Degradation of unsaturated hydrocarbons by methanogenic enrichment cultures

Abstract The biodegradability of hydrocarbons under anaerobic conditions was studied in enrichment cultures using mineral media inoculated with sewage sludge or sediment samples of limnic and marine origin. No indication of methanogenic degradation was obtained with either n -hexane, n -hexadecane, n -heptadecane, 1-hexene, cis -2-hexene, trans -2-hexene, isoprene, 1-hexine, benzene, toluene, xylene, cyclohexene, cycloheptatriene, cyclopentadiene, styrene, naphthalene, azulene, or β-carotene. Squalene was incompletely converted to methane and carbon dioxide. Complete degradation was observed with 1-hexadecene. Methanogenic subcultures were maintained on 1-hexadecene and squalene. Both enrichments contained after several transfers Methanospirillum hungatei and Methanothrix soehngenii as prevalent methanogenic bacteria. Acetate (≤80 μ M) was the only intermediary product detected indicating that degradation proceeded via hydrogen-dependent syntrophic β-oxidations. Short rods on hexadecene and cocci on squalene were found to be associated with substrate degradation. The results indicate that terminal double bonds can be sufficient to allow methanogenic degradation of hydrocarbons whereas branching and terminal ring closures may significantly contribute to hydrocarbon stability in anoxic environments.     

Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.     

Signal enrichment with strain-level resolution in metagenomes using topological data analysis

Background

A metagenome is a collection of genomes, usually in a micro-environment, and sequencing a metagenomic sample en masse is a powerful means for investigating the community of the constituent microorganisms. One of the challenges is in distinguishing between similar organisms due to rampant multiple possible assignments of sequencing reads, resulting in false positive identifications. We map the problem to a topological data analysis (TDA) framework that extracts information from the geometric structure of data. Here the structure is defined by multi-way relationships between the sequencing reads using a reference database.

Results

Based primarily on the patterns of co-mapping of the reads to multiple organisms in the reference database, we use two models: one a subcomplex of a Barycentric subdivision complex and the other a Čech complex. The Barycentric subcomplex allows a natural mapping of the reads along with their coverage of organisms while the Čech complex takes simply the number of reads into account to map the problem to homology computation. Using simulated genome mixtures we show not just enrichment of signal but also microbe identification with strain-level resolution.

Conclusions

In particular, in the most refractory of cases where alternative algorithms that exploit unique reads (i.e., mapped to unique organisms) fail, we show that the TDA approach continues to show consistent performance. The Čech model that uses less information is equally effective, suggesting that even partial information when augmented with the appropriate structure is quite powerful.

     

Variation of carbon isotope fractionation in hydrogenotrophic methanogenic microbial cultures and environmental samples at different energy status

Methane is a major product of anaerobic degradation of organic matter and an important greenhouse gas. Its stable carbon isotope composition can be used to reveal active methanogenic pathways, if associated isotope fractionation factors are known. To clarify the causes that lead to the wide variation of fractionation factors of methanogenesis from H₂ plus CO₂ (), pure cultures and various cocultures were grown under different thermodynamic conditions. In syntrophic and obligate syntrophic cocultures thriving on different carbohydrate substrates, fermentative bacteria were coupled to three different species of hydrogenotrophic methanogens of the families Methanobacteriaceae and Methanomicrobiaceae. We found that C‐isotope fractionation was correlated to the Gibbs free energy change (ΔG) of CH₄ formation from H₂ plus CO₂ and that the relation can be described by a semi‐Gauss curve. The derived relationship was used to quantify the average ΔG that is available to hydrogenotrophic methanogenic archaea in their habitat, thus avoiding the problems encountered with measurement of low H₂ concentrations on a microscale. Boreal peat, rice field soil, and rumen fluid, which represent major sources of atmospheric CH₄, exhibited increasingly smaller , indicating that thermodynamic conditions for hydrogenotrophic methanogens became increasingly more favourable. Vice versa, we hypothesize that environments with similar energetic conditions will also exhibit similar isotope fractionation. Our results, thus, provide a mechanistic constraint for modelling the ¹³C flux from microbial sources of atmospheric CH₄.     

Comparative metagenomics of three Dehalococcoides-containing enrichment cultures: the role of the non-dechlorinating community

