北极斯瓦尔巴群岛冰川流域可培养细菌的系统发育多样性及固氮菌初步研究

从北极斯瓦尔巴群岛冰川流域的上、中、下游采集了5种代表性土壤和冰川融水样品(MT、AT、TN、BT、MS),采用常规LB和严格无氮培养基低温培养方法,对其中的可培养细菌和潜在固氮细菌进行了总菌计数以及菌株的富集、分离与纯化。经菌落菌体形态观察、16S r RNA基因初步测序排重、固氮基因nif H的PCR检测,分别选取48株可培养分离细菌和31株潜在固氮菌进行了基于16S r RNA基因全序列测定的系统发育分析。目的是初步了解该特殊生境中可培养细菌(包括潜在固氮菌)的多样性及其分布状况,为进一步研究它们在此生境中的生态功能奠定基础。实验结果表明,可培养细菌在5个采集样品中的总菌数为BTTNATMT=MS,严格无氮培养基分离菌总菌数为BTATMTTNMS。48株可培养代表菌分属于Proteobacteria、Actinobacteria、Bacteroidetes、Firmicutes 4个门的7纲、14属之中,它们在5个样品中的多样性程度为BT(6纲、6属)MT(4纲、6属)AT、MS(均为3纲、3属)TN(2纲、3属),优势菌群主要分布在γ-proteobacteria纲的Pseudomonas属(20.8%,集中在AT、BT、MS样品中)和β-proteobacteria纲的Janthinobacterium属(35.4%,集中在MS样品中),表现出一定程度的系统发育多样性。潜在固氮菌实验结果表明,能在严格无氮培养基上生长且可检测到nif H的31株代表菌具有极为单一的系统发育多样性。其中90.32%菌株集中在γ-proteobacteria纲的Pseudomonas属,仅在MT和MS样品中有3株菌分布在α-proteobacteria纲的Rhizobium属、β-proteobacteria纲的Janthinobacterium属和Actinobacteria纲的Arthrobacter属,它们与分离自其他极端环境的相关标准菌株具有高度16S r RNA基因同源性。另外,本实验还发现有8株菌的16S r RNA基因序列与相关标准菌株的相似度低于98.50%,推测它们具有成为该特殊生境下新种的潜质。

Preliminary Study on Phylogenetic Diversity of Culturable Bacteria and Nitrogen-fixing Bacteria in Svalbard Glaciers of Arctic

Five different samples of representative soil and meltwater (MT, AT, TN, BT, MS) were collected from the upstream, midstream and downstream of the Arctic Svalbard glaciers. The culturable bacteria and potential nitrogen-fixing bacteria were counted, enriched and isolated by using LB medium and strict nitrogen-free medium at the low temperature, respectively. After colony observation, preliminary 16S rRNA gene sequencing and nifH -PCR, 48 culturable bacterial strains and 31 potential nitrogen-fixing bacterial strains were selected to examine the phylogenetic diversity based on the almost full-length 16S rRNA gene sequences. The purpose was to obtain a preliminary understanding of the diversity and distribution of these bacteria in this special area, and to provide a basis for further research on their ecological functions. The results showed that the populations of the culturable bacteria in the five samples were ordered as BT > TN > AT > MT=MS. The populations of potential nitrogen-fixing bacteria were ordered as BT > AT > MT > TN > MS. Forty-eight culturable bacteria belonged to 4 phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), 7 classes and 14 genera, and their degrees of diversity were ordered as BT(6 classes, 6 genera)> MT(4 classes, 6 genera)> AT=MS(3 classes, 3 genera)> TN(2 classes, 3 genera). Meanwhile, the majority of culturable bacterial isolates were composed of Pseudomonas of γ-proteobacteria (20.8%, isolated from the AT, BT and MS samples) and Janthinobacterium of β-proteobacteria(35.4%, isolated from the MS sample). The experimental results of the potential nitrogen-fixing bacteria showed that the diversity of 31 strains isolated from the five samples was very low. Of the strains, 90.32% belonged mainly to Pseudomonas(γ- proteobacteria), only 3 strains isolated from the MT and MS samples belonged to Rhizobium (α-proteo¬bacteria), Janthinobacterium (β-proteobacteria) and Arthrobacter (Actinobacteria C), respectively. There was high 16S rRNA gene homology among these potential nitrogen-fixing strains and some related standard-type strains isolated from other extreme environment. In addition, eight strains that showed less than 98.6% 16S rRNA gene-sequence similarity with strains of related type may represent potential novel species in this special ecological environment.