## 3 Results and Discussion
### Shifts in Microbial Community Structures with Seasonal Temperature  
High COD removal and biogas production were achieved by the AWFR system in summer, but not in winter. To better elucidate the changes of the microbial community and explore the COD removal mechanism during the whole operation period, the composition and structures of the microbial community were analyzed. Biofilm samples on days 15, 90, 180, 270 and 340, which represented five different periods of the AWFR, were chosen for high-throughput sequencing by the Illumina MiSeq platform. The archaeal community had a lower diversity and most of the archaeal sequences were assigned to the three main orders _Methanosarcinales_, _Methanobacteriales_ and _Methanomicrobiales_ throughout the operation period (Figure 8a). These orders occupied 92.8-96.5% of the total community and were closely related to acetonitrile and hydrogenotrophic methanogen. During the start-up period, the predominant orders _Methanosarcinales_, _Methanobacteriales_ and _Methanomicrobiales_ accounted for 49.3%, 21.4%, and 22.2%, respectively, of the total community. However, the relative abundance of the order _Methanosarcinales_ (34.3%) decreased in spring, whereas the relative abundances of the orders _Methanobacteriales_ (35.0%) and _Methanomicrobiales_ (26.0%) increased. In contrast, the order _Methanosarcinales_ showed an increase in abundance during summer, but a reduction in abundance in winter. The relative abundances of the orders _Methanobacteriales_ and _Methanomicrobiales_ significantly decreased in summer but largely increased in winter. To further validate the function of archaeal community, the microbial distribution at genus level was shown in Figure 8b. The results demonstrated that _Methanosaeta_, _Methanobacterium_ and _Methanolinea_ were the predominant genera throughout the operation period. Among these genera, _Methanosaeta_ was affiliated with acetoclastic methanogen and the remaining genera _Methanobacterium_ and _Methanolinea_ were affiliated with hydrogenotrophic methanogens. _Methanosaeta_ affiliated with the order _Methanosarcinales_ had a high relative abundance (49.2%) in the start-up period, whereas the relative abundances of the genera _Methanobacterium_ and _Methanolinea_ were 18.7% and 18.3%, respectively. During summer, the genus _Methanosaeta_ (61.0%) was the most abundant followed by the genera _Methanobacterium_ (26.1%) and _Methanolinea_ (4.8%). These findings indicated that acetoclastic methanogenesis was the main pathway, with some contribution from the hydrogenotrophic methanogens. These genera may have helped the AWFR system to maintain low VFA level, leading to relatively better COD removal efficiency and higher biogas production in summer. These results were  
Figure 8: Archaeal community shifts at (**a**) order and (**b**) genus level. in accordance with that in a previous study, achieving higher COD efficiency and biogas production in summer in a UASB treating domestic wastewater [8]. Nevertheless, during the winter period, the hydrogenotrophic genus _Methanobacterium_ (33.8%) was the most dominant species followed by the hydrogenotrophic genus _Methanoline_ (29.9%) and the acetoclastic genus _Methanosaeta_ (23.2%). Furthermore, other less abundant hydrogenotrophic genera were also detected in winter, including the genera _Methanospirlum_, _Methanosphaareula_ and _Methanosphaera_, which are capable of using H\({}_{2}\)/CO\({}_{2}\) to produce methane. Hydrogenotrophic genera (>63.7%) made up a large portion of the archaeal community in winter. These findings were in line with the previous observations of methanogen population shifts to favor hydrogen utilization under low temperatures [25; 41; 42; 43]. It might be explained that lower temperature in winter affected the microbial membrane fluidity and inhibited the utilization of acetate than H\({}_{2}\)[44].