Date of Award


Degree Name

MS in Civil and Environmental Engineering


Civil and Environmental Engineering


Nirupam Pal


Biological nutrient removal (BNR) from wastewater, and specifically nitrogen removal, is a growing concern to wastewater dischargers such as municipalities. Excess nutrients in effluent can create problems such as eutrophication, toxicity to aquatic life, and dissolved oxygen depletion in receiving waters. BNR systems have been installed in many locations with success, but their operation presents operational and financial demands greater than conventional biological treatment. Nitrogen removal is typically performed in sequential autotrophic nitrification and denitrification, which increases needed energy input, operational complexity, and therefore cost. Simultaneous nitrification-denitrification (SNdN) achieved in a single system has also been successfully implemented, however operational parameters that compromise between ideals for aerobic nitrification and anoxic denitrification result in decreased reaction rates and removal efficiencies. The application of a product that could potentially enhance SNdN reaction rates and removal efficiencies through bioaugmentation could help ease operational and financial strains.

In contrast to common sequential processes, some heterotrophic Bacillus bacteria have demonstrated SNdN (Kim et al., 2005), (Zhang et al., 2011). However, their application outside of laboratory setting has yet to be established. Aqua™ is a proprietary bioaugmentation product composed of specific Bacillus strains developed by BiOWiSH® Technologies with the intent of improving aerobic, heterotrophic SNdN rates and removal efficiencies. Screening and bench-scale experiments were performed in flasks at 35° C on orbital shakers operated at a range of speeds. Primary wastewater and minimal media were used for the experiment, and inoculation was performed with both specific Bacillus strains and Aqua™.

Rapid total ammonia nitrogen (TAN) removal was observed in initial screening experiments with Aqua™ in sterile wastewater. Bacillus pumilus was identified as the fastest growing organism of the Aqua™ assemblage with the greatest TAN removal 1st order rate constant (0.32/ hr.), decreasing TAN 96% within 10 hours from an initial 48.5 ppm.

The orbital shaker speed that maximized TAN removal was 100 rpm, with reduction 47% and 88% more effective than both the upper (150 rpm) and lower (50 rpm) bound tested speeds, respectively. Visible floc growth centered in flasks, along with optical density data indicated cell growth and the possibility the system could support SNdN. Carbon amendments to minimal media were then evaluated, and sodium succinate improved TAN reduction by 53% compared to dextrose amended systems. This was likely because dextrose metabolism requires glycolysis to produce pyruvate for utilization in the TCA cycle for energy production; while succinate avoids glycolysis and thus is more easily utilized. In another experiment, flasks with supplemental trace minerals had a 59% higher TAN removal than the controls. Additions of supplemental vitamin solution or yeast extract improved TAN removal by 18% and 38%, respectively.

Two 10-day experiments assessed Aqua™ performance in municipal primary clarifier effluent. Nitrogen balance and optical density data showed that Aqua™ dosing at 10 ppm had no effect on nitrogen removal. The second 10-day experiment increased Aqua™ dosing to 50 ppm and evaluated product activation through incubation in growth media prior to inoculation. Nitrogen balance analysis showed no effect from Aqua™ on nitrogen removal during the second 10-day experiment as well. Systems amended with dextrose saw an initial rapid TAN first order removal rate (0.25/ hr.). However, difference between control and inoculated flasks was negligible showing no effect from Aqua™. A lack of total nitrogen losses and a lack of nitrate presence during initial rapid TAN losses confirmed these losses were by assimilation into organic nitrogen.

The above experiments suggest that initial success in TAN removal during screening experiments resulted from lack of competition with other microorganisms, the high 1500 ppm dose of Aqua™, and amended dextrose.