Lipid Accumulation Mechanism In Chlorella Protothecoides Cells By Two-step Cultivation Method With PSH As Carbon Source

Biodiesel is fatty acid methyl ester prepared by lipids or fatty acids and methanolthrough ransesterification. Heterotrophic fermentation of Cholorella protothecoideshas been shown to accumulate high lipids and acids, which are praised as one of themost promising feedstock for sustainable biodiesel production. To increase thebiomass and reduce the cost of microalgal culture, This paper investigated thebiomass and lipid accumulation of heterotrophic C.protothecoides CS-41by potatostarch hydrolysate as carbon source under heterotrophic condition，and proteomicanalysis of lipid accumulation in Chlorella protothecoides cells by heterotrophic Ndeprivation coupling cultivation. The main contents and results were as follows:1. Lipid accumulation of heterotrophic microalgae Chlorella protothecoides byglucose as carbon sourseThe results showed with optimal dosage of glucose and urea (40g/L and3g/L)，thebiomass concentration (15.59g/L) was obtained, and fatty acid yield and total lipidscontent reached1.26g/L and6.407g/L respectively, which provided an excellent routefor biodiesel preparation by fermentation mode of heterotrophic microalgae C.protothecoides.2. Lipid accumulation of heterotrophic microalgae Chlorella protothecoides by potatostarch hydrolysate as carbon sourseThe optimal hydrolysis technology of potato starch by α-amylase andglucoamylase were discussed to provide a reference for reducing the costs of biodiesel.The results showed that the optimum conditions were as follows: temperature,substrate concentration, the best enzyme dosage, reaction time and pH were80℃,0.1g/mL, ratio of enzyme dosage to dry starch of0.6％,4h and pH4.0respectively, andunder this condition, the percent of glucose is highest, which reaches802.9g/L.Potato starch hydrolyzate concentration of glucose40g/L, the chlorella of thebiological indicators achieved optimal results, Chlorella biomass,total lipid yield andfatty acid production rate are20.23g/L,1.66g/L,8.6g/L,respectively.Compared toglucose as carbon source, biomass increased22.94%, the yield of higher total lipidcontent raised25.5%, and fatty acids was24.1%higher.3. Two-step method for heterotrophic microalgae Chlorella protothecoides by potatostarch hydrolysateFollowed by nitrogen deprivation phase, the algal biomass in the late oflogarithmic stage were collected and transferred into N deficiency medium (0,0.15, 0.30,0.45,0.60g/L urea) respectively for lipid accumulation. After72h (3days) Ndeprivation induction, the biomass yields of C. protothecoides varied with differentlevel of urea concentration in the cultivation, and the biomass concentration increasedwith increasing of urea addition. However, when N nutrient ingredient was absolutelyabsent (0g/L), the biomass concentration was slightly decreased. In addition, thehighest lipid content was72%when urea was absolutely absent in medium.4.Proteomic analysis of lipid accumulation in Chlorella protothecoides cells byheterotrophic N deprivation coupling cultivationThe present research identified the proteins differentially expressed during Nstarvation-induced lipid accumulation in the green algae C. protothecoides, includingsome novel proteins. The KEGG pathway analysis show that33altered proteins weresuccessfully identified in algal cells grown in BCM-N medium, including15down-regulated and18up-regulated proteins. Among down-regulated proteins, sevenproteins were involved in photosynthesis (L29, L31, L32, L34, L39, L46, and L67),four proteins (L41, L76, L78, and L79) participate in protein synthesis and folding,comprising PPIase, ribosomal protein L15, two ribosomal protein S12, were alsoobserved to be significantly down-regulated. one protein (L48) on gene regulationwas disappeared in Chlorella cells after N deficiency. More interestingly, acyl-CoAdehydrogenase (L70) involved into lipid metabolism-β-oxidation was down-regulated,indicating a promising strategy for cellular accumulation of lipid. In addition, twohypothetical protein proteins (L1and L11) were down-regulated in N deficiencycells.The up-regulated proteins in Chlorella cells after N deficiency fell into5majorbiological process groups. Seven proteins (H7, H12, H26, and H30, H31, H40, andH47) were implicated in carbohydrate metabolism process. Among them, H31, H40,and H47were involved in pyruvate synthesis which is the final output of glycolysis.Moreover, the up-regulation of transketolase (H2) might have contributed to the totalbiomass yield of C. protothecoides. There were two up-regulated proteins involved inamino acid metabolism (H33and H48). H10, H11, H19, and H38are a kind of stressresponse and defense proteins, their enhanced expression accelerate cell recoveryfrom nutrition limitation conditions. Another significantly up-regulated protein (H44)was CPX1which belongs to secondary metabolite biosynthesis.