Selection of new fungal strains and development of a microbial consortium for the bioremediation of complex hydrocarbon mixtures

The release of hydrocarbons and their derivatives into the environment can have harmful effects on ecosystems and the organisms that populate them. Recently a further problem has arisen concerning the treatment of hydrocarbons in the oil industry, where large tanks are used to store raw materials and intermediate or finished products. Sludge and heavy oil deposits accumulated in tanks have to be cleaned periodically, usually by manual means. The procedure is dangerous, time-consuming, labour-intensive and expensive. Also, substances resulting from cleaning must be disposed of specifically as they are rich in hazardous substances. An alternative method to these methods is bioremediation, which involves using microorganisms to degrade toxic components in a substrate. This technique exploits algae, fungi, and bacteria's normal metabolic activities to modify harmful organic compounds into less complex metabolites, inorganic minerals, H2O and CO2. The biodegradation of hydrocarbons is a complex process that depends on the nature and quantity of the hydrocarbons present; moreover, one of the critical factors limiting the biodegradation of oil pollutants in the environment is their limited bioavailability to microorganisms, in particular, the compounds of oil hydrocarbons that bind to soil components. Many studies have reported how bacteria, yeasts and fungi can degrade hydrocarbons in different environments. In particular, fungi are excellent hydrocarbon degradation agents because they produce extracellular enzymes with low substrate specificity and are active even under micro-aerobic or anaerobic conditions and with low water activity. Many papers have been published on fungal degradation of polycyclic aromatic hydrocarbons (PAHs), among the most toxic components of oil, but few regarding the degradation of hydrocarbon mixtures, mainly resulting from gas or oil production processes. This PhD project aimed to assemble a fungi-bacteria consortium, selecting microorganisms directly from contaminated substrates to be treated to achieve the highest possible degradation rate, reducing the use of chemicals, costs and time required for remediation. From the contaminated substrates from a gas plant (Eni S.p.A.) in Pakistan, 29 fungal strains and 50 bacterial strains have been isolated and identified. The fungal strains were then selected based on their ability to grow using hydrocarbons as the only source of carbon, and from these first tests, 8 strains potentially useful for the project were selected. These fungi were tested in their degradative capacity, by analysing the composition of hydrocarbon substrates before and after treatment with the fungal strains. Qualitative enzymatic tests were also carried out to estimate the enzymatic production of the selected strains and tests to verify the ability to produce biosurfactants, substances useful in increasing the bioavailability of pollutants. Once the results of the various tests had been analysed, the fungi-bacteria consortium was set up, but the fungal strains were first subjected to antibiosis tests to select further only those capable of establishing positive relationships with the others. Therefore, 6 fungal strains contributed to the consortium formation, while the bacterial strains were 28. The consortium was also subjected to tests to verify its degradative capacity, demonstrating an excellent potential, degrading, within a week under laboratory conditions, all the hydrocarbon fractions that could be analysed. The project's last step was to set up an ecotoxicological test to demonstrate the consortium's bioremediation capacity. The results obtained in this work are encouraging and open the door to many applications. Consortia composed of different microorganisms, from the tests carried out, seems to be an effective bioremediation technique with high potential. Unquestionably further investigation is needed to understand how to optimise the consortium's performance.