BACTERIAL COMMUNITY DYNAMICS IN A NOVEL 1,2-DCA DECHLORINATING ANAEROBIC CONSORTIUM

ABSTRACT 1,2-dichloroethane (1,2-DCA) is a chlorinated solvent and a polyvinylchloride (PVC) synthesis intermediate. Its production worldwide sums up to more than 20 million tons per year and due to its relatively high solubility in water, it tends to partition in groundwater aquifers, constituting a permanent source of pollution, being a typical persistent organic pollutant (POP). A groundwater aquifer near to Ferrara (Italy) has been the object of this study, in which we investigated the potential of the indigenous microbial community to carry over remediation of pollutant, following amendment with electron donors suitable to stimulate reductive dehalogenation of 1,2-DCA. In the first park of the work, we investigated response of lower aquifer microbial community to lactate biostimulation, in terms of population shift, through 16s rRNA libraries. Contextually we investigated the functional diversity of the putative reductive dehalogenase genes retrieved in the aquifer, prior and posterior to amendment. The population variation was moderate in terms of Shannon index and community organization shifts, but the population represented changed nearly completely, showing emerging co-dominating population, in which no known 1,2-DCA degrader are present. From the other hand, functional genes analysis showed some redundancy of the function as well, since four distinct, but highly similar among each other dehalogenase sequences were retrieved and compared with sequences found in upper aquifer by Marzorati and colleagues (2007), to find a high identity percentage (99%) among these sequences and the three found in upper aquifer that clusterized with WL rdhA1 sequence retrieved by Grostern & Edwards in a 1,2- DCAdegrading co-culture dominated by Dehalobacter sp.WL. In the second part of the work, we investigated response of upper aquifer communities, proceeding from two distinct piezometers, to amendment with various electron donors, with the aim of obtaining an enriched dechlorinating community and characterize it through transfers. Total diversity and functional genes were monitored through transfers through different techniques: 16S rRNA clone libraries, Illumina MySeq and functional genes PCR-DGGE. Eventually, the enriched culture was monitored through flow cytometry to follow the population growth during 1,2-DCA degradation. The best response, in terms of conservation of dechlorinating activity through transfers, was observed following amendment with a mixture of acetate and formate, which eventually led to a co-dominated community in which a putative dechlorinating Unc. Geobacter sp. grew together with a Pseudomonas aeruginosa strain, with an apparent mutual benefit. Phylogenetic analysis of 16s rRNA and of functional genes was performed. The putative dechlorinator Unc. Geobacter sp. closed characterized relatives appeared to be G. psychrophilus and G. chapellei, while highest identity was found with an Unc. Geobacter sp. retrieved during previous studies on the same aquifer. In the third and final part, we intended to investigate the mechanisms of interaction between the dominating species of the previously enriched culture, stressing the role of P. aeruginosa secondary metabolites, pyocyanin (PYO) and rhamnolipids (RL), whose positive effect in exoelctogenic activity of P. aeruginosa, as well as of other bacteria, has been described in several studies. At this purpose, we set up different parallel replicates in which various combinations of P. aeruginosa metabolites were tested to verify their effect on dechlorination rate as well as on population growth. Results showed a beneficial effect of RL and PYO addiction to culture, but not of PYO alone. Moreover PYO was detected in all microcosms after 1,2-DCA degradation, suggesting RL concentration as a limiting factor for PYO function as putative electron shuttle, in microcosm conditions.