3.2 Microbial communities under well-performed and disturbed states targeting OLR
Linking the microbial dynamics to process perturbations is fundamental in order to understand and deal with process instability. Therefore, two OLRs were chosen in order to investigate the microbial community structure, i.e. during process stability at 10.2 g VS L-1 d-1, and subsequent process disturbances (17 g VS L-1 d-1). In Fig. 2, bacterial community compositions slightly changed under the two OLR conditions although the abundances were different.Thermotogae (phyla Thermotogae ) were the predominant bacterial class, followed by Clostridia (phyla Firmicutes ) and Synergistia (phyla Synergistetes ) at both OLRs. It has been reported that a significant number of members belonging to phylumThermotogae frequently seem to be higher in thermophilic digesters (Shi et al., 2018). This phenomenon may be attributed to their ability to encode thermo-stable enzymes that play a role in conversion processes (Conners et al., 2006). At an OLR of 10.2 g VS L-1 d-1, Defluviitoga(79.7%) dominated the microbial genera (Fig. 2a). At an OLR of 17.1 g VS L-1 d-1, Defluviitoga decreased to 49.8% but there were significant increases in Clostridium andAnaerobaculum (Fig. 2b), implying that these microorganisms were functionally redundant. Accordingly, it was obvious that the increase in OLR resulted in alternation of the microbial abundant during the experiment. Furthermore, the decline in relative abundance ofDefluviitoga was associated with process failure, signifying its significant role in substrate metabolism and process stability.Defluviitoga spp. are able to utilize a wide range of carbohydrate as electron donors (Hania et al., 2012), thus the C-rich straw in the substrate mixture might promote the enrichment of this genus. Defluviitoga spp. frequently exists not only in thermophilic AD (Hania et al., 2012), but also in metabolic association with hydrogenotrophic methanogens (Maus et al., 2016).
In this study, genera Tepidanaerobacter, Clostridium andSyntrophaceticus , which were three possible syntrophic acetate oxidation bacteria (SAOB), were detected at an OLR of 10.2 and 17.1 g VS L-1 d-1. As depicted in Table 3,Clostridium sp. represented 2.8% and 11% of total bacterial at 10.2 and 17.1g VS-1 L-1 d-1 of OLR, respectively. Consequently, the reduction in the relative abundance of most dominant carbohydrate-fermenting species (Defluviitoga ) might be linked to simultaneous increase in relative abundance of Clostridium sp. and meanwhile promoted carbohydrate availability in digester. The increased presence of SAOB indicated the existence of SAOB combined with a hydrogenotrophic methanogenesis (HM) pathway for methane formation. SAOB of generaTepidanaerobacter and Syntrophaceticus were also detected even though they were at less than 0.5% during the two OLRs.Syntrophomonas , which is known to function as a syntrophic microbe to degrade complex organic matters to simple fatty acids (Hansen et al., 1999), was detected with little change in its relative abundance with the OLRs of 10.2 and 17.1 g VS L-1 d-1 (0.7% and 1%, respectively).
Hydrogenotrophic methanogens (including the genera Methanoculleusand Methanothermobacter) represented 88.2% and 53.5 % of all archaea population for OLRs of 10.2 and 17.1 g VS L-1d-1, respectively (Fig. 2c, and 2d). The genus Methanoculleus clearly dominated at OLR of 10.2 g VS L-1d-1 (86.9%) and OLR of 17.1 g VS L-1d-1 (52.8%).Methanoculleus has been reported to be a very efficient hydrogen-utilizing methanogen during the thermophilic AD of chicken manure (Bayrakdar et al., 2017) and it acts as an important partner withClostridium ultunenes (Yin et al., 2018). The increased OLR to 17.1 g VS L-1d-1, as previously stated, reduced the presence of Methanoculleus , and coincided with the VFA accumulation and the reduction of methane production (Fig. 1a and 1c). Methylotrophic Methanomassiliicoccuswere represented at relatively low levels at the two OLRs, however, their relative abundance was obviously enhanced at highest OLR.Methanomassiliicoccus is a H2-dependent methanogen which can consume methylated compounds to produce methane (Liu et al., 2016). Nevertheless, it has still not been identified to act as a hydrogenotrophic partner for SAO and it needs to be further verified (Westerholm et al., 2016). In fact, the important role of both Methanoculleus and Methanomassiliicoccus have been previously reported in AD of lignocellulosic biomass (Li et al., 2018a). The acetoclastic methanogens, Methanosarcina accounted for only 2% and 4% of the total archaea at an OLR of 10.2 and 17.1 g VS L-1d-1, respectively (Fig. 2). In this study, the high abundance of hydrogenotrophic methanogens, and a low representation of acetoclastic methanogens at OLR of 10.2 g VS L-1d-1, alongside a low concentration of acetate (338 mg L-1), strongly indicated that the acetate conversion may be a two-stage process.