3.1 Operation and performance of mesophilic and thermophilic anaerobic reactors
As in Fig. 1, the mesophilic experiment was conducted for 90 days in total. Days 31–60 were defined as the mesophilic stable stage (MStable) and days 61–90 as the mesophilic collapsed stage (MCollapsed). The overall thermophilic experiment was to last for 129 days. The thermophilic stable stage (TStable) occurred on days 39–64, and the thermophilic collapsed stage (TCollapsed) on days 100–129.
In this study, the performance of the mesophilic and thermophilic reactors was reflected by the fluctuation of gaseous parameters (i.e., the methane content and volumetric methane production rate (VMPR)) and liquid parameters (i.e., VFAs, TAN, and pH value). The VMPR and Total VFA (TVFA) are presented in Fig. 1 while the other parameters are summarized in Table 1. The TVFA sharply increased to 2276.84 and 6476.56 mg/L at MCollapsed and TCollapsed stages, respectively, while the VMPR at MCollapsed and TCollapsed stages were both decreased to zero. Both the mesophilic and thermophilic anaerobic reactors experienced process failure caused by the severe accumulation of VFAs. The maximum OLRs for the stable mesophilic and thermophilic reactors were both 1.0 g VS/(L·d). The balance between acidogens and methanogens is indispensable for a stable AD system. However, methanogens are more vulnerable to the acid stress than acidogens, which finally led to the disturbance between the acidogens and methanogens and resulted in severe acidification [13]. In addition, VW is a type of carbohydrate-rich substrate with a weak buffering capacity in the AD process [14]. High temperatures accelerated the degradation of VW; thus, improved the average VMPR value from 0.16 ± 0.03 L/(L·d) at the mesophilic condition to 0.29 ± 0.02 L/(L·d) at the thermophilic condition when the two reactors operated at a stable OLR of 1.0 g VS/(L·d).
Table 1
Performance of mesophilic and thermophilic anaerobic reactors at different stages
| Mesophilic reactor (35 °C) | Thermophilic reactor (55 °C) |
Stage | MStable | MCollapsed | TStable | TCollapsed |
CH4 (%) | 54.31 ± 1.37 | 48.10 ± 4.17 | 55.11 ± 1.38 | 12.02 ± 16.68 |
Total VFAs (mg/L) | 113.38 ± 60.94 | 741.76 ± 653.07 | 72.24 ± 36.14 | 4,703.56 ± 1,037.49 |
Acetate (mg/L) | 89.86 ± 40.88 | 300.45 ± 152.41 | 56.78 ± 28.47 | 2,083.84 ± 319.61 |
Propionate (mg/L) | 15.30 ± 7.10 | 300.94 ± 360.44 | 11.28 ± 2.77 | 450.33 ± 92.40 |
TAN (mg/L) | 371.51 ± 81.13 | 203.16 ± 61.02 | 406.45 ± 66.09 | 264.49 ± 51.76 |
pH value | 6.82 ± 0.11 | 6.32 ± 0.22 | 6.91 ± 0.06 | 4.76 ± 0.64 |
Notes: MStable: Mesophilic stable stage, MCollapsed: Mesophilic collapsed stage. TStable: Thermophilic stable stage, TCollapsed: Thermophilic collapsed stage. |
3.2 Overall microbial diversity
The microbial composition and dynamics of the mesophilic and thermophilic reactors at the stable and collapsed stages were obtained through high-throughput sequencing technology based on the Illumina MiSeq platform. Moreover, the alpha diversity indexes, including the Simpson, Chao, and Simpsoneven indexes, were analyzed in this study to exhibit the differences in the microbial diversity at different temperatures and stages (Table 2). The Simpson index decreased as the microbial diversity increased, while the Chao and Simpsoneven indexes increased as microbial richness and evenness increased, respectively [15]. The Simpson index increased slightly from MStable to MCollapsed, and the Chao index decreased slightly from MStable to MCollapsed. This result reflects that the microbial diversity and richness decreased at MCollapsed. The accumulation of VFAs at MCollapsed resulted in the inhibition of microorganisms, which might lead to the reduction of most types of bacteria and methanogens. The decrease of the Simpsoneven index at MCollapsed also confirmed the aforementioned speculation that the microbial evenness was less in MCollapsed compared with that in MStable. A similar tendency was found for the thermophilic anaerobic system. The accumulation of the total VFAs at TCollapsed was far more than that at MCollapsed (Table 1), indicating that the severe acidification resulted in a clear reduction of most types of microorganisms at the thermophilic collapsed condition. The microbial diversity, richness, and evenness at MStable were slightly higher than those at TStable. Greses et al. found that the microbial diversity was higher at the mesophilic condition in comparison with that at the thermophilic condition using Scenedesmus spp as the substrate [16]. Vanwonterghem et al. also clarified that the increase of temperature from the mesophilic to thermophilic anaerobic conditions resulted in a limited population of microorganisms [17]. Therefore, temperature is one of the most important factors that influenced the microbial diversity.
