PROBIOTICS AND THEIR USE IN AQUACULTURE April 2006 10 th Aquaculture Insurance and Risk Management Conference Presented by: William P. Long Chief Executive Officer Epicore BioNetworks Inc.
PROBIOTICS AND THEIR USE IN AQUACULTURE
April 200610th Aquaculture Insurance and Risk Management Conference
Presented by: William P. Long
Chief Executive OfficerEpicore BioNetworks Inc.
BASIC AQUACULTURE MARKET NEEDS
To survive and make money To overcome problems of:
Pond crashes Farm abandonment Low survival Poor quality animals Low weights Poor feed conversion ratio (FCR)
It’s not a hobby; it’s a business!
MURPHY’S LAW Whatever can go wrong, will go wrong!
Mismanagement Weak PL, bad feed, inadequate oxygen, poor
harvest procedures Natural events
Weather, predators Disease Theft Crop market prices
THE SCIENCE OF SURVIVAL
Through microbiological science aquaculturists can fight Murphy's Law
Probiotics are a natural, environmentally friendly application of microbiology
TRADITIONAL DISEASE CONTROL UNDER FIRE
Many older antibiotics proven unsafe so are banned from all uses
Shrimp with antibiotic residues cannot be sold in many major markets
State of art antibiotics should be reserved for humans
ROLE OF ENVIRONMENTAL MICROBIOLOGY IN AQUACULTURE
Pollution control Bioremediation of wastes Cleaner water & pond bottoms Disease resistance imparted by creating a
lower stress growing environment
Probiotic action Directly protects animals from disease
Pathogenic Vibrio parahemolyticus, Vibrio harveyi bacteria, Aeromonas
Indirectly protects from some viral diseases
POLLUTION CONTROL Waste is major problem in aquaculture pond and
hatchery waters Affects animal health and vigor Lowers harvest yield value Forces frequent water exchange
A biosecurity risk Bacteria are nature’s chosen waste degraders End result of microbial action is biomass - a soil
amendment
Harnessing the power of nature’s microbial
workforce
BIOREMEDIATION MODE OF ACTION Aquaculture wastes are organic in nature Microorganisms “eat” organic wastes
Bacteria + N + C + P + O2 = More Bacteria + H2O + CO2
Microorganisms digest pollutants Ammonia: very toxic to crustaceans and fish Nitrite: very toxic to crustaceans and fish
And is food for disease causing Vibrio Nitrate: too much causes algae crashes Pond bottom waste: release nutrients in older
ponds to promote natural algae bloom without added fertilizer
WHAT IS A PROBIOTIC
A live microbial supplement that benefits the host animal– From the Greek “probios”:
Pro = “for” Bios = “life”
Opposite of antibiotic– A microbial killer or suppressant
PROBIOTIC MODES OF ACTION Competitive exclusion
Out-competes pathogenic bacteria for nutrients Displaces pathogens from gut wall sites Produces acids in gut to inhibit pathogenic
bacteria Bacteriocin (antimicrobial) excretion
Repels or kills pathogenic bacteria Stimulates immune response Aids digestion
Produces enzymes to help digest & absorb feed Reduces toxic amines
PROBIOTIC BENEFITS vs. ANTIBIOTICS
Probiotic use eliminates need for antibiotics Probiotics create no illegal residue Bacteria cannot develop resistance to
probiotics but can to antibiotics Antibiotics lead to slow growth rate of larvae Probiotics boost immune system of shrimp Probiotics also digest pollutants Probiotics promote high survival so are cost-
efficient to use Cheaper to use probiotics than antibiotics
PROBIOTICS AND VIRUSES Anecdotal evidence that probiotics
mitigate pathogenicity of WSSV virus– Pond in Vietnam saved by double does of
probiotic after WSSV outbreak– Pond in Mexico with same experience after
PCR testing and mortality confirmed WSSV presence
Some researchers believe probiotics stimulate shrimp immune response
Some researchers believe that probiotics mitigate WSSV by controlling secondary pathogens
PROBIOTIC COMPOSITION Single strain of live microorganism
Usually bacterial or yeast e.g. Lactobacillus acidophilus, Saccharomyces
cerevisae, Vibrio alginolyticus Lactic bacteria typical of human probiotics
Microbial ecosystems Mixtures of specially selected, live but dormant
microorganisms Synergistic mixtures High strength for economy of use
Usually are dry powders Excellent shelf life if kept dry
WHY MICROBIAL ECOSYSTEMS? Teams work better than individuals
– Ecosystems work better than single strains What makes a good ecosystem?
