Anaerobic bacteria that dechlorinate perchloroethene

In this study, we identified specific cultures of anaerobic bacteria that dechlorinate perchlorethene (PCE). The bacteria that significantly dechlorinated PCE were strain DCB-1, an obligate anaerobe previously shown to dechlorinate chlorobenzoate, and two strains of Methanosarcina. The rate of PCE dechlorination by DCB-1 compared favorably with reported rates of trichloroethene bio-oxidation by methanotrophs. Even higher PCE dechlorination rates were achieved when DCB-1 was grown in a methanogenic consortium.     

Novel uncultured Chloroflexi dechlorinate perchloroethene to trans-dichloroethene in tidal flat sediments

The marine environment represents a rich source of bio- and geogenically produced organohalogens, including the common pollutant perchloroethene (PCE). However, diversity and function of marine chloroethene-dechlorinating microorganisms are largely unknown. Here, we have studied the activity and composition of a tidal flat sediment bacterial and archaeal community from the North Sea exposed to low concentrations of PCE. After 2 weeks of incubation, PCE was rapidly dechlorinated via trichloroethene to dichloroethene (DCE). Unexpectedly, these microcosms produced 3.5-fold more trans-DCE than cis-DCE. The actively dechlorinating microbial populations were traced by stable isotope probing of rRNA with (13)C-labelled acetate for 4 days. Terminal restriction fragment length polymorphism fingerprinting and clone libraries of isotopically enriched, 'heavy'(13)C-labelled bacterial 16S rRNA revealed the populations potentially involved in reductive dechlorination. Major clone groups belonged to the Proteobacteria (50.0%; 22.4% delta-, 12.1% gamma-, 6.9% alpha-, 6.9% beta- and 1.7% epsilon-subgroup) and Chloroflexi (29.3%). Populations represented by the two dominant terminal restriction fragments were affiliated with the Dehalococcoidetes (subphylum II of the Chloroflexi), and were exclusively detected in the heavy fraction of the PCE-dechlorinating incubation. The phylogenetically novel, larger population, designated Tidal Flat Chloroflexi Cluster, was closely related to the recently discovered PCE-dechlorinating Lahn Cluster bacteria from anoxic river sediment but more distantly related to canonical Dehalococcoides spp. (92-94% sequence identity). The second population was closely related to 'Dehalobium chlorocoercia DF-1'. Both populations appear to be responsible for reductive dechlorination of highly chlorinated ethenes to predominantly trans-DCE in tidal flat sediment incubations.     

Anaerobic dechlorination of perchloroethene in an extractive membrane bioreactor

An extractive membrane bioreactor (EMB) is described that used an undefined anaerobic culture to dechlorinate tetrachloroethene (C₂Cl₄) reductively in a synthetic wastewater. Comparable reactors described in the literature use set-ups where the bacteria are in direct contact with the wastewater, and thus would require the addition of significant quantities of nutrients to the wastewater stream in practical application. In the EMB, a silicone rubber membrane separates the microbial culture from the wastewater stream, so that addition of nutrients can be minimised. The EMB was operated continuously for 48 days and dechlorinated 359 mol C₂Cl₄/(l biomedium⁻¹ day⁻¹) on average. Lactate was fed as an electron donor and C₂Cl₄ dechlorination was verified by chloride measurements. Particular attention was paid to the reduction of transmembrane C₂Cl₄ flux caused by a membrane-attached biofilm. Following a start-up period, the reactor operation was stable and remained largely unaffected by biofilm thickness and oxygen contamination from the wastewater. Received: 19 January 1998 / Received revision: 8 May 1998 / Accepted: 8 May 1998     

Anaerobic bacteria from hypersaline environments.

