Unravelling the process of petroleum hydrocarbon biodegradation in different filter materials of constructed wetlands by stable isotope fractionation and labelling studies.

Maintaining and supporting complete biodegradation during remediation of petroleum hydrocarbon contaminated groundwater in constructed wetlands is vital for the final destruction and removal of contaminants. We aimed to compare and gain insight into biodegradation and explore possible limitations in different filter materials (sand, sand amended with biochar, expanded clay). These filters were collected from constructed wetlands after two years of operation and batch experiments were conducted using two stable isotope techniques; (i) carbon isotope labelling of hexadecane and (ii) hydrogen isotope fractionation of decane. Both hydrocarbon compounds hexadecane and decane were biodegraded. The mineralization rate of hexadecane was higher in the sandy filter material (3.6 µg CO2 g-1 day-1) than in the expanded clay (1.0 µg CO2 g-1 day-1). The microbial community of the constructed wetland microcosms was dominated by Gram negative bacteria and fungi and was specific for the different filter materials while hexadecane was primarily anabolized by bacteria. Adsorption / desorption of petroleum hydrocarbons in expanded clay was observed, which might not hinder but delay biodegradation. Very few cases of hydrogen isotope fractionation were recorded in expanded clay and sand & biochar filters during decane biodegradation. In sand filters, decane was biodegraded more slowly and hydrogen isotope fractionation was visible. Still, the range of observed apparent kinetic hydrogen isotope effects (AKIEH = 1.072-1.500) and apparent decane biodegradation rates (k = - 0.017 to - 0.067 day-1) of the sand filter were low. To conclude, low biodegradation rates, small hydrogen isotope fractionation, zero order mineralization kinetics and lack of microbial biomass growth indicated that mass transfer controlled biodegradation.

Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality.

Microbial water quality assessment currently relies on cultivation-based methods. Nucleic acid-based techniques such as quantitative PCR (qPCR) enable more rapid and specific detection of target organisms and propidium monoazide (PMA) treatment facilitates the exclusion of false positive results caused by DNA from dead cells. Established molecular assays (qPCR and PMA-qPCR) for legally defined microbial quality parameters (Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa) and indicator organism group of coliforms (implemented on the molecular detection of Enterobacteriaceae) were comparatively evaluated to conventional microbiological methods. The evaluation of an extended set of drinking and process water samples showed that PMA-qPCR for E. coli, Enterococcus spp. and P. aeruginosa resulted in higher specificity because substantial or complete reduction of false positive signals in comparison to qPCR were obtained. Complete compliance to reference method was achieved for E. coli PMA-qPCR and 100% specificity for Enterococcus spp. and P. aeruginosa in the evaluation of process water samples. A major challenge remained in sensitivity of the assays, exhibited through false negative results (7-23%), which is presumably due to insufficient sample preparation (i.e. concentration of bacteria and DNA extraction), rather than the qPCR limit of detection. For the detection of the indicator group of coliforms, the evaluation study revealed that the utilization of alternative molecular assays based on the taxonomic group of Enterobacteriaceae was not adequate. Given the careful optimization of the sensitivity, the highly specific PMA-qPCR could be a valuable tool for rapid detection of hygienic parameters such as E. coli, Enterococcus spp. and P. aeruginosa.