Improvement Of Lipid Production In Lipidrich Microalgae Through Different Stress Strategies And Analysis Of The Physiological Mechanisms

Due to the limited fossil fuel reserves and the increasing concerns over global environmental protection,more attention has been recently devoted to develop the renewable and sustainable energy source.Microalgae are considered as an ideal feedstock for biomass energy because of its short growth cycle,ease of cultivation,high photosynthetic efficiency,and high lipid concent.However,the material cost for microalgae cultivation is slightly higher and the yield of microalgae lipids is often lower.Therefore,this presents a key scientific challenge in the microalgae commercialization process.In this study,Desmodesmus intermedius Z₈ was used as the test algae species.The effects of different stress conditions such as ultrasonic coupled nitrogen and phosphorus stress,precise two-step regulation,and high temperature stimulation on the growth and lipid production were researched.At the same time,the physiological response of the microalgae and the mechanisms of high lipid production under the different stress conditions were also studied.Finally,the physiological mechanisms of relieving the lignocellulosic hydrolysis inhibitors under the?-aminobutyric acid(GABA)addition condition was also investigated through the analysis of intracellular reducing power and energy charge,and the results of this thesis can provide some reliable theoretical and practical references for reducing the production cost of microalgae cultivation.The main findings are as follows:(1)The effect of ultrasonic coupled nitrogen and phosphorus(UCNP)stress strategy on the growth and lipid production of Desmodesmus intermedius Z₈ was investigated,and the results show that the maximum biomass(5.39 g/L)and maximum lipid concentration(2.33 g/L)were obtained with 20 min of UCNP.Furthermore,under this condition,the fatty acid(FAME)composition analysis showed that the proportion of saturated fatty acids(SFA)and C16-C18 fractions increased by 42.75%and 20.28%,respectively,compared to the control group.This indicates that the UCNP strategy could improve the lipid quality and make it more suitable material for biodiesel production.Meanwhile,a preliminary interpretation of the physiological response mechanisms of the microalgae under this condition was performed through studing the changes of intracellular antioxidant enzyme activity and non-enzymatic antioxidant content.The results indicat that the superoxide dismutase(SOD)and glutathione(GSH)were the most important responders,and their activity was 55.45%and 14.51%higher than that of the control group at day 6.Futhermore,the SOD and GSH would work together with other antioxidant components to ensure efficient lipid production under these stress conditions.(2)The effects of different nitrogen and phosphorus souceses on the growth and lipid production of the algal strain Z₈ were investigated.It was found that the biomass yield of 446.73 mg/L/d was obtained under Nlim&P conditions.At the same time,the lipid yield was also maximum(147.73 mg/L/d)under this conditon.On the other hand,the feasibility of precise regulation of the two-stage culture strategy was further explored,and a brief assessment of the lipid properties and their potential application in biodiesel production was performed.The results show that when the strategy of using Nlim&P as the first stage culture condition and continuing to add 750.0 mg/L nitrogen source and 40.00 mg/L phosphorus source(Group 1)precisely on the second day of culture was used,the algal biomass yield would be enhanced significantly,and its production of 588.73 mg/L/d was obtained at the end of the cultivation.Furthermore,in Group 1,the highest lipid content and yield of 41.56%and 244.69 mg/L/d was achieved,respectively,which were 1.25 and 1.57 times higher than that of the control group.FAME analysis result indicates that the SFA content increased by 31.99%for the Group 1,suggesting that this control strategy could improve the oxidative stability of the lipid to some extent.(3)The effect of extreme high temperature stimulation at 40°C at the end of the logarithm on the growth and lipid production of the algal strain Z₈ was investgated.The results show that the biomass and lipid production of the algal strain Z₈could be effectively enhanced if the method of the continuing culture for 1 day after 40°C stimulation was adopted.Under this condition,the maximum biomass and lipid production of 3.72 g/L and 1.54 g/L was obtained,respectively,which were 11.29%and19.48%higher than that of the control group.In addition,the dynamic changes of the malondialdehyde(MDA)and reactive oxygen species(ROS)contents and the corresponding antioxidant enzyme activities at above cultivation process were also researched,it was found that the MDA and ROS contents were 2.91 and 1.61 folds of which was after 1 day of high temperature culture.At the same time,the intracellular antioxidant enzymes such as SOD,catalase enzyme(CAT),and peroxidase(POD)also actively played a role.The enzyme activities of 176.89 U/g,23.67 U/g/min,and 14.45U/g/min were obtained,respectively,after 1 day of high temperature incubation.These changes ensured the normal growth of cells under high temperature culture conditions.In contrast,intracellular energy charge changes show that the high temperature incubation could decrease the ATP content which was 38.97%lower compared to the control group under the same conditions,indicating that high temperature could accelerate the energy consumption,Therefore,the phenomenon of a greater rate of ATP breakdown than synthesis was encounted.Finally,the lipid synthase activities were also determined in the present experiment.These enzyme activities were increased by26.37%,15.28%and 50.63%,respectively,compared with the control group,which also effectively ensured the effective accumulation of lipids in the algae.(4)We researched the effect of furfural inhibitors in lignocellulose hydrolysis products on the growth of algal strains,and the physiological mechanisms of relieving furfural toxicity under the GABA addition condition was investigated based on the results of the changes of the intracellular redox levels and key enzymes in fatty acid synthesis.The results show that the high concentration of furfural had obvious toxic to the algal plants.The growth of microalgae Z₈ was inhibited when the concentration of furfural was above 0.1 g/L.In particular,if the furfural concentration was 2.0 g/L,the biomass and lipid accumulation of microalgae Z₈was reduced by 93.44%and 96.93%,respectively,compared with the control group.When a certain amount of GABA was added,the inhibition phenomenon could be effectively alleviated.When the GABA addition concentration was 1.5 mmol/L,the biomass and lipid accumulation of algae were 35.23%and 38.10%higher than that of the non-added group.Based on the results of the changes in intracellular reducing power,the NAD⁺levels would be reduced due to the addition of GABA.Furthermore,when the culture was at day 7,the NAD⁺level in the treated group with 1.5 mmol/L GABA was 55.36%lower than that of the non-added group.The behavior indicat that the added GABA could regulate the intracellular redox level and accelerate the conversion of furfural into non-toxic or less toxic substances,and thus relieving the toxic effects of furfural.On the other hand,the GABA addition was also found to improve the activities of NR,phosphatidic acid phosphatase(PAP),FAS,and acetyl coenzyme A carboxylase(ACC).When the addition concentration of GABE was 1.5 mmol/L,the activities of lipid synthesis-related enzymes such as PAP,FAS and ACC were 15.52%,60.01%,33.33%,and 54.27%higher than that of the control group.This indicates that the addition of GABA also could promote the increase of lipid-related synthetase activity,which would effectively accelerate the secretion of microalgal lipids and ultimately enhance the production of microalgal lipids.