Separation Identification And Characterization Of Eukaryotic Microorganisms Of Xylose Metabolism

Fuel ethanol has been officially sanctioned as the most promising renewable and clean energy sources. With the national emphasis on environmental issues, and the cycle energy demand increases, the demand for bio-fuel ethanol market is growing. But now,lignocellulosic hydrolyzate hexose, pentose synchronization anaerobic ethanol fermentation technology is still possible to obtain a significant breakthrough from the actual industrial production, there is a considerable gap. To the effective recombinant strain that had got, many shortcomings still need to study.For xylose fermentation production problems. In this study, we hope to be able to filter out the xylose metabolic strain that had high tolerance in temperature, pH, salinity and ethanol from the sample. And further study the diversity of xylose metabolism eukaryotic microbial in Sichuan fermented grains. In order to provide excellent strain resources for further transformed into industrial production strainsThe lees as samples of this experiment were collected from14liquor factory eight regions of Sichuan Province. About104metabolic xylose eukaryotic microbial strains isolated and purified from those samples.Select strains that colony and cell morphology were different from14liquor factory. A total of23strains were selected. Observation and biochemical identification of the tested strains. It could preliminary identification as Candida rugosa、Candida pararugosa、Debaryomyces hansenii、Meyerozyma guilliermondii、Pichia manshurica、Pichia norvegensis and Pichia kudriavzevii.Molecular identification by26S rDNA D1/D2regions of95strains, to identify strain on the molecular biology level. Contrast to GeneBank, the amplified fragment of26S rDNA that nearly5’of the D1/D2region of homology was more than99%. There were7genera and11species,they were Candida rugosa、Candida pararugosa、 Candida tropicalis、Hansenula anomala、Hannaella sinensis、Torulaspora delbrueckii、Debaryomyces hansenii、Meyerozyma guilliermondii、Pichia kudriavzevii、Pichia manshurica and Pichia norvegensis.The number of strains of Pichia manshurica were22,largest than other strains,the second was Candida rugosa, had19; then the Meyerozyma guilliermondi had14; both Torulaspora delbrueckii and Debaryomyces hanseni were least, had only one. Metabolism of xylose microbial species separated from Shehong up to seven kinds, species diversity was richer than other regions. Both Candida rugosa and Pichia kudriavzevii were separated from four regions, were the most widely distributed, while other species are basically isolated from one region.Select different strains from same place or same kind strains from different parts.A total of40strains selected from the sequencing results of26S rDNA, then ITS cloned and sequenced and further confirmed the strain species. Using MEGA5.0system to establish the phylogenetic tree preliminary analysis of the the system evolutionary relationships between the tested strains. The experimental results showed that40strains were divided into two branches, explained that they may be there was a big difference in the level of ITS region.The experimental microsatellite DNA markers, primers were M13and (GTG)5. Further study the genetic diversity of the isolated75strains by M13,(GTG)5fingerprint analysis. M13and (GTG)5fingerprints showed a more consistent results, more similar both the resources shown in similarity and genetic distance. In the similarity case of each62%and60%, they both display4alkylene branched; In the similarity case of each72%,78%, showing11branch. There were two categories had advantages in the populations in Lees’strains. From the results, M13has a better Efficiency in distinguishing the differences between the strains isolated than (GTG)5.Study of temperature, salinity, PH, ethanol tolerated from strains. The results showed that the four strains of Candida ruqosa, Pichia Kudriarzevii, Candida tropicalis and Pichia norveqensis had a strong temperature tolerance. Candida ruqosa, Candida tropicalis,Hannaella sinensis, Candida pararuqosa and Harnnaella anomala salt tolerance was higher. Pichia Kudriarzevii had a stronger acid resistance. Harnnaella anomala and Pichia Kudriarzevii had a stronger capacity to resist to ethanol. The Candida tropicalis and Pichia Kudriarzevii glowed fastest in medium that xylose as sole carbon source.Experimental screening, isolated from the fermented grains of Sichuan,Pichia kudriavzevii lived more adaptable and had a more strong tolerance in the environment of high temperature, high salt, acid conditions than other strains.And it glowed fastest in medium that xylose as sole carbon source.lt can provid resources for future research.