PRO4000X tablets

Reduces ammonia, vibrio loads and accumulated organic matter. Alters pond ecosystem improving the quality of the production environment.

AquaInTech Inc. microbial products for improving shrimp productivity and farming profitability.

Field proven cost effective science based tools for environmental manipulation using naturally occurring bacteria strains.

Satisfied clients in China, Vietnam, Taiwan, India, Venezuela, Indonesia, Malaysia, Ecuador, Peru, Belize, Philippines and elsewhere.

Biotechnology Benefiting Aquaculture

How our products can help

Cleaner Pond Bottoms

Shorter time between cycles

Less stress

Increased Survivals

Less lime needed to oxidize organic material

Lower levels of hydrogen sulfide

Less water exchange required

Lower levels of vibrios

Less blue green algae

Better Growth

Eliminate antibiotics

Excellent cost benefit due to reduced costs of production

as a result of improved quality of environment

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Complete reduction of ammonia

Stress is the biggest single problem aquaculturists face

• What is stress?

– Production of animals in environments that are notideally suited for optimum animal physiologicalfunctioning is the norm for most shrimpaquaculture rather than the exception

– Result is that there is less then ideal growth,increased susceptibility to diseases, pathogen andenvironment related and less productivity withoverall consequences that result in LOWER PROFITS

What can you do to lessen the stress?

– Improve culture conditions to create a healthier environment

– Increase dissolved oxygen levels, lessen the build up of metabolites such as ammonia and hydrogen sulfide, reduce the organic matter loads to lower nutrient loads that contribute to eutrophication

– Some things are inherent in the production system, others can be readily controlled

– Our products are tools-part of a solution.

Tools for lessening the stress

• PRO4000x family of products

• Bacillus based

– Natural strains selected for specific biochemical properties

– Stable spores indefinite shelf life when kept free of moisture

– Field proven

– Targeted delivery for common sense application

How do the tablets work?

• Each tablet contains more than 60,000,000,000(60 billion) spores of two of our selectedbacterial strains.

• The tablets are thrown into ponds where theysink to the bottom and dissolve

• As they dissolve the spores germinate (fancyword for hatch) and proliferate if theenvironment is favorable

• More is better- the more species included and the high the level the better the impact

REALITY CHECK

Nutrient loads limit bacterial growth. Addingmore spores or lots of different types of bacteriadoes not change this. How well the tabletswork is dependent on nutrient availability.Bacteria grow the best when they are well fed.

The Bacteria

Stable spore forming bacteria. The spores allow the bacteria to survive in environments that kill other bacteria.

Bacillus subtilis• Natural occurring in terrestrial soils 106 spores per gram

• Human digestive tract 104 spores per gram

• Considered to be a normal inhabitant of many animals digestive tracts

• Very large and diverse group of bacteria many different strains

Electron microscope photo Colonies on agar

Bacillus licheniformis

• Produces a variety of enzymes involved in the recycling of organic matter and other nutrients.

• Very tolerant of environmental extremes

Modes of action Direct

• Direct inhibition by the production of bacteriocins.

• Picture below shows inhibition of a vibrio strain (clear zone) by our Bacillus subtilis.

Modes of action Indirect

• Indirect inhibition by the consumption of nutrients. Bacteria will compete against other bacteria and algae in the ponds.

Before After

Miscellaneous Bacillus species

• Form a complex that work together to optimize the impact of the bacteria in in the ponds

• Several different strains with different properties.

Scanning electron micrograph of B. subtilis.

Our product line

• PRO4000X tablets >60 billion CFU/tablet– Mini-tablets > 4 billion CFU/tablet

– For targeted delivery NO ACTIVATION REQUIRED

• AquaPRO B > 4 billion CFU/gram–Nutrients plus spores requires activation before addition to the ponds

• AquaPRO EZ 800 billion to 1 trillion CFU/bag–Nutrients plus spores in biodegradable bags. NO ACTIVATION REQUIRED

• AquaPRO F for oral application. > 4 billion CFU/gram–Five strains of bacillus combined with MOS and enzymes for improved digestion of feed. NO ACTIVATION REQUIRED

PRO4000X tablets

• Nn activation required.

• Direct addition to ponds or tanks.

• Depend 100% on nutrients available at the time they are added.

