Effects of Stepwise Temperature Shifts in Anaerobic Digestion for Treating Municipal Wastewater Sludge: A Genomic Study

In wastewater treatment plants (WWTP), anaerobic digester (AD) units are commonly operated under mesophilic and thermophilic conditions. In some cases, during the dry season, maintaining a stable temperature in the digester requires additional power to operate a conditioning system. Without proper conditioning systems, methanogens are vulnerable to temperature shifts. This study investigated the effects of temperature shifts on CH4 gas production and microbial diversity during anaerobic digestion of anaerobic sewage sludge using a metagenomic approach. The research was conducted in lab-scale AD under stepwise upshifted temperature from 42 to 48 °C. The results showed that significant methanogen population reduction during the temperature shift affected the CH4 production. With 70 days of incubation each, CH4 production decreased from 4.55 L·g−1-chemical oxygen demand (COD) at 42 °C with methanogen/total population (M·TP−1) ratio of 0.041 to 1.52 L·g−1 COD (M·TP−1 ratio 0.027) and then to 0.94 L·g−1 COD ( M·TP−1 ratio 0.026) after the temperature was shifted to 45 °C and 48 °C, respectively. Methanosaeta was the most prevalent methanogen during the thermal change. This finding suggests that the Methanosaeta genus was a thermotolerant archaea. Anaerobaculum, Fervidobacterium, and Tepidanaerobacter were bacterial genera and grew well in shifted-up temperatures, implying heat-resistant characteristics.


Introduction
Water resources and environmental protection policies worldwide have mandated thorough treatment of wastewater prior to discharge into water bodies [1]. Activated sludge treatment is a common wastewater treatment method [2]. However, the main issue regarding this type of treatment is sludge generated from primary sedimentation (PS) and activated sludge (AS). Sludge produced from wastewater treatment plants (WWTP) is produced in large volumes worldwide with up to 8910, 6510, 2960, 650, 580, 550, and 370 thousand metric tons of dry sludge produced annually by EU countries, the United States, China, Iran, Turkey, Canada, and Brazil, respectively [3]. Considering the substantial volumes of waste production, it is not surprising that WWTP sludge management and disposal have become an area of significant concern globally [4]. As a result of its high water content, low dewaterability, and rigorous regulations for sludge reuse and disposal, sludge management is a demanding and complicated issue in wastewater treatment plants [5].
In some countries, landfill is the most favorable disposal method [6]. However, due to the volume of sludge produced and the availability of the land area, attention has shifted to the development of other potential usable products. Currently, the wastewater treatment paradigm has shifted to an environmentally friendly process to reduce the volume of resistant characteristics and possible benefits of AD in WWTP-scale applications, further research regarding thermotolerant microorganisms needs to be performed, especially to investigate their potential for biogas production. Therefore, the objectives of this study were to investigate the potential of biogas production (especially CH 4 ) and to identify the most biogas-producing microorganisms among the cultures after being shifted to several temperature levels.

Inoculum and Substrates
The inoculum (I) originated from anaerobically digested sludge obtained from a municipal sewage treatment plant located in Ube City, Yamaguchi Prefecture, Japan. The inoculum characteristics are listed in Table 1. The inoculum samples were then mixed with the substrate (S) solution before being placed in an incubator and exposed to gradually elevated temperature conditions. The substrate used for biogas production in this research was a glucose-based synthetic wastewater consisting of 1.5 g·L −1 glucose, 2 mg·L −1 NaHCO 3 , 2 mg·L −1 K 2 HPO 4 , 1 g·L −1 yeast extract, 0.7 g·L −1 (NH 4 ) 2 HPO 4 , 0. 75  Glucose was chosen as the ideal carbon source for microbial metabolic transformations in the fermentation process, and is also a readily biodegradable substance abundantly found in municipal wastewater [26,27]. To obtain the maximum CH 4 potential, the appropriate ratio between the microorganisms and substrate must be determined [28]. The CH 4 yield, in theory, is independent of the inoculum to substrate ratio (I·S −1 ), and the I·S −1 ratio should influence only the kinetics of CH 4 production [29]. Table 1. Characteristics of anaerobic sludge as inoculum.