Table 2
Summary of alpha diversity indexes
| Alpha diversity index |
Stage | Simpsona | Chaob | Simpsonevenc |
MStable | 0.088 ± 0.003 | 91.548 ± 5.680 | 0.150 ± 0.015 |
MCollapsed | 0.109 ± 0.046 | 83.824 ± 8.327 | 0.132 ± 0.052 |
TStable | 0.140 ± 0.034 | 71.675 ± 3.103 | 0.119 ± 0.035 |
TCollapsed | 0.586 ± 0.196 | 55.875 ± 12.929 | 0.041 ± 0.017 |
Notes: aDiversity of the microbial community, bRichness of microbial species, cDistribution evenness of the microbial community.
The principal coordinate analysis results of the microorganisms in both the mesophilic and thermophilic anaerobic reactors at different stages are exhibited in Fig. 2. Clusters 1, 2, 3, and 4 represent the samples from MStable, MCollapsed, TStable, and TCollapsed, respectively. The total microbial community showed a 43.04% variation in principal components 1. Under different temperature conditions, clusters 1 and 2 from the mesophilic condition located in the left side of the plot, and clusters 3 and 4 from the thermophilic condition located in the right side of the plot. The clear separation of the microbial communities under different temperature conditions showed that temperature changes significantly affect the microbial community shifting under the AD of VW. The microbial community showed a 23.83% total variation in principal components 2. Under the mesophilic condition, the samples from the stable and collapsed stages were separated into two clusters (clusters 1 and 2). However, the distance between clusters 1 and 2 was quite close, indicating that the microbial community structure was only slightly shifted under the mesophilic condition. However, all the samples from the stable and collapsed stages were distinctly divided into two clusters (cluster 3 and 4) under the thermophilic condition, demonstrating that the severe acidification caused dramatic shifts of the microbial community structure. The final collapse of the mesophilic and thermophilic AD of VW occurred at an OLR of 1.5 g VS/(L·d) and 2.5 g VS/(L·d), respectively. A high OLR led to a high VFA concentration; thus, inhibits the growth of the bacterial community more severe compared with a low OLR.
3.3 Dynamics of microbial community composition on phylum level
The results of the microbial community analysis that revealed the phylum level are shown in Fig. 3(a). Members of the phyla Bacteroidetes (33.33%), Cloacimonetes (19.70%), Chloroflexi (12.95%), and Euryarchaeota (10.70%) were the most abundant within MStable, followed by phyla Firmicutes (9.18%), Actinobacteria (6.39%), Synergistetes (3.28%), Atribacteria (1.68%), and Thermotogae (1.42%). Bacteroidetes (43.28%) and Spirochaetae (16.35%) were the most dominant phyla within the MCollapsed, followed by the phyla Euryarchaeota (7.84%), Chloroflexi (6.54%), Firmicutes (6.33%), Cloacimonetes (6.01%), Proteobacteria (4.89%), Actinobacteria (4.00%), Thermotogae (2.34%), and Synergistetes (1.44%). Among these phyla, Euryarchaeota was the only phylum that functioned as methanogens in the mesophilic reactor. Most genus within the phyla of Spirochaetae, Synergistetes, and Atribacteria functioned as syntrophic organic acid oxidation bacteria in the AD system [18–20]. While the remaining phyla were mainly attributed to the fermentation and degradation processes of the AD system. When the mesophilic anaerobic system collapsed, the abundance of Bacteroidetes notably increased, followed by a slight increase in the Thermotogae, indicating that these two phyla were more tolerant to the acidified condition. The Bacteroidetes and Thermotogae phyla play a significant role in the degradation of cellulose and protein under anaerobic conditions [18, 21]. The phyla of Spirochaetae, which existed uniquely in the collapsed stage, could be used as a microbial early warning indicator for the AD instability of VW.
