THE THREE PATHWAYS FOR THE

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THE THREE PATHWAYS FOR THE REMOVAL OF AMMOIA-ITROGE I AQUACULTURE SYSTEMS James M. Ebeling, Ph.D. Research Engineer Aquaculture Systems Technologies, LLC Michael B. Timmons, Ph.D. Professor Dept. of Bio. & Environ. Eng. Cornell University

Introduction C inorganic / Ratio Ammonia-nitrogen C organic / Ratio Photoautotrophic (green-water systems) Autotrophic (fixed-cell bioreactors) Heterotrophic (zero-exchange systems) H 4+ - C 106 H 263 O 110 16 P Algae H 4+ - O 2- - Ammonia Oxidizing Bacteria O 2- - O 3- - itrite Oxidizing Bacteria H 4+ - C 5 H 7 O 2 Bacteria

ew Paradigm??????? Understanding of the Removal System Photoautotrophic Autotrophic Heterotrophic Some Combination! Impact on Water Quality!!!! Management Strategies! Current Issues in Biofloc Technology Systems AAS-2011

itrogen Removal Pathways C organic H 4+ - CO 2 Alkalinity Trace utrients VSS Algae VSS Auto Extensive Pond O 2 Alkalinity CO 2 O 3- - Photoautotrophic Autotrophic Heterotrophic Other Mysterious Ways VSS Hetero

itrogen Removal Pathways C organic VSS Algae VSSAuto H 4+ - CO 2 Alkalinity Trace utrients Intensive Pond Alkalinity CO 2 Photoautotrophic Autotrophic Heterotrophic Other Mysterious Ways VSS Hetero O 2 O 3- - Algae Based Systems

itrogen Removal Pathways C organic H 4+ - O 2 Alkalinity Trace utrients Recirculation Systems Photoautotrophic Autotrophic Heterotrophic VSS Algae VSS Auto VSS Hetero CO 2 Alkalinity O 3- - O 2- - Denitrification Fixed-film Bioreactors

itrogen Removal Pathways C organic H 4+ - O 2 Alkalinity Trace utrients Zero-exchange Photoautotrophic Autotrophic Heterotrophic VSS Algae VSS Auto CO 2 Alkalinity O 3- - Denitrification VSS Hetero Suspended Growth Systems

Photoautotrophic (algal based systems) Biosynthesis of saltwater algae: itrate as nitrogen source 16 O 3- + 124 CO 2 + 140 H 2 O + HPO 2-4 C 106 H 263 O 110 16 P + 138 O 2 + 18 HCO - 3 Ammonia as nitrogen source 16 H 4+ + 92 CO 2 + 92 H 2 O + 14 HCO 3- + HPO 4 2- C 106 H 263 O 110 16 P + 106 O 2

Photoautotrophic (algal based systems) Consumes C organic C inorganic Consumables Stoichiometry (g) (g) (g) (g) H 4+ - 1.0 ----- ----- 1.0 Carbon Dioxide 18.07 g CO 2 / g 18.07 ----- 4.93 ----- Alkalinity 3.13 g Alk/ g 3.13 ----- 0.75 ----- Yields C organic C inorganic Products Stoichiometry (g) (g) (g) (g) VSS Algae 15.85 g VSS A / g 15.85 5.67 ----- 1.0 Oxygen 15.14 g O 2 / g 15.14 ----- ----- -----

Autotrophic - itrification Biosynthesis of Autotrophic bacteria: H 4+ + 1.83 O 2 + 1.97 HCO 3-0.024 C 5 H 7 O 2 + 0.976 O 3- + 2.9 H 2 O + 1.86 CO 2 The major factors affecting the rate of nitrification include: ammonia-nitrogen and nitrite-nitrogen concentration carbon/nitrogen ratio dissolved oxygen ph temperature alkalinity salinity

Autotrophic - itrification Consumes C organic C inorganic Consumables Stoichiometry (g) (g) (g) (g) H 4+ - 1.0 ----- ----- 1.0 Alkalinity 7.05 g Alk/ g 7.05 ----- 1.69 ----- Oxygen 4.18 g O 2 / g 4.18 ----- ----- ----- Yields C organic C inorganic Products Stoichiometry (g) (g) (g) (g) VSS A 0.20 g VSS A / g 0.20 0.11 ----- 0.024 O 3- - 0.976 g O 3- - /g 0.976 ----- ----- 0.976 CO 2 5.85 g CO 2 / g 5.85 ----- 1.58 -----

