An Engineering Analysis of the An Engineering Analysis of the Stoichiometry Stoichiometry of Autotrophic, Heterotrophic Bacterial of Autotrophic, Heterotrophic Bacterial Control Control of Ammonia-Nitrogen in Zero-Exchange of Ammonia-Nitrogen in Zero-Exchange Production Production James M. James M. Ebeling, Ph.D. Ebeling, Ph.D. Aquaculture Engineer Aquaculture Engineer Michael B. Timmons, Michael B. Timmons, Ph.D. Ph.D. Professor Professor Dept. of Bio. & Environ. Eng. Dept. of Bio. & Environ. Eng. Cornell University Cornell University James J. Bisogni, James J. Bisogni, Ph.D. Ph.D. Professor Professor School of Civil & Environ. Eng. School of Civil & Environ. Eng. Cornell University Cornell University
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. - PowerPoint PPT Presentation
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An Engineering Analysis of the Stoichiometry An Engineering Analysis of the Stoichiometry of Autotrophic, Heterotrophic Bacterial Control of Autotrophic, Heterotrophic Bacterial Control
of Ammonia-Nitrogen in Zero-Exchange Productionof Ammonia-Nitrogen in Zero-Exchange Production
James M. Ebeling, Ph.D. James M. Ebeling, Ph.D. Aquaculture EngineerAquaculture Engineer
Michael B. Timmons, Ph.D.Michael B. Timmons, Ph.D.ProfessorProfessor
Dept. of Bio. & Environ. Eng.Dept. of Bio. & Environ. Eng.Cornell UniversityCornell University
James J. Bisogni, Ph.D.James J. Bisogni, Ph.D.ProfessorProfessor
School of Civil & Environ. Eng.School of Civil & Environ. Eng.Cornell UniversityCornell University
6th International Conference on Recirculating Aquaculture6th International Conference on Recirculating Aquaculture
IntroductionIntroduction
Ammonia-nitrogen
NH4+
-N NO2--N
Ammonia Oxidizing Bacteria
Photoautotrophic(green-water systems)
HeterotrophicHeterotrophic(zero-exchange systems)
NH4+
-N C5H7O2N
BacteriaBacteria
Autotrophic(fixed-cell bioreactors)
NO2- -N NO3
--NNitrite Oxidizing Bacteria
NH4+
-N CC106106HH263263OO110110NN1616PP
AlgaeAlgae
Cinorganic / N Ratio Corganic / N Ratio
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““New Paradigm”New Paradigm”
Zero-exchange Systems “Belize System”Zero-exchange Systems “Belize System”
Biosynthesis of Biosynthesis of Heterotrophic bacteriaHeterotrophic bacteria:: NH4
+ + 1.18 C6H12O6 + HCO3- + 2.06 O2 →
C5H7O2N + 6.06 H2O + 3.07 CO2
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Heterotrophic BacteriaHeterotrophic Bacteria
Consumes C organic C inorganic N
Consumables Stoichiometry (g) (g) (g) (g)
NH4+-N 1.0 ----- ----- 1.0
C6H12O6 15.17 g Carbs/ g N 15.17 6.07 ----- -----
Alkalinity 3.57 g Alk/ g N 3.57 ----- 0.86 -----
Oxygen 4.71 g O2/ g N 4.71 ----- ----- -----
Yield C organic C inorganic N