Members of the phyla Firmicutes (46.98%), Euryarchaeota (33.77%), and Synergistetes (12.60%) were the most abundant within TStable, followed by Bacteroidetes (2.76%) and Atribacteria (2.63%). Meanwhile, Thermotogae (48.93%) and Firmicutes (37.72%) were the most dominant phyla within the TCollapsed, followed by Euryarchaeota (5.48%), Chloroflexi (4.79%), Atribacteria (1.34%), and Bacteroidetes (1.17%). The phyla Firmicutes was dominant in both the thermophilic stable and collapsed stages; however, its abundance in the collapsed stage was lower than it in the stable stage. Thermotogae was the most abundant phyla that existed uniquely in the thermophilic collapsed stage, demonstrating the higher adaptability of Thermotogae to the acidic condition. Thus, Thermotogae might play a key role in relieving the further acidification, which has been reported to degrade various organic substrates under acidic anaerobic conditions [22]. The abundance of the phyla Euryarchaeota and Synergistetes sharply decreased at the collapsed stage, which was consistent with the final failure of AD of VW.
3.4 Dynamics of microbial community composition on genus level
Details regarding the major microbial composition and abundance on the genus level were analyzed to further explore the dynamics of the microbial community (Fig. 3(b)). The most abundant genera from MStable were Candidatus_Cloacamonas (19.7%) and vadinBC27_wastewater-sludge_group (11.05%), followed by unclassified_f__Anaerolineaceae (8.68%), Proteiniphilum (7.78%), Petrimonas (6.55%), Propionimicrobium (6.39%), Methanosaeta (6.10%), norank_f__Anaerolineaceae (4.14%), Methanosarcina (4.08%), and Microbacter (3.43%). In contrast, the most abundant genera from MCollapsed were Bacteroides (15.11%), vadinBC27_wastewater-sludge_group (14.01%), and Sphaerochaeta (10.15%), followed by Candidatus_Cloacamonas (6.01%), Methanosaeta (5.65%), norank_f__V2072-189E03 (5.61%), norank_f__Anaerolineaceae (4.92%), Aeromonas (4.09%), norank_f__Porphyromonadaceae (4.02%), and Propionimicrobium (4.00%). According to previous studies, the specific functions of each major genus from the mesophilic and thermophilic anaerobic reactors are listed in Table 3. At the mesophilic stable stage, the genera Candidatus_Cloacamonas and vadinBC27_wastewater-sludge_group were classified as syntrophic bacteria. Candidatus_Cloacamonas was able to digest cellulose and use hydrolyzing products to produce H2 and CO2, and VadinBC27_wastewater-sludge_group was reported to degrade amino acids in syntrophic association with hydrogenotrophic methanogens. Methanosaeta and Methanosarcina were the only two dominant methanogens in the mesophilic stable stage, which functioned as acetoclastic and mixotrophic (i.e., acetoclastic, hydrogenotrophic, and methylotrophic) methanogens, respectively [18, 23]. Thus, only Methanosarcina has the ability of syntrophic association with Candidatus_Cloacamonas and vadinBC27_wastewater-sludge_group, which mainly uses H2 and CO2 to produce methane. The genera unclassified_f__Anaerolineaceae, Proteiniphilum, Petrimonas, Propionimicrobium, norank_f__Anaerolineaceae, and Microbacter functioned as fermentative and acidogenic bacteria that degraded glucose to produce acetic and propionic acids. The dominant microbes changed clearly from the mesophilic stable to the collapsed stages. The most abundant bacteria shifted from syntrophic bacteria in the mesophilic stable stage to fermentative, acidogenic, and syntrophic bacteria in the mesophilic collapsed stage. Among the three most dominant genera of Bacteroides, vadinBC27_wastewater-sludge_group, and Sphaerochaeta in the mesophilic collapsed stage, two are fermentative and acidogenic bacteria (i.e., Bacteroides and Sphaerochaeta). Bacteroides are reported to have the ability to hydrolyze a complex insoluble substrate, such as polysaccharides, while Sphaerochaeta can ferment carbohydrates to produce acetate, formate, and ethanol (Table 3). Candidatus_Cloacamonas, which was the most abundant syntrophic bacteria, decreased dramatically form the mesophilic stable (19.7%) to the collapsed (6.01%) stage. The