Microbial communities of four lab-scale anaerobic bioreactors in treating real antibiotic production wastewater.

In this study, four lab-scale anaerobic bioreactors were operating continually for more than 100 days to treat real antibiotic production wastewater (avermectin, lincomycin, tylosin and their mixture). The operation performance and their functional microbial communities were analyzed.Five activated sludge microbial samples were collected at different time from the four lab-scale anaerobic bioreactors. Each sample was 100 ml mixture of 3 collections from 10%, 40%, and 70% height of the bioreactor. Sample OR_1 was collected from inoculation sludge used by the four bioreactors on 1st day of the whole study period. Sample AVM_1, LCM_1, TYL_1, and MIX_2 was collected from the bioreactors which treated the production wastewater of avermectin (168th day), lincomycin (137th day), tylosin (90th day) and the mixture (109th day), respectively.Each sample was delivered to the laboratory at below 4 degree centigrade after collection, and the DNA of each sample was extracted within 24 hours. Total DNA of each sample was extracted following the procedure of DNeasy PowerSoil Kit (Qiagen, Germany). Primers of 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3') with different barcodes were used to amplify V3-V4 regions of microbial 16S rDNA genes. 20 microlitre PCR reaction mixture was used for 16S rDNA gene fragment amplification contained 10 ng DNA, 0.2 micromole each primer, 250 micromole each deoxyribonucleotide triphosphate (dNTP), 5 FastPfu buffer (5 microlitre each), 1 U FastPfu Polymerase (TransGen Biotech). The PCR protocol began with an initial denaturation (5 min at 95 degree centigrade), followed by 27 cycles consisting of 30 s at 95 degree centigrade, 30 s at 55 degree centigrade, and 45 s at 72 degree centigrade, and final with one cycle of 10 min at 72 degree centigrade. Amplicons were checked with 2% agarose gels and purified using the KAPA Pure Beads (Roche) according to instructions of the manufacturer.The purified PCR products were quantified using Qubit 4.0 (Life Technology) and mixed with other amplicons with different barcodes. The KAPA Hyper Prep Kit (Roche) was used for the mixed 16S rDNA gene fragment library construction following the kit procedure. The amplicon library was paired-end sequenced (2*250 bp) on an Illumina NovaSeq platform according to the standard protocols.

本研究中，4台实验室规模厌氧生物反应器连续运行超100天，用于处理实际抗生素生产废水（阿维菌素、林可霉素、泰乐菌素及其混合废水），并对其运行性能与功能微生物群落展开分析。从4台实验室规模厌氧生物反应器的不同时间点共采集5份活性污泥微生物样品：每份样品为采集自反应器10%、40%、70%高度的3份子样本混合而成的100mL混合液。其中，样品OR_1采集自整个研究周期第1天4台反应器所用的接种污泥；样品AVM_1、LCM_1、TYL_1及MIX_2分别采集自处理阿维菌素生产废水（第168天）、林可霉素生产废水（第137天）、泰乐菌素生产废水（第90天）以及混合抗生素生产废水（第109天）的对应反应器。每份样品采集后均置于4℃以下条件运输至实验室，并在24小时内完成DNA提取。每份样品的总DNA均按照DNeasy PowerSoil试剂盒（Qiagen，德国）的标准操作流程完成提取。使用带有不同条形码的引物341F（5'-CCTAYGGGRBGCASCAG-3'）和806R（5'-GGACTACNNGGGTATCTAAT-3'）扩增微生物16S rDNA基因的V3-V4可变区。采用20μL的PCR反应体系进行16S rDNA基因片段扩增，体系组成如下：10 ng DNA、0.2 μmol/L各引物、250 μmol/L各脱氧核糖核苷三磷酸（dNTP）、5×FastPfu缓冲液（每体系添加5 μL）、1 U FastPfu DNA聚合酶（TransGen Biotech）。PCR扩增程序为：95℃初始变性5 min；随后进行27个循环，每个循环包含95℃变性30 s、55℃退火30 s、72℃延伸45 s；最终于72℃进行10 min终延伸。扩增产物通过2%琼脂糖凝胶电泳检测，并使用KAPA Pure Beads（Roche）按照制造商说明书进行纯化。纯化后的PCR产物采用Qubit 4.0荧光定量仪（Life Technology）进行定量，随后与带有不同条形码的其他扩增产物混合。使用KAPA Hyper Prep试剂盒（Roche）按照试剂盒操作流程构建混合的16S rDNA基因片段文库。最终采用Illumina NovaSeq测序平台，按照标准流程对该扩增子文库进行双端测序（2×250 bp）。