Enhancement of Microbial Function for a Sulfur Redox Cycle Action in a Sewage Treatment Process Combining an UASB and an Aerobic Filter Reactor

利用16S rRNA基因克隆文库对好氧生物滤池污泥样品的微生物群落结构进行了分析。从文库中随机抽取188个克隆，与硫杆菌和硫thrix相似的克隆分别为10个和7个。生物滤池污泥中约7%的DAPI染色总细胞与硫杆菌群特异性探针Thio840杂交。利用BONE663cF-Thio840R引物，采用SYBR green I实时荧光定量PCR法测定硫杆菌源16S rRNA基因的拷贝数。高浓度的硫化物在30 mg-S/L的进水中强烈增强了从好氧生物滤池污泥中提取的总DNA中基因的丰度，达到10^5拷贝/ng水平。通过克隆和测序技术分析了UASB污泥中产生硫酸盐呼吸关键酶的腺苷-5′-磷酸硫酸还原酶（apsA）基因的多样性，该基因存在于所有硫酸盐呼吸原核生物中。基于apsA氨基酸序列的系统发育树显示，所鉴定的apsA克隆属于4个类群：硫酸盐还原菌群、Syntrophobacteraceae、Thiobacillus和克隆簇群，与先前描述的apsA克隆都有远亲关系。采用四种apsA基因序列特异性引物进行实时荧光定量PCR分析，结果表明，在整个实验期间，UASB污泥中所有硫酸盐还原菌的优势种均为Desulfobulbaceae和Desulfovibrionales。硫酸盐还原菌与硫杆菌、Syntrophobacteraceae与未知的apsA克隆群之间存在apsA基因丰度和行为的良好相关性。

The structure of microbial community was analyzed on sludge samples taken from the aerobic biofilter reactor by using clone libraries of 16S rRNA genes. Clone similar to Thiobacillus and Thiothrix was counted at 10 and 7, respectively, among 188 clones randomly selected from the libraries. Approximately 7% of DAPI stained total cells in the biofilter sludge hybridized with the probe Thio840 specific for Thiobacillus group. Real-time PCR using SYBR green I was employed with BONE663cF-Thio840R primer set to determine the copy number of 16S rRNA gene originated from Thiobacillus. High concentration of sulfide at 30 mg-S/L in the influent strongly enhanced abundance of the gene of 10^5 copies/ng levels in total DNA extracted from the aerobic biofilter sludge. the diversity of adenosine-5'-phosphosulfate reductase (apsA) genes, which produce a key enzyme for sulfate respiration and are present in all sulfate-respiring prokaryotes (SRPs), in UASB sludge was analyzed by cloning and sequencing techniques. The apsA amino acid sequence-based phylogenetic tree showed that the identified clones belonged to four groups : the sulfate-reducing bacterial group, Syntrophobacteraceae, Thiobacillus and the clone cluster group, distantly related to any previously described apsA clone. Real-time PCR quantification which used the primer sets specific for each of the four apsA gene sequences revealed that Desulfobulbaceae and Desulfovibrionales were the predominant species all of the sulfate-reducing bacteria in the UASB sludge throughout the experimental period. An excellent correlation of apsA gene abundance and behavior was found between the sulfate-reducing bacteria and Thiobacillus, as well as between Syntrophobacteraceae and the unknown apsA clone group.