• High titer engineered for maximum bacterial growth with proper nutrient balance.

Hatchery Studies

Reducing vibrio loads is an essential componentof any enlightened biosecurity program and iscritical to improving survival in the hatchery andproducing a healthier, higher quality PL forsurvival in the farm.

• Green vibrios are bad and Yellow vibrios are good

• The ability of vibrios to utilize the sugar sucrose is a useful identification tool

• Thiosulfate-citrate-bile salts-sucrose agar (TCBS) is a selective media that most vibrio species grow on

REALITY CHECKThere is no correlation between the ability to produce disease in shrimp and the ability to breakdown sucrose. Yellow colonies can produce severe disease.

Yellow versus Green

Yellow colonies can kill shrimp as readily as green colonies can

Hatchery studies IndonesiaPRO4000X one tablet/20 MTs daily

• Reduction in TCBS green vibrio (s) in the water

1.00E+00

1.30E+04

0.00E+00

2.00E+03

4.00E+03

6.00E+03

8.00E+03

1.00E+04

1.20E+04

1.40E+04

PRO4000X Control

WaterVibrioGreenonTCBSGreen vibrios reduced by 13000 fold (>99.99%)

Hatchery studies IndonesiaPRO4000X one tablet/20 MTs daily

• Reduction in TCBS green vibrios on the animal

1.00E+00

1.60E+05

0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

1.00E+05

1.20E+05

1.40E+05

1.60E+05

1.80E+05

PRO4000X Control

BodyVibrioGreenonTCBSGreen vibrios reduced by 160000 fold (>99.999%)

Hatchery results in India

• Different hatcheries will have different dosagerequirements (tablets per MT)

• Effectiveness will depend on how clean thewater is. Nutrient loads impact effectiveness.

• Using one tablet daily per 5 MTs of waterreduced vibrio counts significantly as well aspotential coliform counts (measured on LBagar).

Reduction of TCBS green vibrios in hatchery tanks

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PRO4000x reduced vibrio loads in hatchery tanks both yellow and green on TCBS

• Use of PRO4000x on a daily basis reduced TCBS green and yellow vibrio loads

• Effect was rapid and substantial compared to tanks that were not treated with PRO4000x

• Control tank was treated with conventional products

• Proof that PRO4000X is a valuable tool for use in the hatchery to control vibrio loads.

Reduction of TCBS yellow colonies in hatchery tanks

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Reduction of coliforms in hatchery tanks

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Field trial results in India

Four ponds total. Three experimental and one control.

Significant impact on profitability using PRO4000X tablets

• Benefit #1– Farmer was able to close experimental ponds 100% reducing risk of WSSV

• Benefit #2– Shrimp in closed experimental ponds grew faster and were larger at

harvest

• Benefit # 3– FCR’s in treated ponds were significantly lower than in the control pond

• Benefit # 4– Ammonia levels were reduced to almost zero in treated ponds

• Benefit #5– Significant difference in yields

Tablet application rate and frequency for India trial

Shrimp in PRO4000X treated ponds grew faster and were larger at harvest than shrimp from control pond

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DOC 31 38 45 52 59 66 73 80 87 94 101 108

Pond 1

Pond 2

Pond 3

Control

PRO4000x reduced ammonia levels in all treated ponds

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DOC 31 38 45 52 59 66 73 80 87 94 101 108 115

Pond 1

Pond 2

Pond 3

Control

PRO4000X treated ponds had lower levels of TCBS green vibrios

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Intake Canal

Control Pond

Pond 1

Pond 2

Pond 3

PRO4000X treated ponds had lower TCBS yellow vibrio loads

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Intake Canal

Control Pond

Pond 1

Pond 2

Pond 3

• Gradually increasing the number of tablets per application gave the observed results.

• Each application was approximately ten days apart.

• The farmer added a mixture of molasses, yeast and bran that had been allowed to ferment for 24 hours prior to addition to the pond.

What is the best way to use the tablets?

• Start out with low levels of tablets

• Distribute tablets

– Problems areas of the ponds (corners)

– Drainage canals (both inside the ponds and outside canals)

– Sedimentation ponds

• Add a source of carbon if possible, such as molasses to enhance bacterial growth

Best use of tablets

• Every pond is different

• Protocols will vary between ponds and farms

• Products that offer set protocols fail to take this into account

• Targeted delivery to problem areas of selected bacteria is a common sense approach

• Pouring liquids and powders into ponds cannot have the same impact-physics determines this

What is the best way to use PRO4000X?

