consortia was maintained by laboratory transfer for over 2 years. In the methanogenic consortia, the aromatic ring was dechlorinated before mineralization to methane and carbon dioxide. After dechlorination, the chlorophenol consortia converted phenol to benzoate before mineralization. All methanogenic consortia degraded both phenol and benzoate. The 3-chlorophenol and 3-chlorobenzoate consortia also degraded 2-chlorophenol. No other cross-acclimation to monochlorophenols or monochlorobenzoates was detected in the methanogenic consortia. The consortium which required nitrate for the degradation of 3-chlorobenzoate degraded benzoate and 4-chlorobenzoate anaerobically in the presence of KNO(3), but not in its absence. This consortium also degraded benzoate, but not 3-chlorobenzoate, aerobically.

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii.

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of Eubacterium limosum with Carbon Monoxide as the Energy Source.

Eubacterium limosum grew with CO as the sole source of energy and formed acetate and CO(2) as the major products. The generation time on CO was 7 h. Uninhibited growth occurred in cultures containing 50% CO or less, but growth occurred at all concentrations tested (i.e., up to 75% CO). The pH optimum for growth was 7.0 to 7.2, whereas growth was poor at a pH below 6.7. CO(2) stimulated growth on CO. CO was preferentially utilized when both CO and H(2) were present.

Syntrophic Association by Cocultures of the Methanol- and CO(2)-H(2)-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium.

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H(2)-CO(2), and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO(2), and H(2) from pectin. The coculture produced one-third more acetate, and butyrate and CO(2) were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.

Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species.

Eubacterium limosum was isolated as the most numerous methanol-utilizing bacterium in the rumen fluid of sheep fed a diet in which molasses was a major component (mean most probable number of 6.3 X 10(8) viable cells per ml). It was also isolated from sewage sludge at 9.5 X 10(4) cells per ml. It was not detected in the rumen fluid of a steer on a normal hay-grain diet, although Methanosarcina, as expected, was found at 9.5 X 10(5) cells per ml. The doubling time of E. limosum in basal medium (5% rumen fluid) with methanol as the energy source (37 degree C) was 7 h. Acetate, cysteine, carbon dioxide, and the vitamins biotin, calcium-D-pantothenate, and lipoic acid were required for growth on a chemically defined methanol medium. Acetate, butyrate, and caproate were produced from methanol. Ammonia or each of several amino acids served as the main nitrogen source. Other energy sources included adonitol, arabitol, erythritol, fructose, glucose, isoleucine, lactate, mannitol, ribose, valine, and H2-CO2. The doubling time for growth on H2-CO2 (5% rumen fluid, 37 degree C) was 14 h as compared with 5.2 h for isoleucine and 3.5 h for glucose. The vitamin requirements for growth on H2-CO2 were the same as those for methanol; however, acetate was not required for growth on H2-CO2, although it was necessary for growth on valine, isoleucine, and lactate and was stimulatory to growth on glucose. Acetate and butyrate were formed during growth on H2-CO2, whereas branched-chain fatty acids and ammonia were fermentation products from the amino acids. Heat tolerance was detected, but spores were not observed. The type strain of E. limosum (ATCC 8486) and strain L34, which was isolated from the rumen of a young calf, grew on methanol, H2-CO2, valine, and isoleucine and showed the same requirements for acetate as the freshly isolated strains.