Parameters
Anaerobic Sludge Units pH 7.09 pH = −log 10 [a(H + )] Total Solid (TS) 8000 mg/L Volatile Solid (VS) 3000 mg/L Fixed Solid (FS) 5000 mg/L VS/TS ratio 0. 37 -In contrast, previous studies have demonstrated that the I·S −1 ratio can impact both the CH 4 yield and production rate, as significant evidence suggested that the ratio directly influences the microorganism growth pattern [30][31][32]. Referring to the German Standard VDI4630, the I·S −1 ratio should be adjusted to more than two [33]. Other studies have discovered that a higher I·S −1 ratio generates more biogas on a consistent basis during the AD process, while a lower I·S −1 ratio produces less biogas due to the lower pH and accumulation of volatile fatty acids (VFAs) [34,35]. In this study, the I·S −1 ratio maintained at approximately 3.0, which indicates that 1 mL of substrate was added for every 3 mL of inoculum.

Experimental Procedures
Laboratory-scale anaerobic digester containers were later defined as vials. A total volume of 160 mL was prepared. To ensure obligate anaerobic conditions, the vial was filled with pure nitrogen gas to flush the remaining oxygen. Subsequently, the vials were capped with butyl rubber stoppers and aluminum caps. Since the laboratory-scale vials were used as the reactor in this research, there were some potential risks that may emerge, such as lower capacity to contain total biogas production, low sample provision to perform several monitoring parameters, and possibility that the sensitivities and instabilities in the laboratory scale reactors do not represent that in the full-scale digesters [36]. This research was divided into two categories: temperature shifted-up (shift-up) and controlled temperature condition. For the shift-up research, in phase 1, batch experiments were performed by adding 110 mL of sludge as inoculum to a vial mixed with 40 mL of substrate. The mixture was then incubated at 42 • C with shaking at 50 rpm for the first two weeks. This action was intended to enhance the growth of microorganisms and ensure the availability of nutrients during acclimatization.
After the first two weeks, phase 2 was initiated. Every time gas production declined sharply, up to 2 mL of substrate with a chemical oxygen demand (COD) of 2000 mg·L −1 was injected into the vial. This treatment altered the reactor system from a batch reactor to a fed-batch reactor system. The incubation was continued for 70 days of incubation period in the fed-batch reactor system. Every 70 days, the temperature was increased by 3 • C for the shift-up research until it reached 45 • C and 48 • C. Meanwhile, the controlled temperature research was carried out at 45 • C and 48 • C from the beginning of incubation without any temperature shifts. This study was intended to compare the biogas production and microbial communities among the shifted condition and stabilized condition. During the fermentation period, the total gas volume and composition were measured daily using gas chromatography.
A glass syringe was used to measure the volume of the biogas produced. The gas composition of the samples, such as H 2 , N 2 , CH 4 , and CO 2 , was determined using gas chromatography (GC-8APT/TCD; Shimadzu Co., Kyoto, Japan) with 60/80 activated charcoal mesh column (1.5 m × 3.0 mm internal diameter) and argon gas as the carrier gas. During operation, the temperatures of the injector, column, and detector were adjusted to 50 • C, 60 • C, and 50 • C, respectively.

Deoxyribonucleic Acid (DNA) Extraction and Sequencing
DNA was extracted according to the NucleoSpin ® soil manual. Sludge samples were prepared using an MN Bead Tube Type A (MACHEREY-NAGEL GmbH & Co., Düren, Germany). KG was mixed with lysis buffer SL1 and lysed using Enhancer SX. Contaminants were precipitated using lysis buffer SL3, and the lysate was filtered using a NucleoSpin ® Inhibitor Removal Column. Subsequently, the binding conditions were adjusted using Binding Buffer SB. DNA was bound by loading 550 µL sample on the NucleoSpin ® Soil Column. After the binding phase, the silica membrane was washed with binding buffer SB, wash buffer SW1, and SW2. Finally, the DNA was eluted using SE elution buffer. DNA samples were then delivered to the Faculty of Medicine, Yamaguchi University, Japan, for next-generation sequencing.
Next generation sequencing (NGS) was performed to acquire a broad range of genes or gene regions from phylum to genus using the 16 s ribosomal ribonucleic acid (RNA) gene amplicons for the Illumina MiSeq System, wherein DNA or RNA are sequenced using hybrid capture or amplicon-based approaches (previously transcribed into complementary DNA). Using these approaches, the genome (all 3 billion base pairs), all coding genes (exome; 1% of the genome or 30 million base pairs-that is 20,000 genes made of 180,000 exons), all RNA produced from genes (transcriptome), and any subset of these can be sequenced [37].

Microbial Diversity Analysis
Diversity index analysis was conducted to determine possible changes in the microbial communities during the anaerobic digestion process. A diversity index is a numerical measure of how many distinct types (such as species) are present in a dataset (a community), as well as the evolutionary relationships among individuals dispersed throughout those types, such as richness, divergence, and evenness [38]. In this study, Simpson's diversity index, Shannon's diversity index, and Shannon's equitability index were utilized.