Products Stoichiometry (g) (g) (g) (g)
VSSH 8.07 g VSSH / g N 8.07 4.29 ----- 1.0
CO2 9.65 g CO2/ g N 9.65 ----- 2.63 -----
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Impact of C/N Ratio
Autotrophic Heterotrophic
inorganic carbon as alkalinity
organic carbon from the feed
(109 g C organic /kg feed)@ 35% protein
organic carbon from the feed plus supplemental carbohydrates
C/N Ratio
Clabile /N ~ 2.2
C/N ~ 8-10
35.6% Heterotrophic 64.4 % Autotrophic
Clabile /N ~ 6.2
C/N ~ 12-16
Clabile /N ~ 0
Corganic/N ~ small
100% Heterotrophic 100 % Autotrophic
(Recirculation System withexcellent solids removal)
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StoichiometryStoichiometry
Photoautotrophic System
16 NO3- + 124 CO2 + 140 H2O + HPO4
2- → C106H263O110N16P + 138 O2 + 18 HCO3-
50.4 g N * 15.8 g VSS/ g N
= 800 g VSSphotoautotrophic
0.063 gN/gVSSA 0.358 gC/gVSSA
50.4 g NVSS 286 g CVSS
Conversion of 1 kg of feed @ 35% protein
6th International Conference on Recirculating Aquaculture6th International Conference on Recirculating AquacultureConversion of 1 kg of feed @ 35% protein
Consumes C organic C inorganic N
Consumables Stoichiometry (g) (g) (g) (g)
NH4+-N 50.4 ----- ----- 50.4
Carbon Dioxide 18.07 g CO2/ g N 911 ----- 249 -----
Alkalinity 3.13 g Alk/ g N 158 ----- 37.9 -----
Yields C organic C inorganic N
Products Stoichiometry (g) (g) (g) (g)
VSSAlgae 15.85 g VSSA / g N 800 286 ----- 50.4
O2 15.14 g O2/ g N 763 ----- ----- -----
Photoautotrophic (Pond intensive system)
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StoichiometryStoichiometry
50.4 g N * 0.20 g VSS/ g N
= 10.1 g VSSautotrophic
0.124 gN/gVSSA 0.531 gC/gVSSA
1.25 g NVSS 5.35 g CVSS
+ 49.2 g NO3-N + 80.1 g CO2
Autotrophic System
Conversion of 1 kg of feed @ 35% protein
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Autotrophic (Intensive Recirculation System)
Consumes C organic C inorganic N
Consumables Stoichiometry (g) (g) (g) (g)
NH4+-N 50.4 ----- ----- 50.4
Alkalinity 7.05 g Alk/ g N 355 ----- 85.6 -----
Oxygen 4.18 g O2/ g N 211 ----- ----- -----
Yields C organic C inorganic N
Products Stoichiometry (g) (g) (g) (g)
VSSA 0.20 g VSSA / g N 10.1 5.35 ----- 1.25
NO3--N 0.976 g NO3
--N /g N 0.976 ----- ----- 49.2
CO2 5.85 g CO2/ g N 295 ----- 80.1 -----
85.6 g CAlk / 50.5 g N C/N ratio of 1.7 and TOC is very, very small
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Zero-exchange System (no Carbon Supplementation)
Heterotrophic and Autotrophic Components
1 kgfeed * 0.36 kg BOD/kg feed * 0.40 kg VSS/ kg BOD =
= 144 g VSSheterotrophic
0.124 gN/gVSSH 0.531 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 CAlkalinityAssume that the heterotrophic bacteria out compete the autotrophic bacteria.
