荧光定量PCR监测五氯酚对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响

Real time PCR quantification of ammonia-oxidizing bacteria in aerobic granular sludge and activated sludge influenced by pentachlorophenol

摘要:

通过特异引物扩增环境中氨氧化细菌16S rDNAV2保守区域,将该片段克隆到T-easy载体上,PCR产物经测序和定量PCR扩增体系鉴定,证实PCR扩增产物为氨氧化细菌16S rDNA保守序列,以含该序列的重组质粒作为定量PCR监测氨氧化细菌数量的DNA标准品。用荧光定量PCR技术比较了五氯酚(PCP)对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响。结果表明,不加PCP的反应器中,好氧颗粒污泥和活性污泥中氨氧化细菌的数量分别为4.28×10⁷±5.44×10⁶cells/(g干污泥)和2.51×10⁹±8.61×10⁸cells/(g干污泥)。随着PCP浓度的增加(0~50mg/L),PCP对氨氧化细菌数量的影响不大(P>0.05),而且,污泥中氨氧化细菌的数量与氨氮的去除率无直接的正相关关系(P>0.05),PCP主要是抑制氨氧化细菌的代谢活性导致污泥氨氮去除效率降低。

Abstract:

The V2 region of the 16S ribosomal DNA of the ammonia-oxidizing bacteria (AOB) was amplified directly from the environmental sample by using the specific PCR primers. The purified PCR product was cloned into T-vector and was identified as 16S rDNA fragment of AOB by sequencing and Real-time PCR method. Then, the recombined plasmid was used as standard molecule sample in Real-time PCR for AOB quantification. The numbers of the AOB were monitored in samples of both aerobic granular sludge and activated sludge influenced by PCP by using Real-time PCR. The results showed that the numbers of AOB in aerobic granular sludge and activated sludge were 4.28×10⁷±5.44×10⁶cells/g dried sludge and 2.51×10⁹±8.61×10⁸cells/g dried sludge without PCP in the reactors, respectively. With the increase of PCP concentration (from 0mg/L to 50mg/L)，the numbers of AOB in both types of sludge had no obvious change（P>0.05）. The numbers of AOB had no obvious correlation with ammonia removal (P>0.05). The main effect of PCP on AOB in both types of sludge was to inhibit their metabolic activity.