Biogas Production under Shifted-Up Temperature
The fluctuation in daily CH 4 production and cumulative CH 4 production during the first incubation at 42 • C is shown in Figure 1. During the batch anaerobic digestion period, biogas production increased gradually with the cumulative volume of biogas generated being up to 316.5 mL on day 14, with cumulative CH 4 production being up to 120.76 mL CH 4 . The CH 4 content in the biogas increased rapidly in the first 8 days, reaching 69% on the 8th day, and 64.7% on average until day 13. Cumulative CH 4 production increased gradually in the first 3 days, and then showed a substantial increase on day 7 (66.59 mL CH 4 ). On the final day of the batch period, the cumulative methane production reached 120.76 mL CH 4 which was equal to 1.43 L·g −1 COD feed. Theoretically, the energy recovery (expressed as CH 4 production) from digested wastewater sludge through anaerobic process was 0.38 L·g −1 COD [39]. From this study, it took approximately four days of anaerobic digestion to exceed the theoretical CH 4 production with the aforementioned inoculum and substrates, with an I·S −1 ratio of 2.75.
However, on the 14th day, methane production sharply decreased to 0 mL. Declining CH 4 production indicates a dead phase of methanogenic activity [40]. This drawdown in CH 4 production may also be linked to a decrease in pH caused by the interaction of VFAs with other fragmented precursors during oxidative processes [41]. To maintain CH 4 production, 2 mL of the substrate was injected into the vial when CH 4 production started diminishing due to the scarcity of nutrients. As illustrated in Figure 1a, substrate injection led to a spike in CH 4 production as the activity of methanogenic microorganisms increased due to the availability of glucose as a carbon source and other nutrients that expedited microbial growth. Consequently, as shown in Figure 1a*, the cumulative volume of methane produced increased significantly after substrate addition. At the end of the incubation period at 42 • C, the cumulative CH 4 production was observed to increase to 454.5 mL CH 4 , with a yield of 4.55 L·g −1 COD feed.
After incubation for 70 days at 42 • C, the incubation temperature was shifted to 45 • C with the same treatment conditions and hydraulic retention times. Even though the same sample was utilized, the cumulative CH 4 calculation was restarted from 0 mL CH 4 . As presented in Figure 1b, the daily CH 4 production peaked at 17.2 mL CH 4 which was obtained on day 60 after receiving the 4th feed. From the Figure 1b*, the cumulative CH 4 produced and yield after 70 days of incubation was 152.2 mL CH 4 and 1.52 L·g −1 COD feed, respectively. This was less than the volume of CH 4 produced during incubation at 42 • C by a factor of three. There was also a significant difference in CH 4 production behavior after the temperature was increased to 45 • C, e.g., a shorter period required to shift from peak days to trough days, which denotes faster methanogenesis and death phase for methanogenic bacteria. This shorter methanogenesis phase can be attributed to the higher concentration of CO 2 produced during the incubation period.
As shown in Figure 1c, CH 4 production decreased further when the temperature was increased to 48 • C. After 70 days of incubation, only 86.57 mL of CH 4 was produced, with a CH 4 yield of 0.94 L·g −1 COD feed. The CH 4 production trend after the 3rd feed at shiftedup 45 • C is a good illustration of the potential inhibition of methanogenic bacterial activity by the presence of high CO 2 levels ( Figure 2). The high CO 2 levels indicated the occurrence of acetoclastic methanogenesis in the AD process, which later led to the abundant presence of VFAs, particularly acetic acid [42]. The decrease in CH 4 was also parallel to the decrease in overall biogas production consisting of H 2 , N 2 , CH 4 and CO 2, which is illustrated in Figure 3a. The total biogas production decreased from 1161.93 mL (11.61 L·g −1 COD feed) at 42 • C to 672 mL (6.7 L·g −1 COD feed) and then to 505 mL (5.49 L·g −1 COD feed) after the temperature was shifted to 45 • C and 48 • C, respectively. The decreasing CH 4 production volume after being shifted-up to the higher temperature at 45 • C and 48 • C was followed by the decline in CH 4 content in biogas compositions as seen in Figure 3b-d.
At the end of incubation period, the concentration of COD, total suspended solids (TSS), volatile suspended solids (VSS), and pH were measured for each temperature condition. As seen in Table 2, the concentration of COD was increasing up to 3-fold, in contrast to TSS and VSS that significantly decreased every temperature shift. This finding indicates that the number of microbial communities (represented by VSS) declined every upshifted thermal condition and subsequently causing a depletion on microbial activity, which even-tually resulted in the lower organic matter consumed by microorganisms in the reactor. The pH is another parameter that affects digestion process. The pH increased substantially from 7.64 at 42 • C to 8.20 and 8.33 when the temperature was shifted-up to 45 • C and 48 • C, respectively. Subsequently, the biogas production decreased along with the rising pH. This finding supports a research study from Kouzi et al. [43] who discovered that the optimum pH range for sewage sludge AD was 7.0, while the biogas production was considerably lower in the reactors with higher pH of 8.0, 9.0, and 10.0.