Heterotrophic Component: Organic Carbon from Feed
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Zero-exchange System (no Carbon Supplementation)
Heterotrophic and Autotrophic Components
Excess Ammonia-nitrogen:50.4 g NH3-N - 17.9 g NVSS = 32.5
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Heterotrophic Bacteria Consumes C organic C inorganic N
Stoichiometry (g) (g) (g) (g)
NH4+-N 0.356 * NT 17.9 ----- ----- 17.9
C6H12O6 feed 15.17 g Carbs/ g N 272 108.9 ----- -----
Alkalinity 3.57 g Alk/ g N 63.9 ----- 15.4 -----
Autotrophic Bacteria Consumes C organic C inorganic N
Stoichiometry (g) (g) (g) (g)
NH4+-N 0.644 * NT 32.5 ----- ----- 32.5
Alkalinity 7.05 g Alk/ g N 229.1 ----- 55.4 -----
C organic C inorganic N
Total Consumed Consumes (g) (g) (g)
NH4+-N 50.4 g N ----- ----- 50.4
C6H12O6 272 g Carbs 108.9 ----- -----
Alkalinity 293 g Alk ----- 70.8 -----
Zero-exchange System (no Carbon Supplementation)
Heterotrophic and Autotrophic Components
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Heterotrophic Bacteria Yields C organic C inorganic N
Stoichiometry (g) (g) (g) (g)
VSSH 8.07 g VSSH / g N 144 76.5 ----- 17.9
CO2 9.65 g CO2/ g N 174 ----- 47.4 -----
Autotrophic Bacteria Yields C organic C inorganic N
Stoichiometry (g) (g) (g) (g)
VSSA 0.20 g VSSA / g N 6.5 3.45 ----- 0.81
NO3--N 0.976 g NO3-N/g N 31.7 ----- ----- 31.7
CO2 5.85 g CO2/ g N 189 ----- 51.7 -----
C organic C inorganic N
Total Products Yields (g) (g) (g)
VSS 150.5 g VSS 80.0 ----- 18.7
NO3--N 31.7 g NO3-N ----- ----- 31.7
CO2 363.4 g CO2 ----- 99.1 -----
Zero-exchange System (no Carbon Supplementation)
Heterotrophic and Autotrophic Components
460 g Cfeed / 50.5 g N C/N ratio of 9
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Zero-exchange System (no Carbon Supplementation)
Heterotrophic and Autotrophic Components
83%
62%
50%
64%
69%72%
75%77%
23%25%
28%31%
36%
41%
17%
38%
50%
59%
0%
20%
40%
60%
80%
100%
12.4% 15% 20% 25% 30% 35% 40% 45% 50% 55%
Heterotrophic Bacteria
Autotrophic Bacteria
Percent removal of ammonia-nitrogen by Heterotrophic or Autotrophic Process as a function of % Protein
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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/gVSSH 0.531 gC/gVSSH
32.5 g NVSS 139 g CVSS
+ 85 g C CO2 = 225 g C
197 g Cs 28 g CAlkalinity
Carbon Supplement
Zero-exchange System (Carbon Supplementation)
Carbohydrate is 40% Carbon 492 g carbs
6th International Conference on Recirculating Aquaculture6th International Conference on Recirculating Aquaculture(460 g Cfeed + 197 g Ccarb ) / 50.5 g N C/N ratio of 13
Consumes C organic C inorganic N
Consumables Stoichiometry (g) (g) (g) (g)
NH4+-N 50.4 ----- ----- 50.4
C6H12O6 15.17 g Carbs/ g N 765 306 ----- -----
Alkalinity 3.57 g Alk/ g N 180 ----- 43.3 -----
Oxygen 4.71 g O2/ g N 237 ----- ----- -----
Yield C organic C inorganic N
Products Stoichiometry (g) (g) (g) (g)
VSSH 8.07 g VSSH / g N 407 216 ----- 50.4
CO2 9.65 g CO2/ g N 487 ----- 133 -----
Zero-exchange System (Carbon Supplementation)
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Supplemental Carbohydrate as percentage of feed ratefor heterotrophic metabolism of ammonia-nitrogen to microbial biomass
6%
17%
28%
38%
60%
71%
82%
93%
49%
0%
20%
40%
60%
80%
100%
15 20 25 30 35 40 45 50 55
Feed Protein (%)
Supp
lem
enta
l Car
bohy
drat
e .
(% o
f fee
d) .
Zero-exchange System (Carbon Supplementation)
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Research Trial #1 – C/N RatioResearch Trial #1 – C/N Ratio
StockingStocking 3.6 gm mean weight3.6 gm mean weight 150 shrimp / m150 shrimp / m22
Treatment (Sucrose)Treatment (Sucrose) ControlControl 50 % of Feed Rate50 % of Feed Rate 100% of Feed Rate100% of Feed Rate
4x12 Juvenile Production Tanks
DosageDosage 100% carbon Demand100% carbon Demand 200% of carbon Demand200% of carbon Demand
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Research SystemResearch Systemy =0.90 gms/wk
R2 = 0.98
2.0
4.0
6.0
8.0
10.0
12.0
0 10 20 30 40 50 60 70Days
Mea
n W
eigh
t (gm
s) .
