Molecular tools to characterise plant-associated bacterial communities in soils naturally enriched or contaminated with trace metals

Resumo

Soil contamination is a worldwide problem with associated risk for the environment and human health. Trace elements (TE) are considered as one of the most contaminant groups. High concentrations of TE in the soil can be due to the human activity but also to the alteration of naturally TE-rich rocks, such as ultramafic soils. In the ultramafic areas there is often a high degree of plant endemisms, such as Ni-hyperaccumulating species. In recent years much interest has turned towards studying the microorganisms associated with these plants, especially those present in the rhizosphere, and their possible application in phytoremediation techniques. Soil microorganisms are essential for soil functioning and plant nutrition and development. In addition, soil microorganisms are highly sensitive to changes in soil making the study of microbial communities and their responses to changes in soil properties, as for example those induced by the implementation of phytoremediation options in TE contaminated soils, of great interest. The objectives of this thesis were (1) characterise and compare the bacterial communities associated with Ni-hyperaccumulating and metal-excluding plants growing in ultramafic soils (to obtain information about bacteria potentially involved in the mechanisms of TE (hyper)accumulation) and (2) analyse bacterial communities in soils contaminated with TE and treated with phytoremediation strategies (to study the relationships between the bacterial communities and the changes induced in soil properties by phytomanagement) The analysis carried out indicated that the microbial community of the ultramafic soils studied was dominated by members of the phyla Alphaproteobacteria and Actinobacteria (which represented about 2/3 of the pyrosequences obtained). The results obtained also showed that bacterial communities in the non-vegetated ultramafic soils differed from those in the rhizosphere of the Ni-hyperaccumulator and the Ni-excluder plants growing in the same soils. In addition, the rhizosphere of the Ni-hyperaccumulator, Alyssum, and of the Ni-excluder, Dactylis, hosted different bacterial communities. The bacterial communities in the ultramafic sites in the NW and S of Spain were also significantly different and the differences were more pronounced in non-vegetated soil than in the rhizosphere. DGGE and pyrosequencing analysis of 16S rDNA fragments indicated that some relevant members of the bacteria community in the rhizosphere of the Ni hyperaccumulating subspecies of Alyssum growing in ultramafic areas of NW and S of Spain are: Actinobacteria of the family Geodermatophilaceae (known for their modest growth requirements and their ability to colonise poor substrates) and of the genus Pseudonocardia; Alphaproteobacteria of the family Hyphomicrobiaceae (such as members of Devosia and Hyphomicrobium, characterized by prosthecate, that confer them the ability to survive in nutrient poor habitats and/or possess in their genomes genes related to N2 fixation), of the genus Methylobacterium (previously found in association with Alyssum and other plants, and known for their plant growth promoting properties) and of the Rhodobacteraceae family (which includes N2 fixing bacteria and strains involved in Mn cycling); as well as bacteria related to Gemmatimonas. The rhizosphere of the Ni-hyperaccumulating Alyssum serpyllifolium subspecies growing in artificial substrate enriched in ultramafic rock hosted a higher density of Ni-tolerant bacteria than the rhizosphere of the Ni-excluder Dactylis glomerata or the non-planted substrate. The copy number of nccA gene, involved in bacterial tolerance to Ni Co and Cd, was higher in the rhizosphere of Alyssum than in non-vegetated soil. Analysis of phytomanaged soils indicated that the phytoremediation process induced increases in microbial biomass and activity, as well as clear shifts in bacterial community structure and diversity. Actinobacteria were sensitive to changes in TE contaminated soils induced by phytomanagement options that included soil amendments, while α-Proteobacteria and β-Proteobacteria were particularly useful to monitor changes over time in those sites treated with organic and inorganic amendments, respectively. In general, the copy numbers of genes involved in denitrification and nitrification (nirK, nirS, nosZ and amoA) were influenced by phytomanagement techniques implemented. The most pronounced changes were observed in sites where organic amendments were incorporated. The microbiological parameters were highly sensitive to changes induced by phytoremediation at mid and long term.