The relationship between reactor performance and functional microbial communities in the anaerobic digestion of tannery wastewater

Abstract

Tannery wastewater poses severe environmental threats due to its characteristically high organic load and metal content. The remediation of this waste stream is often problematic. While anaerobic digestion (AD) has significant advantages, the process remains enigmatic due to a lack of data about the microbial communities responsible for the success of the process. There is a need for the simultaneous investigation of reactor performance and microbial consortia dynamics in response to changes in operational conditions. This study aimed to quantify copy numbers of methyl coenzyme M reductase (mcrA) and dissimilatory sulfite reductase (dsrB) genes encoding the enzymes that catalyse the terminal processes in AD treatment of ostrich tannery wastewater. It also aimed to correlate these gene copy numbers respectively with the efficiency of methane (CH4) generation and sulfate (𝑆𝑂4 2−) concentrations in anaerobic digesters treating ostrich tannery wastewater. Biochemical methane potential (BMP) tests were conducted in 2 L glass bottles at 37°C. Thirteen reactors were set up based on central composite design at different inoculum to substrate ratios (ISR) of 2 to 5 and different 𝑆𝑂4 2− concentrations ranging from 665 to 2000 mg/L to assess the effect of ISR and 𝑆𝑂4 2− concentration on CH4 generation and biodegradability of ostrich tannery wastewater. To try and maximize AD efficiency, two 20 L anaerobic sequencing batch reactors (ASBR) were operated under similar conditions to those suggested by the BMP results. However, ASBR1 operated at intermittent mixing (300 rpm for 5 to 10 min/day) while ASBR2 operated at continuous mixing at 300 rpm. The study was conducted for 50 days in two different operational runs. The first run at the start-up period of the ASBR operated for 30 days with a 5-day settling period before decanting. During the second run, the ASBRs operated for 20 days. Deoxyribonucleic acid (DNA) was extracted from (i) samples from the BMP tests collected at baseline, when the reactors started and stopped producing biogas, and at the end of the study and (ii) samples from the ASBRs collected at the beginning of the experiment and every week thereafter. Quantitative Real-Time PCR (qRT-PCR) was performed on all the DNA samples, and next generation sequencing (NGS) was conducted on selected DNA samples taken from the BMPs (based on 𝑆𝑂4 2− concentration) and biweekly samples taken from the ASBRs. Based on response surface methodology (RSM), the optimum operating conditions for maximal gas (CH4, biogas) and biodegradability were found to be 983.687 and 3.687 for 𝑆𝑂4 2− concentration and ISR respectively. Results showed that minimal CH4 (<1 mLCH4/ gVS) was produced at high 𝑆𝑂4 2− concentration (≥1960 mg/L) and ISR <3.0, suggesting that pre-treatment is required at high 𝑆𝑂4 2− concentration. In the ASBRs, continuous mixing in ASBR2 was shown to be more efficient than intermittent mixing in ASBR1 by producing high cumulative CH4 in total (1149 and 106 mLCH4/gVS in ASBR2 and ASBR1, respectively). However, a large decrease in CH4 production was observed between successive runs in both ASBRs. It was therefore assumed that biomass washout occurred during the decanting step. From a microbial point of view, the NGS results revealed that 𝑆𝑂4 2− concentration and ISR did not have significant (P >0.05) effects on the methanogenic and sulfidogenic community structure in the BMP tests. However, Desulfofustis glycolicus, known to reduce 𝑆𝑂4 2− to H2S was found at high relative abundance (RA, 15.91%) in the BMP test operating at 𝑆𝑂4 2−≥ 1960 mg/L compared to the other BMP tests (<0.003% RA). It was postulated that the H2S may have inhibited some methanogens in the former. According to the analysis of similarity (ANOSIM), both methanogenic and sulfidogenic community structures were established once biogas generation commenced, and were responsible for ongoing physicochemical changes thereafter, as there was significant difference between the measured physicochemical parameters with factor ‘time’ (initial, start of biogas production and final). The changes in the sulfidogenic community structure were driven mainly by combinations of ammonia (NH3), volatile organic acids (VOA), total organic carbon (TOC), and alkalinity concentrations, as well as VOA:alkalinity and dsrB copy numbers, while changes in the methanogenic structure was driven mainly by pH, NH3, VOA, TOC, alkalinity and nitrogen (N) concentrations. In ASBRs, continuous mixing promoted better survival and high abundance of Methanosarcina mazei in ASBR2 (14.7-31.6%) than in ASBR1 (4.3-6.8%). Quantitative Real-Time PCR (qRT-PCR) results showed that in the BMP tests, the abundance of the mcrA gene ranged from 3.63×105 to 6.46×106 copy numbers/ng DNA and were 1 to 2 order of magnitude higher than the dsrB gene copy numbers (5.13×104 to 8.44×105/ng DNA) indicating the dominance of the former. While in the ASBRs, although the copy numbers of mcrA were higher in ASBR2 (from 8.23×106 to 1.26×107, and 9.32×106 to 1.32×107 in ASBR1 and ASBR2 respectively), the difference was not significant. The selection of M. mazei was therefore associated with the higher CH4 yield in ASBR2. The dsrB gene copy numbers varied between 2.70×105 to 1.12×106 and 2.27×105 to 6.72×105/ng DNA in ASBR1 and ASBR2 respectively, indicating that, in contrast to methanogenesis, sulfidogenesis was more favoured in ASBR1 than ASBR2, and may have contributed to the lower production CH4 in this digester. Positive significant correlations (P <0.05) were observed between mcrA gene copies and specific CH4 yield in the BMPs and the ASBRs, and also between dsrB gene copies and H2S gas in the BMPs and S2- concentration in ASBR1 but not with 𝑆𝑂4 2− (P >0.05). Taken together, results from this study indicate that the knowledge about the selected functional microbial consortia in diverse anaerobic reactor systems is of practical interest in order to comprehensively understand and control the AD process, mitigate process disturbances, and maximize the CH4 yield.