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James M. Ebeling, Ph.D. Aquaculture Engineer

An Engineering Analysis of the Stoichiometry of Autotrophic, Heterotrophic Bacterial Control of Ammonia-Nitrogen in Zero-Exchange Production. James M. Ebeling, Ph.D. Aquaculture Engineer. Michael B. Timmons, Ph.D. Professor Dept. of Bio. & Environ. Eng. Cornell University.

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James M. Ebeling, Ph.D. Aquaculture Engineer

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  1. An Engineering Analysis of the Stoichiometry of Autotrophic, Heterotrophic Bacterial Control of Ammonia-Nitrogen in Zero-Exchange Production James M. Ebeling, Ph.D. Aquaculture Engineer Michael B. Timmons, Ph.D. Professor Dept. of Bio. & Environ. Eng. Cornell University James J. Bisogni, Ph.D. Professor School of Civil & Environ. Eng. Cornell University

  2. Introduction Cinorganic / N Ratio Corganic / N Ratio Ammonia-nitrogen Photoautotrophic (green-water systems) Heterotrophic (zero-exchange systems) Autotrophic (fixed-cell bioreactors) NH4+-N C106H263O110N16P Algae NH4+-N C5H7O2N Bacteria NH4+-N NO2--N Ammonia Oxidizing Bacteria NO2--N NO3--N Nitrite Oxidizing Bacteria 6th International Conference on Recirculating Aquaculture

  3. “New Paradigm” Zero-exchange Systems “Belize System” • Shrimp– high health, selectively bred SPF stock • Feed – high protein feeds combined with carbon supplementation • Water management – zero water exchange, recycling water between crops 6th International Conference on Recirculating Aquaculture

  4. “New Paradigm” Zero-exchange Systems “Belize System” • Shrimp– high health, selectively bred SPF stock • Feed – high protein feeds combined with carbon supplementation • Water management – zero water exchange, recycling water between crops 6th International Conference on Recirculating Aquaculture

  5. “New Paradigm”  ???? ??? Understanding of the ‘Removal System’ • Photoautotrophic • Autotrophic • Heterotrophic • Some Combination! Impact on Water Quality!!!! Management Strategies! 6th International Conference on Recirculating Aquaculture

  6. Ammonia Production In general: PTAN = F * PC * 0.092 where:PTAN = Production rate of total ammonia nitrogen, (kg/day) F = Feed rate (kg/day) PC = protein concentration in feed (decimal value) For marine shrimp: PTAN = F * PC * 0.144 6th International Conference on Recirculating Aquaculture

  7. Nitrogen Removal Pathways NH4+-N CO2 Alkalinity Trace Nutrients Corganic • Photoautotrophic • Autotrophic • Heterotrophic • Other Mysterious Ways Extensive Pond CO2 VSSAlgae Alkalinity VSSAuto O2 NO3--N VSSHetero 6th International Conference on Recirculating Aquaculture

  8. Nitrogen Removal Pathways NH4+-N CO2 Alkalinity Trace Nutrients Corganic • Photoautotrophic • Autotrophic • Heterotrophic • Other Mysterious Ways Intensive Pond VSSAlgae CO2 Alkalinity VSSAuto O2 NO3--N VSSHetero Algae Based Systems 6th International Conference on Recirculating Aquaculture

  9. Nitrogen Removal Pathways O2 NH4+-N Alkalinity Trace Nutrients Corganic • Photoautotrophic • Autotrophic • Heterotrophic • Denitrification Recirculation Systems VSSAlgae Alkalinity VSSAuto NO3--N CO2 VSSHetero NO2--N Fixed-film Bioreactors 6th International Conference on Recirculating Aquaculture

  10. Nitrogen Removal Pathways NH4+-N O2 Alkalinity Trace Nutrients Corganic • Photoautotrophic • Autotrophic • Heterotrophic • Denitrification Zero-exchange VSSAlgae Alkalinity CO2 VSSAuto NO3--N VSSHetero Suspended Growth Systems 6th International Conference on Recirculating Aquaculture

  11. Photoautotrophic(algal based systems) Biosynthesis of saltwater algae: Nitrate as nitrogen source 16 NO3- + 124 CO2 + 140 H2O + HPO42- → C106H263O110N16P + 138 O2 + 18 HCO3- Ammonia as nitrogen source 16 NH4+ + 92 CO2 + 92 H2O + 14 HCO3- + HPO42- → C106H263O110N16P + 106 O2 6th International Conference on Recirculating Aquaculture

  12. Photoautotrophic(algal based systems) 6th International Conference on Recirculating Aquaculture

  13. Autotrophic - Nitrification Biosynthesis of Autotrophic bacteria: NH4+ + 1.83 O2 + 1.97 HCO3-→ 0.024 C5H7O2N + 0.976 NO3- + 2.9 H2O + 1.86 CO2 • The major factors affecting the rate of nitrification include: • ammonia-nitrogen and nitrite-nitrogen concentration • carbon/nitrogen ratio • dissolved oxygen • pH • temperature • alkalinity • salinity 6th International Conference on Recirculating Aquaculture

