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.