Biogas Production under Shifted-Up Temperature
The fluctuation in daily CH4 production and cumulative CH4 production during the first incubation at 42 °C is shown in Figure 1. During the batch anaerobic digestion period, biogas production increased gradually with the cumulative volume of biogas generated being up to 316.5 mL on day 14, with cumulative CH4 production being up to 120.76 mL CH4. The CH4 content in the biogas increased rapidly in the first 8 days, reaching 69% on the 8th day, and 64.7% on average until day 13. Cumulative CH4 production increased gradually in the first 3 days, and then showed a substantial increase on day 7 (66.59 mL CH4). On the final day of the batch period, the cumulative methane production reached 120.76 mL CH4 which was equal to 1.43 L·g −1 COD feed. Theoretically, the energy recovery (expressed as CH4 production) from digested wastewater sludge through anaerobic process was 0.38 L·g −1 COD [39]. From this study, it took approximately four days of anaerobic digestion to exceed the theoretical CH4 production with the aforementioned inoculum and substrates, with an I·S −1 ratio of 2.75.  However, on the 14th day, methane production sharply decreased to 0 mL. Declining CH4 production indicates a dead phase of methanogenic activity [40]. This drawdown in CH4 production may also be linked to a decrease in pH caused by the interaction of VFAs with other fragmented precursors during oxidative processes [41]. To maintain CH4 production, 2 mL of the substrate was injected into the vial when CH4 production started diminishing due to the scarcity of nutrients. As illustrated in Figure 1a, substrate injection led to a spike in CH4 production as the activity of methanogenic microorganisms in-  °C was followed by the decline in CH4 content in biogas compositions as seen in Figure  3b-d.  At the end of incubation period, the concentration of COD, total suspended solids (TSS), volatile suspended solids (VSS), and pH were measured for each temperature condition. As seen in Table 2, the concentration of COD was increasing up to 3-fold, in contrast to TSS and VSS that significantly decreased every temperature shift. This finding  °C was followed by the decline in CH4 content in biogas compositions as seen in Figure  3b-d.  At the end of incubation period, the concentration of COD, total suspended solids (TSS), volatile suspended solids (VSS), and pH were measured for each temperature condition. As seen in Table 2, the concentration of COD was increasing up to 3-fold, in contrast to TSS and VSS that significantly decreased every temperature shift. This finding

Alpha Diversity Analysis
To study the changes in microbial diversity at the upshift temperature, samples from the reactor were used for DNA isolation for bioinformatic analysis using NGS. For comparison, several samples from other fed-batch reactors with controlled temperatures of 42 • C, 45 • C, and 48 • C were examined for their microbial diversity. This action was intended to elucidate the differences among microbial communities and compositions that matured under controlled and shifted-up temperatures. The Shannon diversity index (SDI) and Simpson index were used to measure and compare the richness of the microbiota at a certain temperature, while the Shannon equitability index (SEI) was assigned to approximate the evenness of the microbiota diversity.
As illustrated by Figure 4a,b, the index value for both richness and evenness of the microbiota communities spread within the range 3.2-3.7 and 0.46-0.54, respectively. The Simpson index, as seen in Figure 4c, ranged from 0.89 to 0.96, indicating high diversity for all sample conditions. The deviation of the diversity index between the shifted-up temperature conditions and controlled temperature conditions was not significantly discerned, which signifies that each reactor has a close similarity of microbiota abundance and species to each other. However, compared to the controlled temperature conditions, the shifted-up temperature conditions showed a significant drop in microbiota diversity, with an increase in temperature. The diversity index value declined from 3.72 at 42 • C to 3.22 at 48 • C. This indicated that several bacteria communities were vanished during the temperature shift. This was also confirmed by the decrease in the equitability index value; however, since the equitability values were greater than 0.1, some microorganism colonies managed to acclimatize to this chaotic condition and experienced massive growth while the other colonies became extinct. The effects of temperature on diversity were confirmed using analysis of variance (ANOVA), with significance of p < 0.05. The probability value (p-value) for all diversity indices (SDI, SEI, and Simpson index) was p < 0.001, which signified that there were statistically significant differences in relative abundances and diversity indices between several temperature conditions.

