| Object:More than 80%of the world’s energy is provided in the form of fossil fuels.With the continuous development of the economy,the demand for fossil fuels is increasing day by day,and hence lead to energy shortages and an increase of pollution.In order to match the growing energy demand and protect the environment,it is particularly important to develop other types of renewable and green energy sources.Microalgae have the ability to efficiently convert light energy and CO2into bio-oils,which are currently one of the most potential biodiesel raw materials.Moreover,microalgae have the advantages such as fast growth rate,wide adaptation to the environment,and easy collection of biomass,which leads to broad prospects for development and application.However,synthetic media are still used for the cultivation of most modern microalgae cultures,the biomass of cultured algae cells is limited,and the cost of production is high.Furthermore,a large amount of organic solvents are applied for the oil leaching extraction process,which not only causes environmental pollution,but also leads to a large waste of algae protein resources.The purpose of this thesis is to improve the microalgae nutrition medium by use of the addition of cattle urine from livestock breeding waste to optimize the microalgae culture conditions and obtain a higher cell density.On the other hand,through the use of microbial proteases pretreatment of microalgae protein,the amount of organic solvents applied in the lipid extraction process is effectively reduced,and algae protease hydrolysis products with high reactive oxygen species removal ability are obtained at the same time.Thus,implementation of the comprehensive utilization of microalgae,reduce environmental pollution,and lay a research foundation for the industrial cultivation,production,and utilization of microalgae.Methods:In this study,five different nutrient media were applied to cultivate microalgae Chlorella protothecoides:TAP,BBM,BG-11,Tamiya,BBM（Glu）.Through comparing the specific growth rate,biomass productivity and biomass concentration of microalgae,the BBM（Glu）nutrient medium was selected as the basic nutrient medium for further microalgae cultivation.Thereafter the effects of additional of different concentrations of nitrogen and phosphorus to nutrition medium were studied on the basis of the growth and cell composition of microalgae.Different concentrations of NH4NO3are used as the source of additional nitrogen,different concentrations of K2HPO4are used as the source of additional phosphorus,and different concentrations of cow urine are added to the basic BBM（Glu）medium for comparison of microalgae culture.At the same time,water with addition of different concentrations of cow urine,instead of the basic nutrition medium,for microalgae cultivation was also used.Systematic comparison and analysis of the specific growth rate,biomass productivity,biomass concentration,chlorophyll content,intracellular soluble sugar content,intracellular soluble protein content and intracellular neutral lipid content of microalgae in different media were carried out.The effects of cow urine addition on the microalgae growth,changes in intracellular composition and possible regulatory mechanisms were studied through metabolomics and transcriptome sequencing.Improved microalgae lipid extraction methods include the use of ultrasonic cleavage cell wall destruction and further application of extracellular protease produced by Pseudoalteromonas sp.ZB23-2,Pseudoalteromonas sp.B27-3and Pseudoalteromonas sp.JS4-1 for enzymatic hydrolysis.Obtained microalgae enzymatic hydrolysates with antioxidant activity determined by DPPH free radical scavenging,hydroxyl radical scavenging activity,and oxygen radical absorption capacity.The cytotoxicity of the microalgae enzymatic hydrolysates was determined by the MTT method,and the ability of the enzymatic hydrolysis product to reduce the level of oxygen radical absorption capacity in the cell was detected by the vascular endothelial cells（HUVECs）high sugar model.The lipids were extracted from sediment of microalgae after enzymatic hydrolysis by the acetone-ethanol method,the acetone method,the ethanol method,and the chloroform-methanol method of Kates and Paradise（modified by Bligh and Dyer）.The content and composition of the lipid samples extracted by improved methods were analyzed by gas chromatography-mass spectrometry and compared with the standard Kates and Paradise chloroform-methanol extraction methods to determine the better lipid extraction method.Results:（1）It was determined from five different nutrient media（TAP,BBM,BG-11,Tamiya,BBM+Glu）that the application of BBM（Glu）as the basic nutrient medium,showed the best cultivation effect of microalgae:the specific growth rate reaches 0.71±0.04/d,while the biomass concentration is 1.33±0.06 g/L,and the biomass productivity reaches 0.07±0.09 g/d/L.As next step,the effects of addition of different levels of nitrogen and phosphorus on the growth and cell composition of microalgae were studied.The BBM（Glu）nutrient medium group with addition of 50m L/L cow urine（CU）showed the best culture effect.The growth rate reached 0.71±0.02/d,the biomass concentration 4.67±0.33 g/L,and the biomass productivity 0.18±0.01 g/d/L.The content of chlorophyll a in microalgae cultured using BBM（Glu）+CU（50 m L/L）medium reached 3.35 mg/L,the content of chlorophyll b 3.45 mg/L,the protein 1.37 mg/m L,the carbohydrate 0.28μg/m L,and the accumulated amount of lipids reached 74.31 mg/m L,which was significantly higher than that of other experimental groups and control group.At the same time,studies were also conducted on the cultivation of microalgae using cow urine as the only nutrient source.It was determined that at 100 m L/L concentration of cow urine,the composition of microalgae cells reached 0.91 mg/L chlorophyll a content,chlorophyll b content reached 1.86 mg/L,protein 0.69 mg/m L,carbohydrates 0.30μg/m L,and the cumulative amount of lipids was 33.54 mg/m L,which was not too far from BBM（Glu）nutritional medium lipid results of 39.56 mg/m L,showing that there is big potential to cultivate microalgae with the use of cow urine as the only nutrient source.