| Petroleum as an energy source is widely used, but the soil environment contaminated by oil extraction, transportation and production is also a serious problem bothering the human beings. There are several methods to treat the oil-contaminated soil, including physical, chemical and bioremediation ways. Bioremediation attracts much attention because of its advantages such as safety, economy and no secondary pollution. Oil-contaminated soil, especially the aromatic hydrocarbons in petroleum, is difficult to be decomposed by microorganisms for its complexity of the oil component. Therefore, it is very important to isolate and select the microorganism that can degrade the oil in the contaminated soil, and to study the metabolic characteristics of the degradation microorganism. Besides, using the transcriptome sequencing technology, the degradation-related gene expression of the aromatic hydrocarbon in the fungus is also investigated; the purpose is to provide the technical support for screening and developing the aromatic hydrocarbon degradation microbes, and to improve the bioremediation efficiency of the oil-contaminated soil.In this study,12fungi with efficient oil-degradability were isolated from long term contaminated soil in Daqing. By means of colony morphology, hyphae form, physiological, biochemical testing and molecular identification by ITS sequencing, these fungal species were confirmed:H1, H3, H9, X9and X12belong to the Fusarium sp; H2belongs to the Aspergillus terreus; H4belongs to the Actinomucor elegans; X1belongs to the Fungal endophyte; X3belongs to the Fungal sp; X7belongs to the Bionectria ochroleuca; X11belongs to the Stachy botrys sp; D4belongs to the Trichoderma atroviride.The influence of different pH, nitrogen and phosphorus source to the growth of the12fungi in the liquid culture was researched. The result showed that the12fungi grew vigorous and had good yield in the hyphae dry weight under the condition of pH7. The growth of the fungi H9and XI2is the highest in the nitrogen source of NH4NO3, NaNO3is the best nitrogen source for the fungus H4, the other9fungi have the average harvest in the nitrogen source of KNO3. The12fungi have much high biomass under both KH2PO4and K2HPO4/KH2PO4(1:2), but there is no significant difference on hyphae growth between these sources.There are many factors that affect the oil-degradation rate of the fungi. Our study mainly discussed the influence of culture condition and crude oil concentration on the oil degradation rate of the fungi. Overall, the oil-degradation rate of the fungi in the liquid condition were higher than that in the soil when pH、N/P and NaCl concentration is fixed. Fungi could maintain a much high degradation rate when pH was8to9, though it was the highest when pH was7. The oil-degradation rate of the12fungi decreased along with the increase of the NaCl concentration. The oil-degradation rate of the strain X9and D4was still more than20%when the concentration of NaCl was10%, so that the oil-degrading fungi from Daqing have been considered with a certain resistance to salt and alkalinity, which is accordance with the adaptability of these strains to the saline soil characteristics of Daqing area. Among the12strains, X1show the highest oil-degradation rate in both the liquid and soil culture.The degradation metabolism of the petroleum hydrocarbons by most fungi starts with add single oxygen, but there are many complex enzymes participate in the metabolic process of the different fungal species. In this study, the activity of the main degradation-related oxidordeuctase of the12fungi in1%concentration of the crude oil was measured, the relationship between the activity of Lip、MnP, Lac and the oil-degradation rate in the process of the crude oil degradation by the fungi was discussed. The results showed that the different fungal species produced different enzymes with different activity. The enzyme activity produced by most strains increased under the induction of the crude oil, except Lip produced by X12and Lac produced by H4. there was significant positive correlation between the Lac activity of the fungi and the oil-degradation rate.Through sequencing、filtrating and jointing to the transcriptome of the strain XI who could degrade the aromatic hydrocarbon component in the crude oil, we obtain34003pri-miRNA named as the unigene, in which28660gene segments were annotated in Nt, Nr, GO etc7database. There are16376enzyme genes related with the alkane’s metabolism in Go classification, with the function of oxidordeuctase acts on CH-CH, on bisphenol, and on CH-OH, transferase acts on acyl, transferase acts on alkyl or aryl, C-C splitting. The result of KEGG Pathway showed that the strain X1’s gene mostly focused in carbohydrate metabolic pathways,527in total with the annotated genes12.3%, in which the gene number of the aromatic compounds and heterocyclic metabolic pathways in cells are significant higher than the others.Compared the gene expression difference of the strain XI induced by indene from the control without indene in PDA medium, the highest percentage of the up-regulation genes was found in naphthalene degradation pathway and oxidative phosphorylation pathway from all1216up-regulation genes. Combine with GO classification, we predict that the indene metabolism in cells could have a very similar process with the naphthalene metabolism. |
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