Control
50% C Demand
100% C Demand
4x12 System with sludge settling tank,automatic feeders, bacterial floc
Weekly Growth – about 0.9 gm/wk
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Solids ManagementSolids Management
Solids Management:Solids Management:
• Settling conesSettling cones
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Settling BasinsSettling Basins
Sedimentation: AdvantagesSedimentation: Advantages• Simplest technologiesSimplest technologies• Little energy inputLittle energy input• Relatively inexpensive to install and operateRelatively inexpensive to install and operate• No specialized operational skillsNo specialized operational skills• Easily incorporated into new or existing facilitiesEasily incorporated into new or existing facilities
18
D)(gV
2pp
s
Sedimentation: DisadvantagesSedimentation: Disadvantages• Low hydraulic loading ratesLow hydraulic loading rates• Poor removal of small suspended solidsPoor removal of small suspended solids• Large floor space requirementsLarge floor space requirements• Resuspension of solids and leechingResuspension of solids and leeching
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Settling BasinsSettling Basins
Design to minimize turbulence:Design to minimize turbulence:
vs = 0.0015 ft/sec
Q = flow (gpm)
vo = 0.00076 ft/sec
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Autotrophic/Heterotrophic ModelAutotrophic/Heterotrophic Model
• allocated excess ammonia-nitrogen to autotrophic bacterial consumption, allocated excess ammonia-nitrogen to autotrophic bacterial consumption,
[TAN[TANAA= TAN= TAN
feedfeed – TAN – TANHH]]
• determined VSSdetermined VSSA A [VSS[VSS
AA = TAN = TANAA * 0.20 g VSS * 0.20 g VSS
AA/g N]/g N]
• calculated Total VSS and TSS.calculated Total VSS and TSS.
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Heterotrophic Model Heterotrophic Model (50% feed)(50% feed)
• allocated the daily feed carbon to heterotrophic bacterial production, allocated the daily feed carbon to heterotrophic bacterial production,
• calculated VSScalculated VSSHH, [, [VSSVSSHH = feed g/m = feed g/m33 day * 0.36 g BOD/g feed * 0.40 g VSS day * 0.36 g BOD/g feed * 0.40 g VSSHH / g BOD] / g BOD]
• calculated amount of ammonia-nitrogen sequestered in the VSScalculated amount of ammonia-nitrogen sequestered in the VSSHH, [, [TANTANHH = 0.123 * VSS = 0.123 * VSSHH] ]
• subtracted from the daily TANsubtracted from the daily TANfeedfeed produced, [ produced, [TANTANfeedfeed = feed g/m = feed g/m33 day * (0.35 * 0.16 * 0.9)] day * (0.35 * 0.16 * 0.9)]
• allocated excess ammonia-nitrogen to additional heterotrophic bacterial production, allocated excess ammonia-nitrogen to additional heterotrophic bacterial production,
[[TANTANH+H+= TAN= TANfeedfeed – TAN – TANH H ]]
• determined VSSdetermined VSSH+H+ [VSS[VSSH+H+ = 8.07 g VSS = 8.07 g VSSHH/g N * g N]/g N * g N]
• calculated Total VSS and TSS.calculated Total VSS and TSS.
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Heterotrophic Model Heterotrophic Model (100% feed)(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 VSSdetermined VSSH+ H+ [VSSH+ = g sucrose/m3 day * 0.56 g VSSH/g sucrose]
•calculated Total VSS and TSS.