（2）The addition of cow urine has a significant effect on the growth and content of microalgae.In order to investigate the mechanism of cow urine influence,in this research the Illumina Nova Seq6000 sequencing platform was used for comparative analysis of transcriptome sequencing of C.protothecoides cultivated on BBM（Glu）nutrition medium with 50 m L/L cow urine and BBM（Glu）medium without cow urine used as a control.56854179 and 62781231 raw reads were obtained from the control and cow urine treated group respectively,and 89520,62953 and 28291unigenes were obtained from the control,cow urine treated group and All-unigenes after filtration and assembly.The obtained unigenes was compared with the NR,Swiss-Prot,Pfam,COG（KOG）,GO and KEGG databases,and a total of 88478unigenes were annotated.First of all,the differences of genes expressions in theС.protothecoides control and treatment samples were analyzed,and there were educed13765 unigenes with significant expression differences between the two.The differential expression genes were categorized through GO and KEGG databases,and it was found that there are significant differences between the samples in translation（ribosome）,protein processing in the endoplasmic reticulum,biosynthesis of cofactors,transcription（spliceosome）,oxidative phosphorylation,ubiquitin-mediated proteolysis,ribosome biogenesis and other metabolic pathways.The comparison of transcription factors with the Plant TFDB database shows that the largest got affected groups are the SBP,AP2,ERF,MYB and b ZIP families,and their domains have potential transcriptional regulatory effects on lipid metabolism in microalgae.Most genes involved in lipid metabolism and synthesis are upregulated.Analysis of transcriptomics results showed that after the intervention of cow urine,a large number of genes were differentially expressed in microalgae,mainly in the metabolism of lipids and carbohydrates.The mechanism of increasing the level of lipid accumulation may be related to the upregulation of the expression of genes acsl,acc,desa etc.associated with lipid metabolism.The mechanism of elevated carbohydrate levels may be related to the upregulation of gene expression such as gnl,g6pd,gpi,tkta,tktb,pgm etc.on the pentose phosphorylation pathway.（3）In order to compare the effects of the standard nutrient medium BBM（Glu）and the BBM（Glu）nutrient medium with addition of 50 m L/L cow urine on the cell metabolites of microalgae,a combination of LC-MS was used for metabolomic analysis.Metabolomics data showed that a total of 36,841 metabolites were detected in the two experimental groups.7316 metabolites were subsequently detected and identified through MS/MS data.The metabolite determination results were matched with HMDB,massbank,Lipid Maps,mzclound and KEGG databases,and normalized,retaining only 424 ion peaks with a relative standard deviation（RSDs）of less than30%,and then analyzed with PCA and OPLS-DA models.As result 180 metabolites with significant differences were identified.Enrichment analysis showed that the following metabolic processes were apparently affected by cow urine:the biosynthesis pathway of cofactors and vitamins（flavin biosynthesis）;lipid metabolism（linoleic acid metabolism,unsaturated fatty acid biosynthesis）;sugar metabolism（pentose phosphate pathway）etc.In the pentose phosphate pathway,the non-phosphorylated Entner-Doudoroff pathway was affected,and the amount of glycerate increased by 6.75 times.In the biosynthesis of riboflavin,the amount of simplified riboflavin-5-phosphate（FMNH2）is significantly reduced,causing3.89-fold increase in the amount of riboflavin-5-phosphate（FMN）,which leads to forms flavin adenine dinucleotide（FADH2）.The biosynthesis of unsaturated fatty acids has also increased significantly:Linoleic acid increasing by 12.99 times,Erucic acid by 31.54 times and Oleic acid by 3.02 times.The increase in the content of unsaturated fatty acids in the lipids accumulated by microalgae will help reduce the emissions of CO,hydrocarbons and smoke after combustion.