* By Stephen Newman, Ph.D.
Many products are sold for use in aquaculture, ostensibly to improve outcomes. Considering the global market, all too often many of these products are potentially of limited use and the purveyors often rely on natural pond to pond variability for marketing and claims of efficacy. The handful of products that work consistently regardless of this variability face challenges in terms of how to best use them. The general conclusion, partly common sense and partly experience over 46 years, which appears to be obvious is that how (what, when, where and how much) these products are used impacts efficacy.
When I first began working with aquaculture in 1979, the global production of shrimp was a fraction of what it is today. In 1991, I was working for a publicly traded small company and, using VC money, developed a product, a parabiotic, that provided animals with a cost benefit that was observable in the field. Targeted primarily against vibriosis, working with cooperative partners in the lab and the field, we determined that it was an effective tool for lessening the impact of disease (parabiotics) among other things.
We concluded from our lab and field observations that the impact was limited in duration and non-specific. Under the “right” conditions the benefit noted in lab and field trials was less disease issues (apparently as a result of lowering of specific pathogen loads and reducing certain sources of stress), better growth, less wasted feed, etc. However, when the environment changed so did the benefit.
We observed that under both lab and field conditions with a single exposure the effect seemed to last 60 days or so, but we also noted with repeated pathogen exposure and under what would be deemed to be stressful production conditions (defined as a result of any environmental impact that disrupts the animals’ normal homeostatic mechanisms), the protective benefit did not offer consistency.
We concluded that at the very least a short-term exposure in the hatchery was sufficient to protect animals against modest levels of pathogens for at least 60 days and that additional exposure in the feed could enhance this. When and where and how much dictated the outcome as well as environmental conditions and the presence of stressors on the animal.
Around that time there were a few companies that were selling microbial products for use in bioremediation in aquatic environments (they have been used for many years is waste water management) and some of the reported observations strongly suggested a benefit. Roughly 25 years ago, Dr. Roland Larimore, one of the founding fathers of global shrimp farming and a partner in establishing the efficacy of the aforementioned parabiotic we developed, asked me if I was interested in evaluating a patented product for use in catfish. This was a blend of Bacillus spores that had been evaluated in shrimp farming in the US with some very positive results (Duda Trial Florida, 1999).
Aquaintech Inc. tested this product on a shrimp farm in Belize. At that time, activation, typically incubating the spore-based products in order to produce large numbers of metabolically active cells, was the norm. The first thing I noted, which, as a microbiologist with fermentation experience, was a problem with empowering this approach to people that did not appreciate the large potential downside. While the Bacillus would grow so would other airborne and water borne bacteria, including vibrios, some species of which reproduce in ten minutes or so.
This contamination made this approach risky, although short term activation in clean water with an added carbon source to kick start growth was less of a risk. After the first tests where we did see a modest and weakly statistically significant positive impact, I wondered if there might be a better approach. Knowing that in the wastewater industry there were a few companies marketing tableted (pelleted) spores I decided to test this on the farm.
This approach had none of the risks of activation on pond side and allowed the spores to be placed directly into those areas where the organic matter accumulated. The benefits observed were significant and the use of the tablets became a standard operating procedure in the farm (RMSF Belize PRO4000X benefits). Today this approach is widely used globally.
The vast majority of products marketed for use in shrimp act on the environment. Despite the many published observations which imply that some act as true probiotics (defined as bacteria ingested orally that colonize the gut and via their metabolome impact animal health) lab studies for the most part cannot be used to make claims that products will work in the same manner in the field.
Demonstrating that they are true probiotics is not straightforward as what happens in the lab under controlled conditions is not the same as what one would see in the field. Technically as well, there are some that believe that claims of this nature could be defined as drug claims forcing a much more rigorous registration process.
Based on our earlier experiences with the parabiotic we noted that as we fined tuned the use of the tablets there were critical factors that impacted how well the product worked. Bacillus spore-based products and the few spray dried/freeze dried and metabolically suspended non spore forming organisms sold for use in aquaculture are living organisms that thrive under specific conditions and do not survive for extended periods of time in most environments that they are introduced into.