Microbial Community Structure
Overall, the number of methanogens decreased sharply when the temperature was shifted from 42 °C to 45 °C, as shown in Figure 5.

Microbial Community Structure
Overall, the number of methanogens decreased sharply when the temperature was shifted from 42 • C to 45 • C, as shown in Figure 5. Methanosaeta was the most dominant methanogenic archaea that existed during incubation at 42 • C, shifted up to 45 • C, subsequently shifted up to 48 • C, and also abundant during incubation atcontrolled temperatures of 45 • C and 48 • C. This result implied that Methanosaeta is a thermotolerant methanogen. The relative abundances of methanogens at the order level at various temperatures are shown in Figure 6a Figure 6c). However, during the incubation at a controlled temperature of 48 • C, the dominance of Methanosaeta was lower than under shifted-up temperature (84% of the total methanogens at shifted-up 48 • C and 51% at controlled 48 • C) while Methanosarcina genes were detected up to 27% of the total methanogens. These findings confirmed the results of Figeac et al. [44] who discovered that the family Methanosarcinaceae was the most abundant acetotrophic archaea in the initial thermophilic inoculum, whereas the Methanosaetaceae family was mostly found in the initial mesophilic inoculum. Therefore, the population of Methanosaeta with an initial temperature of 48 • C was considerably lower than that in the upshift condition that was initially acclimatized under mesophilic conditions (at 42 • C). Apart from acetotrophic methanogens, hydrogenotrophic methanogens, such as members of genera Methanobacterium, Methanobrevibacter, and Methanothermobacter, started to grow significantly after the reactor temperature was shifted up to 48 • C. Hydrogenotrophic methanogens favor thermophilic conditions, whereas acetotrophic methanogens cannot resist high temperatures [45].    At the family level, as illustrated in Figure 6b, the methanogen communities were composed of Methanosaetaceae, Methanomicrobiaceae, Methanoregulaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanospirillaceae.  Methanobacterium communities grew from 2.5% to 7.4% of the total methanogen population, and Methanobrevibacter population ranged from 0.76% to 1.64% at a shifted-up temperature of 42-48 • C. This finding contradicts previous research reporting that Methanobacterium genera were mostly found at lower mesophilic temperature (24-35% at 35 • C and 32-45% at 37 • C) and eradicated with increasing the temperature to 55 • C [44,46]. Methanobrevibacter genera were also found to be the dominant methanogens at 24 • C and 35 • C and vanished at 55 • C [44]. However, the researchers did not examine the existence of Methanobrevibacter genera at higher mesophilic temperatures (42-48 • C). Judging from the results of previous studies that showed Methanobacterium and Methanobrevibacter were abundant in the lower mesophilic conditions, our research reported a significant spike in the population of those methanogens after shifting up temperatures to higher mesophilic conditions, signifying that Methanobacterium and Methanobrevibacter genera potentially have heat-resistant characteristics that allow them to compromise the staggering increase in temperature conditions. Lastly, Methanothermobacter population increased from 0.17% to 0.24% relative abundance at 42-48 • C. This is not surprising as Methanothermobacter genera is a thermophilic methanogen that dominated methanogenesis at temperatures of 50 • C and higher [47][48][49][50].
The distribution of non-methanogenic bacteria is also an important factor for determining the influence of several bacteria on CH 4 production. As seen in Figure 7a Clostridia was found to be dominant at both mesophilic (this research) and thermophilic temperatures (at 52 • C) [51], indicating that the Clostridia order belongs to thermotolerant bacteria. According to the experimental results, Synergistia were found at higher mesophilic temperatures, but in some cases, were also found abundantly at low temperatures of 20 • C [52]. This suggested that Synergistia was resistant to both low-and high-temperature environments, leading to the conclusion that temperature had a responsive connection with the microbial community structure. At the family level, Anaerobaculaceae, Clostridiaceae, and Thermoanaerobacterceae dominated the microbial communities, as shown in Figure 7b. At the genus level (see Figure 7c), Anaerobaculum, Fervidobacterium, Tepidanaerobacter, Clostridium, Moorella, Aminiphilus, Carboxydocella, and Methanosaeta are some microorganism genera that exhibited noteworthy growth during the temperature shift. Anaerobaculum and Tepidanaerobacter from the Thermoanaerobacterales family are syntrophic bacteria that have an essential role in converting short-chain fatty acids to methanogenic components such as acetate, H 2 , and formate [53]. Moorella has been identified as an acidogenic bacterium [54], whereas Clostridium is a hydrogen-producing bacterium that plays a key role in the hydrolysis process [46].  