## 3 Results and Discussion
### Effect of Magnetite Loading on Biogas Production and COD Removal  
The AD system was operated with various magnetite doses in a range of 0.25-1.0 g/L with an increment of 0.25 g/L at a constant HRT of 21 days. The graph of cumulative biogas can be useful in investigating bacterial adaptation and growth during AD. Figure 3a shows the performance of each dose in biogas production and bacterial adaptation. Figure 1: An experimental setup consisting of the UASB reactor and the downward displacement gas collection technique. Figure 2: Effect of hydraulic retention time on (**a**) cumulative biogas generation; (**b**) degradation of chemical oxygen demand (COD). It is clear that the production of biogas at the start of the period for all the digester systems varies with the amount of magnetite loaded. The amount of time it takes for microorganisms to adjust to their new environment is determined by the first steady rise of biogas accumulation, which is commonly referred to as the lag phase period (acclimatize) [27]. As evident from the graph in Figure 3a, the control has a lag phase of 5-6 days, suggesting that it takes longer for the microorganisms to fully adapt to the traditional AD system. The use of magnetite is found to reduce the lag phase of the AD process. This is because the use of magnetite in anaerobic digestion improves the interspecies electron transfer, which ultimately enhances biogas production [19; 25; 28]. Due to the high surface area of magnetite at the nanoscale, the reaction is relatively fast [29], making it interact with the sludge cell membrane, causing structural changes in the cells that eventually result in bacteria-permeable membranes. As a result, more bacteria find their way to attack sludge, increasing overall biogas production [16; 28]. The best-performing digesters in terms of lag phase are the 4 g, 6 g, and 8 g MNP digesters, which have lower lag phases, fewer than 3 days. Following the lag phase, all digesters reach the exponential stage where the cumulative biogas output grows significantly due to methanogen exponential growth. The 6 g and 8 g MNPs show the highest maximum growth rate between days 4 and 10. However, due to the very high magnetite concentration, the 8 g digester experiences overloading, which results in a rapid decrease in biogas generation after 9 days. The addition of excess Fe ions to the anaerobic microbial community gives rise to toxicity and excess reactivity, thus, hindering the growth and activity of microorganisms [30]. The trend of the 6 g digester in Figure 3b suggests that there is a disturbance in biogas generation between days 9-10, which sees the 4 g digester surpassing all other digesters with a steady increase in biogas generation. This could be the effect of excess magnetite, which promotes the formation of toxicants and inhibits the sensitive methanogenic activities during AD, thus, meaning a decrease in biogas production up to day 18. The daily biogas production in Figure 3b confirms that the 8 g MNP digester has the highest biogas production, reaching its peak of 25 mL/day within 6 days, however, after 9 days the biogas production is very low. The 6 g digester also reaches its peak production of 23 mL/day on day 7 and a gradual decrease is seen after day 9, which is due to MNP overload. The 4 g digester is at peak production between days 9-12, with a production rate of 20 mL/day, followed by a gradual decrease after day 12, towards completion of organic degradation within the digester. The 2 g and control digesters reach their peak production of 15 and  
Figure 3: Effect of magnetite load on (**a**) cumulative biogas for the duration of AD; (**b**) daily biogas production. 12 mL/day on days 8 and 7, respectively, because of low and no magnetite in the reactor to promote the growth and activity of microorganisms. The five digesters generated cumulative biogas in this order; 4 g > 6 g > 8 g > 2 g > control, with values of 210, 190, 175, 140, and 110 mL, respectively.