| With the deteriorating global environment and the increasing demand for energy,finding a new energy substance that can replace fossil fuels is a key method to solve the problem in the world’s energy demand.Microalgae are considered as an ideal energy raw material for the third generation energy due to the fast growth rate and strong lipid production capacity.Micro algae biodiesel has several advantages such as clean and renewable compared with fossil energy.However,there are some obstacles to achieve commercial cultivation due to the insufficient oil production capacity of existing oil-producing algae species and the high cost of cultivation.Therefore,to promote the commercialization of microalgae biodiesel,obtaining a high-yield algae strain and effectively reducing its production cost are the important ways.In this study,the oil-producing algae species were obtained by the method of plate coating and line separation from water samples collected from parts of the rivers of Jiangxi and Fujian provinces.Furthermore,the oil-producing ability of the screening algal was preliminarily determined through the vanillin phosphate colorimetric and Nile red staining.With the effort,some algal species with high oil-producing potential were obtained.At the same time,the physiological,biochemical characteristics and growth characteristics were also studied,and the algae species were identified by means of molecular biology.In order to improve oil production capacity and explore internal mechanisms,several methods,such as nitrogen metabolism control,cultivation process optimization and two-step cultivation,were investigated.In addition,the feasibility of using industrial and agricultural wastes to cultivate microalgae was also studied.The main research results are as follows:(1)42 microalgae species were isolated by plate coating and line separation from the collected water samples,among which 10 algae species have a high oil-producing capacity.In order to obtain some high growth rate and high oil production capacity from those species,Nile red staining and vanillin phosphate colorimetry were used in this paper and the algae H and Z8 were obtained.At the same time,the algae H and Z8 were identified as Chlorolobion braunii and Desmodesmus intermedius by 18S rDNA molecular compariison.When the alga Z8 was use as the producing algae,the production of total lipid was 1.33 g/L,which was higher than that of the alga H.Furthermore,the alga Z8 has a wide range of carbon source utilization ability and strong pH adaptability,and then it was selected as the producing srtain for next experiment.(2)To research the possibility of microalgae culture from waste residue,three kinds of waste residue hydrolysates,such as sugarcane residue,pueraria residue,and tea seed hulls were investigated.Among the selected materials,pueraria residue hydrolysate was the most suitable for microalgae culture.Futhermore,the fermentation performance was basically the same as that of the normal heterotrophic medium.In order to further enhance the total lipid production,the effect of sodium tungstate on the oil production capacity of microalgae was studied.The results showed that the optimal addition concentration and time were 1 mmol/L and 120 h,respectively.C18:1 in fatty relative percentages was the mose highest after culturing to 168 h under this condition.Specifically,it reached at 45.88%.The relative fatty acid content increased by 30.05%compared with the control group,and the ratio of C18:1 in the total lipid content increased by 28.83%.(3)The changes of malondialdehyde,superoxide anion radical,total sugar,total protein content and energy charge under sodium tungstate treatment were investgated.The results showed that the contents of malondialdehyde and superoxide anion were reduced when sodium tungstate was added into ferementation medium.Furthermore,the lowest concentrations were 0.21 mmol/mL and 0.025 μmol/mL,respectively,which were reduced by 54.60%and 58.86%compared to the control group.The protein and total sugar content were 1.52 mmol/mL and 138.98 μg/mL,respectively,which were 116.57%and 28.57%higher than the control group.In addition,the energy charge of the test group was up to 0.18 μg/mL,which was slightly higher than the control group.In order to investigate the effect of nitrogen starvation conditions on the synthetic fat metabolism pathway,the key enzymes acitivity of nitrogen metabolism pathways,such as nitrate reductase(NR),glutamine synthetasev(GS),glutamate synthetase(GOGAT))and lipid synthesis pathways(Acetyl CoA carboxylase(AACase),fatty acid synthase(FAS)and phosphatidic acidphospholipid phosphate(PAP),were researched.The results showed that the acitivty of NR,GS,and GOGAT decreased by 51.85%,51.78%and 33.97%,respectively,compared to the control group.At the same time,the activity of AACase,FAS and PAP increased by 162.54%,154.55%and 80.41%,respectively,compared to the control group in the fatty acid syntheis-related pathway.These results indicated that the oil production would be improved when the enzyme activities which related to nitrogen metabolism pathways were inhibited.At the same time,the enzyme activities which related to lipid synthesis and stress resistance could be enhanced under the same conditions.(4)In order to further reduce the production cost and improve oil production,Plackett-Burman design and response surface optimization were performed.With the effort,the optimum conditions of alga Z8 from pueraria residue hydrolysate were obtained,and it was 0.75 g/L of NaNO3,0.02 g/L of K2HPO4,and 0.5 mg/L of EDTANa2(the remaining media components were consistent with BG-11).The total lipid production was 2.61 g/L,which was 40.32%higher compared with that orgainal condition.However,the total lipid production was only 2.08 g/L when sewage pueraria residue was used as fermentation medium,which was lower than that of the optimization condition.To enhance the total lipid production from the sewage puerariae residue culture medium,the two-step culture method was performed.The results indicated that the biomass and total lipid amount were higher than that of optimization medium.Specifically,the biomass and total lipid were 1.35 folds and 1.52 folds of which cultured by the optimized pueraria residue medium. |
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