Compared to the other types of microorganisms, methanogenic archaea were shown to have the least portion of the population among the microbial communities. The decrease in the methanogen population accelerated faster in shifted-up temperature conditions than in controlled temperature conditions. However, the number of methanogen populations is unlikely to affect CH4 production. The cumulative CH4 production decreased along with the decline in the methanogen:total population (M·TP −1 ) ratio during the shifted-up temperature period. This result contradicts the outcome of the controlled temperature, in which the cumulative CH4 production increased conspicuously despite the fluctuation in the M·TP −1 ratio (Figure 8a). To date, no particular ratio has been found to be effective in understanding the influence of the microbial ratio (the existence of a particular microorganism) on biogas production. The closest ratio was that of sulphatereducing bacteria (SRB) to methanogens (SRB·M −1 ). Previous research has stated that the Compared to the other types of microorganisms, methanogenic archaea were shown to have the least portion of the population among the microbial communities. The decrease in the methanogen population accelerated faster in shifted-up temperature conditions than in controlled temperature conditions. However, the number of methanogen populations is unlikely to affect CH 4 production. The cumulative CH 4 production decreased along with the decline in the methanogen:total population (M·TP −1 ) ratio during the shifted-up temperature period. This result contradicts the outcome of the controlled temperature, in which the cumulative CH 4 production increased conspicuously despite the fluctuation in the M·TP −1 ratio (Figure 8a). To date, no particular ratio has been found to be effective in understanding the influence of the microbial ratio (the existence of a particular microorganism) on biogas production. The closest ratio was that of sulphate-reducing bacteria (SRB) to methanogens (SRB·M −1 ). Previous research has stated that the existence of SRBs in the AD process may inhibit CH 4 production as it would compete with methanogens for convenient H 2 , acetate, propionate, and butyrate [55]. From the shifted-up temperature experiment, the SRB·M −1 ratio showed harmony with the statement of previous research. The higher the SRB·M −1 ratio, the lower the CH 4 production as the SRB emulated the methanogens in consuming available H 2 (Figure 8b). Nevertheless, the results from the controlled temperature experiments showed that the SRB·M −1 ratio was also ineffective in determining the relationship between the ratio and CH4 production. At controlled 48 °C, the maximum CH4 volume production was observed despite the low M·TP −1 ratio and high SRB·M −1 ratio. Under these conditions, the populations of Methanosarcina and Methanoculleus genera were the most abundant. Methanosarcina genera are known to be the major contributors to CH4 production [56] and manage to perform all methanogenesis pathways (hydrogenotrophic, acetoclastic, and methylotrophic) that help them survive in food competition [57]. In contrast to other methanogens, Methanosarcina was capable of growing significantly under high concentrations of VFAs and ammonia, which are the foremost inhibitors in biogas production [58]. Methanoculleus, in contrast, has been reported to increase in abundance along with elevated sulfate concentration [59]. Thus, both Methanosarcina and Methanoculleus acclimatized well and were attributed to high CH4 production under high inhibitor concentrations.
These findings may explain the phenomenon of increasing CH4 production at a controlled temperature of 48 °C. The influence of Methanosarcina and Methanoculleus on CH4 production was demonstrated by considering the decreasing volume of CH4 production parallel to the decrease in Methanosarcina and Methanoculleus abundance during shifts in temperature. Methanosarcina abundances decreased from 2% to 0.75% and 0.59%, parallel to the Methanoculleus population that declined from 6.52% to 4.96% and 3.81% during the incubation at temperature of 42 °C, shifted up to 45 °C, and shifted up to 48 °C, respectively.

