Study of Bio-based approaches for the biodegradation of petroleum derived hydrocarbons in environmental matrices.

Hydrocarbon polluted soils from a decommissioned oil refinery were subjected to mycoremediation treatment in mesocosms. The concentrations of total petroleum hydrocarbons (TPHs) was reduced to a greater extent in the current experiments, by using lower percentages of fungal inoculum (7% and 1% fresh weight) than were used in previous experiments (where 10% fresh weight inoculum was used). The diversity of bacterial and fungal communities was studied by metabarcoding using Next Generation Sequencing (NGS) over the duration of the bioremediation process. The aim was to identify putative organisms cooperating with fungi in remediation of TPHs, and to isolate them. A diversified approach to culturing microbes from the contaminated soils was applied to isolate key microorganisms, such as Gram-positive bacteria, involved in the humification of the organic matter in soil, generalist species able to utilize several carbon sources at low concentration in the environment, and specialist species able to degrade carbon sources (possibly the contaminants) at high concentrations in environment. Bacterial isolates from different genera, such as Pseudomonas, Limnobacter, Bacillus, and Microbacterium spp., were retrieved. Microbial diversity analyses showed a significant effect of the fungal inoculum density on the bacterial community. The highest density of fungal inoculum accelerated changes in functional bacterial biomarkers in different stages of TPH degradation. In particular, generalist species emerged initially, suggesting a role in both detoxification of the polluted matrix, and in cooperation with fungi, degradation of hydrocarbons. Specialist species appeared in the last phase of the process of hydrocarbon depletion, contributing to the complete TPH depletion. Bacteria belonging both to specialist and generalist groups that might improve the bioaugmentation approach were isolated. The data obtained suggest that mycoaugmentation may be improved by reducing the fungal inoculum density reduction and by co-inoculation of the contaminated matrix with both generalist and specialist species. The importance of generalists in future augmentation approach is innovative, and lead to the isolation of a putative specialist species capable of degrading several different hydrocarbons sources, including BTEX, a mixture of benzene, toluene, ethylbenzene and xylenes. The genome of the isolate was sequenced and annotated, and the isolate was identified as a strain of Pseudomonas putida, and was designated as P. putida AQ8. Analyses of the P. putida AQ8 genome revealed genes associated with BTEX degradation pathway, as well as genes involved in the regulation of gene expression by quorum sensing. The role of the quorum sensing in the regulation of the genes involved in BTEX degradation, was verified by creating a quorum sensing minus mutant. This was accomplished by transformation of P. putida AQ8 with a plasmid that expresses an enzyme that degrades quorum sensing signaling molecules. Quantitative polymerase chain reaction (qPCR) experiments revealed that genes involved in the upper pathway of BTEX depletion were modulated by quorum sensing