Browdy
SHRIMP BIOFLOC TECHNOLOGIES, FEEDS AND GUT HEALTH Craig L. Browdy Executive Manager Aquaculture Research
EMS
OK, Is there anything about shrimp health and bioflocs that we haven’t covered?
Biofilms
Concentrators
Biofilms
Host
Pathogen
Environment
Pathogens Biosecurity
Viral versus bacterial shrimp pathogens • Transfer in shrimp – Live, frozen?
• Obligate host? • Excludable? • Vertical transmission? • Resistance mechanisms / breeding strategies? • Mutation and gene transfer rates
• Other spread – Ballast? Currents?
• Concentrated in environment • Effect of probiotics • Effects of antimicrobials • Quorum sensing • Biofilms
Vibrio fitness and virulence • genomic plasticity • attachment and colonization – biofilm formation: flagella, type IV pili, exopolysaccharide synthesis, quorum sensing
• • • • •
immune evasion virulence factors nutrient acquisition competition survival in unfavorable biotic and abiotic conditions – viable non culturable state, signaling, colonization, transcriptional regulation, antimicrobials, acid and salt tolerance, polysaccharides Johnson 2013 - Microbial Ecology
Host Feeds and Gut Health
Feed cost reduction Fast growing shrimp strains
Feed cost
Economical return for farm: ROI
• • • •
Formulation Raw material cost Operational efficiencies Finance
• Survival: health • FCR & growth • Fish/shrimp quality
Role of Nutritional Additives ?
Feed cost reduction and health “Feed cost reduction is not only about supplying and balancing nutrients for fast growth. Assuring nutrient
availability and improving animal health is equally important” • • • • •
More bioavailable micronutrients Attractants and pallatants Gut health modifiers Immunostimulants, nucleotides Enzymes
Shrimp digestive system • The anatomy of the shrimp digestive system includes barriers to protect against pathogen infection
Gastric Sieve Bell and Lightner 1988 Digestive fluid circulation Ceccaldi 1997
Peritrophic membrane in dissected midgut Wang et al. 2012 Stomach cuticular lining Bell and Lightner 1988
Vibriosis White Feces Disease Nyan Taw 2010
Bacterial colonization in posterior stomach Lightner 1996
Lightner AHPNS presentation NACA 2012
Gut environment modifiers • Antibiotics Antimicrobials • Essential Oil blends • Organic acid blends
Prebiotics
• Oligosacharides
Probiotics
• Bacillus • Pediococcus
Gut Health and Microbial Community Equilibrium
Essential oil blend – NEXT Enhance 150 40 X a
a
a
b
1 mm
Challenge mortality
HP microbial load
active substances found naturally in Oreganum spp Kasetsart University, 2008
Organic acid blends Treatment
Number of total bacteria (CFU/g)
Control
1.52 + 1.21 x 107b
MeraCid 1%
3.06 + 1.95 x 106a
Bacterostatic and bacteriocidal Number of total Vibrio (CFU/g)
Control
1.15 + 1.10 x 107b
MeraCid 1%
7.74 + 4.16 x 105a
120 100 Survival (%)
Treatment
80 60 40 20
control
Meracid 0.5%
0
0
10
20
30 Days
40
50
60
Minimum inhibitory concentration against Vibrio parahemolyticus Vibrio parahemolyticus has been identified as the causative agent of Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Syndrome (EMS/AHPNS) affecting shrimp.