Discussion
In AD processes, temperature has a significant influence on biogas production and microbial ecology [60,61]. There has been a number of research that examined the effects of temperature in mesophilic and thermophilic conditions [18,62,63]. However, to the best of our knowledge, the assessment of biogas production (especially CH4) and microbial community adaptation under multiple rising temperature conditions in a fed-batch reactor has not been widely studied. We expected instability in microbial communities (espe- Nevertheless, the results from the controlled temperature experiments showed that the SRB·M −1 ratio was also ineffective in determining the relationship between the ratio and CH 4 production. At controlled 48 • C, the maximum CH 4 volume production was observed despite the low M·TP −1 ratio and high SRB·M −1 ratio. Under these conditions, the populations of Methanosarcina and Methanoculleus genera were the most abundant. Methanosarcina genera are known to be the major contributors to CH 4 production [56] and manage to perform all methanogenesis pathways (hydrogenotrophic, acetoclastic, and methylotrophic) that help them survive in food competition [57]. In contrast to other methanogens, Methanosarcina was capable of growing significantly under high concentrations of VFAs and ammonia, which are the foremost inhibitors in biogas production [58]. Methanoculleus, in contrast, has been reported to increase in abundance along with elevated sulfate concentration [59]. Thus, both Methanosarcina and Methanoculleus acclimatized well and were attributed to high CH 4 production under high inhibitor concentrations.
These findings may explain the phenomenon of increasing CH 4 production at a controlled temperature of 48 • C. The influence of Methanosarcina and Methanoculleus on CH 4 production was demonstrated by considering the decreasing volume of CH 4 production parallel to the decrease in Methanosarcina and Methanoculleus abundance during shifts in temperature. Methanosarcina abundances decreased from 2% to 0.75% and 0.59%, parallel to the Methanoculleus population that declined from 6.52% to 4.96% and 3.81% during the incubation at temperature of 42 • C, shifted up to 45 • C, and shifted up to 48 • C, respectively.