Heterotrophic Bacteria Biosynthesis of Heterotrophic bacteria: H 4+ + 1.18 C 6 H 12 O 6 + HCO 3- + 2.06 O 2 C 5 H 7 O 2 + 6.06 H 2 O + 3.07 CO 2 The major factors affecting the rate of nitrification include: ammonia-nitrogen carbon/nitrogen ratio dissolved oxygen ph temperature alkalinity salinity

Heterotrophic Bacteria Consumes C organic C inorganic Consumables Stoichiometry (g) (g) (g) (g) H 4+ - 1.0 ----- ----- 1.0 C 6 H 12 O 6 15.17 g Carbs/ g 15.17 6.07 ----- ----- Alkalinity 3.57 g Alk/ g 3.57 ----- 0.86 ----- Oxygen 4.71 g O 2 / g 4.71 ----- ----- ----- Yield C organic C inorganic Products Stoichiometry (g) (g) (g) (g) VSS H 8.07 g VSS H / g 8.07 4.29 ----- 1.0 CO 2 9.65 g CO 2 / g 9.65 ----- 2.63 -----

Impact of C/ Ratio Autotrophic C labile / ~ 0 C organic / ~ small inorganic carbon as alkalinity 100 % Autotrophic (Recirculation System with excellent solids removal) C/ Ratio C labile / ~ 2.2 C/ ~ 8-10 organic carbon from the feed (109 g C organic /kg feed) @ 35% protein 56 % Heterotrophic 44 % Autotrophic C labile / ~ 6.2 C/ ~ 12-16 Heterotrophic organic carbon from the feed plus supplemental carbohydrates 100% Heterotrophic

Autotrophic (Intensive Recirculation System) Autotrophic System Conversion of 1 kg of feed @ 35% protein 32.2 g * 0.20 g VSS/ g = 6.44 g VSS autotrophic 0.124 g/gvss A 0.531 gc/gvss A 0.80 g VSS 3.42 g C VSS + 31.4 g O 3 - + 51.3 g CO 2 Current Issues in Biofloc Technology Systems AAS-2011

Autotrophic (Intensive Recirculation System) Consumes C organic C inorganic Consumables Stoichiometry (g) (g) (g) (g) H 4+ - 32.2 ----- ----- 32.2 Alkalinity 7.05 g Alk/ g 227 ----- 54.7 ----- Oxygen 4.18 g O 2 / g 135 ----- ----- ----- Yields C organic C inorganic Products Stoichiometry (g) (g) (g) (g) VSS A 0.20 g VSS A / g 6.44 3.4 ----- 0.8 O 3- - 0.976 g O 3- - /g 31.4 ----- ----- 31.4 CO 2 5.85 g CO 2 / g 188 ----- 51.3 ----- 54.7g C Alk / 32.2 g C/ ratio of 1.6 and TOC is very, very small Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System (?) (no Carbon Supplementation) Heterotrophic and Autotrophic Components Heterotrophic Component: Organic Carbon from Feed 1 kg feed * 0.36 kg BOD/kg feed * 0.40 kg VSS/ kg BOD = = 144 g VSS heterotrophic 0.124 g/gvss H 0.531 gc/gvss H 17.9 g VSS 76.5 g C VSS + 47.1 g C CO2 = 123.6 g C Assume that the heterotrophic bacteria out compete the autotrophic bacteria. 109 g C feed 15.4 g C Alkalinity Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System (?) (no Carbon Supplementation) Heterotrophic and Autotrophic Components Autotrophic Component: Inorganic Carbon Alkalinity 14.3 g * 0.20 g VSS/ g Excess Ammonia-nitrogen: 32.2 g H 3 - - 17.9 g VSS = 14.3 g A = 2.86 g VSS autotrophic 0.124 g/gvss A 0.531 gc/gvss A 0.35 g VSS 1.52 g C VSS + 14 g O 3 - + 24.3 g C Alk

Biofloc System (?) (no Carbon Supplementation) Heterotrophic and Autotrophic Components Heterotrophic Bacteria Consumes C organic C inorganic Stoichiometry (g) (g) (g) (g) H 4+ - 0.56 * T 17.9 ----- ----- 17.9 C 6 H 12 O 6 feed 15.17 g Carbs/ g 272 109 ----- ----- Alkalinity 3.57 g Alk/ g 63.9 ----- 15.4 ----- Autotrophic Bacteria Consumes C organic C inorganic Stoichiometry (g) (g) (g) (g) H 4+ - 0.44 * T 14.3 ----- ----- 14.3 Alkalinity 7.05 g Alk/ g 101 ----- 24.3 ----- Conversion of 1 kg of feed @ 35% protein

