Comparing Chemoautotrophic-Based

Systems and the Use of Three

Carbohydrates to Promote Heterotrophic-
Based Biofloc Shrimp (Litopenaeus
vannamei) Culture Systems

The National Shellfisheries

Association

Andrew J. Ray*, Verlee M. Breland, Christopher C. Farno,

Kevin S. Dillon, and Jeffrey M. Lotz
Gulf Coast Research Laboratory
The University of Southern Mississippi, Ocean Springs, MS USA
AndrewJRay@gmail.com
 Biofloc Bacteria
• Chemoautotrophic
– NH3 NO2- NO3-
• Establishment
• Toxic compounds 3.0 45

Ammonia/Nitrite-nitrogen (mg/L)
40
• Reliable once established? 2.5
35

Nitrate-nitrogen (mg/L)
• ↓O2 consumption 2.0 30 Ammonia-
nitrogen
25
1.5 Nitrite-
• Heterotrophic 1.0
20
15
nitrogen
Nitrate-
– N assimilation  Protein 0.5
10
5
nitrogen

• ↑ O2 consumption 0.0
1 2 3 4 5 6 7 8 9 10
0

Week
• Solids generation
• Carbohydrate addition (C:N)
– Carbohydrate source?
• This Project
– Autotrophic versus heterotrophic
– Evaluate carbohydrate sources
 Materials and Methods
• 16, 500-L round tanks under a greenhouse
– Water from
established raceway
– 16‰ salinity

• Four Treatments
– 1.) Autotrophic (T-A) = no additional carbohydrate
– 2.) Heterotrophic Sucrose (T-HS)
– 3.) Heterotrophic Molasses (T-HM)
– 4.) Heterotrophic Glycerol (T-HG)
– Four randomly assigned replicate tanks each
 Materials and Methods
• C:N of inputs (feed + carbohydrate) = 25:1
– Added twice daily
– Reduced by 25% during week 5
• Shrimp stocked at 300 m-3
– 6.8 g mean stocking weight
– Grown for 8 weeks
• Particle removal
– Turbidity measured daily beginning week 2
• 15-L settling chambers
• > 150 NTU, run all day
• > 225 NTU, run all day and night (week 4)
 Results
Treatment
Autotrophic Heterotrophic Sucrose Heterotrophic Molasses Heterotrophic Glycerol
Temperature (⁰C)
AM 24.2 ± 0.3 (14.6-31.2) 25.0 ± 0.3 (15.8-30.1) 24.4 ± 0.3 (15.7-30.5) 24.5 ± 0.3 (15.8-29.7)
PM 26.9 ± 0.2 (19.1-31.9) 27.3 ± 0.2 (20.2-31.3) 27.1 ± 0.2 (20.0-31.7) 27.2 ± 0.2 (20.1-31.2)
-1
Dissolved Oxygen (mg L )
AM 8.9 ± 0.1 (6.8-13.1) 8.4 ± 0.1 (6.7-11.6) 8.7 ± 0.1 (6.8-12.3 8.3 ± 0.1 (6.4-11.5)
PM 7.9 ± 0.1 (6.5-11.8) 7.3 ± 0.1 (5.0-11.8) 7.6 ± 0.1 (5.5-13.2) 6.7 ± 0.1 (3.3-12.6)
pH
AM 8.1 ± 0.0 (7.8-8.5) 8.3 ± 0.0 (7.6-8.6) 8.4 ± 0.0 (8.0-8.7) 8.2 ± 0.0 (7.9-8.5)
PM 8.3 ± 0.0 (7.8-8.6) 8.1 ± 0.0 (7.8-8.6) 8.3 ± 0.0 (7.8-8.6) 8.1 ± 0.0 (7.7-8.5)
Salinity (g L-1) 16.4 ± 0.0 (15.4-17.5) 16.3 ± 0.0 (15.4-17.7) 16.4 ± 0.0 (15.6-17.6) 16.3 ± 0.0 (15.5-17.5)
Data are reported as mean ± SE (range)

70 0.9 a
a
a 0.8 a
60 ab
ab
Growth Rate (g week-1)
0.7
50
Percent Survival

0.6
40 0.5
b
30 b 0.4
0.3
20
0.2
10 0.1
0 0.0
Autotrophic Heterotrophic Heterotrophic Heterotrophic Autotrophic Heterotrophic Heterotrophic Heterotrophic
Sucrose Molasses Glycerol Sucrose Molasses Glycerol
 3.0