Discussion
In AD processes, temperature has a significant influence on biogas production and microbial ecology [60,61]. There has been a number of research that examined the effects of temperature in mesophilic and thermophilic conditions [18,62,63]. However, to the best of our knowledge, the assessment of biogas production (especially CH 4 ) and microbial community adaptation under multiple rising temperature conditions in a fed-batch reactor has not been widely studied. We expected instability in microbial communities (especially methanogens) and decreased CH 4 production, along with the temperature shift process owing to perturbations caused by sudden temperature changes. Among the three temperature conditions, there was a noticeable decrease in CH 4 production when the temperature was increased. The cumulative CH 4 production decreased from 454 mL at 42 • C to 152 mL after increasing the temperature to 45 • C and to 86.57 mL after increasing the temperature to 48 • C. This result is consistent with previous findings for the shifted-up temperature [51,64]. Each temperature shift was conducted after 70-day incubation periods in order to allow a period of acclimatization for methanogens and other bacteria. The total methanogen abundance in shifted-up 45 • C and 48 • C were close to that in controlled 45 • C and 48 • C after 70 days operation. Previous studies showed potential steadier operation condition in term of CH 4 production after acclimatization for 100-140 days, yet the risks of instability still exists [51,65].
Beale et al. [64] investigated the effect of upshift temperature shock from 37 • C to 42 • C on the biogas production volume of anaerobically digested sludge. Similarly, the biogas generated after the temperature was increased to 42 • C was persistently lower than that from the controlled digester at 37 • C during the first 32 days of operation. Identical results were also reported by Ziembinska-Buczynska et al. [66] who found a significant decrease in the biogas production rate from 70.5 L·day −1 to 28.6 L·day −1 along with an increase in temperature from 38 • C to 55 • C. Researchers also discovered that there was a decrease in microbiota diversity as the temperature of the digester influenced the evolution from mesophilic to thermophilic conditions. Some methanogens cannot survive at higher temperatures (heat unresistant), e.g., Methanobrevibacter (37-39 • C), Methanogenium (20-25 • C), or Methanobacterium (37-45 • C) [67]. However, our research contradicts the findings of Bouskova et al. [68] who discovered higher CH 4 production after the temperature was shifted from 42 • C to 47 • C, 51 • C, and 55 • C. A possible reason for the observed discrepancies was the characteristics and ratio of the inoculum and substrates. The researchers used an inoculum with TS of 31.24 g·L −1 and vs. of 14.48 g·L −1 . Meanwhile in our study, the inoculum consisted of 8 g·L −1 TS and 3 g·L −1 VS. Previous researchers also utilized a mixture of primary sludge and waste activated sludge as substrates which also contained seeds of microorganisms that maintained the longevity of biogas production.
We also found that the presence of excessive CO 2 in the reactor may have led to lower methane production. Methanogenic bacteria require CO 2 and H 2 to produce CH 4 , which indicates that if the CO 2 volume was greater than CH 4 after substrate feeding, this signifies the failure of these bacteria to consume sufficient quantities of CO 2 and H 2, which consequently would lead to the accumulation of VFAs, lower CH 4 yield, and low pH [69,70]. Low pH is a serious concern as it inhibits methanogenic bacteria due to the increase in the concentration of free acid molecules, which is harmful for microorganisms and impacts enzymatic activity [71]. It has been suggested that microbiota composition and methanogenic pathways are altered when encountering an immediate low pH and high acetate crisis (pH 5.5-6.5, completely hindered at pH 5.0) [72].
Temperature shifts also affected the microbial communities in the reactor, especially the methanogens. The number of methanogens decreased significantly after the temperature was shifted from 42 • C to 45 • C and then stabilized at 48 • C, as shown in Figure 4. This instability supported the study by Westerholm et al. [51] who found that immense perturbation occurred in the interval of 40-44 • C, signifying that the 40-44 • C temperature range had a significant impact on both mesophilic and thermophilic microbial populations. The only conceivable interpretation for this phenomenon is that the temperature range may be greater than the upper threshold for the growth of mesophiles but not sufficiently high for the growth of thermophiles [73]. Because our reactor was initially developed using mesophilic anaerobic sludge, this potentially limits the abundance of thermophilic microorganisms. Tian et al. [74] omitted the 40-44 • C area and still found a transitory decrease in total methanogen concentration after the temperature was shifted (37-55 • C), but then recovered quickly on day 11. Among all the methanogens, Methanosaeta (Methanosaetaceae family) was the most abundant under all temperature conditions in this study. This finding contradicts the findings of Kim et al. [75] who reported that the Methanosaetaceae family started to dominate the microbial structure only at temperatures above 45 • C. A plausible explanation for this difference is that the researchers started cultivation at 35 • C, which was more favorable to the growth of Methanomicrobiales order than Methanosarcinales (the ancestor of Methanosaetaceae). The instability of methanogen populations in anaerobic digestion also led to an increase in some types of bacteria that contributed to H 2 and VFAs consumption, such as SRBs. As illustrated in Figure 7b, our research shows that the increasing SRB/methanogen ratio has a considerable influence on the decrease in CH 4 production under the shifted-up temperature conditions. This result supports the idea from previous studies that reported that sulfide generation by SRBs inhibits methanogenesis, with the latter being the leading rival of methanogens for electron donors and substrates [65,76]. In addition, Beale et al. [64] emphasized that even a small amount of SRBs was enough to inhibit biogas production, as methanogens were vulnerable to the toxicity caused by metabolic products of SRBs.
Although this study characterized the behavior of some microorganisms and their influence on CH 4 production during the shifted-up temperature, there are still several unidentified factors that can potentially affect the outcome of the AD process. Further studies are needed to determine and characterize the mechanism by which shifted temperature may affect the abundance of microorganisms, especially methanogenesis-related microbiota (e.g., methanogens, SRB, methanotrophs, hydrogenotrophs, acetotrophs, nitrogen-fixing bacteria, and sulfate-oxidizing bacteria) and the influence on CH 4 production. Other microbial communities with syntrophic and fermentative behaviors, as well as their metabolic networks, merit study. Minimizing the number of unknown microorganisms may also provide a clearer insight into the relationship between the abundance of microorganisms and CH 4 production, as in this study, we detected a large number of unknown bacteria.

Conclusions
Treating wastewater sludge using anaerobic digestion does not eliminate the risk of temperature instability. Consequently, the effects of shifting the temperature during anaerobic digestion of anaerobic sludge were investigated in this study. The results showed a considerable reduction in the CH 4 cumulative gas production, from 454 mL (4.55 L·g −1 COD) to 152 mL (1.52 L·g −1 COD) then to 86.57 mL (0.94 L·g −1 COD) when the temperature of the reactor was increased from 42 • C to 45 • C and subsequently to 48 • C, respectively. Several factors have been attributed to the decrease in CH 4 production under the shifted-up temperature, such as the decreasing methanogen population (expressed as the M·TP −1 ratio) due to intense food competition, increasing SRB populations over methanogens, and low abundance of major CH 4 producers (e.g., Methanosarcina and Methanoculleus). Methanosaeta was the most dominant methanogen in this study, while Anaerobaculum and Tepidanaerobacter were the most abundant syntrophic bacteria, and Clostridium which are known as hydrogen-producing bacteria. Overall, the diversity of the anaerobic microbial consortium observed in this study altered slightly during the shift in the thermal conditions. This indicated that the majority of the communities belonged to thermotolerant microorganisms.