GROUNDWATER BIOREMEDIATION: MICROBIAL POPULATIONS INVOLVED IN CHLOROETHENES AND BTEX CONTAMINATED AQUIFER PROCESSING

Groundwater plays an important role in water supply around the world. 2 billion of people use aquifers as drinking water. Consequently, contamination of groundwater has a great social and economic impacts. The use of organisms (microorganisms and plants) to remediate contaminated matrices, called bioremediation, is becoming more and more frequent. These techniques are cheaper than chemical and physical remediation techniques. Chloroethenes, aromatic and aliphatic hydrocarbons are widely contaminant compounds because of their intensive use in industrial activity. It is possible to lower their concentration in the environment by means of microbial biodegradation in anaerobic and aerobic conditions. In this study, an aquifer (located near Porto Marghera, Venice, Italy) contaminated by a leaching from a former landfill was analyzed. The contamination comprised chlorinated solvents, benzene, toluene, ethylbenzene and xylenes (BTEX) and aliphatic hydrocarbons. In 1995, an intervention with a pump and treat reactor was installed. Due to low efficiency and high maintenance costs of the physic-chemical treatment, the installation of a biological treatment, based on two permeable reactive biobarriers, was planned. After preliminary characterization of the microbial community at the site in order to evidence the presence of natural microbial populations involved in decontamination processes, in February 2016 a first biobarrier was installed to stimulate bacterial anaerobic organohalide respiration to dechlorinate chloroethenes. The injection of a reducing substrate was set up to create strong reducing conditions to improve the activity of anaerobic bacteria. A second biobarrier was meant to stimulate bacterial aerobic biodegradation of BTEX and aliphatic hydrocarbons. Urea, ammonium phosphate and O2 were planned to be injected in the aquifer. Moreover, this treatment was also forecasted to be used for complete vinyl chloride aerobic biodegradation. In order to define the presence of organo-halide respiring bacteria at the aquifer, laboratory-based anaerobic microcosm study was set up. The effect of the biostimulation intervention (i.e., the addition of a reducing substrate) was also monitored in comparison with natural attenuation processes. Chlorinated ethenes were analyzed through gas-chromatography coupled to mass spectrophotometry (GC-MS). At microcosms scale, the natural organohalide respiration activity was influenced by the presence of reducing substrate, showing an increase of dechlorination of highly chlorinated ethenes, with a concomitant accumulation of vinyl chloride. Landfill active microbial community composition was determined through Illumina 16S rRNA sequencing of cDNA from RNA extracted from groundwater samples. Active organo-halide bacteria were quantified by quantitative Real Time PCR (q-PCR). Phylogenetic bacterial biomarkers for Dehalococcoides, Geobacteriaceae, and functional biomarkers tceA and vcrA, coding for chlorinated ethenes reductases, were applied. The ability of aerobic biodegradation of vinyl chloride, BTEX, aliphatic hydrocarbons and chlorobenzene was studied by the Most Probable Number (MPN) technique and q-PCR of etnC and tbmD genes, coding for alkene and toluene-benzene monooxygenases, respectively. Once established the presence of bacterial natural attenuation activities for all the compounds, chemical and microbiological analyses were performed at field scale in order to monitor the efficacy of the bioremediation treatments. Moreover, the microbial community composition of anaerobic biobarrier was analyzed before and after 22 months of treatment, by 16S rRNA Illumina sequencing. Reducing substrate addition affected the microbial community composition at the site, causing an increase of fermentative bacteria, mainly belonging to Archaea domain, whereas typically recognized bacteria involved in organohalide respiration were not displayed. These data, along with a decrease in chlorinated solvents measured at the site, suggest a possible presence of a still unexplored biodiversity of OHR bacteria and further culturomics efforts will help to elucidate this. At the plume fringe in the aerobic part of aquifer, BTEX, chlorobenzene and aliphatic hydrocarbon degrading bacteria were characterized. Moreover, microbial consortia able to use vinyl chloride as sole carbon and energy form were selected, demonstrating the feasibility to remediate the site from the carcinogenic intermediate of organohalide respiration. The microbiological work carried out during this Doctorate, along with hydrogeochemical data, demonstrated that a bioremediation intervention could successfully decontaminate this historical and naturalistically important site. Since the beginning of 2020, a full-scale biobarrier plant has been established and it is expected to run for 30 years in order to completely remediate the aquifer.