Transcript USAS Las Vegas Feb. 24

Understanding photoautotrophic, autotrophic,
and heterotrophic bacterial based systems
using basic water quality parameters
James M. Ebeling, Ph.D.
Michael B. Timmons, Ph.D.
Research Engineer
Aquaculture Systems
Technologies
Professor
Dept. of Bio. & Environ. Eng.
Cornell University
“New Paradigm”
Zero-exchange Systems “Belize System”
 Shrimp – high health, selectively bred Specific Pathogen Free stock
 Feed – low protein feeds in combination with traditional high protein feeds
 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 Specific Pathogen Free stock
 Feed – low protein feeds in combination with traditional high protein feeds
 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!!!!!
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Nitrogen Removal Pathways
NH4+-N
CO2
Alkalinity
Corganic
Trace
Nutrients
•
• Autotrophic
System
CO2
VSSAlgae
Alkalinity
VSSAuto
O2
Photoautotrophic
• Heterotrophic
• Other Mysterious Ways
NO3--N
VSSHetero
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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 HCO3Ammonia as nitrogen source
16 NH4+ + 92 CO2 + 92 H2O + 14 HCO3- + HPO42- →
C106H263O110N16P + 106 O2
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Photoautotrophic (algal based systems)
The water quality impacts of photosynthesis:
 photosynthesis is a one step process
 for both reactions, the C/N ratio of algal biomass is the same, 5.69 g C/g N
 the net production of biomass is also the same, 15.85 g VSS/ g N
 VSS consist of 36% C and 6.3% N
 both reaction utilize CO2 as their primary carbon source
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Photoautotrophic (algal based systems)
The water quality impacts of photosynthesis:
 both reactions metabolize only a small quantity of phosphorus, 0.14 g P / g N
 alkalinity is consumed when ammonia is the nitrogen source, 3.13 g Alk/ g N
 alkalinity is produced when nitrate is the nitrogen source, 4.02 g Alk/ g N
 pH increases due to consumption of carbon dioxide
 both reactions generate oxygen
 generation times for algal biomass range from 1 to 2 days
 at night or during low light levels, respiration dominates and the stoichiometric
relations are reversed, i.e., pH decreases, oxygen is consumed, carbon dioxide is
released.
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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
The water quality impacts of nitrification:
 nitrification is a two step process, ammonia to nitrite to nitrate
 the second step doesn’t start until the first step is well underway,
i.e., nitrite conversion to nitrate
 the C/N ratio of nitrifying bacterial biomass is 4.29 g C/ g N
 the net production of biomass is very small, 0.20 g VSS/ g N
 VSS consist of 53.1% C and 12.4% N
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Autotrophic - Nitrification
The water quality impacts of nitrification:
 nitrification is a two step process, ammonia to nitrite to nitrate
 the second step doesn’t start until the first step is well underway,
i.e., nitrite conversion to nitrate
 the C/N ratio of nitrifying bacterial biomass is 4.29 g C/ g N
 the net production of biomass is very small, 0.20 g VSS/ g N
 VSS consist of 53.1% C and 12.4% N
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Autotrophic - Nitrification
The water quality impacts of nitrification:
 the reaction utilizes alkalinity as the primary carbon source, 7.05 g Alk / g N
 pH of the system drops as the alkalinity is consumed and carbon dioxide is produced
 actual carbon requirement is 1.69 g C / g N
 oxygen requirement is 4.18 g O2 / g N
 carbon dioxide is produced, 5.85 g CO2/ g N
 generation times for biomass are very slow on the order of 2 to 5 days
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Autotrophic - Nitrification
The water quality impacts of nitrification:
 the reaction utilizes alkalinity as the primary carbon source, 7.05 g Alk / g N
 pH of the system drops as the alkalinity is consumed and carbon dioxide is produced
 actual carbon requirement is 1.69 g C / g N
 oxygen requirement is 4.18 g O2 / g N
 carbon dioxide is produced, 5.85 g CO2/ g N
 generation times for biomass are very slow on the order of 2 to 5 days
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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
The water quality impacts of heterotrophic bacteria:
 heterotrophic conversion is a one step process
 the C/N ratio of bacterial biomass is the same as nitrifiers, 4.29 g C/ g N
 the net production of biomass is 8.07 g VSS/ g N
 VSS consist of 53.1% C and 12.4% N
 the reaction utilize carbon from carbohydrate as the primary carbon source,
6.07 g C /g N or 15.17 g carbohydrate / g N
 pH of the system drops as the alkalinity is reduced, 3.57 g ALK / g N
 oxygen requirement is 4.71 g O2 / g N
 carbon dioxide is produced, 9.65 g CO2/ g N
 generation times for biomass are very fast
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Impact on Water Quality
Photoautotrophic
Autotrophic
Heterotrophic
Start-up
Production
Start-up
Production
Start-up
Production
NH4+-N


 then 



NO2--N


 then 



NO3--N






 (Light) or  (Dark)