% 1.0 0.5
0.25
Product
Minimum inhibitory concentration (%; pH 7.0)
0.125 0.0625
0.063
0.0313 0.0156
0.250
0.0078
V. parahemolyticus used was donated by Dr Dang Thi Hoang Oanh , Can Tho University
Nutritional additives and Health: Farm trial Trial set up: Cage (1x1x1 m) trials in pond. 6 cages per treatment 20 shrimp per cage (20/m²), Initial weight: 10g; 71 days, final weight 23-24 g Treatments: Control Antibiotic treatment (Enroflox) Next Enhance 150 (30 ppm thymol+carvacrol) Meracid ( 5 kg/ton)
In pond cage trial - Growth 1.80
b ab
1.40
ab
a ab
FCT
a 1.35
1.20
1.25
1.00 b a
b
1.40
1.30
90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40%
a
1.60
4500.00
ab
a
Production (kg/ha)
Survival (%)
Growth (g/wk)
1.45
b ab
4000.00 3500.00 3000.00 2500.00 2000.00
a
a
Phagocytosis
Phenoloxidase activity
Respiratory burst
Environment Biofloc systems
Resuspension in intensive aquaculture • Prevents precipitation of organic particles to the bottom of the pond • Enhances growth of heterotrophic bacteria using ammonia • Organic matter-bacteria protozoa complex is a source of natural food
Solar Aquafarms - ODAS
Aerated Zero Exchange Biofloc Systems • • • • • • • •
↓ eutrophication ↓ sedimentation ↓ escapement ↑ nitrogen assimilation ↑ water quality stability ↑ growth factors ↓ production costs ↓ pathogen introduction
Microbial Community • Heterotrophs – Nitrogen uptake – lower salinities – Floc substrates – Sludge degradation
• Chemoautotrophs – Nitrogen cycling – Nitrification – Denitrification
• Photoautotrophs – Growth enhancement – photosynthesis – Nitrogen uptake
Research Questions • What are the best tools for measuring and describing the complex microbial floc community • How do we establish a diverse and stable community • What is the optimal microbial floc community composition – For competitive exclusion of pathogens – For target crop growth – For water quality management
• How do we manipulate the community to maintain optimal composition – Fertilization - Filtration – Sterilization – Inoculation, probiotics – Habitat - Environment
Solids Management and Microbial Communities 800
35
700 Fishmeal
25
Fishmeal Settled
20
Plant
15 Plant Settled
10 5
600
500 400 300
1
2
3
4
5
6
7
8
9
Branched and Odd Chain Fatty Acids (µg L-1)
1600
1.5
1.0
0.5
0.0
8
0
2.5
2.0
Fishmeal Fishmeal Settled
Plant
Plant Settled
Fishmeal Fishmeal Settled Plant Plant Settled
10
Plant Plant Settled
10 11 12
Week
Mean Photosynthetic Oxygen Production (mg L-1 h-1)
Fishmeal Settled
100
0
12
Fishmeal
200
Weight (g)
30
Chlorophyll-a (µg L-1)
PAR Extinction Coefficient
40
Fishmeal Fishmeal Settled Plant Plant Settled
1400 1200
6 4 2
1000 800
0 0
600
1
2
3
4
5
6
7
8
9
10
11
12
Week 400
200 0 1
2
3
4
5
6
7
8
9
10
11
12
Week
• Effects of settling chambers – 47% ↓photosynthetically active radiation extinction coefficient – 200% ↑ photosynthetic oxygen production – 65% ↓ final chlorophyll-a – 80% ↓ fatty acid bacterial indicators
• Shrimp Production – – – –
No difference in survival 28% ↑ growth rate 41% ↑ biomass 26% ↓ FCR
Rat et al 2012 - Aquaculkture
Biofloc Influence on Shrimp Growth Diatoms in a bacterial matrix
1.60 1.40
RW1Mean Wt
1.20
RW2 Mean Wt
1.00 0.80 0.60 0.40 0.20 0.00 4/18
4/25
5/2
5/9
5/16
5/23
5/30
6/6
6/13
Growth rates converged as biofloc communities converged
Synechococcus dominated (cyanobacteria)
Recent results from Texas A&M EXP
27.22 ± 0.85
28.80 ± 1.84
Growth (g/wk)
2.05 ± 0.13
2.16 ± 0.31
Yield (kg/m3)
8.21 ± 0.31
7.79 ± 1.13
FCR
1.59 ± 0.01
1.72 ± 0.08
93.14 ± 3.13a
83.35 ± 2.69b
Final Weight (g)
Survival (%) 35
30
Average weight (g)
• Low level mortality from enteric and systemic vibriosis. 16S rRNA sequencing: showed presence of Vibrio parahaemolyticus, V. owensii, V. communis, V. alginolyticus • No significant differences in performance except for survival • Shrimp fed the HI-35 feed had higher survival than those fed the EXP potentially due to the presence of Zeigler VPak® and possible contribution to control of Vibrio
HI-35
25 20 15 10 5 0 0
20
40
60
Samocha et al. Unpublished
80
• Control vs sucrose (0.5kg/kg feed) as carbohydrate source • No water exchange
• Periodic probiotic additions to water
Performance
With Sucrose
Control
Weight Gain
11.33±0.02a
9.98±0.025b
FCR
1.67±0.11a
1.8±0.17b
Survival
65.7±4.6a
52.3±6.1b
• DGGE analysis • Bacillus dominant with sucrose (27.71%±2.83%) • Vibrio sp. Dominant in control group (22.65%±4.49%).
