Extracellular Enzymatic Activities And Diversity Of Yeasts Isolated From Three Plateau Lakes In Central Yunnan Province

Dianchi Lake,Fuxian Lake,and Xingyun Lake belong to the three plateau lakes in central Yunnan Province,and they all have abundant yeast resources.Plateau lakes are a type of special environments due to the conditions of high altitude,high ultraviolet radiation,low air pressure and lower oxygen content,and yeasts isolated from plateau lakes can secrete extracellular enzymes to adapt to the special environment.Extracellular enzymes have special physiological properties and are widely used in many fields,such as environmental protection,high-concentration organic wastewater treatment,food processing industry,pharmaceutical industry,detergent industry and genetic engineering.In recent years,researcher has focused on lake pollution and governance on Dianchi Lake,Fuxian Lake and Xingyun Lake.Until then,there was not report on diversity and extracellular enzymatic activities of the yeasts isolated from the three lakes,there was not any report on the correlation between the extracellular enzyme-producing yeast diversity and abiotic factors of its habitats.The qualitative screening and quantitative studying on extracellular enzyme-producing yeasts in the plateau lakes were performed to evaluate the correlation between the enzyme-producing yeast diversity and abiotic factors,and to explore the ecological roles of yeasts in decomposition and circulation of organic matter.These provided the theoretical basis for the in-depth and systematic research,protection,development and utilization of yeast resources from Yunnan plateau lakes.At the same time,these provided a certain theoretical basis for the protection and the treatment of the surrounding environmental pollution of the plateau lakes.Culturable yeast diversity was studied by using classical methods of isolation,purification,classification and identification.One thousand two hundred and fifty-five yeast strains were obtained from the three plateau lakes,and eleven extracellular enzymes were detected by using screening plate method at 5 °C and 25 °C,respectively.The results showed that the rich diversity of yeasts with extracellular enzymatic activities existed in the three lakes.There were 430 strains(14 genera and 21 species)from Dianchi Lake,506 strains from Fuxian Lake(52 genera and 52 species),and 319 strains from Xingyun Lake(17 genera and 38 species).The enzyme-producing yeasts isolated from Dianchi Lake was mainly composed of Ascomycetes,while the enzyme-producing yeasts isolated from Fuxian Lake and Xingyun Lake were mainly Basidiomycetes.Rhodotorula was the dominant genus shared by three lakes,and Rhodotorula mucilaginosa was the dominant species shared by three lakes.All yeast strains from the three lakes exhibited at least one extracellular enzymatic activity,and mainly exhibited activities of phytase,inulase and amylase;the yeast strains isolated from Dianchi and Xingyun Lakes did not exhibited laccase activity,and thirty-two yeast strains isolated from Fuxian lake exhibited laccase activity.In this study,the abiotic factors(sediment: moisture,organic carbon,total nitrogen,total phosphorus,ammonium nitrogen,nitrate nitrogen,nitrite nitrogen;lake water: pH,total phosphorus,total nitrogen,organic carbon,total hardness,turbidity,conductivity)of samples from the three lake were determined,and the spatial distribution characteristics of abiotic factors in three lakes with different spots and regions were investigated by using classical analysis methods.The results showed that there were certain differences in the spatial distribution of abiotic factors from the three lakes.Overall,the concentrations of abiotic factors(moisture,organic carbon,total nitrogen,total phosphorus,ammonium nitrogen,nitrate nitrogen)from Caohai was higher than those from the Waihai,and the abiotic factors from Fuxian Lake and Xingyun Lake showed a trend of decreasing from south to north.The correlations among yeast diversity,the ratio of strains with extracellular enzymatic activities and abiotic factors of the three lakes were performed by using SPSS software.The results showed that the major enzyme-producing yeast diversity from the three lakes showed a certain correlation with abiotic factors,but most of them were not significantly correlated.There was also a certain correlation between the ratio of active strains producing different extracellular enzymes and the abiotic factors.The ratio of active strains of some extracellular enzymes was significantly correlated with abiotic factors such as total nitrogen,total phosphorus,and organic carbon.The reason for that may be that some yeasts can use nitrates and phosphates as their nitrogen sources for growth,while others cannot.The two hundred and seventy-eight yeast strains with higher enzyme activities were selected from the three lakes for assessments of extracellular lipase(70 strains),amylase(55 strains),manganese-dependent peroxidase(72 strains),lignin peroxidase(72 strains)and laccase(9 strains)activities.The results showed that the yields of lipase,amylase,lignin peroxidase,and manganese-dependent peroxidase produced by yeast strains from Fuxian Lake were higher than those from Dianchi and Xingyun Lakes,the yields of enzymes were the lowest from Dianchi.There were two high-yield yeast strains Papiliotrema flavescens Ym26764(25 °C;Xingyun Lake strain)and Rhodotorula glutinis Ym27053(15 °C;Fuxian Lake strain).The fermentation conditions(carbon source,nitrogen source,initial pH,temperature,inoculation quantity)of Pa.flavescens Ym26764 and Rh.glutinis Ym27053 were optimized by single-factor experiments and orthogonal experiments based on lipase-producing fermentation medium.The results showed that the optimal enzyme production conditions for Pa.flavescens Ym26764 were: sucrose 7.5 g/L,urea 25.0 g/L,initial pH 8.0,inoculum size 4 %,incubation temperature 25 °C,incubation time 48 h;lipase activity up to 451.09 U/mL,and 6.0 times higher than that at previous optimization conditions.The optimal enzyme production conditions of Rh.glutinis Ym27053 were: sucrose 2.5 g/L,nitrogen source urea 25.0 g/L,initial pH 9.0,inoculum 4 %,incubation temperature 15 °C,incubation time 96 h;the enzyme activity reached 244.11 U/mL,which was 3.3 times higher than that at previous optimization conditions.