only methanogen that existed in the top ten genera at the mesophilic collapsed stage was Methanosaeta, compared with the two dominant methanogens (i.e., Methanosaeta and Methanosarcina) at the stable stages. The decrease of methanogens corresponded with the reduction of syntrophic bacteria at the mesophilic collapsed stage. These results reveal that fermentative and acidogenic bacteria were dominant occupants in the mesophilic collapsed stage, compared with syntrophic bacteria in the stable stage. When the performance of the AD of VW changed from the stable to collapsed state, caused by the accumulation of VFAs, the abundance of fermentative and acidogenic bacteria considerably shifted. All the fermentative and acidogenic bacteria of unclassified_f__Anaerolineaceae, Proteiniphilum, Petrimonas, and Microbacter were eliminated from the top ten genera at the mesophilic collapsed stage, and instead comprised the genera of Bacteroides, Sphaerochaeta, norank_f__V2072-189E03, Aeromonas, and norank_f__Porphyromonadaceae. This result indicates that the genera of Bacteroides, Sphaerochaeta, norank_f__V2072-189E03, Aeromonas, and norank_f__Porphyromonadaceae prefer to thrive under acidic environmental conditions, while the genera of unclassified_f__Anaerolineaceae, Proteiniphilum, Petrimonas, and Microbacter are vulnerable to high concentration of VFAs.
Table 3
Functional descriptions of the dominant genera that existed in the mesophilic and thermophilic anaerobic reactors
Genus | Functional description | Reference |
VadinBC27_wastewater-sludge_group | Syntrophic bacteria; degrade proteins and carbohydrates in syntrophic association with hydrogenotrophic methanogens under anaerobic condition. | [26] |
Candidatus_Cloacamonas | Syntrophic bacteria; digest cellulose, propionate, and amino acid to produce H2 and CO2. | [22] |
Bacteroides | Secrete different hydrolyzing enzymes, such as cellulase, to hydrolyze a complex insoluble substrate, such as polysaccharides. | [27] |
Sphaerochaeta | Ferment carbohydrates to produce acetate, formate, and ethanol. | [28] |
Unclassified_f__Anaerolineaceae | Anaerobic fermentative and acetogenic bacteria. | [29] |
Propionimicrobium | Growth under anaerobic conditions and produce propionic acid. | [30] |
Norank_f__Anaerolineaceae | Convert glycerol into VFAs. | [31] |
Proteiniphilum | Ferment organic substrates to produce acetic and propionic acids. | [32] |
Petrimonas | Anaerobic fermentative and acetogenic bacteria. | [29] |
Norank_f__V2072-189E03 | Ferment polysaccharides to produce acetate, formate, and ethanol. | [33] |
Microbacter | Fermentative bacteria in the anaerobic digestion system. | [34] |
Aeromonas | Ferment glucose to produce acetate in the anaerobic digestion system. | [35] |
Norank_f__Porphyromonadaceae | Ferment glucose to produce lactate, acetate, butyrate, and isobutyrate. | [23] |
Defluviitoga | Degrade biomass to produce acetate. | [36] |
Thermoanaerobacterium | Degrade lignocellulose to produce acetic acid, butyric acid, and lactic acid. | [37] |
Anaerobaculum | Ferment a range of amino acids to acetate, propionate, and hydrogen. | [38] |
Clostridium_sensu_stricto_8 | Acetogens; conduct extracellular electron transfer and reduce Fe3+. | [39] |
Clostridium_sensu_stricto_1 | Acetogens; conduct extracellular electron transfer and reduce Fe3+. | [39] |
Ruminiclostridium | Hydrolyze cellulosic substrates and ferment the resulting sugars to ethanol. | [40] |
Ruminiclostridium_1 | Hydrolyze cellulosic substrates and ferment the resulting sugars to ethanol. | [40] |
Mobilitalea | Hydrolyze celluloses, peptides and fermenting amino acids, and carbohydrates to produce acetate, butyrate, lactate, CO2, and H2. | [41] |
Bacillus | Fermentative bacteria; functioned as lignin degraders as they can decompose lignin with little consumption of cellulose. | [42] |
Anaerolinea | Use carbohydrates to produce hydrogen and acetic acid. | [43] |
Candidatus_Caldatribacterium | Thermophilic bacteria; produce acetate from sugar fermentation. | [44] |
Norank_o__D8A-2 | Syntrophic acetate-oxidizing bacteria (SAOB). | [45] |
