Study On Fermentation Process For Microbial Lipid Using Cassava As Feedstock

In the past years, biodiesel has received increasing attention because of the airpollution and energy crisis worldwide. At present, the main materials for biodieselproduction are crop oils. Although biodiesel has been already used in EU, US andmany other places, the high cost restricts its wide application. Therefore, the keyfactor for the industrialization and wide application of biodiesel is to find a cheap andsustainable feedstock. Microbial oils, produced by oleaginous microorganisms, arenow believed as a promising potential feedstock for biodiesel production due to theirsimilar composition of fatty acids to that of vegetable oils. Trichosporon cutaneum B3has been screened out and proved to be able to accumulate lipid with cassava starchhydrolysate in our lab previous study, and the methods to drastically improve thetechno-economics of microbial oils production processes was explored in thisdissertation, and the effects of related factors on the fermentation were systematicallyexamined. In order to realize low cost and high-density cell culture, we conductedfed-batch fermentation in2L and30L stirred-tank fermenter, respectively. Moreover,simultaneous saccharification and fermentation for microbial oils produce fromcassava starch and recycle of spent cells from microbial oil fermentation wereinvestigated to reduce the cost of microbial oils production.With initial total sugar concentration of60g/L and corn steep liquorconcentration of6ml/L, a biomass of15.1g/L, lipid yield of5.95g/L and lipidcontent of39.4%could be achieved after batch culture for144h in shake-flask. Underthe optimized conditions, batch cultures in a2L stirred-tank fermenter were run for44h and resulted in dry biomass, lipid yield and lipid content of28.7g/L,12.27g/Land42.8%, respectively.Different substrate feeding strategies were tried in2L stirred-tank fermenter toenhance lipid productivity. Improved biomass (105.3g/L) and lipid content (47.9%)have been achieved by control the media total sugar concentration at5g/L~30g/L. Apilot-scale fed-batch culture using a30L stirred-tank fermenter was conducted, thebiomass, lipid yield and lipid content reached a maximum of108.5g/L,51.5g/L and47.5%, respectively, after104h fermentation. In particular, the lipid production ratereached0.50g/(L·h).To reduce the cost of microbial oils production, this work deals with theoptimization of process parameters of simultaneous saccharification and fermentation(SSF) for microbial oils produce from cassava starch using response surface methodology (RSM). RSM based on the Box–Behnken design was applied todetermine factors’ optimal levels and their mutual interactions for microbial oilsproduction using SSF process. The predicted results showed that oil yield of10.33g/Lwas obtained with optimal condition of fermentation temperature (27.4°C), α-amylase(13.5U/g cassava starch), and glucoamylase (1462.8U/g cassava starch) in the flaskfermentations. The experiment was scaled up in a2L fermenter and oil yield of39.37g/L was achieved in96h using fed-batch fermentation.Spent cells of Trichosporon cutaneum B3recovered from microbial lipidfermentation were used as nitrogen source for microbial lipid production. When theenzymatic hydrolysate of spent cells was added to reach a total nitrogen concentrationof0.3g/L, the oil yield was4.41g/L. However, when3.0ml/L corn steep liquor wassupplemented with the enzymatic hydrolysate of spent cells, the biomass and oil yieldreached18.5g/L and7.09g/L, respectively. The biomass, lipid yield and lipid contentreached a maximum of111.6g/L,50.9g/L and45.6%, respectively, in2L fermenterusing this compound nitrogen source.The chemical compositions of biodiesel prepared from lipids of T. cutaneum B3mainly included palmitic acid methyl ester, stearic acid methyl ester, oleic acid methylester and linoleic acid methyl ester etc., and its main physicochemical properties werein compliance with relevant national diesel standards and most of them comply withthe limits established by ASTMD related to biodiesel quality. Therefore, the biodieselprepared from lipids of T. cutaneum B3can serve as a potential fossil fuelalternatives.