  14. Autotrophic - Nitrification 6th International Conference on Recirculating Aquaculture

  15. Heterotrophic Bacteria Biosynthesis of Heterotrophic bacteria: NH4+ + 1.18 C6H12O6 + HCO3- + 2.06 O2 → C5H7O2N + 6.06 H2O + 3.07 CO2 • The major factors affecting the rate of nitrification include: • ammonia-nitrogen • carbon/nitrogen ratio • dissolved oxygen • pH • temperature • alkalinity • salinity 6th International Conference on Recirculating Aquaculture

  16. Heterotrophic Bacteria 6th International Conference on Recirculating Aquaculture

  17. Impact of C/N Ratio C/N Ratio Clabile /N ~ 0 Corganic/N ~ small Clabile /N ~ 2.2 C/N ~ 8-10 Clabile /N ~ 6.2 C/N ~ 12-16 Autotrophic Heterotrophic organic carbon from the feed plus supplemental carbohydrates inorganic carbon as alkalinity organic carbon from the feed (109 g C organic /kg feed) @ 35% protein 100 % Autotrophic (Recirculation System with excellent solids removal) 100% Heterotrophic 35.6% Heterotrophic 64.4 % Autotrophic 6th International Conference on Recirculating Aquaculture

  18. Stoichiometry Photoautotrophic System 16 NO3- + 124 CO2 + 140 H2O + HPO42- → C106H263O110N16P + 138 O2 + 18 HCO3- 50.4 g N * 15.8 g VSS/ g N = 800 g VSSphotoautotrophic 0.063 gN/gVSSA0.358 gC/gVSSA 50.4 g NVSS 286 g CVSS 6th International Conference on Recirculating Aquaculture Conversion of 1 kg of feed @ 35% protein

  19. Photoautotrophic (Pond intensive system) Conversion of 1 kg of feed @ 35% protein 6th International Conference on Recirculating Aquaculture

  20. Stoichiometry Autotrophic System 50.4 g N * 0.20 g VSS/ g N = 10.1 g VSSautotrophic 0.124 gN/gVSSA0.531 gC/gVSSA 1.25 g NVSS5.35 g CVSS + 49.2 g NO3-N + 80.1 g CO2 6th International Conference on Recirculating Aquaculture Conversion of 1 kg of feed @ 35% protein

  21. Autotrophic (Intensive Recirculation System) 85.6 g CAlk / 50.5 g N  C/N ratio of 1.7 and TOC is very, very small 6th International Conference on Recirculating Aquaculture

  22. Zero-exchange System (no Carbon Supplementation) Heterotrophic and Autotrophic Components Heterotrophic Component: Organic Carbon from Feed 1 kgfeed * 0.36 kg BOD/kg feed * 0.40 kg VSS/ kg BOD = =144 g VSSheterotrophic 0.124 gN/gVSSH0.531 gC/gVSSH 17.9 g NVSS 76.5 g CVSS + 47.1 g CCO2 = 123.6 g C 108.2 g Cfeed 15.4 g CAlkalinity Assume that the heterotrophic bacteria out compete the autotrophic bacteria. 6th International Conference on Recirculating Aquaculture

  23. Zero-exchange System (no Carbon Supplementation) Heterotrophic and Autotrophic Components Autotrophic Component: Inorganic Carbon Alkalinity Excess Ammonia-nitrogen: 50.4 g NH3-N - 17.9 g NVSS = 32.5 g NA 32.5 g N * 0.20 g VSS/ g N = 6.5 g VSSautotrophic 0.124 gN/gVSSA0.531 gC/gVSSA 0.80 g NVSS 3.45 g CVSS + 31.7 g NO3-N+ 55.4 g CAlk 6th International Conference on Recirculating Aquaculture

  24. Zero-exchange System (no Carbon Supplementation) Heterotrophic and Autotrophic Components 6th International Conference on Recirculating Aquaculture

  25. Zero-exchange System (no Carbon Supplementation) Heterotrophic and Autotrophic Components 460 g Cfeed / 50.5 g N  C/N ratio of 9 6th International Conference on Recirculating Aquaculture

  26. Zero-exchange System (no Carbon Supplementation) Heterotrophic and Autotrophic Components Percent removal of ammonia-nitrogen by Heterotrophic or Autotrophic Process as a function of % Protein 6th International Conference on Recirculating Aquaculture

  27. Zero-exchange System (Carbon Supplementation) Carbon Supplement Excess Ammonia-nitrogen: 50.4 g NH3-N - 17.9 g NVSS = 32.5 g NA 32.5 g N * 8.07 g VSS / g N = 262 g VSSheterotrophic 0.124 gN/gVSSH0.531 gC/gVSSH 32.5 g NVSS 139 g CVSS + 85 g C CO2 = 225 g C 197 g Cs 28 g CAlkalinity Carbohydrate is 40% Carbon 492 g carbs 6th International Conference on Recirculating Aquaculture

  28. Zero-exchange System (Carbon Supplementation) (460 g Cfeed + 197 g Ccarb ) / 50.5 g N  C/N ratio of 13 6th International Conference on Recirculating Aquaculture

