Production and Biomineralization Performance Assessment of Biogranules Simultaneously Conducting Urea Hydrolysis and Denitrification

Abstract

Biomineralization, particularly calcium carbonate precipitation through microbial pathways, is gaining attention as an environmentally friendly alternative for various applications, including bioremediation, bioconsolidation, and the production of bio-based materials. Calcium carbonate precipitation naturally occurs through various metabolic pathways, such as urea hydrolysis and nitrate reduction. However, most studies have focused on axenic (pure) cultures and single metabolic processes, leaving the potential of combined metabolic activities unexplored. Using single metabolic pathways and axenic cultures resulted in biomineralization performances under either aerobic or anoxic conditions. Urea hydrolysis and nitrate reduction are complementary to achieving biomineralization under both aerobic and anoxic conditions. This should be investigated to enhance the overall biomineralization performance. This thesis presents the production of non-axenic biogranules capable of conducting urea hydrolysis and nitrate reduction metabolisms. These biogranules could extend the occurrence of the biomineralization process in both aerobic and anoxic conditions and enhance the overall calcium carbonate precipitation. An SBR was operated for 560 days under alternating anoxic-aerobic periods and biogranule production under minimal nutrient conditions was optimized. Produced biogranules were compared with a previously reported nitrate-reducing granular culture in terms of microbial activity and biomineralization performance. Biogranules in the size range of 0.2-0.7 mm could be produced continuously during the steady production period between operation days 270 and 560. Metagenomic analyses revealed a consistent microbial community dominated by ureolytic and denitrifying bacteria, maintaining a high degree of similarity across samples taken at different time intervals from the bioreactor. The produced biogranules achieved maximum specific urea hydrolysis and nitrate reduction activities of 232 mg urea.h-1.g-1 VSS and 9.25 mg NO3-N.h-1.g-1 VSS, respectively. In 9-day batch tests, the biogranules precipitated up to 642.5 mg CaCO3 in calcite form, which was significantly higher than the 137 mg CaCO3 obtained by the reference nitrate-reducing granular culture. The desiccated biogranules demonstrated 73% nitrate reduction and 67% urea hydrolysis efficiency on average in the biomineralization test. However, reference nitrate-reducing granular culture showed 34% nitrate reduction and 2% urea hydrolysis efficiency on average. To see the shelf-life effect on MICP performance another biomineralization test was conducted after 1.5 years of storage of the desiccated biogranules. When comparing the yeast-containing batches B1 and C1 with the yeast-free batches B3 and C3, it was found that biogranules exhibited a 30% and 164% higher MICP performance, respectively, compared to the reference nitrate-reducing granular culture in the second biomineralization test. It was observed that the storage period negatively impacted the MICP performance of the desiccated biogranules. Overall, it was revealed that complementary microbial metabolisms could enhance the total amount of CaCO3 precipitated in a single application which can be beneficial for improving the efficiency of biomineralization applications.