These products were developed for the environment. Their purpose is to accelerate the rate of biodegradation of organic matter beyond what the naturally occurring bacteria can do as well as denitrifying nitrates, detoxifying ammonia and reducing the loads of some types of heterotrophic bacteria, specifically some of the more virulent vibrio strains and some may function as non-specific immune stimulants.
What Is in the Product?
Strains make the product work, not a given genus and species. Strains with weak or poor enzyme profiles will not work as efficiently, if at all, as strains with strong profiles that produce high levels of a variety of enzymes. It is a common misconception that all members of a given species invariably have the same properties. As an example, consider Vibrio parahaemolyticus.
As with most other species there are a range of strains within the taxon. These strains have a common metabolic and structural base which allows taxonomists to classify them as a given species, but they can vary greatly in the presence of certain genes that produce proteins that are virulence determinants (part of the metabolome). Their regulation and expression determine how virulent a given strain is.
There are strains of V. parahaemolyticus that can kill humans quickly and others that kill shrimp, fish, etc. Many are however benign. There is a very wide range of enzymatic abilities among the dozens of species classified today as Bacillus and the many hundreds that have been reclassified (Paenibacillus, Lysinbacillus, etc.).
Many commercial products for aquaculture contain strains of Bacillus, facultative aerobes (they can grow in the presence or absence of oxygen) spore forming gram-positive bacteria that can be sold in a dried form with an indefinite shelf life. There are a number of major factors, above and beyond the specific enzyme profiles, that are essential for ensuring that the user will see the maximum benefits.
How Much Is Used?
This is perhaps the most difficult aspect of ensuring optimum benefit. Failure to use enough and not using it when and where it needs to be used is highly likely to result in the product not appearing to perform as expected. All too commonly farmers ignore this. From day one I advocated that farmers start with smaller amounts and gradually increase the amount ensuring that the observed benefit occurs by real time monitoring.
This is why the first farm we worked with tablets saw the benefits that they did. These are biological products that perform based on the nature of the environment you put them in. They are not widgets that perform the same independently of where you use them.
Some of the more important considerations are:
1.Ponds when they are first stocked usually have little organic matter present. Dumping high levels of spores in them is wasteful ($$). Since spores germinate over time, the spores that germinate later will die off quickly for lack of nutrients. Most heterotrophic bacteria use organic matter as nutrients with metabolic by products so there is already competition present.
2. Adding high levels of spores to start can make it harder for Bacillus species to establish themselves. Starting with lower dosages allows them to form biofilms (this is environment dependent) and ensures that with subsequent dosing you replenish them in a manner that minimizes the system from taking dramatic steps to lower them. Many things can act against them aside from competing heterotrophs. One example is that of bacterial viruses, known as phages. They can be specific and target certain species ensuring that under most circumstances no one bacterial species dominates. Creating an environment that ensures high levels of these will make it harder for the added Bacillus to survive and do their jobs.
3. As the cycle progresses the amount of organic matter present increases. Organic matter is typically composed largely of uneaten feed (often in the form of fines as the shrimp (Litopenaeus vannamei) grind feed twice before it enters their digestive tracts), feces, molts, dead and dying bacteria, and the phytoplankton and zooplankton that are always present in healthy outdoor open systems. In RAS systems organic matter also increases although without all of these components.
4. Proper management of ponds requires monitoring them. Typically, this is resource and experience dependent. In general water needs to be sampled for pH and DO levels. NH3-N and metabolic products including NO2 and NO3, pond odor, physical appearance such as water color, clarity, and outgassing from anaerobic sediments should be monitored. There should be less black sludge if adequate dosing has been used. Animals need to be sampled regularly to ensure that feed is being consumed and not wasted, that they are growing as expected and that no disease processes are impacting the population that can be mitigated. These are all useful in telling the user that the product is working and adjusting dosages if needed.
Starting out with a low dosage and increasing it as the cycle progresses coupled with observations during and post cycle and adjusting the dosage as needed is the most economical and efficient way to use these products.
