Date of Award
Master of Science
The present study investigated the feasibility of using Nile red fluorescent method for measuring cellular neutral lipid content as well as the capability of Cryptococcus curvatus to utilize sugars in hydrolysates of sweet sorghum bagasse- an agriculture byproduct- for the production of lipids. Nile red is a fluorescent dye which is specific for intracellular neutral lipids. To generate biodiesel from microbial lipids through alkaline transesterification, only neutral lipids are readily convertible. Knowing the accurate content of neutral lipids in microbial cells is important for evaluating amount of biodiesel that can be produced. To develop the Nile red protocol, different wavelengths for excitation and emission were investigated together with different cell numbers and cell-dye contact time. In this study, Nile red was proven to be capable of serving as an excellent dye for quantifying cellular neutral lipid content in C. curvatus. The obtained protocol can certainly be applied for other purposes, for example, high throughput screening of oleaginous species with high lipid content and measuring neutral lipid concentration in any other microbial cells. Cryptococcus curvatus, one of the most efficient yeast candidates for lipid production, can accumulate more than 60% of dry biomass as lipids on a broad range of mono- and disaccharides and sugar alcohols. In separate batch studies,Cryptococcus curvatus was grown and monitored on both pure sugar substrate and sorghum hydrolysates. Like other oleaginous yeast species, C. curvatus can uptake glucose and xylose simultaneously. Interestingly, this yeast can also utilize cellobiose. Sweet sorghum, a C4 plant possessing high photosynthetic efficiency, high sugar yield but low requirements for fertilizer and water has been identified as an excellent biofuel feedstock. After sweet sorghum juice is extracted, the left bagasse poses a disposal problem and its usage as fodder for animals is not a sufficient approach. Sweet sorghum juice has been reported for ethanol and biodiesel production due to the high sugar content. However, no special attention has been given so far to the utilization of the sweet sorghum bagasse for biodiesel production through microbial fermentation. The main components of sweet sorghum bagasse are cellulose, hemicellulose, and lignin. To release fermentable sugars from sorghum bagasse, we have tested lime-assisted microwave pretreatment. The pretreated material was then subjected to enzymatic hydrolysis using commercial enzymes. The hydrolysates obtained were used for lipid accumulation by yeast Cryptococcus curvatus. With sweet sorghum hydrolysates derived from microwave pretreatment with lime, the maximal yeast cell dry weight and lipid content were 10.83 g/l and 73.26%, respectively. For hydrolysates developed from microwave pretreatment without lime, these two parameters were 15.50 g/l and 63.98%, respectively. As a result, higher lipid yield of 0.11 g/g bagasse or 0.65 ton/hectare of land was achieved from bagasse pretreated by microwave followed by enzymatic hydrolysis while 0.09 g/g bagasse or 0.51 ton/hectare of land was attained from the same process but with lime during microwave pretreatment. With sweet sorghum hydrolysates derived from oven pretreatment with lime, maximal cell biomass dry weight as 6 g/l was achieved in 5 days. Maximal neutral lipid content as 2.6 g/l was observed by day 3. Neutral lipid yield (g neutral lipid/g sugar) was calculated as 0.19 which is close to the theoretical value. This research shows that the hydrolysates of sweet sorghum bagasse can be utilized by Cryptococcus curvatus to yield substantial quantities of lipids. Based upon the results revealed from the batch stage studies, large scale lipid production from this agricultural by-product could be a reality in the near future. This production process will: 1) produce high-value lipids in an environmentally friendly, economical, and sustainable way, and 2) provide lipid feedstocks for various industrial applications.
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