PROBIOTICS AND THEIR USE IN

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
 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”

 Opposite

Bios = “life”

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 costefficient 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
EPICIN
(mg/l)
4.0

0

error bars represent +/- sd

TAN (mg/l)

3.0

10

2.0

50

1.0

0.0
0

1

2

3

4

5

Time (Days)

• Probiotics take time to work
• Ammonia digestion is dose sensitive


SALINITY TOLERANCE
4

TAN (ppm)

3.5
3
2.5

48
37
24
12
0

2
1.5
1
0.5
0

0

1

2
Time (days)

3


ORGANIC CARBON IMPORTANCE
4

TAN (mg/l)

3.5
3
2.5
2
1.5
1
0.5
0

0

22

43

64

86

Total 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 GROWTH
Cell Count (billion cfu/ml)

HYDRATION IN POND WATER
600
500
400
300
200
100
0
0

2

4

6

8

10

12

Grow-up time (hours)

Growth reverses unless special growth media is used


PROBIOTIC GROW-UP WITH MEDIA
In FRESH and SEA WATER

4.0E+08
3.5E+08

cfu/ml

3.0E+08
2.5E+08
Fresh Water
Sea Water

2.0E+08
1.5E+08
1.0E+08
5.0E+07
0.0E+00

0.0 hrs

4.0 hrs

8.0 hrs

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