摘要
多年冻土作为占据地球陆地总面积26％的生态环境，因其长期的低温和地磁辐射、严重缺乏液态水和可获取的营养，对生存于其中的生物是极大的挑战，是极端环境的一个重要代表。上世纪90年代，因研究低温环境下的生命特征和进化起源、作为地外生命研究的模型和开发低温环境下微生物资源的需要，冻土微生物的研究得到了大力支持而长足进展。然而，青藏高原作为一个特殊多年冻土环境的代表，其微生物学研究开展甚少；而随着全球气候变暖，青藏高原多年冻土正在急剧退化，这使研究其中微生物的工作显得重要且紧迫。
本工作以取自青藏高原腹地北麓河流域的冻土样品为材料，重点研究了青藏高原多年冻土区可培养微生物的理化性质和多样性分布，并对一些细菌的新种和一些有应用前景的菌株做了进一步研究，主要结果如下：
1．青藏高原冻土中可培养的细菌，其数量在10~2-10~7个/克土壤之间(干重)，该数量随着冻土年龄的增加而减少；在低温恢复培养的过程中，发现在温度升高冻土消融的过程中，可培养细菌的数量增加而多样性却逐渐减少；通过系统发育学研究和分类分析，该细菌群落包含15个属，归于六个系统发育组：即高G+C革兰氏阳性细菌，低G+C革兰氏阳性细菌，α-proteobacteria，β-proteobacteria，γ-proteobacteria和CFB群细菌；通过生理生化研究，发现分离出来的细菌群落具有耐低温的特性，但真正的嗜冷菌却非常少。
2．通过在培养基中添加碳酸氢钠的办法，恢复出一群数量在10~2-10~5个/克土壤之间的细菌群落，分离出的48株菌分属于6个属3个系统发育组。对其生理性质研究发现，这是一个具有耐冷嗜碱特性的细菌类群：在温度在10-15℃、pH为9-9.5的时获得最佳生长状态。这是首次对冻土中的嗜碱菌群进行了的研究，并发现一些能产生可能是新型的冷活性碱性蛋白酶的菌株，为进一步研究该类群细菌在生物科技产业的应用打下基础。
3．通过比较已采用的几种培养方案，发现不同培养基对相同样品有不同的培养效果，而同一批样品经不同pH和温度下培养后，分离出来的菌群差异很大。本工作也对几种功能类群的细菌和放线菌的培养和初步研究成果，分析了使用多种培养方案对研究和开发微生物资源的重要意义。
4．通过筛选发现有一部分细菌菌株能产生具低温活性胞外酶类，其中有一株来自地下5.7米处永冻层的Brevundimonas属细菌产冷活性淀粉酶和纤维素酶能力比较强，继而对其产酶最佳发酵条件进行了研究，为开发冻土中的微生物应用价值作了初步探索。
5．通过常规鉴定手段和系统发育学研究，发现青藏高原冻土中的细菌有很大比例的细菌很可能是新的细菌物种代表，通过进一步研究，已完成鉴定了一个CFB类群的细菌新种，将其命名为Hymenobacter psychrotolerans，目前尚有数个能作为细菌新种的菌株正在研究鉴定中。
通过以上研究表明，独特的青藏高原多年冻土中孕育着一个独特的微生物群落，很多微生物的新种等待被认识，更多有应用前景的微生物资源等待开发。
The permafrost environment represents 26% of terrestrial soil ecosystems, is considered extreme because indigenous microorganisms must survive prolonged exposure to subzero temperatures and background radiation for geological time scales in a habitat with seriously lack of liquid water reachable nutrition. In the last century 90's, with the purpose to study the life characteristic and evolution under the low temperature environment, as model to research extraterrestrial life and to exploration of the microorganism resources under the low temperature environment, research of permafrost microbiology obtained it's vigorously support and considerable achievements. The Qinghai-Tibet Plateau represents a unique and important permafrost environment. However, its microbiology remains nearly unexplored to date. In addition, the permafrost degradation is very severe in the Qinghai-Tibet Plateau under global warming scene. Thus, there is an urgent need of investigations concerning microorganisms in this extreme and unique soil.
In this study, samples were collected from Beilu river basin, the hinterland of Qinghai-Tibet Plateau. And the investigations were concentrated in the characteristics, distribution and diversity of the viable microorganisms from the Qinghai-Tibet Plateau permafrost region including with the study concerning the description of novel bacteria species and the study of some candidate strains that they might be of potential value for biotechnological exploitation. The results obtained here were summarized as follows:
1. The numbers of colony forming unit (CFU) were ranged between 0 and 10~7/g dry soil. Phylogenetic analyses indicated that all the isolates belong to 19 genera and fell into six categories: high G+C Gram-positive bacteria, low G+C Gram-positive bacteria,α-proteobacteria,β-proteobacteria,γ-proteobacteria and CFB group bacteria. These results of characterization suggested that the isolated population is psychrotolerant but owns very less psychrophiles.
2. The viable bacteria on PYGV agar modified by adding sodium bicarbonate were varied between 10~2 and 10~5 CFU/g of dry soil. Forty-eight strains were gained from 18 samples. Through phylogenetic analyses, these isolates fell into three categories and six genera. The strains could grow best at pH values ranging from 9 to 9.5 with the optima temperatures ranging from 10℃to 15℃. These results indicate that there are populations of nonhalophilic alkaliphilic psychrotolerant bacteria within the permafrost of the Qinhai-Tibet plateau. The abilities of some strains to produce cold active extracellular protease suggest that they might be of potential value for biotechnological exploitation.
3. The comparisons of the utilized isolation procedures suggest that, though the same batch of samples used, the different media resulted in the different isolated populations and the same medium with different pH or different incubation temperatures could isolate very different bacteria populations. Combined with the results of cultivating some functional kinds of bacteria and actinomyces, the important significances of using multiple strategies to investigate and explore microorganism resource were analysed.
4. A part of strains can produce cold active extracellular hydrolytic enzymes. The result showed that a Brevundimonas strain, isolated from a permafrost layer sample with the depth of 570cm, had relatively stronger ability to produce cold active extracellular amylase and cellulose. Subsequently, the further researches were focus on the optimal conditions to produce the enzymes for biotechnological exploitation of the permafrost organisms.
5. With using the routine methods and phylogenetic analyses, results indicate that a good part of strains isolated from the Qinghai-Tibet Plateau permafrost region might potentially be new taxa. Subsequently, investigation work concerning description a CFB group bacteria strain as a novel species was completed and the scientific name Hymenobacter psychrotolerans was proposed. In addition, some candidate strains might potentially be new taxa was still under recent study.
The results above indicate that the unique permafrost on the Qinghai-Tibet Plateau accommodates a very special original microbial assemblage. There are many novel taxa of microorganisms need to be investigated and a great potential value of microbial resources ready to be explored.
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