Feed programs • Feeds are the driver of nutrient inputs into the system – – – – –
Physical characteristics, leaching Nutrient quality, digestibility Nutrient density, formulations Ingredient costs Feeding timing, frequency, amounts
Biofloc Feed Formulation Strategies • Design feeds to drive biofloc management – Focus on microbial community, C:N ratios, low protein
OR • Design feeds to efficiently meet shrimp requirements – – – –
High protein nutrient dense formulations Tight control of feeding rates Supplemental carbon addition as necessary Avoid waste nutrient buildup, Phosphorus, Minerals etc.
Approaches • Improve makeup of the exogenous microbial community by actively maintaining a healthy and biosecure biofloc system • Maintain shrimp pond environment quality and shrimp performance by providing balanced nutrient dense feeds in highly efficient feeding programs • Explore opportunities to enhance shrimp fitness and immunocompetence while managing gut biota, structure and function through the effective use of selected feed supplements
Integrated strategies • Understanding of the pathogens – laboratory models, sensitive diagnostics, sharing of data and isolates
• Innovative strategies for assuring shrimp fitness – Feed quality, breeding, resistance, nutrition, gut microbial health , theraputants
• Improving culture systems – intensive nursery systems – micro and meiofaunal community management
Thank You!
37
EMS
OK, Is there anything about shrimp health and bioflocs that we haven’t covered?
Biofilms
Concentrators
Biofilms
Host
Pathogen
Environment
Pathogens Biosecurity
Viral versus bacterial shrimp pathogens • Transfer in shrimp – Live, frozen?
• Obligate host? • Excludable? • Vertical transmission? • Resistance mechanisms / breeding strategies? • Mutation and gene transfer rates
• Other spread – Ballast? Currents?
• Concentrated in environment • Effect of probiotics • Effects of antimicrobials • Quorum sensing • Biofilms
Vibrio fitness and virulence • genomic plasticity • attachment and colonization – biofilm formation: flagella, type IV pili, exopolysaccharide synthesis, quorum sensing
• • • • •
immune evasion virulence factors nutrient acquisition competition survival in unfavorable biotic and abiotic conditions – viable non culturable state, signaling, colonization, transcriptional regulation, antimicrobials, acid and salt tolerance, polysaccharides Johnson 2013 - Microbial Ecology
Host Feeds and Gut Health
Feed cost reduction Fast growing shrimp strains
Feed cost
Economical return for farm: ROI
• • • •
Formulation Raw material cost Operational efficiencies Finance
• Survival: health • FCR & growth • Fish/shrimp quality
Role of Nutritional Additives ?
