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Considerations regarding the use of bacteria in aquaculture for bioremediation/bioaugmentation

Bacteria are everywhere.   There are very few environments on Earth where there are none.  They can be highly resilient with some forms persisting for eons.  Spores from Bacillus species have been isolated that were viable more than 250 million years after being trapped in salt.  Some years ago, a few mainstream microbiologists theorized that we had characterized all the bacteria that there are based on our ability to grow them on culture media.  The genetic revolution revealed that this was at best misguided.  Today we understand that most bacteria cannot be cultured and there are many species and strains whose presence cannot be determined by growing them on media (in fact, the vast majority of bacteria).  Molecular biology has resulted in a much better understanding of how ubiquitous bacteria are and their wide-ranging impacts.  We are only at the beginning of unraveling this complexity.



Bacteria are ubiquitous.


While this understanding is a positive development for science, unfortunately many who are exploiting bacteria for money are making claims about what might be occurring without the science to support their assertions.  The standard taxonomic classification of bacteria ends with the species.  Within a given species there are variants that, while they are taxonomically the same species, possess different enzymes and other abilities.  These are known as strains, and their enzymes (substrates they degrade, under what conditions and quantities produced) determine how effective a given strain will be in bioremediation or in other aspects of their abilities that one wishes to exploit.  Using bacteria with a narrow range of enzymatic activities to address specific environmental concerns, i.e. to degrade a specific pollutant, is the norm.  Often an entrepreneur will select microbes based on their genus/species and ready availability without ensuring that their metabolic abilities are suited for the environment and application that they are being used in.  Most bacteria that are present normally in aquatic environments do not produce a wide range of degradative enzymes at high levels.  Understanding what substrates are present and choosing the right bacterial strains for the job is possible.  Aquaintech Inc., is focused on the Bacillus genus because they have the ideal properties needed for maximum impact and benefit. 



Bacillus subtilis colony on agar



In a recent article, I pointed out how the practice of growing bacteria up on pond side is risky at best.  It is rarely biosecure and certainly not without appropriate oversight and QC by a qualified microbiologist.  




It can lead to animal health challenges by introducing both potential and obligate pathogens into the production environment.  Furthermore, non-characterized and uncontrolled mixtures of bacteria encourage the exchange of genetic material which can generate strains that contain toxins (and other properties) that they never had before.  Bacteria readily pick up genes from other bacteria that can give them an added competitive advantage.  They are always at war with each other fighting for limited resources.  They have many different tools for this, including antibiotics such as antimicrobial peptides.  Sometimes toxin genes that are not normally present in a given species/strain may be moved.  While this is not necessarily straightforward, the presence of the PirA and PirB toxins in the strains of V. parahaemolyticus that cause EMS/AHPNS/AHPND are potentially one example.  The recent appearance of Tc toxin genes in other strains of this species, responsible for transparent glass shrimp disease in SE Asia, is also potentially another example.  


AHPNS (top) affected shrimp

 

Transparent shrimp disease


The practice of adding uncontrolled mixtures of microbes to production environments poses a serious threat.  Creating environments that are conducive to the ready exchange of genetic material between genera and species of bacteria is not consistent with sustainability.  Many genera (including vibrios) of bacteria degrade organic matter from a variety of sources.  Focusing on shrimp (and some fish) ponds for the sake of this discussion, the organic matter typically consists of fecal material, undigested feeds, plant materials (from algae), dead and dying bacteria, dead and dying shrimp and fish, crabs, etc. and other animals that enter into ponds.  This is a heterogenous mixture of materials.  There are a variety of nutrients that can act as pollutants and create environmental perturbations that favor shifts in microbiomes and their respective metabolomes in an unfavorable manner.  A common example is when soluble P and N levels stimulate algal blooms that can ruin a crop.  Not all of the metabolic properties of given strains are desirable. 

