PROBIOTICS AND THEIR USE IN

ROLE OF ENVIRONMENTAL MICROBIOLOGY IN AQUACULTURE Pollution control  Bioremediation of wastes  Cleaner water & pond bottoms  Disease resistance imparted by creating a lower stress g

PROBIOTICS AND THEIR USE IN

AQUACULTURE

April 2006

10th Aquaculture Insurance and

Risk Management Conference

Presented by:

William P Long Chief Executive Officer Epicore BioNetworks Inc.

BASIC AQUACULTURE

MARKET NEEDS

 To survive and make money

 Poor feed conversion ratio (FCR)

It’s not a hobby; it’s a business!

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

 Bacteria + N + C + P + O 2 = More Bacteria + H 2 O + CO 2

 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

 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

 Aids digestion

 Produces enzymes to help digest & absorb feed

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 efficient to use

cost- 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

stimulate shrimp immune response

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?

– Ecosystems work better than single strains

– 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

error bars represent +/- sd

• Ammonia digestion is dose sensitive

ORGANIC CARBON IMPORTANCE

0 0.5 1 1.5 2 2.5 3 3.5 4

Total Organic Carbon (mg/l)

Works best with organic carbon present

 Neutralize disinfectants before adding PROBIOTICS

 Aeration: use air stone or stir frequently

 Time: follow literature for hydration time

PROBIOTIC CELL GROWTH

HYDRATION IN POND WATER

PROBIOTIC GROW-UP WITH MEDIA

In FRESH and SEA WATER

Slightly more growth in fresh water;

BGM media allows more growth

PROBIOTICS BENEFIT THROUGHOUT THE LIFECYCLE

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

 Better growth & survival

 Larger animals or shorter cycles

 Higher yield

 Probiotic, beneficial microbial environment

 Better growth & survival

 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

SEMI-INTENSIVE VANNAMEI POND RESULTS

Ecuador 1995-1998

10.4 10.6

Annual yield (kg/ha)

121 117

Avg Cycle (days)

16.9 18.2

Avg S.D (PL/sq m)

8.7 3.25

Avg Size (ha)

26 20

Ponds (#)

Control Probiotic

Parameter

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 RESULTS

Brazil 2001

69.5% 73.5%

Avg S.R.

4,750 8,470

Cycle yield (kg/ha)

11.6 12.0

Avg weight (gm)

95 114

Avg cycle (days)

64.3 95.4

Avg S.D (PL/sq m)

2.25 2.25

Avg size (ha)

4 3

Pond Cycles

Control Probiotic

Parameter

Probiotic allowed higher stocking

and gave 78% yield increase

PROBIOTIC ADDITION TO FEED

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

 Use throughout cycle to avoid problems

 Special grades for hatcheries & for zoea stage

 Pass mixture through a 100 micron mesh and discard the remaining solid material

 Follow supplier literature

microbiocides

PROBIOTIC HYDRATION

AND APPLICATION

Larvipac 3 Year Data Comparison

62.21 47.46

EPICORE SYSTEM TRIAL

NORAMAC HATCHERY - ECUADOR

AVERAGE

3 TANKS WITHOUT PROBIOTIC

Probiotics improve all liquid feed results.

FLORIDA KEYS FISH

HATCHERY

U OF MIAMI SEA TROUT STUDY

Mean sea trout lengths 21 days after hatching using lumped trial data

U OF MIAMI SEA TROUT STUDY

Mean Sea Trout Survival 21 days after

PROBIOTICS MAKE A DIFFERENCE

Probiotics always come out on top!

PROBIOTICS FOR AQUACULTURE

The science of survival

Help to improve aquaculture

yield and profits Thanks for your interest.

© 2006 Epicore BioNetworks Inc.