# Influences of Temperature and Substrate Particle Content on Granular Sludge Bed Anaerobic Digestion
## 3 Result and Discussion  
### Biogas Production  
The experiment was carried out in four phases based on substrate type and temperature. Stable temperatures at the new levels were achieved 4-5 hours after each temperature controller change (Table 1). In Phase 1, with RF at 35 \({}^{\circ}\)C, the biogas production rate increased from the start until the sixth day (Figure 3), followed by stable 3.7-3.9 L/day production. An immediate decrease in biogas production rate was observed in Phase 2, from day 11, when the temperature was reduced to 25 \({}^{\circ}\)C (from 3.9 L/day to 1.4 L/day). However, it gradually increased and stabilized around 2.5 L/day until the end of Phase 2. The biogas production rate did not noticeably decrease from Phase 2 to Phase 3 at day 26 when RF was replaced by CF (at 25 \({}^{\circ}\)C), even though COD\({}_{\text{total}}\) of CF was significantly lower than that of RF. Furthermore, the biogas production rate was more stable in Phase 3 compared to Phase 2. In Phase 4, where CF was used at 35 \({}^{\circ}\)C, the biogas production rate increased but with significant fluctuations and an average (11%) less than the stable production in Phase 1. From Phase 1 to 2, the biogas production decreased roughly by 42%. The decrease can be explained by temperature decline (10 \({}^{\circ}\)C within a matter of a few hours), which affects the rate of hydrolysis, as well as reaction kinetics. This is comparable to the 44% decrease in total biogas production observed by [PERSON] et al. (2019) when the temperature was reduced from 35 to 25 \({}^{\circ}\)C. The solubility of methane and other gases also increases as temperature decreases, resulting in a lower amount of biogas in the gas phase ([PERSON] et al., 2019). The decline in biogas production seemed to recover in two steps, with the first one being a sharp increase that lasted for a few hours, followed by a gradual increase that stabilized toward the end of Phase 2. Effluent VFA\({}_{\text{total}}\) before and immediately after temperature decrease was 0.54 g/L (80% acetate) and 1.6 g/L (62% acetate), respectively, suggesting a negative effect on both acetoclastic methanogenesis and acetogenesis. The concentration of VFA\({}_{\text{total}}\) in the effluent increased to 2.3 g/L after few days in Phase 2 and seemed to stabilize at that concentration. Since methanogens are sensitive to temperature changes, process instability (VFA accumulation) during temperature decline is expected. However, the decrease in the proportion of oxidized acids indicates that it may also affect the oxidation of propionic acid to acetate and/or an increase in hydrolysis rate. Figure 2: Anaerobic digestion model no.1 (ADM1) simulation scheme for Mode 1 and 2 simulations. A gradual increase in biogas production was also observed in Phase 2. [PERSON] et al. [31] observed a similar trend. They observed the recovery of biogas production rate with no lasting damage to the performance after an initial decline due to sharp temperature decrease (temperature shock). However, their observed biogas yield decline was less pronounced than what we observed (17% decline from 35 to 25 \({}^{\circ}\)C compared to the 42% decline in our study). The reason for the gradual increase in biogas production may be linked to gradual microbial adaptation to temperature change. In addition, solid particulates entrapped in the granular sludge may have been slowly disintegrated and hydrolyzed, contributing to the increase in biogas production throughout Phase 2. However, the disintegration and hydrolysis of solid particulates at 25 \({}^{\circ}\)C do not seem to be carried out to an appreciable degree. In Phase 3, even with the decrease in CODtotal and solid content of the feed, the biogas production rate did not decrease in any appreciable way compared to Phase 2 (Tables 5 and 6). Experimental results of total and suspended solid contents from influent and effluent samples showed that most of the feed solid content ended up in the effluent.