The Isolation, Identification And Lipid-production Capacity Of Yeast Belong To Genus Lipomyces From Soil In Hainan Island

Yeast of Lipomyces is an important oleaginous yeast, and there is no report about isolation and identification of wild strains in our country. In this study, directional separation method of yeast belong to genus Lipomyces was established. Focusing on the isolation and identification and the lipid production capacity of Lipomyces from soil in Hainan province were intend to enrich our oleaginous yeast strain repository and screen strains that could use mixed sugar of glucose and xylose as carbon source producing lipid,in order to provide basis and foundation for converting the mixture of sugar and lignocellulose hydrolysate into microbial oils.The results were as follows:(1) 55 wild yeast strains were separated from 47 soil samples collected from different ecological regions in Hainan Island by using direct separation and enrichment separation methods in N-free+C separation plate medium. The 55 strains were primarily identified as 9 species of Lipomyces and 1 species asexual genus of Lipomyces by the morphology feature, 26S rDNA D1/D2, ITS and EF-1? domain sequence analysis. There are 2 strains of Lipomyces chichibuensis and Lipomyces tetrasporus accounting for 3.7% of the total strains, respectively. 12 strains of Lipomyces doorenjongii accounting for 21.8% of the total. 1 strain of Myxozyma geophila, Lipomyces spencermartinsiae and Lipomyces yamadae accounting for 1.9% of the total, 23 strains of Lipomyces kononenkoae accounting for 41.8% of the total, 3 strains of Lipomyces orientalis and 4 strains of Lipomyces yarrowii accounting for 5.5%, 7.4% of the total, respectively. 6 strains of Lipomyces starkeyi accounting for 10.9% of the total. Lipomyces kononenkoae was the dominant species in the soil of Hainan, which is widely distributed in the nine areas of Hainan Island.(2) A comparative analysis was made of between the weight method, sulfo-phospho-vanillin reaction and oxidase method: Weight method and sulfo- phospho-vanillin reaction determine total lipid of yeast. Weight method is high in accuracy and precision, but it is inefficient, labor-consuming. Sulfo-phospho-vanillin reaction has an average recovery rate of 93%, and is relatively good in accuracy and precision,time-saving, the result has to be matched with the corresponding standard curve of lipid.The oxidase method determines intracellular triglyceride, which has an average recovery rate of 98.03% and is accurate and precise, convenient and time-saving. The oxidase method need cells disruption and it should be with the standard curve of triglyceride. In practical application, Weight method and sulfo-phospho- vanillincould be chosen in the determination of total lipid of yeast and the oxidase method could be chose in the determination of intracellular triglyceride acconding to the experimental conditions.(3) Strain YP-18, YP-23, YP-32 could use mixed sugar of glucose and xylose as carbon source producing lipid were screened from 55 wild yeast strains that from the soil of Hainan Island and efficiently convert three kinds of mixed sugar (glucose: xylose 2:1,1:1, 1:2) into intracellular lipid. The biomass, lipid concentration and lipid content of the strain YP-32 reached 22.33 g/L, 13.39 g/L and 60.42% respectively by fermenting with 2:1 of glucose and xylose as carbon source. Simultaneous, Lipid-acid component of 3 strains was analyzed, there is almost no difference in lipid-acids component by using different mixed sugar ratios as carbon source and C16 and C18 series are main fatty acids.Oleic acid content is the highest in lipid-acid component, next are palmitic acid and stearic acid in all samples and the sum account for over 90%.(4) Strain YP-18, YP-23, YP-32 could efficiently assimilate cassava hydrolyate accumulating intracellular lipid.The oil production capacity of strain YP-32 is the highest.The fermentation process analysis shows that: When it reched 120h of the fermentation,the biomass, the lipid production, the lipid content and lipid coefficient are all reache the maximum and they are 16.68 g/L?7.15 g/L?42.88%?10.58g/100g, respectively.