Field distribution and activity of chlorinated solvents degrading bacteria by combining CARD-FISH and real time PCR

Abstract

Nowadays several advanced molecular techniques are applied for quantifying bacteria involved in contaminant degradation processes. However, despite the fact that significant efforts have been taken to make these tools more reliable and specific, their application for the analysis of field samples is hardly ever applied. In this study, a combination of three methods (CARD-FISH, qPCR and RT-qPCR) was successfully applied to evaluate the distribution and the activity of known chlorinated solvent dechlorinating bacteria in a contaminated site where no remedial actions have been undertaken. CAtalysed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) specifically provided the cell densities of known dechlorinating bacteria and was found to be more sensitive than quantitative PCR (qPCR) for the quantification of 'Dehalococcoides' cell numbers in the aquifer. Among the screened dechlorinators, 'Dehalococcoides' spp. were mainly found and nearly homogenously distributed in the aquifers at concentrations ranging from 8.1×10(5)±1.2×10(5) to 2.5×10(7)±5.6×10(6)cells per liter of groundwater (with a relative abundance out of the total Bacteria of 0.7-15%). Further, the dechlorination potentialities of 'Dehalococcoides' species living in the aquifer were evaluated by analyzing the abundance and the expression of 16S rRNA genes and reductive dehalogenase (RDase) encoding functional genes by qPCR and Reverse Transcription qPCR (RT-qPCR). 'Dehalococcoides'tceA gene, known to be associated to strains capable of reducing chlorinated solvents beyond cis-DCE, was found and expressed in the field. Overall, this study proved the existence of a well-established dechlorinating microbial community able to use contaminants as substrates for their metabolic activity and indicated the occurrence of reductive dechlorination at the site.

A study of chlorinated solvent contamination of the aquifers of an industrial area in central Italy: a possibility of bioremediation

“…Groundwater analyses revealed the presence of high concentrations of PCE and also TCE, cis -DCE and VC (in traces), which are known to be PCE dechlorinating intermediates. The presence of cis -DCE suggests the occurrence of intrinsic dechlorinating activity at the site, because this compound is formed from the microbially catalyzed hydrogenolysis of TCE ( Matturro et al, 2012 ). This hypothesis is confirmed by statistic and geostatistic analysis.…”

Polychlorinated biphenyl (PCB) anaerobic degradation in marine sediments: microcosm study and role of autochthonous microbial communities

“…Cells extracted from sediment particles were filtered through a 0.2-μm polycarbonate membrane (Millipore, 25-mm diameter) by gentle vacuum (<0.2 bar) and were analyzed by CARD-FISH as reported in Matturro et al (2012). CARD-FISH probes targeting 16S rRNA or 23S rRNA were used to detect total Bacteria (Eub338I, Eub338II, Eub338III),…”

Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation

“…Such additional stimulation can cause further competing processes such as nitrate and sulfate reduction and methanogenesis (Sun and Cupples, 2012 ; Révész et al, 2014 ). Hence, development of processes based on sequential stimulation of anaerobic-aerobic biodegradation has attracted significant attention and has been demonstrated (Thullner et al, 2012 ; Matturro et al, 2013 ) followed by further electro-bioremediation approaches (Rossetti et al, 2008 ; Matturro et al, 2012 ). Alternatively, the complete dechlorination of TCE to ethene of the Dehalococcoides mixed culture, which already had TceA and VcrA, was further enhanced by additional inoculation of Dehalococcoides strain BAV1, which contains BvcA enzyme for dehalogenation of cis -DCE and VC to ethene, using anaerobic biotrickling filters (Futagami et al, 2011 ).…”