Granulation of sulfur-oxidizing bacteria for autotrophic denitrification

Sulfur-oxidizing bacteria (SOB) was successfully employed for effective autotrophic denitrification and sludge minimization in a full-scale application of saline sewage treatment in Hong Kong. In this study, a Granular Sludge Autotrophic Denitrification (GSAD) reactor was continuously operated over 600 days for SOB granulation, and to evaluate the long-term stability of SOB granules, microbial communities and denitrification efficacy. Sludge granulation initiated within the first 40 days of start-up with an average particle size of 186.4 μm and sludge volume index (SVI5) of 40 mL/g in 5 min. The sludge granules continued to grow reaching a nearly uniform size of mean diameter 1380 ± 20 μm with SVI5 of 30 mL/g during 600 days of GSAD reactor operation at hydraulic retention time of 5 h and nitrate loading rate of 0.33 kg-N/m3/d. The GSAD reactor with SOB granular sludge achieved 93.7 ± 2.1% nitrogen and complete sulfide removal with low sludge yield of 0.15 g-volatile suspended solids (VSS)/g-N, and much lower nitrous oxide (N2O) emission than the heterotrophic denitrifying process. Microbial community analysis using fluorescence in situ hybridization (FISH) technique revealed that granules were enriched with SOB contributing to autotrophic denitrification. Furthermore, 16S rRNA analysis showed diverse autotrophic denitrification related genera, namely Thiobacillus (32.6%), Sulfurimonas (31.3%), and Arcobacter (0.01%), accounting for 63.9% of total operational taxonomic units at the generic level. No heterotrophic denitrification related genera were detected. The results from this study could provide useful design and operating conditions with respect to SOB sludge granulation and its subsequent application in a full-scale autotrophic denitrification in the Sulfate reduction-Autotrophic denitrification-Nitrification Integrated (SANI) process.