Biodiesel generated from lipid transesterification can be used as a replacement or blended with for petroleum diesel in any proportion. Current processes to produce biodiesel mostly use plant oil as feedstocks, which keep the costs at high levels and result in a shortage of edible oil in food market. Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. The production of biodiesel from lignocellulosic biomass and other waste materials would have both economic and environmental benefits. This research focused on the screening and identifying novel oleaginous fungal strains with both high lipid content and capability of cellulase enzyme production, the study of the characteristics of selected strain in lipid accumulation process, and the utilization of selected strain in converting lignocellulosic biomass to fungal lipids for biodiesel production. The first step of this research is the screening and identifying fungi from soybean and soil surrounding to soybean plants, and analyzing fungal community of the screened isolates. From two sets of screenings, 33 fungal isolates were obtained from soybean samples collected in August and 17 fungal isolates were obtained from soybean samples collected in October. Meanwhile, the screening from soil surrounding to soybean plant roots obtained 38 fungal isolates. Soybean samples and soil samples showed a great difference in the isolated fungal community, and difference of fungal diversity was also detected from soybean samples collected in August and samples collected in October. Also, different sampling locations had differences in fungal community. These results demonstrated an impact of environment on fungal community, and also indicated a change of plant-associated fungal community through time in soybean samples. For soybean samples, Fusarium and Alternaria were the dominant genera, and other frequently detected genera included Penicillium, Nigrospora, Cercospora and Epicoccum. For soil samples, Trichoderma, Fusarium, Mucor and Talaromyces were most frequently isolated genera. The second step of this research is the bioprospecting of fungal strains isolated from soybean and soil for lipid accumulation to identify strains with high lipid content and cellulase production. Among 33 fungal isolates screened from soybean plant, 13 were oleaginous fungi (lipid content>20% dry biomass weight); among 38 fungal isolates screened from the surrounding soil, 14 were oleaginous fungi. A considerable amount of fungi were identified as oleaginous fungi, and fungi with highest lipid content (>40%) belong to Fusarium genus. One of the strains was selected as the most promising strain was Fusarium equiseti UMN-1 strain. This strain has high lipid content (>56%) and high fatty acid methyl ester (FAME) content (98% in total lipid), also produces cellulase. In addition, it can utilize a wide range of substrates and has promising oil composition for biodiesel production. The F. equiseti UMN-1 strain offers great potential for biodiesel production by directly utilizing lignocellulosic biomass as feedstocks, and was used in the following studies. The third step of this research is the investigation on the characteristic of F. equiseti UMN-1 strain and optimization of cultivation conditions for higher lipid production. Characteristic study of this strain determined the optimal temperature as 27 °C and agitation speed as 150 rpm. The best C:N ratio for lipid accumulation was 80, strong light during cultivation was not suggested, and 6 to 8 day’s culture was sufficient for this strain to reach a high level of lipid production. This fungal strain obtained higher lipid production when using fructose and mannose as carbon source, but it also can grow well on a variety of carbon sources. Most suitable nitrogen source for lipid production was the combination of (NH4)2SO4 and yeast extract. According to the response surface analyses results of the central composite design (CCD), the optimal growth condition for flask culture was 23.7 °C, 37.39 g/L glucose and 0.236 g/L nitrogen (N). The maximum lipid production was predicted as 3.91 g/L, and 3.89 g/L lipid production was verified from flask culture under the optimized conditions. The fourth step of research is to explore the application of F. equiseti UMN-1 strain in lignocellulosic lipid production and study different fermentation process to improve lipid accumulation. When directly using lignocellulosic biomass for lipid production, this strain achieved a lipid yield of 59.1±2.7 mg/g from soybean hulls and 61.1±2.6 mg/g from corn stover through solid state fermentation with 90% moisture content. Application of pretreatment and cellulase hydrolysis further increased the lipid yield to 69.2±5.0 mg/g from corn stover in integrated fermentation. F. equiseti UMN-1 strain was shown to have the capability of lipid accumulation from a variety of materials, and it could be a potential candidate as a lipid source in the production of lignocellulosic biodiesel.
University of Minnesota Ph.D. dissertation. November 2016. Major: Bioproducts/Biosystems Science Engineering and Management. Advisor: Bo Hu. 1 computer file (PDF); xii, 167 pages.
Study of Oleaginous Fungi Screened from Oil-Rich Plants for Improved Lipid Production.
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