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

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Lynn Potter
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1 PRO4000X tablets Reduces ammonia, vibrio loads and accumulated organic matter. Alters pond ecosystem improving the quality of the production environment.

2 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 Complete reduction of ammonia Cleaner Pond

organic material Lower levels of hydrogen sulfide Less water exchange

vibrios Less blue green algae Excellent cost benefit due to reduced

3 How our products can help Complete reduction of ammonia Cleaner Pond Bottoms = Shorter time between cycles Less lime needed to oxidize organic material Lower levels of hydrogen sulfide Less water exchange required Increased Survivals Better Growth Less stress Lower levels of vibrios Less blue green algae Excellent cost benefit due to reduced costs of production as a result of improved quality of environment Eliminate antibiotics

4 Stress is the biggest single problem What is stress? aquaculturists face Production of animals in environments that are not ideally suited for optimum animal physiological functioning is the norm for most shrimp aquaculture rather than the exception Result is that there is less then ideal growth, increased susceptibility to diseases, pathogen and environment related and less productivity with overall consequences that result in LOWER PROFITS

5 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.

6 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

7 How do the tablets work? Each tablet contains more than 60,000,000,000 (60 billion) spores of two of our selected bacterial strains. The tablets are thrown into ponds where they sink to the bottom and dissolve As they dissolve the spores germinate (fancy word for hatch) and proliferate if the environment is favorable

8 More is better- the more species included and the high the level the better the impact REALITY CHECK Nutrient loads limit bacterial growth. Adding more spores or lots of different types of bacteria does not change this. How well the tablets work is dependent on nutrient availability. Bacteria grow the best when they are well fed.

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

inhabitant of many animals digestive tracts Very large and diverse group

10 Bacillus subtilis Natural occurring in terrestrial soils 10 6 spores per gram Human digestive tract 10 4 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

11 Bacillus licheniformis Produces a variety of enzymes involved in the recycling of organic matter and other nutrients. Very tolerant of environmental extremes

12 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.

13 Modes of action Indirect Indirect inhibition by the consumption of nutrients. Bacteria will compete against other bacteria and algae in the ponds. Before After

14 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.

15 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

16 PRO4000X tablets

17 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.

18 Hatchery Studies Reducing vibrio loads is an essential component of any enlightened biosecurity program and is critical to improving survival in the hatchery and producing a healthier, higher quality PL for survival in the farm.

19 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 CHECK There is no correlation between the ability to produce disease in shrimp and the ability to breakdown sucrose. Yellow colonies can produce severe disease.

20 Yellow versus Green Yellow colonies can kill shrimp as readily as green colonies can

21 Hatchery studies Indonesia PRO4000X one tablet/20 MTs daily Reduction in TCBS green vibrio (s) in the water 1.40E+04 Water Vibrio Green on TCBS 1.30E+04 Green vibrios reduced by fold (>99.99%) 1.20E E E E E E E E+00 PRO4000X Control

Hatchery studies Indonesia PRO4000X one tablet/20 MTs daily Reduction in TCBS green vibrios on the animal 1.80E+05 1.60E+05 1.40E+05 1.20E+05 1.

22 Hatchery studies Indonesia PRO4000X one tablet/20 MTs daily Reduction in TCBS green vibrios on the animal 1.80E E E E E E E E E E+00 Body Vibrio Green on TCBS 1.60E E+00 PRO4000X Control Green vibrios reduced by fold (>99.999%)

23 Hatchery results in India Different hatcheries will have different dosage requirements (tablets per MT) Effectiveness will depend on how clean the water is. Nutrient loads impact effectiveness. Using one tablet daily per 5 MTs of water reduced vibrio counts significantly as well as potential coliform counts (measured on LB agar).

24 NAUPLII -1-3 NAUPLII -4-5 Z 1, Z2, Z3 M1 M2 M3 PL1 PL2 PL3 PL4 PL5 PL6 PL7 PL8 PL9 Vibrio load (CFU/ml) Reduction of TCBS green vibrios in hatchery tanks Control Experimental

25 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.

26 NAUPLII -1-3 NAUPLII -4-5 Z 1, Z2, Z3 M1 M2 M3 PL1 PL2 PL3 PL4 PL5 PL6 PL7 PL8 PL9 Vibrio load (cfu/ml) Reduction of TCBS yellow colonies in hatchery tanks Control Experimental

27 NAUPLII -1-3 NAUPLII -4-5 Z 1, Z2, Z3 M1 M2 M3 PL1 PL2 PL3 PL4 PL5 PL6 PL7 PL8 PL9 Vibrio load (CFU/ML) Reduction of coliforms in hatchery tanks Control Experimental 5 0-5

28 Field trial results in India Four ponds total. Three experimental and one control.

29 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

30 Tablet application rate and frequency for India trial

31 Shrimp in PRO4000X treated ponds grew faster and were larger at harvest than shrimp from control pond Pond 1 Pond 2 Pond 3 Control DOC

32 PRO4000x reduced ammonia levels in all treated ponds Pond 1 Pond 2 Pond 3 Control DOC

33 PRO4000X treated ponds had lower levels of TCBS green vibrios Intake Canal Control Pond Pond 1 Pond 2 Pond

34 PRO4000X treated ponds had lower TCBS yellow vibrio loads Intake Canal Control Pond Pond 1 Pond 2 Pond

35 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.

36 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

37 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

38 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

39 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.

40 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.

41 Who uses PRO4000X? Shrimp farms, semi intensive Intensive shrimp farms Royal Mayan Shrimp Farm, Belize Vietnam Tilapia Nursery Indonesia This versatile product works in farms, any pond, lake, reservoir or other similar body of water, golf course ponds, koi ponds, hatchery tanks, etc.

of organic matter resulting in improvement of water quality.

42 TABLET 16 gram >64 billion CFU TABLET 16 gram >64 billion CFU TABLET 16 gram >64 billion CFU TABLET 16 gram >64 billion CFU TABLET 16 gram >64 billion CFU TABLET 16 gram >64 billion CFU TABLET 16 gram >64 billion CFU 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 will depend on your application rates and your environment After

43 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.

44 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 the milling process. They dissolved and the components are mixed with the feed. One bag per MT results in approximately 10 6 spores per gram of feed. The spores do not germinate the first time in the shrimp guts as the transport time is too short, although it is known that in the hepatopancreas of the shrimp residence times 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 anything but a short term colonization as shrimp do not appear to have stable microbial flora.

45 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

46 AquaPro B Trials Florida pounds more from treated ponds than controls With Without

47 Field trial with activated product Four 0.5 acre ponds Stocked at 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

48 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

49 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.

The Presentation. Definitions Concepts Commentary PRO 4000 X Discussion Outlook and Conclusions. January 2009 Aqua In Tech Inc. 2

The Presentation. Definitions Concepts Commentary PRO 4000 X Discussion Outlook and Conclusions. January 2009 Aqua In Tech Inc. 2 The Presentation Definitions Concepts Commentary PRO 4000 X Discussion Outlook and Conclusions January 2009 Aqua In Tech Inc. 2 Ph.D in marine microbiology (vibrio). Developed vaccines for commercially