Development of effective molecular approach for determining of the biodegradation potential of petroleum contaminated soil

Despite an increase in the production of renewable energy, petroleum products are still the main source of energy for industry and domestic consumption. World production of crude oil is more than three billion tons per year. Because of the fact that most of petroleum consumer counties are not major oil producers, huge amount of produced oil is transferred via pipeline and water ways. Vast amount of petroleum enters to the environment through exploration, extraction, production, transportation, refining and storage activities. Petroleum wastes involve toxic, mutagenic and carcinogenic compounds which cause health problems and disorders to human life and ecosystems. The release of these materials to the environment is restricted with regulations. Petroleum sludge has a complicated structure including water, oil emulsion, metals, tars and numerous petroleum hydrocarbon compounds. Improper disposal of oily sludge leads to serious soil, water and groundwater pollutions. Some physicochemical methods such as soil vapor extraction, soil washing and dispersion are common methods for treatment of petroleum polluted sites and remediation of oil sludge, however, these methods are not able to complete decompose petroleum pollution and may lead to increase the toxicity of contaminated sites. Bioremediation as a cost-effective and environmental friendly method overcomes these problems. During this process pollutant compounds are converted to nontoxic materials or completely degraded to CO2 and H2O. Indigenous microorganisms or inoculated ones in the bioremediation sites can be stimulated to metabolize organic pollutants using nutrients, oxygen, or other amendments. Bioaugmentation as an enhanced bioremediation type refers to improvement of the biodegradation potential of bioremediation site by adding of microbial strains. This process has remarkable effect on the degradation of various contaminants. However, concerns about using of genetically engineered microorganisms and their probable impacts on the environment are commonly raised. Biostimulation as an alternative for bioaugmentation and as a sustainable bioremediation method represents a more efficient method of soil bioremediation than bioaugmentation. Monitoring of the efficiency of this process and the evaluation of the biodegradation manner in the contaminated site is one of the important steps in biostimulation practice. An effective monitoring could reduce the bioremediation cost and treatment duration. Development of an effective molecular monitoring approach for evaluation of the biostimulation process and biodegradation potential of petroleum contaminated soil was the mission of this thesis. The evaluation of the microbial community structure and functional gene dynamics during the biostimulation process was also the mission of this thesis. Setting up aerobic microcosms test, the biodegradation manner during biostimulation process, microbial community structure and functional gene dynamics were evaluated. The occurrence of horizontal transfer of functional genes within microbial community was also analyzed. For this purpose, petroleum contaminated soil samples were collected from petroleum storage pit and characterized. Dominant bacterial species of collected samples were determined using Illumina sequencing. Obtained petroleum polluted samples were mixed with clean forest soil samples in different ratios and weighted in glass vessels. Obtained mixtures were amended with different N:P ratios and cultivated at 24 ºC for 90 days. Genomic DNA, total RNA, and plasmids were extracted. Quantitative real time PCR with TaqMan probe was used for the quantifying of alkane and poly aromatic hydrocarbons (PAHs) degrading bacteria and alkB, phnAc and nah functional genes. As a post-treatment method treated contaminated soil was bioaugmented with Trametes versicolor and Bjerkandera adusta fungi following the biostimulation practice. The effects of mentioned fungi on the indigenous microbial community and, phnAc and nah functional genes through 60 days bioaugmentation practice at 29 ºC were evaluated. Throughout the biodegradation test, maximum biodegradation rate was obtained when C:N:P ratio was arranged to 100:15:1. The most total organic carbon (TOC) removal rate was 18% and obtained from setup with initial TOC amount of 15%. Microbial analysis of collected petroleum polluted soil samples showed that gram-negative bacteria were dominant bacterial group. γ-proteobacteria, Chloroflexi, Firmicutes and δ-proteobacteria were dominant bacterial phyla. These bacterial groups covered more than 65% of the determined bacterial strains. Biodegradation examination clearly revealed that biostimulation as a sustainable bioremediation approach enhances the biodegradation of petroleum contaminated soil. Quantitative analysis of indigenous microbial community in the biostimulation process showed that gram-negative bacteria have a competitive advantage over gram-positive bacteria in studied petroleum polluted soil. Dominance of this bacterial group in the petroleum contaminated soil samples and samples obtained from experimental phase demonstrates that due to a long term acclimation to the petroleum hydrocarbons in the polluted region these bacteria could overcome other indigenous bacteria of the biodegradation vessels which were originated from clean forest soil. Any failure in stability of these bacterial will lead to a decrease in alkanes and PAHs biodegradation process. A successful horizontal transfer of alkB and phnAc functional genes was detected. Detection of the occurrence of horizontal gene transfer (HGT) phenomenon during the biostimulation process shows that, HGT can be used a helpful biomarker for monitoring of bioremediation process. Furthermore, evaluation of HGT in a contaminated site can present the broader estimation of bioremediation potential of contaminated site and positive signs about the occurrence of HGT in a petroleum-contaminated site will indicate that biostimulation is a suitable choice for bioremediation of the contaminated site. Results obtained from the post-treatment phase showed that T. versicolor and B.adusta fungi cannot fully biodegrade the petroleum residues remained from biostimulation phase. However, based on the qPCR results T. versicolor is effective than B. adusta in biodegradation of the petroleum residues. During post-treatment phase, the expression level of alkB, phnAc and nah functional genes were increased synergistically. Any significant change in the amounts of active bacterial cells, gram-negative and gram-positive bacteria were observed. For further studies bioremediation studies using anaerobic microorganism because of their efficiency in the biodegradation of toxic compounds is recommended. Combination of different strains of anaerobic fungi and combination of anaerobic fungi and bacteria for maximum biodegradation of petroleum contaminated soil are also suggested. Horizontal transfer of functional genes between inoculated microorganisms and indigenous ones during bioaugmentation process can also be tested as an efficient biomarker for monitoring of this enhanced bioremediation approach.