BIOREMEDIATION OF A POLYCHLORINATED BIPHENYL (PCB) POLLUTED SITE: DEGRADING POTENTIAL OF SOIL MICROBIOTA AND EXPLOITATION OF PLANT-BACTERIA INTERACTIONS FOR ENHANCED RHIZOREMEDIATION
Creators: name: VERGANI, LORENZO
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: The release of xenobiotic chemicals into the environment has dramatically increased over the last century following industrialization, with a consequent impact on the ecosystems and human health. Polychlorinated biphenyls (PCB), in particular, are among the twelve chlorinated organic compound families initially listed as persistent organic pollutants (POPs) by the Stockholm Convention on POPs. PCB, due to their chemical properties and high stability, have been widely used by industries in the twentieth century as dielectric and coolant fluids. Despite their production has been banned since the 1970s-1980s, these pollutants contaminate soils and waters and affect the ecosystems worldwide, being widespread global contaminants. Due to their high lipophilicity, PCB are recalcitrant to biodegradation, persist in the environment and bioaccumulate in the lipids of animals and humans, biomagnifying in the food web. It has been proved that PCB have relevant toxic effects on human health, including carcinogenic activity. The remediation of PCB-contaminated soils represents therefore a primary issue for our society; nonetheless, the available physical-chemical technologies have strong environmental and economic impact and are unsuitable for in situ soil remediation in extended contaminated areas. Rhizoremediation is a type of phytoremediation that relies on the capability of soil microbes responding to plant biostimulation, to degrade pollutants. This strategy appears as the most suitable for the detoxification of large-scale PCB-polluted soils. Among soil contaminants, rhizoremediation of PCB is specifically relying on the positive interactions between plants and microorganisms in the rhizosphere. In fact, several organic aromatic compounds released through root deposition can promote the activation of the biphenyl catabolic pathway that is responsible for the microbial oxidative PCB metabolism, thereby improving the overall PCB degradation performance in aerobic conditions in soil. Moreover, plant-growth promoting (PGP) microorganisms selected in the rhizosphere can sustain plant growth under stressed conditions typical of polluted soils, in turn enhancing the plant biostimulation. Nevertheless, the efficiency of this biotechnology in situ has been poorly assessed in the scientific literature, since the upscaling from laboratory to greenhouse conditions to the field was rarely implemented. Site-specific environmental conditions still represent a major challenge for an efficient in situ rhizoremediation intervention, especially when it comes to understand how the pollution fingerprint affects the autochthonous degrading bacterial populations and whether these are able to establish positive interactions with the introduced plant species. This PhD project focused on the Site of National Priority (SIN) Caffaro, a large site located in Northern Italy historically polluted by chlorinated POPs and metals. Aim of the work was to study the phylogenetic and functional diversity of the soil microbiota, assessing the correlation between diversity and pollutant profiles as a proxy to evaluate the biodegradation potential in the SIN Caffaro soil. A further aim of the thesis was to focus on the plant rhizosphere microbiome in order to setup in situ rhizoremediation strategies by evaluating the plant species with the higher potential for biostimulation. The soil microbiome of three former agricultural fields within the SIN Caffaro was investigated with molecular ecology ?16S rRNA metagenomic sequencing and DNA fingerprinting- and biochemical ?fluorescein hydrolyses- approaches. The results revealed that the bacterial communities? structure, their phylogenetic diversity and the soil microbial activity were related with the soil physical and chemical parameters, both along the soil depth profile and across the surface of the area of collection. These findings suggest the adaptation of the microbial communities to the high xenobiotics concentrations in the soil, possibly resulting in PCB biodegradation abilities. To assess the natural attenuation potential of autochthonous rhizosphere bacteria, we studied bacterial communities along a soil gradient from the non-vegetated to the root-associated soils of three different plant species spontaneously established in the most polluted field within the SIN Caffaro. The overall bacterial community structure of the non-vegetated and root-associated soil fractions was described by 16S rRNA metagenomic sequencing, and a collection of rhizobacteria isolates able to use biphenyl as unique carbon source was assayed for plant growth promotion (PGP) traits and bioremediation potential. The three plant species differentially affected the structure of the bacterial communities in the root-associated soil fractions, establishing the well known so-called rhizosphere effect. Nonetheless, the similar phylogenetic composition of the communities in all the soil fractions and the ubiquitous presence of the degrading potential, assessed by the presence of the bphA gene in the soil metagenome, leads to speculate that the soil contamination was one of the drivers for the enrichment of populations potentially able to sustain the process of natural attenuation. In vitro screening showed that biodegradation and PGP potential were widespread in the rhizosphere cultivable microbiome and the results of in vivo test on model plants suggested that two Arthrobacter sp. strains could be further investigated as bioenhancers on plant species of interest for rhizoremediation. To assess the rhizoremediation potential of different plant species and soil treatments, a microcosm-scale experiment was set up with the SIN Caffaro soil in greenhouse conditions, and the biostimulation effect was studied on the soil microbiota at different sampling times for 24 months. Plant species and treatments were identified basing upon an extensive literature screening, aimed to select the species/treatments that in previous studies showed to be effective in PCB rhizoremediation. The results of bacterial DNA fingerprinting and biochemical analysis of the soil surrounding the plant roots revealed that all the plants, when compared with unplanted control microcosms, significantly changed the bacterial communities? structure and stimulated the overall degrading activity in the soil. The stimulation of the soil microbiota leading to i) a shift in phylogenetic composition and ii) an increase in the organic matter hydrolytic activity, which may contribute to enhance PCB bioavailability and in turn their degradation in the polluted soil, is an indication of a potential positive rhizoremediation effect. The results need nevertheless to be substantiated by chemical analysis which will confirm the effective decrease of pollutants in soil surrounding roots and/or a change in the pollutants fingerprint. From the biostimulated soil a collection of Actinobacteria strains displaying in vitro biodegradation and PGP-related traits was also obtained. Three strains belonging to the genus Rhodococcus were in particular characterized for their PCB degradation capacity and for the ability to promote Arabidopsis thaliana growth and root development under laboratory conditions. Since A. thaliana root exudates previously showed to promote PCB degradation by a Rhodoccoccus bacterial isolate, these results open future research perspectives on the investigation of plant-bacteria interaction for PCB rhizoremediation. Overall, the results disclosed the existence of a PCB natural attenuation potential within the autochthonous microbial communities of the SIN Caffaro soil, and pointed out that rhizoremediation could be an effective strategy to enhance soil detoxification. Further research should be focused to better characterise the degrading microbiome inhabiting the soil and to identify the best plant-treatment combination with the support of chemical investigations assessing the rate of PCB removal from soil. Also, in vivo test with PCB-degrading and PGP bacterial strains are required to assess their potential as bioaugmentation tools to sustain a rhizoremediation intervention.