Evidence for cometabolic transformation of weathered toxaphene under aerobic conditions using camphor as a co-substrate.

AIMS: Toxaphene is a persistent organic pollutant, composed of approximately 1000 highly chlorinated bicyclic terpenes. The purpose of this study was to evaluate if camphor, a structural analogue of toxaphene, could stimulate aerobic biotransformation of weathered toxaphene. METHODS AND RESULTS: Two enrichment cultures that degrade camphor as the sole carbon source were established from contaminated soil and biosolids. These cultures were used to evaluate aerobic transformation of weathered toxaphene. Only the biosolids culture could transform compounds of technical toxaphene (CTTs) in the presence of camphor, while no transformation was observed in the presence of glucose or with toxaphene as a sole carbon source. The transformed toxaphene had lower concentration of CTTs with longer retention times, and higher concentration of compounds with lower retention times. Gas chromatography with electron capture negative ion mass spectrometry (GC/ECNI-MS) showed that aerobic biotransformation mainly occurred with Cl8 - and Cl9 -CTTs compounds. The patterns of Cl6 - and Cl7 -CTTs were also simplified albeit to a much lesser extent. Seven camphor-degrading bacteria were isolated from the enrichment culture but none of them could degrade toxaphene. CONCLUSION: Camphor degrading culture can aerobically transform CCTs via reductive pathway probably by co-metabolism using camphor as a co-substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: Since camphor is naturally produced by different plants, this study suggests that stimulation of aerobic transformation of toxaphene may occur in nature. Moreover plants, which produce camphor or similar compounds, might be used in bioremediation of contaminated soils.

Effect of the extraction and purification of soil DNA and pooling of PCR amplification products on the description of bacterial and archaeal communities.

AIMS: This study evaluated the effects of DNA extraction method, DNA purification and pooling of PCR amplification products on the description of bacterial and archaeal diversity. METHODS AND RESULTS: Soil DNA was extracted by the Power Soil DNA extraction kit and a customized Griffiths' protocol. Both methods are based on cell disruption by bead beating. In total, we used three soils and six independent extractions from each soil obtained by each of the two methods. Then, three of the six extracts of each treatment were further purified by spin columns filled with Sepharose 2B and polyvinylpolypyrrolidone (PVPP). The V4 hypervariable region of the 16S rRNA gene was amplified from each extract using the 515F/806R primer pair in four independent reactions. Three amplification products were combined and sequenced as a pooled sample, while the additional amplification product was sequenced individually. The resulting 72 amplification products were sequenced by Illumina MiSeq platform. DNA extraction method had a statistically significant effect on the estimation of the composition of microbial communities that might overwhelm differences in microbial communities from distinct soils. On the other hand, a further DNA purification step or pooling of PCR amplification products had a minor effect on the description of bacterial and archaeal communities. CONCLUSIONS: DNA extraction had the strongest effect on the description of bacterial and archaeal communities; low concentration of impurities, which allow PCR amplification, can still generate a minor additional bias, while PCR stochastic variability had the lowest effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it is well known that methodological factors affect the description of microbial communities, the relative importance of each step is still unknown. The present study determined that of the factors tested, the DNA extraction method had the strongest effects on the description of bacterial and archaeal communities.

Screening for biosurfactant production by 2,4,6-trinitrotoluene-transforming bacteria.

AIMS: To isolate and identify TNT-transforming cultures from explosive-contaminated soils with the ability to produce biosurfactants. METHODS AND RESULTS: Bacteria (pure and mixed cultures) were selected based on their ability to transform TNT in minimum media with TNT as the sole nitrogen source and an additional carbon source. TNT-transforming bacteria were identified by 16S rRNA gene sequencing. TNT transformation rates were significantly lower when no additional carbon or nitrogen sources were added. Surfactant production was enabled by the presence of TNT. Fourteen cultures were able to transform the explosive (>50%); of these, five showed a high transformation capacity (>90%), and six produced surfactants. CONCLUSIONS: All explosive-transforming cultures contained Proteobacteria of the genera Achromobacter, Stenotrophomonas, Pseudomonas, Sphingobium, Raoultella, Rhizobium and Methylopila. These cultures transformed TNT when an additional carbon source was added. Remarkably, Achromobacter spanius S17 and Pseudomonas veronii S94 have high TNT transformation rates and are surfactant producers. SIGNIFICANCE AND IMPACT OF THE STUDY: TNT is a highly toxic, mutagenic and carcinogenic nitroaromatic explosive; therefore, bioremediation to eliminate or mitigate its presence in the environment is essential. TNT-transforming cultures that produce surfactants are a promising method for remediation. To the best of our knowledge, this is the first report that links surfactant production and TNT transformation by bacteria.

Characterization of an enrichment culture debrominating tetrabromobisphenol A and optimization of its activity under anaerobic conditions.

AIM: To study the effects of incubation conditions on the microbial community structure and activity of a TBBPA-debrominating enrichment culture composed of bacterial and archaeal species. METHODS AND RESULTS: The effects of the methanogen inhibitor 2-bromoethanesulfonate (BES), of the antibiotic ampicillin, of substrate (tetrabromobisphenol A, TBBPA) omission and availability of different electron donors on microbial community structure and activity were examined under anaerobic conditions. Debromination of TBBPA was blocked in the presence of ampicillin, while long-term incubation with BES resulted in delayed debromination activity. The results suggest that the bacterial species responsible for the debromination of TBBPA, while archaeal species involved in electron donor metabolism. The enrichment culture lost its debromination activity after cultivation for 9 months without TBBPA, concomitantly with the disappearance of two DNA bands in a denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments corresponding to Pelobacter carbinolicus and Sphaerochaeta sp. TQ1 that were present in the original culture. When butyrate was used as an electron donor, TBBPA debromination activity was attenuated. When acetate was used as the electron donor, no debromination was observed and in addition, there was a decrease in the abundance of the mcrA gene. CONCLUSIONS: The results indicate that to maintain a high rate of TBBPA debromination activity, it is essential to preserve the microbial community structure (bacterial and archaeal members) of this culture and supply an electron donor that produces high amounts of hydrogen when fermented. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides important information for the management of cultures to be used in bioremediation of TBBPA contaminated sites.