Results 2.5

2.0

mg TAN L-1
1.5

• TAN 1.0

– ↑ in T-HM (P ≤ 0.03)
0.5

0.0
10
9 Autotrophic
8 Heterotrophic-Sucrose
Heterotrophic-Moleasses
• NO2-N 7

mg NO2-N L-1
6
Heterotrophic-Glycerol

– T-A > T-HG (P = 0.05) 4

3
2
1
0
180

• NO3-N 160
140

– Final Sample Date

mg NO3-N L-1 120
100

– ↑ T-A (P ≤ 0.01)
80
60
40
20
0
0 1 2 3 4 5 6 7 8
Week
 Results
• PO4 (dissolved, reactive) 90
80

– ↑ in T-A (P ≤ 0.01) 70
60

mg PO4 L-1
50
40
30

• Alkalinity 20
10

– T-HM > T-HS, THG > T-A 0

600
Autotrophic
(P ≤ 0.01)
Alkalinity (as mg CaCO3 L-1)
Heterotrophic-Sucrose
500
Heterotrophic-Molasses
Heterotrophic-Glycerol
400

300

200

100

0
0 1 2 3 4 5 6 7 8
Week
 8.5

Oxygen 8.0
Sucrose

Dissolved Oxygen (mg L-1)

7.5
600
Autotrophic
7.0
Heterotrophic Sucrose
500 Heterotrophic Molasses
Heterotrophic Glycerol 6.5
400
6.0
BOD5

300 5.5

200 5.0
8.5
100 Molasses
8.0

Dissolved Oxygen (mg L-1)

0 7.5
4 5 Week 6 7
7.0

6.5

• BOD5 (biochemical oxygen demand) 6.0

5.5

• T-HS, T-HG > T-HM > T-A 5.0

(P ≤ 0.04)
8.5
Glycerol
Dissolved Oxygen (mg L-1)
8.0

• Oxygen Depletion (N ≥ 6) 7.5

7.0

– T-HS = 24% in 25 min. 6.5

6.0

– T-HM = 24% in 15 min. 5.5

5.0

– T-HG = 25% in 60 min. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Minutes
 Solids
1000
900
800
• Total suspended solids 700

mg TSS L-1
600

(TSS) 500
400

– ↓ in T-A (P ≤ 0.02)
300 Autotrophic
200 Heterotrophic Sucrose
100 Heterotrophic Molasses
Heterotrophic Glycerol
0
1 2 3 4 5 6 7 8 9
Week

• Removed Solids 90

Settled Material Removed (L)

80

– T-HS, T-HG > T-A, T-HM 70

60

(P ≤ 0.01) 50
40
30
20
10
0
Autotrophic Heterotrophic Heterotrophic Heterotrophic
Sucrose Molasses Glycerol
 Stable Isotope Values

Shrimp Flesh
•Apparent direct 10.0

9.5
a

ab
ab
nitrogen enrichment 9.0 b

8.5
from feed in T-A

δ 15N
8.0

shrimp 7.5

7.0

6.5 Feed δ 15N

•Lower δ 15N values in 6.0

Autotrophic Heterotrophic Heterotrophic Heterotrophic
Sucrose Molasses Glycerol
heterotrophic
treatments
Different N sources?
• Shrimp Production Summary
– No significant differences between autotrophic and heterotrophic
– Very poor in molasses treatment
• Inorganic N
– Carbohydrate stimulated N uptake
– NH3 spike in T-HM, NO2 spike in T-A,
NO3 accumulation in T-A
• PO4 ↑, Alkalinity ↓ in autotrophic treatment
• Oxygen
– BOD ↓ in T-A, highest in T-HG, T-HS
– DO drops quickly, recovers in T-HS, T-HM; gradual in T-HG
• Solids
– Significantly less in T-A
• Stable Isotopes
– Interesting, need further exploration
 Thank You
Heterotrophic Heterotrophic Heterotrophic
Autotrophic
Glycerol Molasses Sucrose

• This work was funded by The National Shellfisheries

Association’s Michael Castagna Student Grant for Applied
Research and the United States Department of Agriculture’s US
Marine Shrimp Farming Program