（4）The traditional method of lipid extraction from microalgae requires a large amount of organic solvents to remove protein,which not only causes a waste of algae protein,but also brings environmental pollution impact from the extensive use of organic solvents.In this research,the extracellular proteases of the three strains of Pseudoalteromonas（Pseudoalteromonas sp.ZB23-2,Pseudoalteromonas sp.B27-3and Pseudoalteromonas sp.JS4-1）was applied as tool enzymes for enzymatic hydrolysis pretreatment of biomass in order to break the cell walls to remove the most of the protein and to obtain the hydrolysates.Inhibitor-substrate immersing zymography showed that bacterial extracellular proteases EB27-3 and EJS4-1 are mainly metalloproteases and serine proteases,and the extracellular proteases secreted by Pseudomonas ZB23-2 are mainly metalloproteases.After breaking cell walls the microalgae was hydrolyzed by protease,which significantly improve DPPH free radical scavenging ability and hydroxyl radical scavenging ability of the enzymatic hydrolysates,reaching 33.47±0.68%and 46.81±2.38%,respectively.The ORAC analysis of the enzymatic hydrolysates of the protease EZB23-2 and EJS4-1 showed stronger capability of removing hydrogen peroxide free radicals,which reached6.42±0.32 mmol·TE/mmol and 7.35±0.37 mmol·TE/mmol,respectively.The cellular antioxidant activity of the enzymatic hydrolysates was further tested,and the MTT results showed that when the concentration of the microalgae hydrolysates reached0.3 mg/m L,it was still not toxic for HUVECs,and it can even slightly stimulate cell growth.The high-sugar stimulation model of HUVECs cells was used to detect the ROS scavenging ability of the microalgae enzymatic hydrolysates.It was discovered that even at the concentration of 0.01 mg/m L,the all of three enzymatic hydrolysates of microalgae could basically return the intracellular fluorescence of the high-sugar stimulated HUVECs cells to the fluorescence level of underlying normal culture conditions,indicating that the protease enzymatic hydrolysates of microalgae have high antioxidant activity effect on cells.（5）The use of bacterial protease enzymatic hydrolysis to remove most of the protein from microalgae cells results in reduction of the microalgae sediment weight by nearly twice.Therefore,the amount of organic solvents used for microalgae lipid extraction has been reduced by 36.34～45.48%.The enzymatic hydrolysis residues of microalgae cells were extracted by the acetone-ethanol method,the acetone method,the ethanol method,and the chloroform-methanol method of Kates and Paradise（modified by Bligh and Dyer）.The results of different lipid extraction methods were compared,and it was discovered that the use of proteases EJS4-1 and EB27-3 for enzymatic hydrolysis pretreatment of microalgae allow obtain bigger variety and the amount of extracted lipids,and can reach 249.55±2.50mg/g and 222.40±2.36 mg/g,respectively.Further combination with the improved extraction methods,such as use of protease EJS4-1 as pretreatment with the combination of ethanol extraction method allowed to extract the highest lipid content of microalgae and reached 291.06±1.70mg/g,while the control Kates and Paradis extraction method only obtained 171.77±0.25 mg/g of lipids.Mass spectrometry analysis of the composition and quality of lipids obtained by different extraction methods was carried out,and the results show that compared with the traditional Kates and Paradise extraction methods,all three improved lipid extraction methods with protease hydrolysis pretreatment increases amount and variety of obtained lipids.Lipids extracted by acetone-ethanol method with microalgae pretreatment by protease EJS4-1 enzymatic hydrolysis increased the amount of extracted polyunsaturated omega-6 octadecenoic acid（（Z,Z）-9,12-octadecenoic acid）up to 41.83%,and 1,2-15,16-diepoxyhexaetane up to 10.79%.Amount of hexadecenoic acid in microalgae lipids,extracted by the Kates and Paradis methods combined with EJS4-1 proteolysis has reached 66.79%.Acetone lipid extraction with EB27-3 protease pretreatment of biomass allowed to extract lipids with high Phytol content 23.76%.By applying organic solvents standard lipid extraction,without enzymatic hydrolysis pretreatment by protease,phytol was not obtained,which shows that protease hydrolysis helps to further release a variety of lipids from microalgae.Conclusion:The research results of the thesis show that the culture of C.protothecoides with high biomass and high oil yield can be achieved under the application of cow urine.The use of a nutrition medium with addition of cow urine to cultivate microalgae is more suitable for the production of biodiesel,and can also significantly increase the content of chlorophyll a and b,soluble sugars,and soluble protein.Considering that the cow urine contains the large amount of nitrogen（6.8～23.3 g/L）and carbon（6.87 g/L）,the cow urine might mainly affect the metabolic pathways of microalgae by regulating the carbon-nitrogen ratio of the culture medium,including stress response,by activating and inhibiting the transcription of multiple target genes which affects the growth and development of microalgae,through changes in photosynthesis,endocytosis,glycosphingolipid biosynthesis and other metabolic pathways,thereby stimulating the upregulation of metabolic pathways related to the lipids and carbohydrates biosynthesis.The above results show that the use of cow urine as a nutrition medium has good potential,and at the same time can reduce the environmental pollution caused by its emission.On the other hand,protease enzymatic hydrolysis pretreatment reduces the weight of microalgae cell sediments,and at the same time,microalgae protein enzymatic hydrolysis products with high antioxidant capacity can be obtained,which can effectively reduce the cost of biofuel production and reduce the protein waste.Moreover,the introduction of enzymatic hydrolysis pretreatment steps to the lipid extraction methods also helps to extract more variety of lipids.The enzymatic pretreatment combined with less toxic extractants for lipid leaching contributes to the comprehensive utilization of microalgae and is more environmentally friendly.Figures 56,Tables 22,References 268... |
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