Screening,Fermentation Condition Optimization,and Mutation Breeding Of Oil-producing Endophytic Fungi From Taxus Chinensis Var.mairei

With the increasing shortage of fossil fuels and environmental pollution,the importance of renewable energy becomes increasingly prominent.Among them,biofuel has attracted more and more attention due to its environmental friendliness,high safety,renewability,easy biodegradation,and low toxicity.The use of microorganisms to produce biofuel is one of the most popular technological routes.Due to the wide source of raw materials,short production cycle,simple production process,high biological safety,and less influence by climate and geographical location,microbial oils and fats have been extensively studied by domestic and foreign scholars.The microorganisms producing oil mainly include microalgae,filamentous fungi,bacteria,and yeast.Filamentous fungi have attracted much attention due to their high yield of polyunsaturated fatty acids,but the growth traits and lipid production capacity of wild-type filamentous fungi strains are still limited.Therefore,breeding of strains is a common and effective method to obtain strains with good production traits.Mutagenesis breeding is widely used due to its relatively simple operation and high probability of obtaining mutant strains with high yield.At the same time,the effects of different fermentation conditions on the growth and oil accumulation of the strain were investigated.The main contents and results of this study are as follows:(1)Screening and identification of oil-producing endophytic fungi.A total of 18endophytic fungi were isolated and purified from the stem,leaves and bark of Taxus chinensis plants.These endophytic fungi were fermented by shaking bottle,and the oil yield was determined by Soxhlet extraction.The oil content of 8 endophytic fungi was higher than 15%,and the oil content of 3 endophytic fungi was higher than 20%.The isolated endophytic fungi were identified by morphology and molecular biology based on ITS(Internal Transcribed Spacer)gene sequence.It is mainly divided into the following 10 genera:Fusarium sp.,Colletotrichum sp.,Penicillium sp.,Trichoderma sp.,Pestalotiopsis sp.,Talaromyces sp.,Lasiodiplodia sp.,Phomopsis sp.,Diaporthe sp.,Cladosporium sp.(2)Six strains with high oil accumulation including 2.S33y,2.L43,S321,S331,B322,and B121 were selected for fatty acid composition analysis.The results showed that the oils of these six strains were mainly composed of C16 and C18 fatty acids.(3)The oil composition from the 18 strains of endophytic fungi were analyzed by~1H NMR.The results showed that the oils from the 18 strains of endophytic fungi mainly contained triglycerides,diglyceride,and free fatty acids.The content of triglycerides was higher than that of diglyceride and free fatty acids,and a small amount of monoglyceride.The oils from the strain S431(Cladosporium sp.)had the highest diglyceride content,reaching 19.20 wt%,but the diglyceride yield of this strain was 102.91 mg/L.The content of diglyceride of the oil from the strain 2.S33Y(Fusarium sp.)was 18.51 wt%,and the highest yield of diglyceride was 335.22 mg/L.(4)The fermentation conditions of the strain 2.S33Y were optimized using the oil yield as the evaluation index.The response surface optimization of fermentation time,inoculation amount,and C/N ratio was carried out by BOX-Behnken experiment design.Under the optimal fermentation conditions,the oil yield of the strain 2.S33Y reached 2.84 g/L.It was 55%higher than that before optimization of fermentation condition(1.83 g/L).(5)UV mutagenesis was performed on the spore suspension of strain 2.L43(Colletotrichum gloeosporioides).The optimal mutagenesis time was 120 s,and the fatality rate was 82.5%at this dose.With the addition of the lipase inhibitor leptin as the screening agent,a large growing colony on the medium was selected and inoculated on a new PDA plate,and then the liquid fermentation oil production was screened.Finally,a mutant strain 2.L43-24 with high oil production capacity and stable genetics was obtained,and its oil content and yield were 21.48 wt%and 2.68g/L,respectively.Compared with the original strain,they increased by 31.13%and60.65%,respectively.Using the oil yield as evaluation index,a three-factor and three-level experimental design was designed for fermentation time,inoculation amount,and C/N ratio.The mathematical model between oil yield and three factors was established,and the optimal fermentation culture condition was obtained.Under these conditions,the oil yield of mutant could reach 3.26 g/L,which was 1.22 times of that before optimization(2.68 g/L).