Role of plant-associated bacteria in the remediation of contaminated soils

Resumo

The global objective of this doctoral thesis consisted in the study and optimisation of the plant-soil-microorganism system within phytoremediation strategies for soils contaminated with organic and inorganic compounds. Phytoremediation can be defined as a group of techniques based on the use of plants and their associated microorganisms for the clean-up of contaminated soils. In recent years, studies aimed at optimising the efficiency of these techniques have been increasingly more focused on the importance of the role of plant-associated bacteria in these processes. It is well known that bacteria are commonly associated with plants, either surface-dwelling (epiphytic bacteria) or in the plant interior (endophytic bacteria). Exploiting these plant-bacterial associations in the field of phytoremediation has been centred in two areas. On one hand, these approaches use bacterial strains which are capable of influencing the general status of the plant, enhancing their establishment and growth in the contaminated soils. This type of bacteria are denominated as plant growth-promoting bacteria (PGPB) and influence plant growth via various mechanisms, such as through an increase in nutrient availability (e.g. nitrogen, iron or phosphorus), or through the synthesis of phytohormones (e.g. indoleacetic acid, IAA). On the other hand, bacterial strains can be employed which act directly on the contaminant in question, for example, strains capable of degrading contaminants (in the case of organic pollutants), or those which can modify the bioavailability of contaminants (e.g. through the production of biosurfactants), thus affecting contaminant fate and/or absorption by the plant. The principal objective of the first phase of this thesis was to obtain and characterise a collection of bacterial isolates associated with plants growing spontaneously in areas affected by a high concentration of trace metals or in areas contaminated with isomers of hexachlorocyclohexane (HCHs). In total, more than 500 bacterial strains were isolated. These were characterised genetically (BOX-PCR and rRNA 16S sequencing) and physiologically (for their metal tolerance, or ability to produce siderophores, indoleacetic acid or biosurfactants, or ability to solubilise phosphorus). In a second phase, selected isolates were used for various studies focused on the improvement of phytoremediation techniques. Regarding metalliferous soils, several studies were carried out in ultramafic soils (naturally rich in trace metals) using distinct populations of two nickel (Ni)-hyperaccumulating subspecies of Alyssum serpyllifolium, an endemic to the Iberian Peninsula and to this type of substrate. Additionally, a mine spoil was selected with an elevated concentration of Cd, Pb and Zn, and at this site three plant species were studied (Festuca rubra, Cytisus scoparius y Betula pendula). A collection of 420 bacterial isolated associated with these plant species was established. A select number of these isolates were then used to determine their influence on metal availability in the soil and their effects on plant growth. Various isolates were identified with the ability to produce metabolites enhancing the mobilisation of soil metals, a phenomenon which could directly affect metal absorption by the plant. Additionally, several bacterial strains were identified with the capacity to improve the growth of plant species, including those plants which are commonly used in the restoration of contaminated sites. Both characteristics are useful from a phytoremediation point of view. Regarding soils affected by organic contaminants, bacterial strains associated with a population of Cytisus striatus growing in a HCH-contaminated area were studied. In this case a collection of 146 bacterial strains was established. Following on from this, various studies were carried out in which the aim was to improve the growth of C. striatus in substrates contaminated with HCHs, and additionally to promote the degradation of this contaminant. As a result, a very promising a combination of bacterial strains (Rodococcus erythropolis ET54b and Sphingomonas sp. D4) was identified. When C. striatus was inoculated with this bacterial combination both plant growth and HCH degradation were significantly improved.