Microbial community analysis in developing biogas reactor technology for Norwegian agriculture

Aims Norway’s goal is to be climate neutral within the year 2030 according to Stortingsmelding nr. 39 (Det Kongelige Landbruks- og Matdepartementet 2008-2009) and agriculture needs to be a part of the solution. This includes livestock manure that amounts to ~40 % of the total energy potential for biogas production in Norway of which only an insignificant fraction of this is exploited today. The aim of this thesis was to contribute to sustainable energy production from manure by evaluating the microbial communities in compact anaerobic digestion sludge bed reactors intended for implementation into existing farm infrastructure for manure slurry treatment. Materials and methods Two long term experiments were run; the high rate experiment and the high ammonia experiment. In the high rate experiment, the filtrate of sieved dairy cow manure was used as influent for a period of 96 days in four reactors. Another four reactors were fed pig manure slurry supernatant for 106 days with unadapted and adapted microbial communities. HRT decreased by 5% per day. A PCR/denaturing gradient gel electrophoresis (DGGE) strategy was employed to characterize the microbial communities, and to evaluate the time needed for adaptation of the granular inoculum to the conditions in the manure-fed anaerobic digestion (AD) reactors. In the high ammonia experiment, four laboratory scale upflow anaerobic sludge bed (UASB) reactors treating pig manure slurry supernatant were operated at different ammonia concentrations and at variable temperatures over a time period of 358 days. High-throughput sequencing of 16S rRNA gene amplicons was applied for both bacterial and archaeal communities to investigate the microbial community dynamics in response to operational variables. Results and discussion Multiple operational parameters were tested, like different influents (dairy cow or pig manure supernatant), a wide range of loading rates (up to 400 g COD L-1 reactor d-1), extremely low hydraulic retention times (HRTs; 1.7h), high levels of total ammonia nitrogen (TAN; 3.7 ± 0.2 g NH4-N L-1) and extreme levels of free ammonia nitrogen (FAN; 1.2 ± 0.3 g NH3-N L-1). Overall, the reactors performed very well under extreme conditions. No foaming and no significant drop or increase in pH were observed even without pH control. The reactors showed remarkable stability and adaptation to changes in loading rate. The process did not fail even at the highest organic loading rate (OLR) tested, implying that supernatant AD is a very robust process. The methane yields obtained at high pig manure OLRs compared to the more particle rich dairy manure indicated that pig manure supernatant was more suitable than dairy manure filtrate as UASB influent. The reactors run on dairy manure needed longer time to stabilize the methane yield, acetate removal and propionate removal, which may be a consequence of the higher fraction of slowly degradable particles in dairy manure. The results illustrated that microbial communities in the reactor sludge seemed to be more similar to the communities in the influent than those associated with the granular sludge inoculum. This suggests that the influent had a higher impact on microbial community composition in the reactors than the granular sludge inoculum. Also, non-granular particles accumulated in the reactors and eventually made up a considerable portion of the solid fraction in the reactors. The non-granular particles most likely served as both slowly degradable substrate and as carriers for biofilm growth, ensuring stable operation of the reactors. The observation that methane production significantly exceeded the biogas potential of dissolved organics implies that the feed particles were efficiently retained and degraded. Unique bacterial communities evolved in the reactor liquids despite low HRT, shaped by the selection pressure and conditions in the reactors. Ammonia inhibition was much reduced after around 200 days of adaptation, allowing methane production at a rather extreme FAN level. The methane production, COD, propionate and acetate removal increased substantially in the high ammonia (HA) reactors from around day 300. The bacterial and archaeal communities adapted into new unique microbiota as a response to the feed composition and elevated concentrations of ammonia. The archaeal operational taxonomic unit (OTU) no. 2, classified as Methanosaeta, was the second most dominating OTU across all solid fraction reactor samples and its abundance increased towards the end of the high ammonia experiment, contradicting previous studies. A possible reason for the apparent high ammonia tolerance we observed for Methanosaeta in this study could be protection obtained by growing in aggregates with other microbes in the granules and colonized non-granular particles. We found that a defined group of OTUs representing archaeal taxa outside the Euryarchaeota was highly abundant in the UASB reactors at high ammonia concentrations. We speculate that they have a functional role, either directly as undiscovered methanogenic archaea or indirectly through syntrophic associations. No indications were found of previously described syntrophic acetate oxidizing bacteria (SAOB) associated with methane production in the HA reactors, though we cannot rule out that putative SAOB could be hiding among the numerous unclassified OTUs