Application Of Microbial Molecular Ecological Technology In The Remediation Of Petroleum Contaminated Soil

This dissertation started with an overview of the recent advancement ofbioremediation techniques and their applications in the remediation ofpetroleum-contaminated soil. The application of molecular biological techniques toprobe the microbial ecological changes in terms of composition and quantity of bothmicrobial community and functional genes was highlighted. The prospects of thesetechniques as enabling tools in the design, implementation and assessment ofbioremediation practice were discussed.The variation of microbial society occurred to a bioaugmentation of petroleumcontaminated soil using Enterobacter cloacae as an inoculant was monitored bydenaturing gradient gel electrophoresis (DGGE) of16S rDNA of the bacteria. It wasshown that the addition of wheat straw in the augmentation enhanced the growth ofbacterium and fungi in comparison to that obtained with solely inoculant. A maximumconcentration of4.6×10~7and5.5×10~3, for bacterial and fungal species respectively, wasobtained in case of adding wheat straw, which also resulted in a highest dehydrogenaseactivity (0.79) and degradation rate (56%in56d). The remediated soil with differenttreatment presented different bacterial composition, as shown by PCR-DGGE.The variations in the microbial ecological system of remediated soil (R) andpetroleum contaminated soil (P) based on comparison with soil that has not beencontaminated (N) was profiled using a cloning library of taxonomic genes (16S rDNAfor bacterium and18S rDNA for eukaryotes). The genotype number of prokaryote andeukaryote found in the N soil was122and34, respectively. Proteobacterial,Acidobacteria, Bacteroidetes, and Arthropoda were the predominant bacterial phylum,while Incertae seids, Dikarya and Cercozoa accounted for predominant ones ineukaryotic composition. The contamination with crude oil led to a reduction ofgenotype number to96(bacteria) and21(fungi), respectively, and, moreover, analteration in composition and proportion of both bacterial and eukaryotic phylum in theP soil. The R soil, as treated using a microbial consortium of Enterobacter cloacae andCunninghamella echinulata as inoculant, gave a genotype number of115(bacteria) and30(fungi), and moreover, a more close distribution of predominant bacterial andeukaryotic phylum with reference to the N soil. The restoration of microbial ecology, as indicated by aforementioned results, confirmed the effectiveness of the remediationpractice.The change of soil function due to the bioagumentation using a microbialconsortium of Enterobacter cloacae and Cunninghamella echinulata as inoculant wasevaluated using three genes encoding the key enzyme in nitrogen fixation, nitrificationand ammoniation as target genes, i.e., nifH, amoA and narG. For nifH gene, while thepetroleum contamination led to a reduction of genotype number in the P soil from25to15, the R soil presented a genotype number of21, being close to that of the N soil.Moreover, the cluster analysis showed the existence of six subclasses in the three soilsample, which was Actinbacteridae, α-,γ-,β-, δ-Proteobacteria and unclassified group,though the relative density of each subclass varied, particularly that of the contaminatedsoil. Similar results were found for amoA and narG gene, the latter was absent in the Psoil, a direct evidence of the imperfection of soil nitrogen cycle due to petroleumcontamination. The remedation restored the above mentioned genotype and gaveenhanced narG gene diversity with reference to the normal soil. These confirmed theremediation of the soil in terms of functional genes. The results obtained by the presentstudy provided a microbial ecological insight that can be used to aid the design andimplementation of bioaugmentation.