ABSTRACT: BACKGROUND: The Dehalococcoides are strictly anaerobic bacteria that gain metabolic energy via the oxidation of H2 coupled to the reduction of halogenated organic compounds. Dehalococcoides spp. grow best in mixed microbial consortia, relying on non-dechlorinating members to provide essential nutrients and maintain anaerobic conditions. A metagenome sequence was generated for the dechlorinating mixed microbial consortium KB-1. A comparative metagenomic study utilizing two additional metagenome sequences for Dehalococcoides-containing dechlorinating microbial consortia was undertaken to identify common features that are provided by the non-dechlorinating community and are potentially essential to Dehalococcoides growth. RESULTS: The KB-1 metagenome contained eighteen novel homologs to reductive dehalogenase genes. The metagenomes obtained from the three consortia were automatically annotated using the MG-RAST server, from which statistically significant differences in community composition and metabolic profiles were determined. Examination of specific metabolic pathways, including corrinoid synthesis, methionine synthesis, oxygen scavenging, and electron-donor metabolism identified the Firmicutes, methanogenic Archaea, and the delta-Proteobacteria as key organisms encoding these pathways, and thus potentially producing metabolites required for Dehalococcoides growth. CONCLUSIONS: Comparative metagenomics of the three Dehalococcoides-containing consortia identified that similarities across the three consortia are more apparent at the functional level than at the taxonomic level, indicating the non-dechlorinating organisms' identities can vary provided they fill the same niche within a consortium. Functional redundancy was identified in each metabolic pathway of interest, with key processes encoded by multiple taxonomic groups. This redundancy likely contributes to the robust growth and dechlorination rates in dechlorinating enrichment cultures.     

2,4-Dichlorophenoxyacetic acid degradation in methanogenic mixed cultures obtained from Brazilian Amazonian soil samples

Biodegradation - 2,4-Dichlorophenoxyacetic acid (2,4-D) is the third most applied pesticide in Brazil to control broadleaf weeds in crop cultivation and pastures. Due to 2,4-D’s high mobility...     

Phosphonate utilization by bacterial cultures and enrichments from environmental samples

A selection of axenic microbial strains and a variety of environmental samples were investigated with respect to the utilization of a series of natural and xenobiotic phosphonates as the sole phosphorus source for growth. Phosphonate degradation was observed only with bacteria and not with eucaryotic microorganisms. All representatives of the phosphonates examined supported bacterial growth, with the exception of methylphosphonate diethylester. Yet, distinctly different phosphonate utilization patterns were noted between phosphonate-positive strains. C-P bond cleavage by a photosynthetic bacterium is reported for the first time; growing photoheterotrophically, Rhodobacter capsulatus ATCC 23782 was able to utilize 2-aminoethylphosphonate and alkylphosphonates. Bacteria with the potential to utilize at least one of the phosphonate moieties from the xenobiotic phosphonates Dequest 2010, Dequest 2041, and Dequest 2060 were detected in all environments, with only two exceptions for Dequest 2010. Phosphonate P utilization to an extent of 94 and 97%, for Dequest 2010 and Dequest 2041, respectively, provided evidence that a complete breakdown of these compounds with respect to the C-P bond cleavage can be achieved by some bacteria. The results suggest that phosphonate-utilizing bacteria are ubiquitous, and that selected strains can degrade phosphonates that are more complex than those described previously.     

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

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

Identification,enrichment, and isolation ofThiothrix spp. from environmental samples

We have developed a method to enrich, isolate, and identifyThiothrix spp. in environmental samples. This procedure employs low concentrations of organic compounds, the addition of reduced sulfur compounds (sulfide or thiosulfate), and preparation with spring water that containsThiothrix spp. The enrichment enhanced identification ofThiothrix spp. by promoting deposition of intracellular sulfur granules and inhibiting overgrowth by other bacteria. The relatively high calcium content of the spring water contributed to the culture procedure. With this technique,Thiothrix spp. were observed in two activated sludge systems, a municipal water storage tank, three springs, and four underground freshwater caves in the phreatic zone of the Floridan aquifer. Two differentThiothrix cultures have been isolated from a freshwater cave and a water storage tank by this procedure. It appears that media prepared with spring water known to supportThiothrix spp. can be designed to provide highly selective methods for isolation ofThiothrix spp. from a wide range of environments.Florida Agricultural Experimentation Station, Journal Series Number R-03446.     

Characterization of acid-tolerant H/CO-utilizing methanogenic enrichment cultures from an acidic peat bog in New York State

Two methanogenic cultures were enriched from acidic peat soil using a growth medium buffered to c. pH 5. One culture, 6A, was obtained from peat after incubation with H(2)/CO(2), whereas culture NTA was derived from a 10(-4) dilution of untreated peat into a modified medium. 16S rRNA gene clone libraries from each culture contained one methanogen and two bacterial sequences. The methanogen 16S rRNA gene sequences were 99% identical with each other and belonged to the novel "R-10/Fen cluster" family of the Methanomicrobiales, whereas their mcrA sequences were 96% identical. One bacterial 16S rRNA gene sequence from culture 6A belonged to the Bacteroidetes and showed 99% identity with sequences from methanogenic enrichments from German and Russian bogs. The other sequence belonged to the Firmicutes and was identical to a thick rod-shaped citrate-utilizing organism isolated from culture 6A, the numbers of which decreased when the Ti (III) chelator was switched from citrate to nitrilotriacetate. Bacterial clones from the NTA culture clustered in the Delta- and Betaproteobacteria. Both cultures contained thin rods, presumably the methanogens, as the predominant morphotype, and represent a significant advance in characterization of the novel acidiphilic R-10 family methanogens.     