Strictly anaerobic halophiles, namely fermentative, sulfate-reducing, homoacetogenic, phototrophic, and methanogenic bacteria are involved in the oxidation of organic carbon in hypersaline environments. To date, six anaerobic fermentative genera, containing nine species, have been described. Two of them are homoacetogens. Six species belong to the family Haloanaerobiaceae, as indicated by their unique 16S rRNA oligonucleotide sequences. Desulfohalobium retbaense and Desulfovibrio halophilus represent the only two moderately halophilic sulfate reducers so far reported. Among anoxygenic phototrophic anaerobes, a few purple bacteria with optimal growth at salinities between 6 and 11% NaCl have been isolated from hypersaline habitats. They belong to the genera Rhodospirillum, Chromatium, Thiocapsa, and Ectothiorhodospira. The commonest organisms isolated so far are Chromatium salexigens, Thiocapsa halophila, and Rhodospirillum salinarum. Extremely halophilic purple bacteria have most commonly been isolated from alkaline brines and require about 20 to 25% NaCl for optimal growth. They belong to the family Ectothiorodhospiraceae. Their osmoregulation involves synthesis or uptake of compatible solutes such as glycine-betaine that accumulate in their cytoplasm. The existence of methanogens in hypersaline environments is related to the presence of noncompetitive substrates such as methylamines, which originate mainly from the breakdown of osmoregulatory amines. Methanogenesis probably does not contribute to the mineralization of carbohydrates at NaCl concentrations higher than 15%. Above this concentration, sulfate reduction is probably the main way to oxidize H2 (although at rates too low to use up all the H2 formed) and occupies a terminal function kn the degradation of carbohydrates. Three genera and five species of halophilic methylotrophic methanogens have been reported. A bloom of phototrophic bacteria in the marine salterns of Salins-de-Giraud, located on the Mediterranean French coast in the Rhone Delta, is also described.     

Anaerobic fecal bacteria of the baboon.

The predominant bacterial genera of baboon feces were enumerated and identified by established procedures. The predominant genera isolated were Lactobacillus, Eubacterium, Streptococcus, and Bacteroides.     

Anaerobic fecal bacteria of the baboon.

The predominant bacterial genera of baboon feces were enumerated and identified by established procedures. The predominant genera isolated were Lactobacillus, Eubacterium, Streptococcus, and Bacteroides.     

Anaerobic bacteria from the large intestine of mice.

Anaerobic bacteria from the colon of laboratory mice were enumerated and isolated using strict anaerobic techniques. Direct microscopic counts revealed 4.4 X 10(10) organisms in each gram (wet weight) of colon contents. Actual cultural counts averaged 3.2 X 10(10) organisms, which was 73% of the direct microscopic count. The tentatively identified genera were Bacteroides, Eubacterium, Fusobacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium. Strains of Fusobacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium were biochemically homogeneous. Strains of Bacteroides and Eubacterium, on the other hand, were biochemically heterogeneous and were subdivided into several distinct groups. The data indicate that many of the isolates are different from previously described species of the respective genera and may belong to new species.     

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.     

Anaerobic bacteria in clinical infections

The findings of 275 cultures from routine clinical specimens obtained from lesions in different sites of body, during a period of 11 months, are presented. The clinical specimens were obtained from surgical wounds, abdominal infections, orthopaedic operations, biliary tract infections and pleuropulmonary infections. The total number of positive cultures including both aerobes and anaerobes was 203 out of 275 (73.8%). Of the 38 cultures positive for anaerobes, 29 (76.3%) grew both aerobic and anaerobic bacteria, while in nine (23.7%) cultures only anaerobes were found. A total of 42 strains of anaerobic bacteria were isolated. The majority of them were found in clinical specimens obtained from abdominal infections (62%), while a low percentage (3.6%) was found in specimens from orthopaedic operations. Strains belonging to the genus Bacteroides were the most frequently isolated anaerobes, accounting for 35.7% of the total, followed by Clostridia 28.5%, Peptostreptococci 23.8% and Prevotella 12%.     

Anaerobic bacteria as producers of antibiotics

Anaerobic bacteria are the oldest terrestrial creatures. They occur ubiquitously in soil and in the intestine of higher organisms and play a major role in human health, ecology, and industry. However, until lately no antibiotic or any other secondary metabolite has been known from anaerobes. Mining the genome sequences of Clostridium spp. has revealed a high prevalence of putative biosynthesis genes (PKS and NRPS), and only recently the first antibiotic from the anaerobic world, closthioamide, has been isolated from the cellulose degrading bacterium Clostridium cellulolyticum. The successful genetic induction of antibiotic biosynthesis in an anaerobe encourages further investigations of obligate anaerobes to tap their hidden biosynthetic potential.     