Norank_o__MBA03 | SAOB. | [46] |
Gelria | SAOB. | [12] |
Syntrophaceticus | SAOB | [47] |
Defluviitalea | Acid-producing bacteria. | [28] |
Norank_f__Syntrophomonadaceae | Syntrophic butyrate oxidizers in anoxic environments. | [48] |
Norank_f__Synergistaceae | Can degrade amino acids into volatile fatty acids and contribute to acidogenesis and acetogenesis via syntrophic relationships with methanogens. | [49] |
Norank_f__Lentimicrobiaceae | Denitrification under anaerobic condition. | [50] |
Coprothermobacter | Protein-degrading bacteria that established a syntrophy with hydrogenotrophic methanogens. | [51] |
Norank_p__Atribacteria | Specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate. | [52] |
Mesotoga | Anaerobic mesophilic acetogens. | [53] |
Hydrogenispora | Ferment glucose to produce acetate, ethanol, and hydrogen. | [54] |
Norank_f__Ruminococcaceae | Fermentative bacteria with the ability of fiber degradation. | [55] |
Methanosaeta | Acetoclastic methanogens that use acetate to produce methane. | [23] |
Methanosarcina | Mixotrophic methanogens that can perform acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. | [23] |
Methanothermobacter | Hydrogenotrophic methanogens that can use H2 as a precursor to produce methane. | [56] |
The most abundant genera from TStable were Methanosarcina (30.58%) and Anaerobaculum (12.59%), followed by Bacillus (9.65%), Syntrophaceticus (4.84%), Defluviitalea (4.66%), Mobilitalea (3.97%), Ruminiclostridium (3.65%), norank_o__D8A-2 (3.29%), Methanothermobacter (3.17%), and norank_f__Syntrophomonadaceae (3.08%). For TCollapsed, the most abundant genera were Defluviitoga (48.93%) and Thermoanaerobacterium (29.08%), followed by Clostridium_sensu_stricto_8 (4.92%), Anaerolinea (3.46%), Methanosarcina (2.77%), Methanothermobacter (2.70%), Candidatus_Caldatribacterium (1.33%), norank_f__Anaerolineaceae (1.29%), Coprothermobacter (1.27%), and norank_f__Lentimicrobiaceae (1.12%). Based on the summary of functional microorganisms in Table 3, the genera of Anaerobaculum, Bacillus, Defluviitalea, Mobilitalea, and Ruminiclostridium functioned as fermentative bacteria, and Syntrophaceticus, norank_o__D8A-2, and norank_f__Syntrophomonadaceae were classified as syntrophic bacteria. Methanothermobacter acted as hydrogenotrophic methanogen that can use H2 as a precursor to produce methane. Syntrophaceticus and norank_o__D8A-2 have been reported to be syntrophic acetate-oxidizing bacteria (SAOB), which possess the syntrophic acetate-oxidizing ability in cocultivation with a hydrogen-utilizing methanogen. The coexistence of Methanothermobacter, Syntrophaceticus, and norank_o__D8A-2 showed that a syntrophic acetate-oxidizing association was developed in the thermophilic stable stage. When the process failure of the thermophilic AD of VW occurred at the TCollapsed, the genus Defluviitoga were dominant, and the abundance increased from 4.66% at TStable to 48.93% at TCollapsed. Defluviitoga was revealed to act as acetogens that degraded various biomass to produce acetate under the anaerobic condition. Another dominant genus was Thermoanaerobacterium, which uniquely existed in TCollapsed. Meanwhile, Thermoanaerobacterium exhibited the ability to degrade lignocellulose to produce acetic, butyric, and lactic acids, and was classified as fermentative and acidogenic bacteria. These results indicate that both Defluviitoga and Thermoanaerobacterium were more tolerant to a highly acidic environment. Furthermore, the dominant functional microbial consortium changed from methanogens and fermentative bacteria at the stable stage to fermentative bacteria at the collapsed stage. All the methanogens decreased at the collapsed stage, particularly for the abundance of Methanosarcina that decreased from 30.58% at the stable stage to 2.77% at the collapsed stage. The sharp decrease of methanogens and the sharp increases of fermentative and acidogenic bacteria both explain the occurrence of the severe accumulation of organic acids in the thermophilic collapsed stage. These results also verify that methanogens are vulnerable to the accumulation of VFAs.