Phosphonate utilization by bacterial cultures and enrichments from environmental samples.

A selection of axenic microbial strains and a variety of environmental samples were investigated with respect to the utilization of a series of natural and xenobiotic phosphonates as the sole phosphorus source for growth. Phosphonate degradation was observed only with bacteria and not with eucaryotic microorganisms. All representatives of the phosphonates examined supported bacterial growth, with the exception of methylphosphonate diethylester. Yet, distinctly different phosphonate utilization patterns were noted between phosphonate-positive strains. C-P bond cleavage by a photosynthetic bacterium is reported for the first time; growing photoheterotrophically, Rhodobacter capsulatus ATCC 23782 was able to utilize 2-aminoethylphosphonate and alkylphosphonates. Bacteria with the potential to utilize at least one of the phosphonate moieties from the xenobiotic phosphonates Dequest 2010, Dequest 2041, and Dequest 2060 were detected in all environments, with only two exceptions for Dequest 2010. Phosphonate P utilization to an extent of 94 and 97%, for Dequest 2010 and Dequest 2041, respectively, provided evidence that a complete breakdown of these compounds with respect to the C-P bond cleavage can be achieved by some bacteria. The results suggest that phosphonate-utilizing bacteria are ubiquitous, and that selected strains can degrade phosphonates that are more complex than those described previously.     

Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries

The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.     

Novel approach for the development of axenic microalgal cultures from environmental samples

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.     

Nitric oxide reductase (norB) genes from pure cultures and environmental samples

A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria. norB database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB). The latter oxidizes cytochrome c, and the gene is localized adjacent to norC. While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains. These primer sets also specifically amplified norB from freshwater and marine sediments. Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB. Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set. Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms. cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.     

Towards an efficient phenotypic classification of fungal cultures from environmental samples using digital imagery

The use of the analysis technique proposed here, based on functions of the digital imagery software eCognition professional 4.0, provides an objective and effective method for the assessment of fungal diversity in the context of environmental screening projects. It is demonstrated that strains of cultivated fungi can be quantitatively segregated with regard to specific false-color patterns, which reflect even the merest differences in pigment composition, indicating genotypic or phylogenetic disparities. Due to resolving subtle differences of phenotypic traits, a rapid recognition of (duplicate) genotypes is possible which allows the direct inference of the mycobial diversity of given environmental samples and a semi-quantitative or qualitative estimation of the fungal community structure. Two sets of image data from cultures were used in the current study: a minor set being applied for the definition of color classes and for usage in an image reference array, and a second, extended dataset for method validation. An objective assignment, based on false-color classification, was carried out by cluster analysis. High reproducibility using standardized methods makes this design an effective pre-screening option in the field of microbial environmental research. The application of false-color imagery may therefore be applied in fungal monitoring studies as a meaningful procedure supplementing molecular analyses by the identification of new strains irrespective of their relatedness.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Molecular analysis of enrichment cultures of marine ammonia oxidisers

Abstract Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the β -rsub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured β -sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira , which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the β -proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.     

Diversity of bacteria and Archaea in sulphate-reducing enrichment cultures inoculated from serial dilution of Zostera noltii rhizosphere samples

We have analysed the diversity of culturable sulphate-reducing bacteria (SRB) in Zostera noltii colonized sediments from Bassin d'Arcachon (France). Four organic substrates have been tested as well as the combination of H2 and CO2 to select for lithotrophic SRB. All energy sources were supplied in parallel cultures that were amended with yeast extract plus NH4+ and prepared without a source of combined nitrogen, the latter to select for diazotrophic SRB. The 10 different enrichment media were inoculated from serial dilution of rhizosphere samples. The highest dilution cultures yielding positive growth (i.e. 10-7) were studied by molecular techniques (16S rDNA clone libraries, RISA and ARDRA). Lactate as a single organic substrate in combination with a source of combined nitrogen resulted in selection of members of the Desulfovibrionaceae. Surprisingly, when lactate was added without a source of combined nitrogen, Desulfobacteriaceae were selected. A strong influence of the presence or absence of combined nitrogen was also observed for the substrates sucrose and fructose. Whereas the liquid culture growing on sucrose and NH4+ systematically yielded 16S rDNA clones related to an environmental unidentified green sulphur bacterium (OPS185), on plates we were able to isolate a SRB related to Desulfovibrio dechloracetivorans, which likely represents a non-described species. Under diazotrophic conditions, sucrose selected for SRB clones related to the cluster formed by Desulfovibrio zosterae, Desulfovibrio salexigens and Desulfovibrio bastinii. The corresponding isolate obtained on plates showed only low sequence similarity with this closest neighbour (93.8%), and we suggest that it also represents a non-described species. Surprisingly, a 16S rDNA sequence corresponding to an archaeon, i.e. a non-extremophile Crenoarchaeota, was retrieved from several of the SRB enrichment cultures even after subsequent transfers.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.