– Enough “players” on the “team” High cell count (cfu = colony forming units)
– Enough of the right types of “players” More than one type (i.e. strain) Too many strains is bad (poor QC, low individual
count)– High quality “players” of each type
Best strain for each intended purpose (e.g. enzyme production or specific waste digestion)
Safe microorganisms
TYPICAL AQUACULTURE PROBIOTIC Selected live microorganisms like:
Bacillus spp., Lactobacillus spp., Yeast Soil-based, non-toxic, not genetically modified Not close relative of pathogens
Minimum strength = 2E+09 CFU/gm i.e. 2,000,000,000 colony forming units per gram
Fortified with a broad spectrum of free enzymes to start waste digestion
Contains proprietary growth stimulants Free of pathogens and toxins Biodegradable carrier
TYPICAL PROBIOTIC PRODUCT
PROBIOTIC SUCCESS FACTORS Existence of water quality or disease problem Use proper dosage & treatment frequency
Follow literature or ask local distributor Time to work
Bacteria, unlike chemicals, take time to work For curative application allow 24-48 hours
Organic matter present Add biological growth media to new or lined ponds
Proprietary media or home-made (e.g. sucrose-based) Salinity
Good probiotics are tolerant but best in salinity <40 ppt Pond pH – tolerant but best at 6-10 Temperature: Slower at low temperatures
AMMONIA DIGESTION
Ammonia (TAN) Concentration Over Time at Various Dose Rates of EPICIN
0.0
1.0
2.0
3.0
4.0
0 1 2 3 4 5Time (Days)
TAN
(mg/
l)
0
10
50
EPICIN (mg/l)
error bars represent +/- sd
• Probiotics take time to work• Ammonia digestion is dose sensitive
SALINITY TOLERANCE
00.5
11.5
22.5
33.5
4
TAN
(ppm
)
0 1 2 3Time (days)
483724120
ORGANIC CARBON IMPORTANCE
00.5
11.5
22.5
33.5
4
TAN
(mg/
l)
0 22 43 64 86Total Organic Carbon (mg/l)
Works best with organic carbon present
PROBIOTIC HYDRATION Best to hydrate (or “wake up”) dormant
bacteria Success factors
Water quality: use clean or disinfected pond water
Neutralize disinfectants before adding PROBIOTICS Aeration: use air stone or stir frequently Time: follow literature for hydration time
Growth media Specially designed media offers control and
optimum growth Pond-side grow-up lowers treatment costs
PROBIOTIC CELL GROWTHHYDRATION IN POND WATER
0
100
200
300
400
500
600
0 2 4 6 8 10 12
Grow-up time (hours)
Cel
l Cou
nt (b
illio
n cf
u/m
l)
Growth reverses unless special growth media is used
PROBIOTIC GROW-UP WITH MEDIAIn FRESH and SEA WATER
0.0E+005.0E+071.0E+081.5E+082.0E+082.5E+083.0E+083.5E+084.0E+08
cfu/
ml
0.0 hrs 4.0 hrs 8.0 hrs
Fresh WaterSea Water
Slightly more growth in fresh water;BGM media allows more growth
PROBIOTICS BENEFIT THROUGHOUT THE LIFECYCLE In maturation
– To produce the eggs In hatcheries
– To produce the juveniles In grow-out
– To produce the adults
PROBIOTICS IN GROW-OUT PONDS
TYPICAL GROW-OUT POND
PROBIOTIC APPLICATION TO POND
GROW-OUT SHRIMP
BENEFITS OF POND PROBIOTICS Better water quality
Less toxic environment for culturing animals Higher stocking densities
Reduced animal stress Better growth & survival
Larger animals or shorter cycles Higher yield
Probiotic, beneficial microbial environment Better growth & survival
