Phylogenetic Diversity And Growth Characteristics Of Microorganisms From Alpine Frozen Soils In The Tianshan Mountains

Microbes in frozen soils are unique biological resources and precious research materials, which is conferred upon human being by the nature. My study was established in resources superiority of alpine permafrost in western China. In this work, we have firstly performed multiple studies on the isolation, phenotypic traits, growth characteristics and phylogenetic diversity of microorganisms from the frozen soils in the Tianshan Mountains. The major results were obtained as follows:1. Aerobic 2.5-6.0×10~5 CFU/gdw (CFU per 1 gram of dry weight) on PYGV medium were recovered from alpine frozen soils at 4℃; among these, 94 bacterial isolates with different morphotypes were characterized by phenotypic properties, such as Gram staining, morphology, colony pigmentation, sporulation and temperature range of growth. Most of these isolates were Gram-positive rod-shaped and 41.5% were observed to form pigmented colonies (peachblow, lemon yellow, pink and orange). Only TSBY79 was an endospore former. A majority of the isolates from alpine frozen deposits were psychrotrophic, while only two isolates were psychrophilic. A few isolates grew over a wide temperature range. The existence of these isolates might be a challenge for the definition of cold-adapted microorganisms and helpful to the study of microbial growth at low temperature.2. 51 representative isolates possessing distinct ARDRA patterns selected for subsequent 16S rDNA sequencing and phylogenetic analysis. The phylogenetic trees placed these isolates in four major groups: the high-G+C Gram-positives, the low-G+C Gram-positives, Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. The most abundant and diverse isolates were members of Gram-positive bacteria, particularly the genus Arthrobacter as a dominant group in culturable populations. The next most abundant and relatively diverse of the 15 organisms belonging to the Proteobacteria fell into three major lineages: alpha, beta and gamma subdivisions. Eight isolates belonging to the CFB phylum were the least diverse and particularly concentrated in the phylum Bacteroidetes, including the classes Flavobacteria and Sphingobacteria. Results of the Jukes-Cantor evolutionary distance matrix suggested that the vast majority of the isolates were different strains of known species, and three may represent new species within the genus Chryseobacterium of the CFB phylum. From this study, it is proposed that alpine frozen sediments in the Tianshan Mountains provide a specific ecological niche for prolonging survival of diverse microbial lineages.3. Molecular diversity of archaea in frozen soils was analyzed by PCR amplification and sequencing of archaeal 16S rDNA libraries using a universal archaeal primer set. Our results indicated that no archaeal sequences were found in our clone libraries by reason of low-specificity primer binding. All sequences obtained from the archaea clone libraries were closely related to uncultured Verrucomicrobia bacteria.4. A total of 33 bacterial strains were derived from the frozen soils in the Tianshan Mountains, which were able to produce cold-active enzymes including cold-active protease (10 strains), cold-active amylase (4 strains), cold-active lipase (14 strains) and cold-active cellulase (13 strains). Of them, 8 strains were found to secrete two studied cold-active enzymes. Protease and amylase from TSBY44 had been characterized as being cold-active. TSBY86 can produce extracellular protease and lipase, moreover, the activity of extracellular enzymes is very high at low temperature. The remaining 6 strains (TSBY2, TSBY13, TSBY30, TSBY61, TSBY80 and TSBY93) had the activity of cold-active lipase and cellulase. Based on physiological-biochemical characteristics analysis, the majority of strains cannot metabolize some sugar alcohols for growth. It is possible that frozen soils were characterized by low nutrition content, in which cold-active enzymes producing bacteria could obtain energy from more substances as nutrients to prolong their survival time.5. To reveal species richness and resource productivity of function-like bacterial population in frozen soils of the Tianshan Mountains, the phylogenetic affiliation of bacteria producing cold-active enzymes was determined by sequencing and analysis of 16S rDNA fragments. Bacteria producing cold-active protease fell in four phylogenetic groups: Bacteroidetes, Firmicutes, Actinobacteria andβ-proteobacteria. They were concretely related to the genus Chryseobacterium, Epilithonimonas, Flavobacterium, Exiguobacterium, Arthrobacter, Rhodoglobus, Massilia and Janthinobacterium, of which some species exhibited the activity of cold-active protease haven't been detected yet. A few isolates related toβ-proteobacteria and Bacteroidetes, were able to degrade gelatin at low temperature. Cold-active lipase producing bacteria were members of the most abundant and diverse species in organisms producing cold-active enzymes. They were affiliated with five distinct phylogenetic clades:α-,β-,γ-proteobacteria, Bacteroidetes and Actinobacteria. Bacteria producing cold-active cellulase were related toα-,γ-proteobacteria and Actinobacteria. Our results suggested that there are more bacteria producing cold-active enzymes in alpine frozen soils of the Tianshan Mountains. They having complementary advantages, play an important role in the biogeochemical cycle in this environment by synergic action.6. Freeze-thaw experiment was performed by modeling freeze-thaw cycles to analyze and interpret community-level physiological profiles in microbial ecology from permafrost table sample K5, and assess diversity and dynamics of the microbial population during freeze-thaw cycles. Dominant strains from sample treated or not with freeze-thaw cycles, were selected for subsequent 16S rDNA sequencing and phylogenetic analysis. The results found that freeze-thaw cycles as one of major extreme factors, had much effect on structure of microbial community. In sample K5 treated with a few freeze-thaw cycles, the diversity of Gram positives has the trends to decrease, but to increase in that of Gram negatives. Dominant taxonomic groups in sample K5 treated with a few freeze-thaw cycles were Actinobacteria, Bacteroidetes andα-,β-proteobacteria. Predominant bacteria in treated frozen deposits belonged to Bacteroidetes andα-,β-proteobacteria, which clearly point towards the opposite conclusion of that in original sample K5. During a series of freeze-thaw treats, bacterial diversity in treated sample K5 decreased with the number of freeze-thaw cycles increasing. Microflora in sample treated with more times freeze-thaw cycles were dominated by Gram-positive strains.The data obtained in this study on genetic and physiological diversity of culturable bacteria from the frozen soils in the Tianshan Mountains expand our knowledge on the extent of bacterial diversity in the cold-adapted realm and improve our understanding of the biogeographic distribution and function of prokaryotes in alpine environments.