Feed cost reduction and health “Feed cost reduction is not only about supplying and balancing nutrients for fast growth. Assuring nutrient
availability and improving animal health is equally important” • • • • •
More bioavailable micronutrients Attractants and pallatants Gut health modifiers Immunostimulants, nucleotides Enzymes
Shrimp digestive system • The anatomy of the shrimp digestive system includes barriers to protect against pathogen infection
Gastric Sieve Bell and Lightner 1988 Digestive fluid circulation Ceccaldi 1997
Peritrophic membrane in dissected midgut Wang et al. 2012 Stomach cuticular lining Bell and Lightner 1988
Vibriosis White Feces Disease Nyan Taw 2010
Bacterial colonization in posterior stomach Lightner 1996
Lightner AHPNS presentation NACA 2012
Gut environment modifiers • Antibiotics Antimicrobials • Essential Oil blends • Organic acid blends
Prebiotics
• Oligosacharides
Probiotics
• Bacillus • Pediococcus
Gut Health and Microbial Community Equilibrium
Essential oil blend – NEXT Enhance 150 40 X a
a
a
b
1 mm
Challenge mortality
HP microbial load
active substances found naturally in Oreganum spp Kasetsart University, 2008
Organic acid blends Treatment
Number of total bacteria (CFU/g)
Control
1.52 + 1.21 x 107b
MeraCid 1%
3.06 + 1.95 x 106a
Bacterostatic and bacteriocidal Number of total Vibrio (CFU/g)
Control
1.15 + 1.10 x 107b
MeraCid 1%
7.74 + 4.16 x 105a
120 100 Survival (%)
Treatment
80 60 40 20
control
Meracid 0.5%
0
0
10
20
30 Days
40
50
60
Minimum inhibitory concentration against Vibrio parahemolyticus Vibrio parahemolyticus has been identified as the causative agent of Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Syndrome (EMS/AHPNS) affecting shrimp.
% 1.0 0.5
0.25
Product
Minimum inhibitory concentration (%; pH 7.0)
0.125 0.0625
0.063
0.0313 0.0156
0.250
0.0078
V. parahemolyticus used was donated by Dr Dang Thi Hoang Oanh , Can Tho University
Nutritional additives and Health: Farm trial Trial set up: Cage (1x1x1 m) trials in pond. 6 cages per treatment 20 shrimp per cage (20/m²), Initial weight: 10g; 71 days, final weight 23-24 g Treatments: Control Antibiotic treatment (Enroflox) Next Enhance 150 (30 ppm thymol+carvacrol) Meracid ( 5 kg/ton)
In pond cage trial - Growth 1.80
b ab
1.40
ab
a ab
FCT
a 1.35
1.20
1.25
1.00 b a
b
1.40
1.30
90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40%
a
1.60
4500.00
ab
a
Production (kg/ha)
Survival (%)
Growth (g/wk)
1.45
b ab
4000.00 3500.00 3000.00 2500.00 2000.00
a
a
Phagocytosis
Phenoloxidase activity
Respiratory burst
Environment Biofloc systems
Resuspension in intensive aquaculture • Prevents precipitation of organic particles to the bottom of the pond • Enhances growth of heterotrophic bacteria using ammonia • Organic matter-bacteria protozoa complex is a source of natural food
Solar Aquafarms - ODAS
Aerated Zero Exchange Biofloc Systems • • • • • • • •
↓ eutrophication ↓ sedimentation ↓ escapement ↑ nitrogen assimilation ↑ water quality stability ↑ growth factors ↓ production costs ↓ pathogen introduction
Microbial Community • Heterotrophs – Nitrogen uptake – lower salinities – Floc substrates – Sludge degradation
• Chemoautotrophs – Nitrogen cycling – Nitrification – Denitrification
• Photoautotrophs – Growth enhancement – photosynthesis – Nitrogen uptake
Research Questions • What are the best tools for measuring and describing the complex microbial floc community • How do we establish a diverse and stable community • What is the optimal microbial floc community composition – For competitive exclusion of pathogens – For target crop growth – For water quality management
• How do we manipulate the community to maintain optimal composition – Fertilization - Filtration – Sterilization – Inoculation, probiotics – Habitat - Environment
