2.4 16S rRNA sequences
Samples were collected directly from anaerobic digester within steady-state conditions (the 200th and 213th days) for microbial community analyses at OLR of 10.2 and 17.1 g VS L-1 d-1, respectively (first stage). In addition, the microbial samples were also obtained during the shortening of the HRT from 5 to 1.5d (second stage) after 3 HRTs during process stability. Samples were stored at -20°C until being analysed. DNA extraction, 16S rRNA amplification, PCR procedures and sequencing libraries were operated according to previous studies (Yin et al, 2018). Representative sequences were filtered for each operational taxonomic unit (OUT). Three alpha diversity i.e. Chao1 (microbial richness), Shannon and Simpson (microbial diversity) were calculated with rarified OTU table.
Total bacteria and three abundant methanogen orders, Methanobacteriales, Methanosarcinales and Methanomicrobiales, were detected by using Quantitative polymerase chain reaction (qPCR) to analyze the microbial community dynamics when shortening the HRT from 5 to 1.5d. Primers were designed according to the 16s rRNA gene sequence as a previous study (Shi et al., 2018). Slurry sample at HRT of 5d was amplified by PCR using four sets of primers. The fragments were recovered from the gel, ligated to the PUC-T vector, and then transformed into E. coliDH5a for cultivation. Positive clones were selected to extract plasmids, and nucleic acid detectors were used to determine the concentration and purity. The concentrations were converted to copies and serially diluted. The purified PCR products were then sequenced, and verified. Real Time PCR system (EDC-810, China) was performed as qPCR. A qPCR mixture (20μL) containing 1μL of slurry sample, 0.4μL of each forward and reverse primer (10μmol/L), 10μL of 2×sybr MIX (with ROX), sterile water to a total of 20μL, and Power SYBR Green (Baygene BG-Power600, China) were used to prepare template DNA (20ng). The amplification processes of the total bacteria and the order Methanobacteriales were as follows: A cycle consisted of 3 min at 94°C; 94°C for 15s with totally 40 cycles, 20s at 60°C, 72°C for 20s, 2 min at 72°C; and extension at 72°C for 20s. The additional steps for the orders Methanosarcinales and Methanomicrobiales were carried out at 94°C for 15s with 40 cycles, i.e. 52°C, 72°C and 72°C for 20s 30s and 2 min, respectively; and extension at 20s at 72°C. For each primer and probe set, a control without the equivalent template DNA was consisted in every qPCR assay. Triplicate standard samples were constructed for each primer set, of which one was selected to draw a standard curve.