| Microbial oils, namely single cell oils (SCOs), can be produced by oleaginous microorganisms and have fatty acids composition that is similar to those of plant oils. Microbial oils production has been suggested to be an important research topic to biodiesel fields by the National Renewable Energy Laboratory-(NREL) of U.S. Department of Energy. Exploration of inexpensive culture media and low-cost fermentation systems to lower the cost of microbial lipid is one of the key points for its development and wide application. In the present work, two strategies for conversion of straw wastes to microbial oils were designed and studied. With regard to conversion of straw to microbial oils by fermentation using multiple fungi, we focused on artificial construction and characterization of fungal consortia that produce cellulolytic enzyme system with strong wheat straw saccharification. According to problems that exist in the conversion of straw to microbial oils by multiple fungi, we further investigated the feasibility of direct bioconversion of straw into microbial lipid (CBP) by a single microorganism. The fermentation system for CBP was constructed on the basis of the isolation of fungi capable of direct bioconversion of wheat straw into microbial lipid. Besides, fermentation optimization, CBP behaviors analysis and genetic modification were performed using Penicillium sp. P-2and Aspergillus oryzae A-4as the major research material. The main results are listed as follows:(1) Artificial construction and characterization of a fungal consortium that produces cellulolytic enzyme system with strong wheat straw saccharificationA consortium-APcT2composed of92%of Trichoderma sp. T-1,6.7%of P. chrysosporium and1.3%of A. oryzae A-4that produces cellulolytic enzyme system with strong wheat straw saccharification was constructed using Taguchi design combined with variance analysis (ANOVA).The finally optimized consortium-APcT2yielded805.12mg gds-1sugars from1g alkali pretreated straw with85%of crude fiber utilized within20hours, which was26.98%higher than the pure Trichoderma sp. T-1. Protein profile analysis of the cellulolytic enzyme systems, sugar composition analysis of the hydrolysates and compatibility evaluation of the fungal constituents showed that the enhanced straw saccharification of the consortium-APcT2could be mainly attributed to the enhancement of the co-cultivation for enzyme production and the synergistic action of different types of enzymes in the hydrolysis process. To simplify the preparation of fermentable sugar and the evaluation of the hydrolytic ability of fungi, the enzyme production and pretreated straw saccharification were combined together in a system without enzyme purification and concentration. Production of SCOs by T. fermentans was conducted using the enzymatic hydrolysate (EH) produced from consortium-APcT2treatment and wheat straw sulphuric acid hydrolysate (SAWSH) as carbon source, respectively. The maximum lipid yields of1.7g1-1and2.3g l-1was respectively obtained by the fermentations of SAWSH and EH.(2) Exploration of fermentation fungi capable of conversion of cellulose into microbial oils by one stepTwo isolation strategies were applied to isolate fermentation strains capable of direct conversion of cellulose into microbial oils:1) isolated from cellulolytic fungi;2) isolated from endophytic fungal strains. Aspergillus oryzae A-4, Penicillium sp. P-2and Trichoderma sp. T-1were isolated from cellulolytic fungi with the lipid content of21.50%,17.50%and11.22%, respectively. Besides, four endophytic fungal strains Fusarium oxysporum, Fusarium solani, Fungal endophyte sp. and Mucor sp. with both strong cellulose decomposition and high lipid accumulation were isolated from plant samples Vernicia Montana and Celtis vandervoetiana. The lipid content of Fusarium oxysporum and Fusarium solani was26.6%and29.75%, respectively.(3) Direct conversion of cellulose into lipids by a newly isolated Penicillium sp. and its CBP behaviors studyPenicillium sp. was selected for further fermentation with a high lipid accumulation. Both submerged fermentation (SmF) using cellulose and solid-state fermentation (SSF) using wheat straw and bran mixture showed that P-2was able to produce SCOs efficiently using cellulosic substrates by CBP, releasing a maximum lipid yield of0.65g/L and40.13mg/gds, respectively. Single factor experiment results showed the condition with moisture of4:2, pH of6, temperature of30℃, and0.2%of tween-80 was optimal for total cellulase (FPA activity) secretion of P-2as well as the optimal one for lipid production. Besides, analysis of lipid accumulation and cellulase secretion of P-2under SmF conditions suggested that cellulase secretion of P-2might play a key role in the lipid production. SmF with exogenous cellulase addition by P-2then showed that higher cellulase dosages lead to higher lipid production, and the lipid yield increased to0.83g/L when24FPU/gds cellulase was added.(4) Microbial oils production of Aspergillus oryzae A-4by solid state fermentation using wheat straw and bran mixture as substrateThe lipid yield obtained from solid-state fermentation of P-2was relatively low with straw as the substrate, although its cellulose decomposition and lipid accumulation were found to be strong. Thus, another strain A. oryzae A-4with a good growth performance on straw was selected for further study. In submerged fermentation, A. oryzae A-4accumulated lipid to15%-18.15%of biomass when pure cellulose was utilized as the sole substrate. Then, CBP behaviors analysis and fermentation optimization were performed in solid state fermentation experiments using straw and bran mixtures. The conversion process of cellulose into lipids in A-4in the SSF was also described. The lipid yield reached62.87mg/gds in SSF on the6th day under the optimized conditions from Plackett-Burman design (PBD) without exogenous cellulase addition. Cellulase secretion of A. oryzae A-4was found to influence the lipid yield. Addition of some agro-industrial wastes to the straw could enhance lipid production of A. oryzae A-4.(5) Construction of recombinant strains with efficient lipid production by the transformation and expression of cellulase gene in Aspergillus oryzae A-4An efficient lipid produced strain A-4was genetic engineered to impove SCOs production according to the effect study results of cellulase on lipid production. A recombinant plasmid of pPTRI-Tamy-gene carrying the hlyA promoter, the amylase terminator and the PT resistance gene, which can express exogenous gene in the oleaginous A. oryzae A-4, can be constructed through overlap PCR and homologous recombination. Then, the above-mentioned transformation system was proved to be feasible using egfp report gene. Based on this transformation system, we constructed cellulase expression cassettes carrying the cloned cellulase gene. Four kinds of cellulase expression cassettes were obtained containing celA, celB, celD and celC, respectively. After cellulase activities determination and PCR screening,5positive transformants with greatly improved cellulase activity was isolated. Among the5isolated transformants, A2-2and D1-B1presented significantly higher lipid yields than the wild-type strain under both submerged and solid-state conditions. A lipid yield of63.30mg/gds was obtained after4days fermentation under unoptimized solid-state condition using wheat straw and bran mixtures. Comparison of fermentation performances of A2-2, D1-B1and WT on different carbon sources indicated the cell growth of D1-B1and A2-2were inhibited by the transformation and expression of cellulase genes, while their lipid accumulation were promoted on the contrary. Beside, the inhibitory effect of gene insertion on cell growth of transformants lowered or disappeared when using cellulosic materials as the main carbon source. |
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