Solids Management and Microbial Communities 800
35
700 Fishmeal
25
Fishmeal Settled
20
Plant
15 Plant Settled
10 5
600
500 400 300
1
2
3
4
5
6
7
8
9
Branched and Odd Chain Fatty Acids (µg L-1)
1600
1.5
1.0
0.5
0.0
8
0
2.5
2.0
Fishmeal Fishmeal Settled
Plant
Plant Settled
Fishmeal Fishmeal Settled Plant Plant Settled
10
Plant Plant Settled
10 11 12
Week
Mean Photosynthetic Oxygen Production (mg L-1 h-1)
Fishmeal Settled
100
0
12
Fishmeal
200
Weight (g)
30
Chlorophyll-a (µg L-1)
PAR Extinction Coefficient
40
Fishmeal Fishmeal Settled Plant Plant Settled
1400 1200
6 4 2
1000 800
0 0
600
1
2
3
4
5
6
7
8
9
10
11
12
Week 400
200 0 1
2
3
4
5
6
7
8
9
10
11
12
Week
• Effects of settling chambers – 47% ↓photosynthetically active radiation extinction coefficient – 200% ↑ photosynthetic oxygen production – 65% ↓ final chlorophyll-a – 80% ↓ fatty acid bacterial indicators
• Shrimp Production – – – –
No difference in survival 28% ↑ growth rate 41% ↑ biomass 26% ↓ FCR
Rat et al 2012 - Aquaculkture
Biofloc Influence on Shrimp Growth Diatoms in a bacterial matrix
1.60 1.40
RW1Mean Wt
1.20
RW2 Mean Wt
1.00 0.80 0.60 0.40 0.20 0.00 4/18
4/25
5/2
5/9
5/16
5/23
5/30
6/6
6/13
Growth rates converged as biofloc communities converged
Synechococcus dominated (cyanobacteria)
Recent results from Texas A&M EXP
27.22 ± 0.85
28.80 ± 1.84
Growth (g/wk)
2.05 ± 0.13
2.16 ± 0.31
Yield (kg/m3)
8.21 ± 0.31
7.79 ± 1.13
FCR
1.59 ± 0.01
1.72 ± 0.08
93.14 ± 3.13a
83.35 ± 2.69b
Final Weight (g)
Survival (%) 35
30
Average weight (g)
• Low level mortality from enteric and systemic vibriosis. 16S rRNA sequencing: showed presence of Vibrio parahaemolyticus, V. owensii, V. communis, V. alginolyticus • No significant differences in performance except for survival • Shrimp fed the HI-35 feed had higher survival than those fed the EXP potentially due to the presence of Zeigler VPak® and possible contribution to control of Vibrio
HI-35
25 20 15 10 5 0 0
20
40
60
Samocha et al. Unpublished
80
• Control vs sucrose (0.5kg/kg feed) as carbohydrate source • No water exchange
• Periodic probiotic additions to water
Performance
With Sucrose
Control
Weight Gain
11.33±0.02a
9.98±0.025b
FCR
1.67±0.11a
1.8±0.17b
Survival
65.7±4.6a
52.3±6.1b
• DGGE analysis • Bacillus dominant with sucrose (27.71%±2.83%) • Vibrio sp. Dominant in control group (22.65%±4.49%).
Feed programs • Feeds are the driver of nutrient inputs into the system – – – – –
Physical characteristics, leaching Nutrient quality, digestibility Nutrient density, formulations Ingredient costs Feeding timing, frequency, amounts
Biofloc Feed Formulation Strategies • Design feeds to drive biofloc management – Focus on microbial community, C:N ratios, low protein
OR • Design feeds to efficiently meet shrimp requirements – – – –
High protein nutrient dense formulations Tight control of feeding rates Supplemental carbon addition as necessary Avoid waste nutrient buildup, Phosphorus, Minerals etc.
Approaches • Improve makeup of the exogenous microbial community by actively maintaining a healthy and biosecure biofloc system • Maintain shrimp pond environment quality and shrimp performance by providing balanced nutrient dense feeds in highly efficient feeding programs • Explore opportunities to enhance shrimp fitness and immunocompetence while managing gut biota, structure and function through the effective use of selected feed supplements
Integrated strategies • Understanding of the pathogens – laboratory models, sensitive diagnostics, sharing of data and isolates
• Innovative strategies for assuring shrimp fitness – Feed quality, breeding, resistance, nutrition, gut microbial health , theraputants
• Improving culture systems – intensive nursery systems – micro and meiofaunal community management
Thank You!
37