  29. Zero-exchange System (Carbon Supplementation) Supplemental Carbohydrate as percentage of feed rate for heterotrophic metabolism of ammonia-nitrogen to microbial biomass 6th International Conference on Recirculating Aquaculture

  30. Research Trial #1 – C/N Ratio 4x12 Juvenile Production Tanks • Treatment (Sucrose) • Control • 50 % of Feed Rate • 100% of Feed Rate • Dosage • 100% carbon Demand • 200% of carbon Demand • Stocking • 3.6 gm mean weight • 150 shrimp / m2 6th International Conference on Recirculating Aquaculture

  31. Research System Weekly Growth – about 0.9 gm/wk 4x12 System with sludge settling tank, automatic feeders, bacterial floc 6th International Conference on Recirculating Aquaculture

  32. Water Quality Parameters • Dissolved Oxygen • Salinity • Temperature • pH • Alkalinity • TSS/TVS • TAN • NO2 –N • NO3 –N • Total Nitrogen • Total Organic Carbon 6th International Conference on Recirculating Aquaculture

  33. Water Quality Summary 6th International Conference on Recirculating Aquaculture

  34. Water Quality Parameters - Dissolved Oxygen 6th International Conference on Recirculating Aquaculture

  35. Water Quality Parameters - Temperature 6th International Conference on Recirculating Aquaculture

  36. Water Quality Parameters - pH 6th International Conference on Recirculating Aquaculture

  37. Water Quality Parameters - TAN 6th International Conference on Recirculating Aquaculture

  38. Water Quality Parameters – NO2- N 6th International Conference on Recirculating Aquaculture

  39. Water Quality Parameters – NO3- N 6th International Conference on Recirculating Aquaculture

  40. Water Quality Parameters – Alkalinity 6th International Conference on Recirculating Aquaculture

  41. Solids Management • Solids Management: • Settling cones 6th International Conference on Recirculating Aquaculture

  42. Settling Basins Sedimentation: Advantages • Simplest technologies • Little energy input • Relatively inexpensive to install and operate • No specialized operational skills • Easily incorporated into new or existing facilities • Sedimentation: Disadvantages • Low hydraulic loading rates • Poor removal of small suspended solids • Large floor space requirements • Resuspension of solids and leeching 6th International Conference on Recirculating Aquaculture

  43. Q = flow (gpm) vo = 0.00076 ft/sec vs = 0.0015 ft/sec Settling Basins Design to minimize turbulence: 6th International Conference on Recirculating Aquaculture

  44. Autotrophic/Heterotrophic Model 6th International Conference on Recirculating Aquaculture

  45. Heterotrophic Model (50% feed) 6th International Conference on Recirculating Aquaculture

  46. Heterotrophic Model (100% feed) 6th International Conference on Recirculating Aquaculture

  47. TSS Production Model • Solids Production Model: • autotrophic/heterotrophic • heterotrophic 6th International Conference on Recirculating Aquaculture

  48. Autotrophic/Heterotrophic Model • allocated the daily feed organic carbon to heterotrophic bacterial production, • calculated VSSH, [VSSH = feed g/m3 day * 0.36 g BOD/g feed * 0.40 g VSSH / g BOD] • calculated amount of ammonia-nitrogen assimilated in the VSSH, [TANH = 0.123 * VSSH] • subtracted TANH from the daily TANfeed produced, [TANfeed = feed g/m3 day * (0.35 * 0.16 * 0.9)] • allocated excess ammonia-nitrogen to autotrophic bacterial consumption, [TANA= TANfeed – TANH] • determined VSSA [VSSA = TANA * 0.20 g VSSA/g N] • calculated Total VSS and TSS. 6th International Conference on Recirculating Aquaculture

  49. Heterotrophic Model (50% feed) • allocated the daily feed carbon to heterotrophic bacterial production, • calculated VSSH, [VSSH = feed g/m3 day * 0.36 g BOD/g feed * 0.40 g VSSH / g BOD] • calculated amount of ammonia-nitrogen sequestered in the VSSH, [TANH = 0.123 * VSSH] • subtracted from the daily TANfeed produced, [TANfeed = feed g/m3 day * (0.35 * 0.16 * 0.9)] • allocated excess ammonia-nitrogen to additional heterotrophic bacterial production, [TANH+= TANfeed – TANH ] • determined VSSH+[VSSH+ = 8.07 g VSSH/g N * g N] • calculated Total VSS and TSS. 6th International Conference on Recirculating Aquaculture

  50. Heterotrophic Model (100% feed) • allocated the daily feed carbon to heterotrophic bacterial production, • calculated VSSH, [VSSH = feed g/m3 day * 0.36 g BOD/g feed * 0.40 g VSSH / g BOD] • assumed all of the sucrose carbon was converted into bacterial biomass • (sufficient nitrogen available) • determined VSSH+ [VSSH+ = g sucrose/m3 day * 0.56 g VSSH/g sucrose] • calculated Total VSS and TSS. 6th International Conference on Recirculating Aquaculture

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