An Engineering Analysis of the Stoichiometry of Autotrophic, Heterotrophic Bacterial Control of Ammonia-Nitrogen in Zero-Exchange Production

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
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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

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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

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New Paradigm ? ????

• ??? Understanding of the Removal System
• Photoautotrophic
• Autotrophic
• Heterotrophic
• Some Combination!
• Impact on Water Quality!!!!
• Management Strategies!

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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

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Nitrogen Removal Pathways
NH4-N
CO2
Alkalinity
Trace Nutrients

• Photoautotrophic
• Autotrophic
• Heterotrophic
• Other Mysterious Ways

Corganic
Extensive Pond
CO2
VSSAlgae
Alkalinity
VSSAuto
O2
NO3--N
VSSHetero
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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
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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
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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
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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

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Photoautotrophic (algal based systems)
Consumes C organic C inorganic N
Consumables Stoichiometry (g) (g) (g) (g)
NH4-N 1.0 ----- ----- 1.0
Carbon Dioxide 18.07 g CO2/ g N 18.07 ----- 4.93 -----
Alkalinity 3.13 g Alk/ g N 3.13 ----- 0.75 -----

Yields C organic C inorganic N
Products Stoichiometry (g) (g) (g) (g)
VSSAlgae 15.85 g VSSA / g N 15.85 5.67 ----- 1.0
Oxygen 15.14 g O2/ g N 15.14 ----- ----- -----
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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

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Autotrophic - Nitrification
Consumes C organic C inorganic N
Consumables Stoichiometry (g) (g) (g) (g)
NH4-N 1.0 ----- ----- 1.0
Alkalinity 7.05 g Alk/ g N 7.05 ----- 1.69 -----
Oxygen 4.18 g O2/ g N 4.18 ----- ----- -----
Yields C organic C inorganic N
Products Stoichiometry (g) (g) (g) (g)
VSSA 0.20 g VSSA / g N 0.20 0.106 ----- 0.025
NO3--N 0.976 g NO3--N /g N 0.976 ----- ----- 0.976
CO2 5.85 g CO2/ g N 5.85 ----- 1.59 -----
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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

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Heterotrophic 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
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) _at_ 35 protein
100 Autotrophic (Recirculation System
with excellent solids removal)
100 Heterotrophic
35.6 Heterotrophic 64.4 Autotrophic
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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/gVSSA 0.358 gC/gVSSA
50.4 g NVSS 286 g CVSS
Conversion of 1 kg of feed _at_ 35 protein
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Conversion of 1 kg of feed _at_ 35 protein
Photoautotrophic (Pond intensive system)
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 ----- ----- -----
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Stoichiometry
Autotrophic System
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
Conversion of 1 kg of feed _at_ 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
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/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 CAlkalinity
Assume that the heterotrophic bacteria out
compete the autotrophic bacteria.
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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/gVSSA 0.531 gC/gVSSA
0.80 g NVSS 3.45 g CVSS 31.7 g
NO3-N 55.4 g CAlk
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Zero-exchange System (no Carbon
Supplementation) Heterotrophic and Autotrophic
Components
Heterotrophic Bacteria Heterotrophic Bacteria Heterotrophic Bacteria Consumes C organic C inorganic N
Stoichiometry Stoichiometry (g) (g) (g) (g)
NH4-N 0.356 NT 0.356 NT 17.9 ----- ----- 17.9
C6H12O6 feed 15.17 g Carbs/ g N 15.17 g Carbs/ g N 272 108.9 ----- -----
Alkalinity 3.57 g Alk/ g N 3.57 g Alk/ g N 63.9 ----- 15.4 -----

Autotrophic Bacteria Autotrophic Bacteria Autotrophic Bacteria Consumes C organic C inorganic N
Stoichiometry Stoichiometry (g) (g) (g) (g)
NH4-N 0.644 NT 0.644 NT 32.5 ----- ----- 32.5
Alkalinity 7.05 g Alk/ g N 7.05 g Alk/ g N 229.1 ----- 55.4 -----

C organic C inorganic N
Total Consumed Consumes  Consumes  Consumes  (g) (g) (g)
NH4-N 50.4 g N 50.4 g N 50.4 g N ----- ----- 50.4
C6H12O6 272 g Carbs 272 g Carbs 272 g Carbs 108.9 ----- -----
Alkalinity 293 g Alk 293 g Alk 293 g Alk ----- 70.8 -----