3.5 Dynamics of methanogen communities
The dynamics of methanogens among the archaea at different temperatures are presented in Fig. 4. The acetoclastic methanogen of Methanosaeta (59.16%) and the mixotrophic methanogen of Methanosarcina (34.73%) were the dominant methanogens at the mesophilic stable condition, reflecting that the most dominant methanogen in the mesophilic reactor was acetoclastic methanogen, which uses acetate to produce methane. Meanwhile, the most dominant methanogens that existed in the thermophilic reactor were Methanosarcina and Methanothermobacter. Methanothermobacter was reported to function as a hydrogenotrophic methanogen, which can use H2 as a precursor to produce methane (Table 3). The relative abundance of Methanosarcina accounted for 90.71% at the thermophilic stable condition, but sharply decreased to 63.61% when the severe acidification occurred at TCollapsed. Nonetheless, the relative abundance of Methanothermobacter dramatically increased from 9.16% at TStable to 36.01% at TCollapsed, indicating that Methanothermobacter is more favorable to survive under a severe acidic environment. These results demonstrate that temperature can change the methanogenesis pathway in a large extent. Furthermore, methanogenesis might shift from acetoclastic in the mesophilic stable stage to a hydrogenotrophic pathway in the thermophilic stage. However, this result should be further confirmed via genomic and metabonomics analyses.
3.6 Differences in microorganisms at mesophilic and thermophilic stable stages
The differences in the microorganisms at the mesophilic and thermophilic stable stages were compared to determine the effect of temperature on the distribution of dominant genera at the stable operational condition of the AD of VW (Fig. 5). The abundances of Candidatus_Cloacamonas (P < 0.01), Propionimicrobium (P < 0.01), Proteiniphilum (P < 0.05), unclassified_f__Anaerolineaceae (P < 0.05), and Petrimonas (P < 0.05) were significantly higher in the mesophilic stable stage. In contrast, the abundances of Anaerobaculum (P < 0.01) and Mobilitalea (P < 0.05) were significantly higher in the thermophilic stable stage. All of these genera were classified as fermentative and acidogenic bacteria (Table 3). Candidatus_Cloacamonas functioned as both syntrophic and fermentative bacteria. Propionimicrobium, Proteiniphilum, unclassified_f__Anaerolineaceae, and Petrimonas were all reported to ferment biomass to produce acetate. Anaerobaculum and Mobilitalea were both found to hydrolyze celluloses and other polysaccharides, peptides and fermenting amino acids, and carbohydrate to produce acetate, butyrate, lactate, CO2, and H2. The abundances of syntrophic bacteria of Syntrophaceticus (P < 0.01) and norank_f__Syntrophomonadaceae (P < 0.05) were both significantly higher in the thermophilic stable condition. Norank_f__Syntrophomonadaceae functioned as syntrophic butyrate oxidizers, and Syntrophaceticus belonged to SAOB, which are highly associated with hydrogen-utilizing methanogens via syntrophic acetate-oxidizing to produce methane [24]. The abundance of Methanosaeta (P < 0.05) was significantly higher in the mesophilic stable condition, while the abundance of Methanosarcina (P < 0.05) was significantly higher in the thermophilic stable condition.
An overall comparison of the functional microbial consortium is exhibited in Fig. 6. The microorganisms were classified into five functional types, including fermentative and acidogenic bacteria, syntrophic organic acid oxidation bacteria, SAOB, homoacetogenic bacteria, and methanogens. The syntrophic acetate-oxidizing relationship between the syntrophic bacteria of Syntrophaceticus and the hydrogenotrophic methanogens of Methanothermobacter and Methanosarcina developed under the thermophilic stable condition. For the mesophilic reactor, acetoclastic methanogenesis was the dominant process in the overall stable operation. Pap et al. indicated that a high temperature will accelerate the degradation of most types of organic substrate; thus, speeding up the accumulation of VFAs [25]. Generally, Methanosaeta was more vulnerable to the stress of VFAs compared to Methanosarcina [13]. Therefore, the dynamic shifts of the functional microbial community from acetoclastic methanogenesis to syntrophic acetate-oxidizing coupled with hydrogenotrophic methanogenesis resulted in the accumulation of VFAs under the thermophilic condition.