Inadequacy of enrichment culture technique for assessing the structure of methanotrophic communities in peat soil
Inadequacy of enrichment culture technique for assessing the structure of methanotrophic communities in peat soil
复制标题
富集培养技术评估泥炭土甲烷氧化菌群落结构的不足
DOI：
10.1134/s0026261708040188
复制
发表时间：
2008
期刊：
影响因子：
1.5
通讯作者：
S. Dedysh
中科院分区：
文献类型：
--
作者：
A. V. Vorob’ev;S. Dedysh
504富集培养技术是用于评估自然和人为生态系统中甲烷氧化菌群落组成的常规方法之一[1]。必须使用这种技术，因为样品中异养细菌的细胞数量通常超过甲烷氧化菌的数量，使其检测和鉴定显着复杂化。使用富集培养物与甲烷作为选择性生长基质的结果在甲烷的微生物群落的甲烷营养组分的主要发展，并允许定性分析it. The问题，如何在实验室条件下形成的富集培养物的组成对应于甲烷营养群落在原始样品的组成是至关重要的。众所周知，不同的pH值、培养基中的总盐含量或孵育温度会导致从相同样品中获得不同组成的富集培养物[2 - 4]。迄今为止，人们普遍接受的是，最充分的信息的甲烷氧化菌群落的组成，在原地获得，介质的pH值和培养温度应接近那些在生态系统研究。同时，对富集培养设置的其他条件没有给予太多关注，例如培养基中结合氮源的存在和性质以及培养通风条件。进行这项调查，以测试这些因素的实验室甲烷营养菌富集的组合物上的效果，以及后者如何对应于在原始样品中的甲烷营养菌组合物。采用16 SrRNA特异性荧光标记寡核苷酸探针（FISH）原位杂交技术作为最客观的微生物鉴定方法。泥炭藓泥炭于2006年7月从托木斯克州Bakcharskoe寡中养泥炭藓沼泽（56°51 ′ N，82°51 ′ E）的10 - 20 cm层中取样。通过用无菌剪刀切割使泥炭均质化并充分混合。根据先前描述的方法[5]，用0.4%甲醛固定2 g泥炭部分，以通过FISH进行进一步分析，剩余材料用于培养基接种。在M2培养基（pH 5.0）[6]中启动富集培养，采用三种改良方法：（a）不含结合氮源;（B）添加200 mg l-1的aNe 3;（c）添加200 mg l-1的（NH 4）2 SO 4。培养基以20 ml por
504 The enrichment culture technique is one of the conventional approaches used to assess the composition of communities of methanotrophic bacteria in natural and anthropogenic ecosystems [1]. This technique has to be used because the number of cells of heterotrophic bacteria in samples usually exceeds the number of methanotrophs, significantly complicating their detection and identification. The use of enrichment cultures with methane as a selective growth substrate results in predominant development of the methanotrophic component of the microbial community and allows qualitative analysis of it. The question as to how the composition of the enrichment cultures formed under laboratory conditions corresponds to the composition of methanotrophic communities in the original sample is of fundamental importance. It is known that varying pH, the total salt content in the medium, or the incubation temperature results in obtaining enrichment cultures of different composition from the same samples [2‐4]. To date, it is commonly accepted that for the most adequate information about the composition of the methanotrophic community in situ to be obtained, the pH of the medium and the cultivation temperature should be approximated to those in the ecosystem studied. At the same time, not much attention is given to other conditions of enrichment culture setup, such as the presence and nature of a source of bound nitrogen in the medium and the culture aeration conditions. This investigation was undertaken to test the effect of these factors on the composition of laboratory methanotrophic enrichments and on how the latter corresponds to the methanotroph composition in the original sample. The direct molecular method for microorganism identification, the in situ hybridization with 16S rRNA-specific fluorescently labeled oligonucleotide probes (FISH), was used as the most objective referent method. Sphagnum peat was sampled from a 10‐20-cm layer of Bakcharskoe oligomesotrophic Sphagnum bog ( 56°51 ′ N, 82°51 ′ E) in Tomsk oblast in July 2006. The peat was homogenized by cutting with sterile scissors and mixed thoroughly. A 2-g portion of peat was fixed with 0.4% formaldehyde according to the method described earlier [5] for further analysis by FISH, and the remaining material was used for medium inoculation. Enrichment cultures were initiated in M2 medium (pH 5.0) [6] used in three modifications: (a) without a source of bound nitrogen; (b) with aNe 3 in an amount of 200 mg l –1 ; and (c) with (NH 4 ) 2 SO 4 in an amount of 200 mg l –1 . The media were dispensed in 20-ml por