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.