De novo anaerobic granulation with varying organic substrates: granule growth and microbial community responses

Anaerobic granulation from dispersed inoculum is recognized as a slow process. However, studies under saline conditions have shown that adding complex proteinaceous substrates can accelerate this process. To explore whether this holds true also under non-saline conditions, we conducted a 262-days experiment with four lab-scale upflow anaerobic sludge blanket reactors inoculated with digested sewage sludge. Each reactor received a synthetic feed containing varying amount of carbohydrate/protein substrate: glucose (R_Glu), acetate/tryptone (R_Ac+Try), glucose/tryptone (R_Glu+Try), and glucose/starch (R_Glu+Sta). Development of granules with different influent composition was monitored with macroscopy, analysis of the extracellular polymeric substances, and microbial diversity. Granulation was faster in reactors R_Glu+Try and R_Glu+Sta. Increasing granule diameters positively correlated with the occurrence of bacteria from Muribaculaceae and Lachnospiraceae families, suggesting their involvement in de novo granulation. Granules of R_Glu+Try also had high relative abundances of both fermenting bacteria (e.g. Lactococcus, Streptococcus, Trichococcus) and bacteria involved in the oxidation of volatile fatty acids (Smithella, Acetobacteroides). The results of this study provide a basis for strategies to enhance the sludge granulation rate in practice when granular inoculum is not available. Specifically, supplementing small amounts of waste protein during reactor start-up can be effective.