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4x12 System: Total Suspended Solids Management - Control
y = 14.7 mg/L day
R2 = 0.92
y = 14.3 mg/L day
R2 = 0.93
y = 14.4 mg/L day
R2 = 0.87
y = 13.8 mg/L day
R2 = 0.94
50
150
250
350
450
550
0 7 14 21 28 35 42 49 56 63
Days
TS
S (
mg/
L)
Autotrophic/Heterotrophic ModelAutotrophic/Heterotrophic Model
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4x12 System Total Suspended Solids - 50% of Feed
y = 29.1mg/L dayR2 = 0.93
y = 23.5 mg/L day R2 = 0.88
y = 27.4 mg/L dayR2 = 0.93
y = 35.5 mg/L dayR2 = 0.99
y = 21.8 mg/L dayR2 = 0.91
y = 38.1 mg/L dayR2 = 0.95
50
150
250
350
450
550
0 7 14 21 28 35 42 49 56 63
Days
TS
S (
mg/
L)
Heterotrophic Model Heterotrophic Model (50% feed)(50% feed)
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4x12 System Total Suspended Solids - 100% of Feed
y = 53.7 mg/L day9
R2 = 0.96
y = 87.8 mg/L day
R2 = 0.96
y = 77.1 mg/L day
R2 = 0.99
y = 81 mg/L day
R2 = 0.99
y = 52.2 mg/L day
R2 = 0.98
50
200
350
500
650
0 7 14 21 28 35 42 49 56 63
Days
TS
S (
mg/
L)
Heterotrophic Model Heterotrophic Model (100% feed)(100% feed)
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Water Quality Management – C/N RatioWater Quality Management – C/N Ratio
0.0
2.0
4.0
6.0
8.0
10.0
7/18/04 7/25/04 8/1/04 8/8/04
Suga
r (k
g)
0.0
0.5
1.0
1.5
Nit
rite
(mg/
L-N
) .
Sugar (kg)
Nitrite (mg/L)
Impact of carbon supplementation (or lack of) on nitrite-nitrogen
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Water Quality Parameters - Water Quality Parameters - TOCTOC
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Water Quality Parameters - Water Quality Parameters - TNTN
0
50
100
150
200
250
300
0 10 20 30 40 50 60 70
Nit
roge
n (m
g/L
-N)
.
T otal Nitrogen - Model
T otal Nitrogen - FeedT otal Nitrogen - Experimental Data
Measured T otal Nitrogen
0
50
100
150
200
250
0 10 20 30 40 50 60 70
Nit
roge
n (m
g/L
-N)
.
Total Nitrogen - Model
Total Nitrogen - FeedTotal Nitrogen - Experimental Data
Measured Total Nitrogen
Mass balance on nitrogen for the autotrophic/heterotrophic system without carbon supplementation and with periodic harvesting of excess bacterial biomass
Impact of carbon supplementation at 50% of the feed as sucrose on the system with excess bacterial biomass and nitrogen being periodically removed from the system
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Conclusions
Further work is needed to characterize the impact on Further work is needed to characterize the impact on production system performance at various C/N ratios.production system performance at various C/N ratios.
Alternative forms of Carbon need to be evaluated for Alternative forms of Carbon need to be evaluated for effectiveness and economics.effectiveness and economics.
Fundamental research is needed on carbon assimilation and Fundamental research is needed on carbon assimilation and conversion efficiency for heterotrophic bacteria.conversion efficiency for heterotrophic bacteria.
Development of optimal strains of bacteria for zero-exchange Development of optimal strains of bacteria for zero-exchange systems. systems.
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AcknowledgementsAcknowledgements
Research was supported by the Agriculture Research ServiceResearch was supported by the Agriculture Research Service of the United States Department of Agriculture, of the United States Department of Agriculture,
under Agreement No. 59-1930-1-130under Agreement No. 59-1930-1-130 and Magnolia Shrimp LLC, Atlanta Georgiaand Magnolia Shrimp LLC, Atlanta Georgia
with special thanks to Miami Aqua-culture, Inc., Dan Spotts.with special thanks to Miami Aqua-culture, Inc., Dan Spotts.
Opinions, conclusions, and recommendations are of the authorsOpinions, conclusions, and recommendations are of the authors and do not necessarily reflect the view of the USDA.and do not necessarily reflect the view of the USDA.
All experimental protocols involving live animals were in complianceAll experimental protocols involving live animals were in compliance with Animal Welfare Act (9CFR) and have been with Animal Welfare Act (9CFR) and have been
approved by the Freshwater Institute Animal Care and Use Committee.approved by the Freshwater Institute Animal Care and Use Committee.
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