Biofloc System (?) (no Carbon Supplementation) Heterotrophic and Autotrophic Components Heterotrophic Bacteria Yields C organic C inorganic Stoichiometry (g) (g) (g) (g) VSS H 8.07 g VSS H / g 144 76.5 ----- 17.9 CO 2 9.65 g CO 2 / g 173 ----- 47.1 ----- Autotrophic Bacteria Yields C organic C inorganic Stoichiometry (g) (g) (g) (g) VSS A 0.20 g VSS A / g 2.86 3.45 ----- 0.35 O 3- - 0.976 g O 3 -/g 31.7 ----- ----- 13.9 CO 2 5.85 g CO 2 / g 83.4 ----- 22.8 ----- Conversion of 1 kg of feed @ 35% protein Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System (?) (no Carbon Supplementation) Heterotrophic and Autotrophic Components C organic C inorganic Consumes (g) (g) (g) H 4+ - 32.2 g ----- ----- 32.2 C 6 H 12 O 6 272 g Carbs (feed) 109 ----- ----- Alkalinity 165 g Alk ----- 40 ----- C organic C inorganic Yields (g) (g) (g) VSS 147 g VSS 80 ----- 18.2 O 3- - 31.7 g O 3 - ----- ----- 13.9 CO 2 256 g CO 2 ----- 70 ----- Conversion of 1 kg of feed @ 35% protein Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System (?) (no Carbon Supplementation) Heterotrophic and Autotrophic Components 100% 80% 97% 78% Heterotrophic Bacteria Autotrophic Bacteria Percent Removal 60% 40% 65% 56% 35% 22% 57% 51% 49% 44% 43% 39% 61% 35% 65% 20% 0% 3% 20% 25% 30% 35% 40% 45% 50% 55% Feed Protein (%) Percent removal of ammonia-nitrogen by Heterotrophic or Autotrophic Process as a function of % Protein Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System Carbon Supplement Excess Ammonia-nitrogen: 32.2g H 3 - - 17.9 g VSS = 14.3g A 14.3 g * 8.07 g VSS / g = 115 g VSS heterotrophic 0.124 g/gvss H 0.531 gc/gvss H 14.3 g VSS 61.1 g C VSS + 37.6 g C CO2 = 98.7 g C Carbohydrate is 40% Carbon 216 g carbs 86.4 g C s 12.3 g C Alkalinity

Biofloc System (Carbon Supplementation) Consumes C organic C inorganic Consumables Stoichiometry (g) (g) (g) (g) H 4+ - 32.2 ----- ----- 32.2 C 6 H 12 O 6 15.17 g Carbs/ g 488 196 ----- ----- Alkalinity 3.57 g Alk/ g 115 ----- 27.7 ----- Oxygen 4.71 g O 2 / g 152 ----- ----- ----- Yield C organic C inorganic Products Stoichiometry (g) (g) (g) (g) VSS H 8.07 g VSS H / g 260 138 ----- 32.2 CO 2 9.65 g CO 2 / g 311 ----- 85 ----- Conversion of 1 kg of feed @ 35% protein Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System (Carbon Supplementation) Supplemental Carbohydrate. (% of feed). 60% 50% 40% 30% 20% 10% 0% 50% 43% 36% 29% 22% 15% 8% 0% 1% 15 20 25 30 35 40 45 50 55 Feed Protein (%) Supplemental Carbohydrate as percentage of feed rate for heterotrophic metabolism of ammonia-nitrogen to microbial biomass Current Issues in Biofloc Technology Systems AAS-2011

Biofloc System (Carbon Supplementation) 500 VSS (g/kg feed) 400 300 200 100 144 148 185 222 259 296 333 370 407 0 15 20 25 30 35 40 45 50 55 Feed Protein (%) VSS production as percentage of feed rate for heterotrophic metabolism of ammonia-nitrogen to microbial biomass Current Issues in Biofloc Technology Systems AAS-2011

Conclusions Engineering Sustainability Organic Carbon + itrogen Bacterial Biomass Current Issues in Biofloc Technology Systems AAS-2011

Questions? Current Issues in Biofloc Technology Systems AAS-2011