Reduced water exchange Greater biosecurity Less pollution output
Lower fertilizer costs in older ponds
PROOF OF EFFICACY
In vitro tests– Lab ammonia digestion test– Microbiological competitive growth
tests vs. pathogenic bacteria In vivo tests
– Shrimp Vibrio challenge test– Field tests
COMPETITIVE GROWTH
EPICORE SHRIMP CHALLENGE TESTS
•24 hour probiotic pre-treatment of five PL30 P. vannamei• Vibrio parahemolyticus challenge (approx. 2E+04 cfu/ml)• Record survival after 24 hours
-40
-20
0
20
40
60
80
100
120
0 5 10 15 20 25
EPICIN:Vibrio Cell ratio
Surv
ival
Incr
ease
(%)
Probiotic protection is dose sensitive
SEMI-INTENSIVE VANNAMEI POND RESULTSEcuador 1995-1998
10.410.6Avg. weight (gm)34.2%39.5%Avg. survival rate15241720Annual yield (kg/ha)
121117Avg. Cycle (days)16.918.2Avg. S.D. (PL/sq m)8.73.25Avg. Size (ha)2620Ponds (#)
ControlProbioticParameter
Probiotic gave 12.9% average higher yield
Microbiological Assay of 1995 Ecuadorian Shrimp Ponds
Pond 51 - CONTROL POND 58 – Probiotic + BGM TREATED
INTENSIVE VANNAMEI POND RESULTSBrazil 2001
69.5%73.5%Avg. S.R.4,7508,470Cycle yield (kg/ha)
11.612.0Avg. weight (gm)95114Avg. cycle (days)
64.395.4Avg. S.D. (PL/sq m)2.252.25Avg. size (ha)
43Pond CyclesControlProbioticParameter
Probiotic allowed higher stocking and gave 78% yield increase
PROBIOTIC ADDITION TO FEED
0.0%10.0%20.0%30.0%40.0%50.0%60.0%
Surv
ival
Control 2.2 g/kg feed 4.4 g/kg feed
EPICIN Treatment Rate
Ecuadorian Pond Survival Results
Very efficient for low SD ponds
TYPICAL SHRIMP HATCHERY
TYPICAL SHRIMP HATCHERY TANKS
POSTLARVAL SHRIMP
PROBIOTICS IN HATCHERIES Prevention better than curative approach
Use throughout cycle to avoid problems Product grade selection
Special grades for hatcheries & for zoea stage Product hydration important
Pass mixture through a 100 micron mesh and discard the remaining solid material
Use extra when conditions dictate Follow supplier literature DO NOT USE with antibiotics or
microbiocides
PROBIOTIC HYDRATION AND APPLICATION
Larvipac 3 Year Data Comparison
62.21
47.46
33.42100,000200,000300,000400,000500,000
2000 2001 2002
Prod
uctio
n '0
00
30
40
50
60
70
Surv
ival
%
EPICORE SYSTEM TRIALNORAMAC HATCHERY - ECUADOR
0%
10%
20%
30%
40%
50%
60%
70%
1
Ave
rage
Su
rviv
al %
AVERAGE 9 TANKS USING
PROBIOTIC
AVERAGE 3 TANKS WITHOUT
PROBIOTIC
Probiotics improve all liquid feed results.
NON-SHRIMP SPECIES
Probiotics work in fish aquaculture as well as in shrimp– In hatcheries– In grow-out
Research on several species– Fish– Crabs– Oysters
FLORIDA KEYS FISH HATCHERY
U. OF MIAMI SEA TROUT STUDY
Mean sea trout lengths 21 days after hatching using lumped trial data for analysis
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
Lumped Probiotics Lumped Control
Treatment
leng
th (m
m)
p = 0.08(NS)
U. OF MIAMI SEA TROUT STUDYMean Sea Trout Survival 21 days after
hatching
0
200
400
600
800
1000
1200
Probiotics ControlTreatment
Cou
nt p = 0.21
PROBIOTICS MAKE A DIFFERENCE
Probiotics always come out on top!
PROBIOTICSFOR AQUACULTURE
The science of survivalHelp to improve aquaculture
yield and profits
Thanks for your interest.
© 2006 Epicore BioNetworks Inc.