Each pond is unique-like a human fingerprint. One can have two ponds right next to each other built at the same time using the same approach and configuration and stocked with PLs that are from the same parents and reared in the same hatchery tanks where one sees two completely different outcomes for no apparent readily discernable reasons. The most effective approach in ponds that start out “clean” is to start out using levels and a frequency to effectively reduce the accumulated levels of organic matter. Start out low and gradually increase the levels as the cycles progress.
No product will consistently permanently colonize highly variable production environments. All must be added repeatedly. As we tested and developed the use of tablets, we fined tuned their use and our suggested approach is based on field observations in many environments in shrimp and fish ponds.
When the Product Is Used?
Waiting until there are high levels of accumulated organics ensures that the bacteria will not be as effective as they can be when they are used from the onset of the cycle (or before). Gradually increasing the dosage and frequency of application is the best approach as the cycles progress. Ponds that have never been treated and that have been used more or less continuously may have very high levels of accumulated organic matter that no bacteria can possibly digest in a reasonable amount of time unless one is willing to use them without any added sources of organics for an extended period of time, i.e. in ponds with no animals in them.
Using the product based on how it is working in your ponds will ensure that you see the maximum possible cost benefits.
Where the Product Is Used?
The tablets were developed for targeted delivery to those areas in the ponds where organic matter accumulates. There are now tablets that are added to automatic feeders that disperse the spores around the feeding areas (ask if you want more information). The bacteria in our products are motile, i.e. they have flagella and move of their own volition through the environment. Being small they are also readily moved with any water movement, such as aeration or animal movement. As the tablets dissolve the bacteria that germinate in contact with the sediment can move through the sediment.
Some clients will push them into the moist sediment where the entire spore content will germinate and act on the anerobic sediment around them reducing hydrogen sulfide levels and degrading organic matter resulting in healthier sediments. The environment that the products are used in impacts how they should be used.
For example, if the paradigm is old earthen ponds stocked at low to moderate densities (let’s assume 30 or less per m2) that are allowed to fallow for weeks to months ensuring that the sun bakes the remaining organic matter and destroys it or it is physically removed, how best to use the product is not going to be the same as it would be if one were using it in a lined pond stocked at higher densities with sumps. No two ponds are identical in terms of their microbial composition so the user must be open to adjusting how they use these microbial products to ensure the maximum cost benefit.
Use the Bacillus where they are needed the most. Most farmers can tell you exactly where they see the worst accumulations post-harvest.
Any product that is used in aquaculture must have a cost benefit. Directly impacting and reducing the overall costs of production by favorably impacting those factors that contribute to profitability is the goal. Using tableted bacteria weekly or every other week at low levels in ponds that have high densities and high levels of accumulating organic matter is not generally going to result in the best results.
Under most circumstances starting out with lower loads and increasing them as the cycle progresses and letting your pond observations tell you what is going on and if you need to use more is the best approach to take.
To summarize, spore-based products should be added at low levels to is to start with gradual increases in the dosage levels and frequencies as the cycle progresses. This is the best way to determine what usage rates will
give the best results and ensure cost beneficial usage. The amount of organic matter in your system should determine utility not a formulaic approach that advocates using the products at the same levels and frequencies throughout the production cycle. As we saw in the trials in Belize, and many elsewhere, proper use can have a wide range of benefits that one can expect to see consistently once the product usage rates are fine tuned for one’s specific environment.
* Stephen G. Newman has a bachelor’s degree from the University of Maryland in Conservation and Resource Management (ecology) and a Ph.D. from the University of Miami, in Marine Microbiology. He has over 40 years of experience working within a range of topics and approaches on aquaculture such as water quality, animal health, biosecurity with special focus on shrimp and salmonids. He founded Aquaintech in 1996 and continues to be CEO of this company to the present day. It is heavily focused on providing consulting services around the world on microbial technologies and biosecurity issues.
sgnewm@aqua-in-tech.com www.aqua-in-tech.com
www.bioremediationaquaculture.com
www.sustainablegreenaquaculture.com.
