1. Nitrogen Removal Project HJR Incorporated Heather Nelson Jeannette Harduby Rudi Schuech For Sparkling Clean Water Every Time

2. Objectives Test Bardenpho Method for efficiency in nitrate removal Test Bardenpho Method for efficiency in nitrate removal Test nitrate concentrations in waste using method of Karlsson et al. Test nitrate concentrations in waste using method of Karlsson et al. Test the nitrate concentrations in waste using Hach cadmium test kit Test the nitrate concentrations in waste using Hach cadmium test kit Test for nitrate accumulation in the aeration stage Test for nitrate accumulation in the aeration stage

3. Background Nitrogen Removal Steps organic nitrogen (proteins, aka casein) → ammonia NH 4 + Ammonia → nitrite NO 2 - Nitrite → nitrate NO 3 - Nitrate → nitrogen gas N 2

4. Nitrifying Bacteria Chemolithotrophic – “burn” ammonia and nitrite for energy Chemolithotrophic – “burn” ammonia and nitrite for energy Cannot use organic carbon or nitrogen Cannot use organic carbon or nitrogen Many are non-motile Many are non-motile May require a solid substrate to attach themselves to for optimum growth May require a solid substrate to attach themselves to for optimum growth Secrete a sticky slime matrix for adhesion Secrete a sticky slime matrix for adhesion Clump together to avoid light (vampiric?!) Clump together to avoid light (vampiric?!)

5. Nitrifying Bacteria Continued… Obligate aerobes - Require DO above 2 mg/L Obligate aerobes - Require DO above 2 mg/L Slow growth and long generation times Slow growth and long generation times In the time it takes one Nitrosomonas to divide, one E. coli will produce 35 trillion offspring! In the time it takes one Nitrosomonas to divide, one E. coli will produce 35 trillion offspring! Very pH sensitive - thrives at pH slightly above 7 Very pH sensitive - thrives at pH slightly above 7

6. Still More on Nitrifying Bacteria Optimal temperature between 77-86°F Optimal temperature between 77-86°F Growth rate decreased by 50% at 64°F Growth rate decreased by 50% at 64°F Ahh! The light, it burns!

7. Bardenpho Process

8. Barnard Sequencing Batch Reactor BOD 0 TKN 0 3 h 2 h0.33 h0.67 h BOD 0 TKN 0 Biomass ammonia + organic N

9. HJR Original State Times State Time (hr) Total fill time (syn. waste + water) 0.114 Anoxic 1 3 Aerobic 1 3 Anoxic 2 2 Aerobic 2 0.33 Settling Time 0.67 Supernatent removal time 0.1 HRT9.214

10. Karlsson et al Method Make standard curve of nitrate absorbancies at 220 nm vs. concentration Make standard curve of nitrate absorbancies at 220 nm vs. concentration Take samples of effluent waste water Take samples of effluent waste water Filter samples Filter samples Run samples through UV spectrophotometer Run samples through UV spectrophotometer Use correction factor to find [NO 3 - ] of samples Use correction factor to find [NO 3 - ] of samples Correction factor = 2 (absorbance @ 275) Correction factor = 2 (absorbance @ 275)

11. Karlsson et al results Absorbance at 220 nm Absorbance at 275 nm CorrectedabsorbanceCorrection factor % of 220 nm absorbanceCalculatedconcentration Trial10.64840.26140.125780.61%1.57E-01 Trial 2 0.28940.08070.128155.74%1.64E-01 Trial 3 0.53780.22160.094782.39%7.23E-02 Trial 4 0.48350.20520.073184.88%1.31E-02

12. Karlsson et al. conclusions Since the correction factor @ 275 nm was greater than 10% of the absorbance @ 220 nm, this test is inaccurate for our wastewater Since the correction factor @ 275 nm was greater than 10% of the absorbance @ 220 nm, this test is inaccurate for our wastewater

13. Cadmium Test Method Take samples of waste water periodically throughout different stages Take samples of waste water periodically throughout different stages Settle and filter sample Settle and filter sample Dilute 0.5 mL of sample to 5 mL with distilled water (to 25 mL for 50X dilution) Dilute 0.5 mL of sample to 5 mL with distilled water (to 25 mL for 50X dilution) Mix in contents of cadmium test packets Mix in contents of cadmium test packets Run through spectrophotometer Run through spectrophotometer Concentrations calculated from 542 nm absorbancies Concentrations calculated from 542 nm absorbancies

14. Anaerobic One Nitrate Concentrations

15. Aerobic One Nitrate Concentrations

16. Long Aeration Test for Nitrate Accumulation

17. Cadmium Test Reality Check Results from separate nitrite, nitrate, and total tests for waste samples Nitrite Concentration (mg/L) Nitrate Concentration (mg/L) Total Concentration Experimental (mg/L) Total Nitrite + Nitrate (mg/L) Trial One 17.91.37.919.2 Trial Two 18.11.112.419.2

18. Cadmium Test Reality Check continued… Results from separate nitrite, nitrate, and total tests for known standards Nitrite Concentration (mg/L) Nitrate Total Concentration Experimental (mg/L) Total Nitrite + Nitrate (mg/L) 3.13.06.26.1 Using a 10 mg/L nitrate standard

19. Email to Hach HJR Inc. told Hach that their testing kit was both difficult to use and inaccurate We suggested a safer packaging method such as capsules rather than packets and reported the discrepancies obtained in our results through their test kit.

20. Hach’s Response and follow up Claims working in a fume hood is not necessary according to MSDS. But… Claims working in a fume hood is not necessary according to MSDS. But… –MSDS calls cadmium compounds carcinogenic and “highly toxic if inhaled” - PEL – 0.05 mg/m 3 air –Hach said, “Best practice is to work in a still air environment” so powder doesn’t blow away A capsule does not work in terms of longevity and shipping. A capsule does not work in terms of longevity and shipping. Indicated that inaccuracy may be due to not enough reagent getting into sample Indicated that inaccuracy may be due to not enough reagent getting into sample Asked for documentation of our results. Asked for documentation of our results.

21. Conclusions New way for testing nitrate concentrations needs to be researched New way for testing nitrate concentrations needs to be researched It likely took many hours for nitrate to form because the population of nitrifying bacteria was too small It likely took many hours for nitrate to form because the population of nitrifying bacteria was too small Method of Karlsson et al. did not work for this experiment. Method of Karlsson et al. did not work for this experiment.