 (NH4+-N) or  (NO3--N)




pH
Alkalinity
VSS
O2
CO2












 (Light) or  (Dark)




TOC






TN






growth rate
fast
slow
very fast
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Recirculating System (low salinity)
Ammonia-nitrogen
Nitrite-nitrogen
12.0
Nitrate-nitrogen
Typical Long-term
average values for largescale recir. system
Concentration (mg/L)
.
10.0
8.0
6.0
4.0
TAN:
NO2-N:
NO3-N:
1.16 mg/L
0.035 mg/L
22.0 mg/L
DOC:
DN:
3.7 mg/L
34.3 mg/L
2.0
DOC/DN:
0.19
0.0
0
7
14
21
28
35
VSS:
3.9 mg/L
Days
Hint: Classic start-up

Autotrophic System
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Experimental Design
Three treatments – Three replicates
• Photoautotrophic - 7.4g NH4CL; 10.4NaHCO3
• Heterotrophic - 7.4g NH4CL; 11.8g NaHCO3; 72.2g sugar
• Autotrophic - 7.4g NH4CL; 23.4g NaHCO3
(Daily Addition of Ammonia Chloride and stoichiometric
Alkalinity and organic carbon requirement)
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Experimental Design
Nine flat bottom fiberglass tanks
–
–
–
–
–
–
1 meter diameter
250 L (66 gal)
Immersion heaters
multiple air stones
Autotrophic Covered
Heterotrophic Covered
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Experimental Design
Daily Water Quality Monitoring
• Dissolved Oxygen
• Temperature
Daily Water Quality Analysis
•
•
•
•
TAN
NO2
NO3
Alkalinity
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Experimental Results - pH
Autotrophic
9.0
Heterotrophic
pH Value .
Photoautotrophic
8.5
8.0
7.5
0
7
14
21
28
35
42
Days into Study
49
56
63
70
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Experimental Results - TAN
12.0
Autotrophic
Heterotrophic
Photoautotrophic
TAN (mg/L-N)
10.0
8.0
6.0
4.0
2.0
0.0
0
7
14
21
28
35
42
49
56
63
70
Days into Stdy
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Experimental Results – Nitrite-nitrogen
NO2 (mg-N/L) .
Autotrophic
60
Heterotrophic
50
Photoautotrophic
Ammonia Addition
40
30
20
10
0
0
7
14
21
28
35
42
49
56
63
70
Days into Study
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Experimental Results – Nitrate-nitrogen
125
NO3 (mg/L-N)
100
Autotrophic
Heterotrophic
Photoautotrophic
75
50
25
0
0
7
14
21
42
35
28
Days into Study
49
56
63
70
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Experimental Results – Alkalinity
Autotrophic
Heterotrophic
Photoautotrophic
Alkalinity (mg/L) .
200
175
150
125
100
75
0
7
14
21
42
35
28
Days into Study
49
56
63
70
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Real World (?)
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Zero-exchange
(Pond intensive system)
Five high-intensity (120 shrimp/m2) shrimp ponds
Belize Aquaculture Ltd. (Burford, et al., 2003)
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Zero-exchange
Pond #3 (recirculated water)
Shrimp: 3.0 g
(Pond intensive system)
TAN:
NO2-N:
NO3-N:
0.13 mg/L
0.06 mg/L
0.01 mg/L
DOC:
DN:
48.1 mg/L
5.4 mg/L
DOC/DN:
VSS:
14.3
62.5 mg/L
Ammonia uptake (g/L hr)
Light/Dark:
Hint: Low Nitrogen, High DOC/DN Ratio, Moderate VSS, Equal Light/Dark Ratio

24.7/27.8
Heterotrophic System
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Zero-exchange
(Pond intensive system)
Pond #18 (seawater water)
Shrimp 5.5 g
TAN:
NO2-N:
NO3-N:
0.15 mg/L
0.01 mg/L
0.01 mg/L
DOC:
DN:
14.2 mg/L
2.5 mg/L
DOC/DN:
5.8
VSS:
64.0 mg/L
Ammonia uptake (g/L hr)
Light/Dark:
Hint: Low Nitrogen, Moderate DOC/DN Ratio, Moderate VSS, High Light/Dark Ratio
60.1/19.5

Photoautotrophic System
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Zero-exchange
(Pond intensive system)
Pond #9 (seawater water)
Shrimp 8.2 g
TAN:
NO2-N:
NO3-N:
1.26 mg/L
2.48 mg/L
0.39 mg/L
DOC:
DN:
17.5 mg/L
7.7 mg/L
DOC/DN:
VSS:
2.3
33.5 mg/L
Ammonia uptake (g/L hr)
Light/Dark:
84.7/40.5
Hint: High TAN & Nitrite, Low DOC/DN Ratio, Low VSS, Moderate Light/Dark Ratio  Conversion to Autotrophic System
Low nitrate values and Light/Dark Ratio suggest still a significant component of Photoautotrophic System
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Zero-exchange
Pond #7 (seawater water)
Shrimp 10.1 g
(Pond intensive system)
TAN:
NO2-N:
NO3-N:
2.76 mg/L
0.32 mg/L
0.11 mg/L
DOC:
DN:
22.8 mg/L
8.8 mg/L
DOC/DN:
VSS:
2.6
47.2 mg/L
Ammonia uptake (g/L hr)
Light/Dark:
Hint: Moderate TAN, Low DOC/DN Ratio, Moderate VSS, High Light/Dark Ratio