Development and characterization of stable sediment-free anaerobic bacterial enrichment cultures that dechlorinate aroclor 1260

We have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarily meta dechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H(2). Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 mug/ml of Aroclor 1260 at 22 to 24 degrees C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 mug/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H(2). Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence of Thauera-like Betaproteobacteria, Geobacter-like Deltaproteobacteria, Pseudomonas species, various Clostridiales, Bacteroidetes, Dehalococcoides of the Chloroflexi group, and unclassified Eubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications.     

A Desulfitobacterium strain isolated from human feces that does not dechlorinate chloroethenes or chlorophenols

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron acceptors such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. 13C-NMR labeling experiments showed homoacetogenic 13C-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 degrees C, respectively. The growth rate under these conditions was approximately 0.10 h(-1). Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor.     

Anaerobic degradation of 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria.

Three strains of anaerobically benzoate-degrading, denitrifying bacteria of the genus Pseudomonas were able to grow on 2-fluorobenzoate as the sole carbon and energy source. Fluoride ion release was stoichiometric, and the reduction of dissolved organic carbon indicated total degradation. Cells grown anaerobically with benzoate were adapted for immediate growth with 2-fluorobenzoate, and both compounds were substrates for an inducible benzoyl-coenzyme A synthetase, the initial enzyme of anaerobic degradation. It is proposed that fluoride is eliminated gratuitously by a regioselective reaction in a sequence common to both carbon sources. Benzoate, but not 2-fluorobenzoate, was oxidized by aerobically grown cells.     

Anaerobic degradation of cresols by denitrifying bacteria

The initial reactions in anaerobic metablism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o-or p-cresol as sole organic substrate with a generation time of 11 h; o-or p-cresol was completely oxidized to CO₂ with nitrate being reduced to N₂. A denitrifying Pseudomonas-like strain oxidized m-or p-cresol as the sole organic growth substrate completely to CO₂ with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps:(1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol 4-OH-benzaldehyde 4-OH-benzoate 4-OH-benzoly-CoA benzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating).(2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH₃-benzoyl-CoA as central intermediate as follows: o-cresol 4-OH-3-CH₃-benzoate 4-OH-3-CH₃-benzoyl-CoA 3-CH₃-benzoyl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between ¹⁴CO₂ and the carboxyl of 4-OH-3-CH₃-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH₃-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH₃-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH₃-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH₃-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH₃-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH₃-benzoate and further to 3-CH₃-benzoyl-CoA.(3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4-and 3,4-dimethylphenol were oxidized to 4-OH-3-CH₃-and 4-OH-2-CH₃-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.The hydroxyl and methyl groups are abbreviated as OH-and CH₃-, respectively     

Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.     

Anaerobic utilization of essential oils bydenitrifying bacteria

Plant volatile organic compounds are a major carbonsource in nature. We studied the degradability ofthese substances by anaerobic microorganisms inenrichment cultures with representative essential oilsas organic substrates and nitrate as electronacceptor. Lemon and pine needle oil supportedmicrobial growth in the presence of pure oil, whereasparsley seed, camphor, sage, fennel, and mint oilsupported growth only when the essential oils weredissolved in an overlying phase of2,2,4,4,6,8,8-heptamethylnonane. Thyme oil did notsupport denitrification. Analyses of the microbiallydegraded oils revealed the disappearance ofmonoterpenes, of several monoterpenoids, and ofmethoxy-propenyl-benzenes, including apiole andmyristicin. Most-probable-number determinations fordenitrifying communities in sewage sludge and forestsoil yielded 10⁶ to 10⁷monoterpene-utilizing cells ml^-1, representing0.7 to 100% of the total cultivablenitrate-reducing microorganisms. The utilization ofessential oils together with the common occurrence ofthis metabolic trait are indications for anenvironmentally important, but currently unexploredanaerobic turnover of plant volatile organic compoundsin soil.