Isolation?Identification And Fermentation Characteristics Of D-xylose Fermenting Yeasts From Rotten Wood

Xylose is a five-carbon backbone molecule of the hemicellulose component of plant cell walls and the second largest component of lignocellulose that is smaller than glucose.In the world,xylose is also the richest and cheapest source of renewable carbon.The key to economic and sustainable production of lignocellulosic biofuel is to screen the microbes that are able to yield fuel ethanol from xylose efficiently and rapidly off from the population.Under hot and humid circumstance,microbes can decompose rotten wood into saccharides which can be used by yeast,but they also produce some noxious products,such as acetic acid and furfural,which restrict the growth of yeast.Due to the factors mentioned above,inhibitor-tolerant yeasts that can ferment xylose may exist in the habitat of rotten wood.These yeasts are significant to the production of cellulosic fuel ethanol.The main research results are as follows:1.43 rotten wood samples were collected from Xishuangbanna,Yunnan Province from July to October in 2016;126 rotten wood samples were collected from Baotianman and Laojieling Natural Reserve,Henan Province.546 strains of yeast were isolated and purified from the 169 samples by using the screening culture medium containing xylose as the only source of carbon.After conducting Durham tubes fermentation experiments and aerobic batch growth,the results showed that 33 strains of yeast ferment xylose and produce ethanol.2.Extracting DNA from these 33 yeast strains and amplifying the fragments in different regions?ITS?26S rDNA D1/D2?with PCR revealed that these 33 yeast strains can be classified into 7 genera and 10 species.6 species of them are already known.They are Candida pseudorhagii?5 strains?,Candida tropicalis?8 strains?,Kluyveromyces marxianus?6 strains?,Meyerozyma guilliermondii?2 strains?,Scheffersomyces stipites?1 strain?,and Sugiyamaella lignohabitans?1 strain?.4 species are suspected as new species.Eventually,the 4 new yeast were authenticated as:Hemisphaericaspora sp.NYNU 16746^T,Spathaspora sp.NYNU 16747^T,Sugiyamaella sp.NYNU 161041^T,Sugiyamaella sp.NYNU 161059^T.3.Research of the characteristics of yielding ethanol by fermenting xylose with these4 species indicates that under the same condition,NYNU 16746^T and NYNU 16747^T have better fermentation performance.The ethanol produced by strain NYNU 16746^T yielding from glucose and xylose are Y_E/glu=0.32±0.02 and Y_E/xyl=0.14±0.03 correspondingly.The ethanol produced by strain NYNU 16747^T yielding from glucose and xylose are Y_E/glu=0.34±0.02 and Y_E/xyl=0.18±0.03.4.After analyzing the inhibitor tolerance of 4 new species,we found that under the same condition,NYNU 16747^T is inconspicuous in acetic acid tolerance,but it has high inhibitor furfural tolerance.This strain can tolerate a concentration of 0.4 g/L of furfural.The strain of NYNU 16746^T,NYNU 161041^T and NYNU 161059^T are inconspicuous in terms of furfural tolerance,however they can survive in acetic acid with a 2 g/L concentration.Our research builds a foundation for the research of diversity of yeast in the rotten wood habitat in China.The discovery of new species of yeast suggests that there might be a large number of them still remain undiscovered from the rotten wood habitats.The 4 new strains isolated in this research provide high-quality resources of improving the capability of yeast to ferment xylose and their tolerance of inhibitor for possible genetic engineering process in the next phase.In addition,they contribute to the directional cultivation of the yeast with high tolerance as excellent supporting strain resources.The research has also laid the foundation to improve the advantages of yeast strains.