焦化废水处理厂接触氧化池中降酚菌群的苯酚羟化酶大亚基基因多样性

研究了某焦化废水处理厂接触氧化池中降酚菌群的苯酚羟化酶大亚基基因(thelargestsubunitofthemulti-componentphenolhydroxylase,LmPH)的多样性。通过温度梯度凝胶电泳(temperaturegradientgelelectrophoresis,TGGE)对比分析了氧化池4个区段(O1—O4)中降酚菌群LmPH的组成。它们的TGGE图谱完全一样,相似性为100%,表明该处理池中不同区段的降酚菌群的功能基因组成是高度相似的。以O4段的菌群为代表建立LmPH基因克隆文库,从中挑选了49个克隆测序。依据LmPH基因的DNA序列所推测的氨基酸序列完全相同的归为一类的原则,49个克隆被分为16种类型,其中优势LmPH基因主要有5种类型(多于4个克隆),而另外11种类型都只有1个克隆。与已知基因同源性超过90%的有7种类型,低于80%的有2种类型。基于氨基酸序列的系统进化树分析表明,LmPH文库中绝大部分的类型都属于低亲和常数(low-Ks)的LmPH,占所有克隆的92%。只有一个类型属于高亲和常数(high-Ks)的。因此,处理焦化废水的工业装置中不仅具有丰富多样的苯酚羟化酶基因类型,而且以编码低亲和常数的占优势地位,而过去报道的通过富集培养分离得到的降酚菌则多带有高亲和常数的酶。这提示我们传统的富集培养方法并不能筛选到生态环境中的真正优势功能菌。

Diversity analysis of the largest subunit of the multi-component phenol hydroxylase (LmPH) gene from the phenol degrading microbial communities of an aeration tank of a coking wastewater treatment plant

The diversity of the largest subunit of the multi-component phenol hydroxylase (LmPH) gene in four compartments of the aeration tank (O1-O4) of a coking wastewater treatment plant in Shanghai was analyzed via PCR-TGGE (temperature gradient gel electrophoresis) analysis. TGGE fingerprints were identical in all four compartments, suggesting that the phenol-degrading microbial populations in the different compartments of the aeration tank were highly similar. A LmPH gene clone library from the fourth compartment (O4) was constructed and the nucleotide sequences of 49 randomly selected clones were determined. The 49 clones were classified into 16 groups (amino acid sequences with 100% similarity were defined as one group) based on the deduced amino acid sequences. Five predominant LmPH groups were identified, each of which had more than four clones. Each of the other 11 groups were represented by one clone. Seven LmPH groups had more than 90% similarity with their nearest LmPHs in the GenBank, while 2 groups had less than 80%. Phylogenetic analysis of the partial amino acid sequences of the LmPHs indicated that most of the clones in the library (92%) were affiliated with low-Ks (affinity constants) LmPH, while only one clone (L-SJ32) was affiliated with high-Ks LmPH. These results suggest that LmPH genes were very diverse in the coking wastewater and that the low-Ks LmPHs were the predominant type involved in phenol degradation. Since most of the previously isolated phenol-degrading bacteria had high-Ks LmPHs, this indicates that traditional enrichment culture methods may not have identified the key functional bacteria from this environment.