• Each environment is unique. Ponds are never the same. Differences include biomass, native bacterial populations, waste products from shrimp culture (nitrogen in the form of ammonia and fecal material), uneaten feed, etc.

• Optimum usage determined by trial and error

• Guidelines provided

• As the farmer you know how this product will work the best for you

Suggested usage for more than 70 animals per square meter

Start out slow and gradually increase the number of tablets. Focus on known problem areas based on pond history, exits and inlets and and drainage areas where sludge can accumulate. Add remainder to pond bottoms.

Add Molasses or another suitable carbon source-it helps the bacteria grow. You can soak the tablets in it or just add it to the pond at 10 to 20 liters per ha per application.

Suggested usage for less than 70 animals per square meter

Start out slow and gradually increase the number of tablets. Focus on known problem areas based on pond history, exits and inlets and and drainage areas where sludge can accumulate. Add remainder to pond bottoms.

Add Molasses or another suitable carbon source-it helps the bacteria grow. You can soak the tablets in it or just add it the pond at 10 to 20 liters per ha per application.

Who uses PRO4000X?Shrimp farms, semi intensive

Royal Mayan Shrimp Farm, Belize

Intensive shrimp farms

Tilapia Nursery Indonesia

Vietnam

This versatile product works in farms, any pond, lake, reservoir or other similar body of water, golf course ponds, koi ponds, hatchery tanks, etc.

Degradation of organic matter resulting in improvement of water quality.

Used properly can compete against other bacteria (vibrios) and blue green algae. Note that the impact that you see willdepend on your application rates and your environment

TABLET16 gram

>64 billion CFU

After

TABLET16 gram

>64 billion CFU

TABLET16 gram

>64 billion CFU

TABLET16 gram

>64 billion CFU

TABLET16 gram

>64 billion CFU

TABLET16 gram

>64 billion CFU

TABLET16 gram

>64 billion CFU

Bacteria alter water composition

Before

After

Note the color difference

Other products for other applications

Biodegradable bags with a minimum of one trillion (1,000,000,000,000) CFU per bag. Use in

stubborn areas. (AquaPro EZ)

Requires water to be calm. Bags settle to the bottom and dissolve with nutrients and spores

AquaPro EZ Can be easily incorporated into feed. Bags can be added during themilling process. They dissolved and the components are mixed with the feed.One bag per MT results in approximately 106 spores per gram of feed. The sporesdo not germinate the first time in the shrimp guts as the transport time is tooshort, although it is known that in the hepatopancreas of the shrimp residencetimes can be days. After the shrimp defecate the spores germinate in the feces.They are readily re-ingested as shrimp forage. It is not likely that there is anythingbut a short term colonization as shrimp do not appear to have stable microbialflora.

AquaPRO B

• For those clients who want to use a traditional approach, AquaPRO B is formulated with nutrients and our two proprietary Bacillus strains

• Requires activation before adding to the ponds

• Field Proven

AquaPro B Trials Florida 19995600 pounds more from treated ponds than controls

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With Without

Field trial with activated product

• Four 0.5 acre ponds

• Stocked at 50-55 per square meter

• 28 weeks of culture

• Required 20 kgs of product per pond

• Increased yields by 40%

• No sludge accumulation in treated ponds compared with significant accumulation in control ponds

AquaPRO F for application in or on the feed

• Contains 5 bacillus strains, Bacillus subtilis, B. licheniformis, B. pumilus, B. megaterium and B. polymxya

• Contains Mannan oligosaccharide (MOS) –a yeast cell wall extract high in glucan and proven to improve animal health and digestion

• Contains enzymes to facilitate feed digestion, cellulase, amlyase, protease and lipase

Conclusions

• Targeted delivery of tablets to pond bottoms ensures bacteria are in the problem areas.

• Bacteria will grow and degrade the nutrients immediately present and spread out from sediments into water column until nutrients become limiting and they die off.

• Adding more bacteria and carbon ensures optimum performance.

• Clients have reported a variety of impacts.

• How many tablets and how often to apply them is derived from experience and the provided guidelines.

• We offer a variety of products for different application philosophies.