75.0/11.7
Photoautotrophic System
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Zero-exchange
Pond #5 (seawater water)
Shrimp 14.7 g
(Pond intensive system)
TAN:
NO2-N:
NO3-N:
0.19 mg/L
1.85 mg/L
8.62 mg/L
DOC:
DN:
20.0 mg/L
15.9 mg/L
DOC/DN:
VSS:
1.3
48.7 mg/L
Ammonia uptake (g/L hr)
Light/Dark:
Hint: Low TAN, Decreasing Nitrite, Low DOC/DN Ratio, Moderate VSS, Moderate Light/Dark Ratio
although high light to dark ratio suggest significant photoautotrophic component
85.7/29.2

Autotrophic System
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Conclusions
By carefully monitoring the water quality in the
production systems, it is possible to
characterize the type or removal process that is
dominating removal of ammonia-nitrogen.
4x12 Tanks: Alkalinity (mg/L CaCO3)
Ammonia-nitrogen (mg/L
8.0
6.0
350
300
GW - 4 ppt
GW - 12 ppt
ZE - 4 ppt
ZE - 12 ppt
250
40
200
GW - 4 pptl
GW - 12 ppt
150
ZE - 4 ppt
Control
ZE - 12 ppt
50% Feed
100
4.0
100% Feed
50
5/17/04
5/27/04
6/6/04
6/16/04
6/26/04
7/6/04
Nitrate-nitrogen (mg/L) .
Alkalinity (mg/L CaCO3) .
400
30
20
7/16/04
10
2.0
0
0
0.0
0
5
10
15
20
Days into Trial
25
30
35
Final Exam
5
10
15
20
25
30
Days into Trial
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Conclusions
Things to look for during the start-up and production phase of an
photoautotrophic nitrification process:
•
ammonia-nitrogen concentration remains relatively constant, at
moderate levels
•
very little nitrite-nitrogen or nitrate-nitrogen is generated
•
alkalinity is consumed when ammonia-nitrogen is consumed and
produced when nitrate is the nitrogen source
•
pH increases
•
substantial quantities of dissolved oxygen are generated
•
relatively rapid growth rate for the algal biomass
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Conclusions
Things to look for during the start-up and production phase of an
autotrophic nitrification process:
•
initial increase, peak and the falloff in ammonia-nitrogen
concentration as nitrification by autotrophic AOB bacteria begins to
convert ammonia-nitrogen to nitrite-nitrogen
•
rapid increase in nitrite-nitrogen concentration and then falloff as
autotrophic NOB convert nitrite-nitrogen to nitrate-nitrogen
•
finally, increase in nitrate-nitrogen
•
over time a decrease in pH, and alkalinity
•
very slow bacterial growth rate, thus little increase in TSS
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Conclusions
Things to look for during the start-up and production phase of an
heterotrophic nitrification process:
•
ammonia-nitrogen concentration remains relatively constant, at
moderate levels
•
very little nitrite-nitrogen or nitrate-nitrogen is generated
•
heterotrophic bacteria utilize carbon from the feed or supplemental
sources
•
pH of the system remains relatively constant
•
carbon dioxide is produced
•
heterotrophic bacteria out compete autotrophic bacteria
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Final Exam
Thus similar to reading a medical chart, one can determine the overall state
of health of a nitrification system, determine what pathway is dominate
and if necessary take corrective action if the systems strays from the
anointed path.
4x12 Tanks: Alkalinity (mg/L CaCO3)
Four Tanks: Ammonia-nitrogen
Two with supplemental Carbon
Two without
300
250
200
150
100
8.0
GW - 4 pptl
Ammonia-nitrogen (mg/L
GW - 12 ppt
6.0
Four Tanks: Nitrate-nitrogen
Two with supplemental Carbon
Two without
350
ZE - 4 ppt
50
5/17/04
Control
50% Feed
100% Feed
5/27/04
6/6/04
6/16/04
6/26/04
7/6/04
ZE - 12 ppt
Three Tanks: Alkalinity
Two with supplemental Carbon
One without
4.0
2.0
GW - 4 ppt
GW - 12 ppt
ZE - 4 ppt
ZE - 12 ppt
40
Nitrate-nitrogen (mg/L) .
Alkalinity (mg/L CaCO3) .
400
30
7/16/04
20
10
0
0
0.0
0
5
10
15
20
25
30
35
5
10
15
20
25
30
Days into Trial
Days into Trial
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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
Opinions, conclusions, and recommendations are of the authors
and do not necessarily reflect the view of the USDA.
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Questions?
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