Can Microbial Acclimation Work to Avert Inhibition During Fog Co-Digestion?

INTRODUCTION Anaerobic digestion is utilized by water resource recovery facilities (WRRFs) to stabilize wastewater residuals and recover renewable energy in the form of biogas. Recently, anaerobic co-digestion of wastewater residuals with high strength organic waste has gained much popularity in the wastewater industry due to it's potential to improve methane yield. Fats, oils and grease (FOG) is one of the desirable co-substrates due to its higher methane production potential and degradability (compared to wastewater residuals) (Salama et al., 2019). However, some studies (Amha et al., 2017; Wu et al., 2018) have shown that higher FOG loadings can be inhibitory to the digestion process or the complex consortium of microorganisms involved. Therefore, it is critical to explore and understand how FOG impacts the co-digestion process and evaluate methods to avoid inhibition. One of such methods examined in this work was to evaluate if microbial acclimation through stepwise introduction of the co-substrate can avoid inhibition during FOG co-digestion. The goal of this study was 1) to determine the impact of co-digestion of waste activated sludge (WAS) with FOG on methane yield and microbial communities using short term Biomethane Potential (BMP) tests and long-term bench-scale reactors; and 2) to determine if microbial acclimation through stepwise introduction of FOG can be implemented to avert inhibition by comparing step-load and shock-load conditions in bench-scale reactors. METHODOLOGY Substrate and inoculum collection: Anaerobic inoculum was obtained from the Nashville Metro Biosolids WRRF's anaerobic digester which treats primary sludge and secondary scum. The WAS was obtained from City of Cookeville WRRF. The FOG used in this study was pure fats and grease obtained directly from a pork roasting grill at a local restaurant. BMP tests design and operation: Three experimental phases were carried out in 160 mL serum bottles at 35 °C. Phase 1 involved mono-digestion of WAS, as well as the co-digestion of WAS with FOG fractions of 25%, 50% and 75% (volatile solids basis). Phase 2 involved the co-digestion of 50% FOG with the 25% FOG digestate from Phase 1 as inoculum, while Phase 3 involved the co-digestion of 75% FOG with digestate from Phase 2 as inoculum. Phase 1 lasted for 20 days, while the phase 2 and 3 lasted for 13 days each. Bench-scale experimental design and operation: The experiments were conducted using two identical 10 L bench-scale reactors with a working volume of 6 L and operated at 35 °C. During the startup phase, all the reactors were fed with 100% WAS until they reached steady state. The designated control reactor was fed with WAS throughout all experimental phases, while the test reactor received various fractions of FOG. Phases 1 to 3 were conducted at volatile solids loading rate (VSLR) of 2 g-VS/L/d, and involved co-digestion of 25%, 50% and 75% (VS basis) of the co-substrates, respectively. Due to digester failure in Phase 3, Phase 4 was experimented as a recovery phase by feeding the test digester with only WAS. Phase 5 involved the co-digestion of 75% co-substrate at higher VSLR of 4 g-VS/L/d without prior exposure to the co-substrate, and therefore without prior microbial acclimation. Reactors were operated at 20 days SRT and each experimental phase was run for 3 SRTs. The digesters were monitored weekly by measuring parameters such as biogas volume and methane content, pH, and VS removal. The methanogenic community present within the different experimental phases were also examined through 16s rRNA gene sequencing to understand the impact of the FOG fractions on the methanogenic communities. RESULTS & DISCUSSION Results from the study revealed that compared to the mono-digestion of WAS, co-digestion with FOG can improve specific methane yield (i.e the total methane volume per gram VS added) up to 16.5-fold. The specific methane yields of all the tested FOG fractions were higher at the BMP level than the ones observed in the bench-scale as shown in Table 1. In the BMP study, inhibition was not observed even at the 75% FOG fraction; however, 75% FOG was found to be inhibitory in the bench-scale experiment as seen in the decline in weekly average biogas production rate in Figure 2. The BMP tests were loaded only once, which made it less likely to cause inhibition as compared to the continuous loading in the bench-scale experiment which resulted in organic overload and subsequent inhibition. At the microbial scale, the overall methanogenic community was dominated by Methanolinea, a hydrogenotrophic methanogen, and its abundance remained similar across all the tested FOG fractions at the BMP level. However, significant reduction of methanogenic population was observed during the inhibitory conditions at 75% FOG fractions in the bench-scale experiment, as shown in Figure 3. The results suggest that bench-scale experiments provide more realistic methane yields with detectability of inhibitory thresholds of FOG as well as reveal methanogenic community response to different FOG fractions under stable and unstable conditions. The bench-scale experiment also showed that step-wise increment of the FOG could not achieve microbial acclimation to avert inhibition in this study. Further, FOG co-digestion was only viable with up to 50% FOG addition at the 2gVS/L/d loading rate. At the higher loading rates, inhibition occurred due to accumulation of volatile fatty acids above 2,000 mg/L and decline in pH. It should be noted that the 50% FOG threshold observed in this study may have been influenced by the inoculum, the FOG characteristics or lack of continuous pH adjustments within the experimental phases. CONCLUSION The study shows that FOG can be used as a co-substrate to improve methane yield when up to 50% FOG fraction is used at an organic loading of 2gVS/L/d. The study also provides insights about the influence of operational modes and stepwise acclimation strategy on FOG co-digestion and highlights the need for other strategies to avoid inhibition in FOG co-digestion systems.