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Zero-exchange System (no Carbon
Supplementation) Heterotrophic and Autotrophic
Components
Heterotrophic Bacteria 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 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  Yields (g) (g) (g)
VSS 150.5 g VSS 150.5 g VSS 80.0 ----- 18.7
NO3--N 31.7 g NO3-N 31.7 g NO3-N ----- ----- 31.7
CO2 363.4 g CO2 363.4 g CO2 ----- 99.1 -----
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
Percent removal of ammonia-nitrogen by
Heterotrophic or Autotrophic Process as a
function of Protein
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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/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
Carbohydrate is 40 Carbon ? 492 g carbs
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Zero-exchange System (Carbon Supplementation)
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 -----
(460 g Cfeed 197 g Ccarb ) / 50.5 g N ? C/N
ratio of 13
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Zero-exchange System (Carbon Supplementation)
Supplemental Carbohydrate as percentage of feed
rate for heterotrophic metabolism of
ammonia-nitrogen to microbial biomass
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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

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Research System
Weekly Growth about 0.9 gm/wk
4x12 System with sludge settling tank, automatic
feeders, bacterial floc
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Water Quality Parameters

• Dissolved Oxygen
• Salinity
• Temperature
• pH
• Alkalinity
• TSS/TVS

• TAN
• NO2 N
• NO3 N
• Total Nitrogen
• Total Organic Carbon

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Water Quality Summary
DO Temp Salinity pH TAN NO2-N NO3-N Alkalinity
  (mg/L) (Cº) (ppt)   (mg/L) (mg/L) (mg/L) (mg/L)
Control 6.1 29.5 4.8 7.78 1.15 0.13 54.7 183
StDev 0.4 0.5 0.4 0.20 1.06 0.15 29.0 49

50 of Feed 5.7 29.8 4.5 8.15 1.06 0.38 7.7 328
StDev 0.9 0.9 0.4 0.14 0.26 1.0 3.3 22

100 of Feed 5.3 29.4 4.7 8.19 1.36 0.60 1.9 360
StDev 1.5 0.2 0.2 0.18 0.81 1.1 0.8 24
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Water Quality Parameters - Dissolved Oxygen
35
Water Quality Parameters - Temperature
36
Water Quality Parameters - pH
37
Water Quality Parameters - TAN
38
Water Quality Parameters NO2- N
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Water Quality Parameters NO3- N
40
Water Quality Parameters Alkalinity
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Solids Management

• Solids Management
• Settling cones

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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

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Settling Basins

• Design to minimize turbulence

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Autotrophic/Heterotrophic Model
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Heterotrophic Model (50 feed)
46
Heterotrophic Model (100 feed)
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TSS Production Model

• Solids Production Model
• autotrophic/heterotrophic
• heterotrophic

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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.

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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.

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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.

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Autotrophic/Heterotrophic Model
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Heterotrophic Model (50 feed)
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Heterotrophic Model (100 feed)
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Water Quality Management C/N Ratio
Impact of carbon supplementation (or lack of) on
nitrite-nitrogen
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Water Quality Parameters - TOC
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Water Quality Parameters - TN
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
Mass balance on nitrogen for the
autotrophic/heterotrophic system without carbon
supplementation and with periodic harvesting of
excess bacterial biomass
57
Conclusions

• Further work is needed to characterize the impact
  on production system performance at various C/N
  ratios.
• Alternative forms of Carbon need to be evaluated
  for effectiveness and economics.
• Fundamental research is needed on carbon
  assimilation and conversion efficiency for
  heterotrophic bacteria.
• Development of optimal strains of bacteria for
  zero-exchange systems.

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Conclusions
Engineering Sustainability

• New Paradigm
• Zero-exchange systems
• Mixed-cell raceways
• SPF, low salinity growout

Organic Carbon Nitrogen ? Bacterial Biomass
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Acknowledgements
Research was supported by the Agriculture
Research Service of the United States
Department of Agriculture, under Agreement
No. 59-1930-1-130 and Magnolia Shrimp LLC,
Atlanta Georgia with special thanks to Miami
Aqua-culture, Inc., Dan Spotts. Opinions,
conclusions, and recommendations are of the
authors and do not necessarily reflect the view
of the USDA. All experimental protocols
involving live animals were in compliance with
Animal Welfare Act (9CFR) and have been approved
by the Freshwater Institute Animal Care and Use
Committee.
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Questions?