【作者】 白玉；

【导师】 安黎哲；

【作者基本信息】 兰州大学 ， 植物学， 2007， 博士

【摘要】 冻土微生物是自然界赋予人类的独特的生物资源和极其珍贵的科研素材。本项研究立足于中国西部高山多年冻土的资源优势，选取天山冻土为介质，在国内首次开展高山冻土微生物多样性及生长特性的研究，获得了以下主要结论：1．用PYGV培养基在4℃条件下，恢复得到2.5～6.0×10~5 CFU／gdw。选取94株菌落形态差异较大的天山冻土细菌进行表型特征，如菌落形态，色素产生，革兰氏染色，孢子形成和生长温度范围的研究。结果表明，天山冻土可培养细菌以不产芽孢的革兰氏阳性杆菌为主。产色素菌的比例较高，占所研究细菌总数的41.5％，菌落颜色呈桃红、柠檬黄、粉红、橘黄等。分离得到的天山冻土细菌绝大多数为耐冷菌，还有两株嗜冷菌和一些能够在较广温度范围内生长的菌株，这些菌株不仅给低温微生物的分类提出了新的问题，同时还是我们深入研究其生长特性的良好材料。2．从中选出51株具有不同ARDRA类型的细菌进行16S rDNA序列测定和系统进化分析。这些细菌在系统进化树上聚类为四个主要的系统分类群：高G+C mol％革兰氏阳性细菌(High-G+C gram-positives)，低G+C mol％革兰氏阳性细菌(Low-G+C gram-positives)，变形菌门(Proteobacteria)和CFB菌群(噬纤维菌-黄杆菌-拟杆菌群，Cytophaga-Flavobacteria-Bacteroides)。丰度和多样性最高的是革兰氏阳性菌群，其中节杆菌属(Arthrobacter)为天山冻土中的优势细菌类群。变形菌门是天山冻土可培养细菌中仅次于革兰氏阳性细菌多样性和数量较多的第二大发育谱系，由15个菌株组成，聚类于α-，β-，γ-proteobacteria三大纲。在最小的发育谱系—CFB菌群中，天山冻土代表菌株集中于拟杆菌门(Bacteroidetes)的黄杆菌纲(Flavobacteria)和鞘脂杆菌纲(Sphingobacteria)。利用Jukes-Cantor法计算得到的遗传距离结果指出大部分天山冻土可培养细菌为已知种属的菌株，但有三株细菌可能是CFB菌群中金黄杆菌属的新种。此次研究表明天山地区的高山冻土环境为多种微生物的保存提供了一个特殊的生境。3．采用16S rDNA克隆文库技术对天山冻土样品中的古细菌进行系统多样性分析。由于扩增古细菌引物的低保守性，在作者的文库中并未发现有古细菌16S rDNA序列的存在。测序的5条16S rDNA序列都与疣微菌门(Verrucomicrobia)不可培养微生物有较高的序列相似性。4．本研究经过对大量天山冻土微生物的筛选，获得了33株产低温酶的适冷细菌，其中有10株产低温蛋白酶，4株产低温淀粉酶，14株产低温脂肪酶，13株产低温纤维素酶。8株具有产两种低温酶特性的细菌，其中TSBY44是唯一既能产低温蛋白酶又能产低温淀粉酶的菌株；TSBY86不仅能够产低温蛋白酶和脂肪酶，并且所产低温酶的活性还很高。其余的6个菌株(TSBY2、TSBY13、TSBY30、TSBY61、TSBY80和TSBY93)都能够产低温脂肪酶和纤维素酶。在生理生化试验中，许多产低温酶菌株不能以某些糖醇类物质作为生长所需的营养。这些特性的出现可能与冻土中有限的营养成分有关，分泌多种低温酶可以使低温菌利用环境中更广泛的营养物质，更有利于在冻土中生存。5．用16S rDNA序列测定、分析的方法对产低温酶菌株的种属多样性进行研究，从而在某一功能性细菌的水平上揭示天山冻土微生物的多样性和微生物资源的丰富性。天山冻土产低温蛋白酶细菌分别归属于：拟杆菌门(Bacteroidetes)、壁厚菌门(Firmicutes)、放线菌门(Actinobacteria)和β-变形菌纲(β-proteobacteria)中的金黄杆菌属(Chryseobacterium)，Epilithonimonas，黄杆菌属(Flavobacterium)，微小杆菌属(Exiguobacterium)，节杆菌属(Arthrobacter)，赤球菌属(Rhodoglobus)，Massilia和紫色杆菌属(Janthinobacterium)，其中有些种属至今尚无产低温蛋白酶菌株的报道。产低温淀粉酶菌株的数量和种类最少，它们分别隶属于β-变形菌纲(β-proteobacteria)和拟杆菌门(Bacteroidetes)。产低温脂肪酶的细菌是研究的四种产低温酶菌群中数量最多、遗传多样性最丰富的一种，属于α-，β-，γ-变形菌纲(α-，β-，γ-proteobacteria)，拟杆菌门(Bacteroidetes)和放线菌门(Actinobacteria)。产低温纤维素酶的细菌分别属于：α-，γ-变形菌纲(α-，γ-proteobacteria)和放线菌门(Actinobacteria)三大类群。以上研究表明了天山冻土中蕴藏着极其丰富多样的产低温酶微生物资源，这些产低温酶微生物群体之间相互协同作用，发挥出优势互补的效果，在天山冻土生物地球化学循环中发挥着重要作用。6．冻融试验以天山冻土冻融界面样品为材料，模拟冻融循环，研究冻土中微生物种群生态学和群落生态学特征，探索在冻融循坏中微生物多样性及数量的动态变化规律。通过对天山冻土样品K5冻融处理和直接分离培养获得的优势菌株进行16S rDNA序列测定和系统发育分析，发现冻融作为冻土一个主要的极端因子对其中的细菌群落结构有很大程度的影响。少数几次冻融处理会降低冻土样品K5中革兰氏阳性细菌的多样性，提高革兰氏阴性细菌的多样性。经过少数几次冻融处理后，冻土样品K5中的优势类群主要为Actinobacteria、Bacteroidetes及α-，β-proteobacteria。与冻土样品未经冻融处理直接分离培养的细菌群落结构最大的不同之处在于，有60％的菌株聚类于革兰氏阴性细菌类群中的Bacteroidetes和α-，β-proteobacteria，表明经过少数几次冻融处理后冻土冻融界面的样品中革兰氏阴性菌群为优势类群。在一系列冻融处理中，冻融循环次数不断增加，天山冻土样品K5中的细菌多样性也随之减少。在多次冻融处理的冻土样品中，可培养细菌在种类和数量方面又以革兰氏阳性细菌为主。天山冻土微生物遗传和代谢多样性的研究拓展了低温生物领域的研究范围，揭示了原核生物在高山环境生物地球化学作用中的分布与功能，为进一步的应用研究提供了理论依据和科学支撑。更多还原

【Abstract】 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.更多还原