 

The list of bacteria and fungi that are being sold for use in aquaculture is lengthy.  Dozens if not hundreds of different genera/species/strains are being sold with a long list of claims about benefits.   Some make claims that they can improve animal health and/or mitigate the impact of diseases.    There are many publications that suggest that under controlled conditions, as seen in aquaria and microcosm studies, there may be a range of benefits.  However, a majority of these organisms are poorly suited for use in pond-based production environments and when used in real world production environments are a waste of money.  Their enzyme profiles, the levels of enzymes being produced, how they are produced and under what biochemical conditions, etc. are not conducive to an optimal impact.

 

What specifically should one expect to accomplish by adding metabolically active microorganisms to production environments?  This will likely vary depending on the production environment, stocking densities and biosecurity considerations.  There are many paradigms that are in use.  The benefits seen will vary as much as the environments do which makes it even more important to work with strains that function across a wide spectrum and deliver consistent results.

 

Added bacteria can act in several ways.  They compete for niches with each other. Aquaintech’s tableted Bacillus spore product (PRO4000x) has been shown to influence the species of vibrios present where they are applied.  With consistent application in hatcheries, maturation, and RAS systems this appears to last for the duration of the application.  In large ponds where economics are such that the product is best used pulsed, if there is too much time between applications, this impact can wane.



Certain vibrio species, notably toxin containing strains of Vibrio parahaemolyticus are obligate pathogens affecting farmed shrimp



The organisms in these products may colonize animal surfaces both internally and externally.   They have specific niches in the environment that they tend to favor although these may not be readily accessible (something is already present).  Thus, the need for them to produce biochemical tools that give them an advantage.  Under some conditions, usually requiring exposure to very high levels, they can also stimulate protective immunity, typically nonspecifically.   

     

A widespread practice is adding a variety of products at once.  The idea is “the more the better” (which pond side culture showed was not the case).  This is more likely to result in inhibition rather than enhancement as well as an undesirable mixing of genes that can cause non pathogens to become pathogens.  This is the nature of bacteria and fungi.       

 

Many widely sold additives are not necessarily the best suited for their stated and/or intended purpose.  While they may have enzyme profiles that are suitable for specific applications, very few of them have the wide range of enzymes that ensure that the required broad range of metabolic activity needed to maximize waste digestion in-situ is present.

 

Typical production environments are open systems.  Deleterious metabolites (such as polluting nutrients) and pathogens are flushed out at various levels.  These systems are open to the air and the environment.  This paradigm is gradually evolving into more efficient smaller systems with varying degrees of biosecurity.  Animals are held in small, sometimes covered ponds where, ideally, a balance of nutrient inputs and in-situ bioremediation allows them to remain largely closed and still remain profitably productive.  These systems require significant investments up front and in the case of shrimp, require the use of strains of shrimp that are able to tolerate the stresses of being reared at high densities and that are free of all pathogens including obligate and many typical opportunistic pathogens from the onset.  These systems are highly controlled and offer an interesting paradigm shift away from the larger ponds that are open to the environment.  Added bacteria can be helpful in these systems but this is limited by the rates of waste accumulation at the high densities required for these systems to be profitable.  Some ex-situ degradation and retention of waste streams likely will be needed to ensure responsible disposal. 



Semi intensive Central America                                                Intensive Vietnam

 


The genus of bacteria that are the best suited for waste stream managements are the Bacillus.   There are other strains from other species that may also be useful, although the genus Bacillus has some distinct advantages.  They are gram positive bacteria many of which can grow in both the presence and absence of oxygen.  Their ability to form spores   allows for a shelf stable (thus the long shelf life) and consistent product without the need for specialized conditions to ensure vegetative cell viability.  Viable vegetative cell products die in the absence of conditions that they need to be in to grow.  While there are tools to stabilize them to some degree as soon as they are added to an environment or improperly handled prior to being used they die.  The favored approach of companies that sell these is to add levels far beyond what they claim to be present such that even with 99% of the bacteria dying some remain viable.  



Bacillus species are enzyme workhorses.  They account for more than 50% of all of the commercial sources of enzymes.  The proprietary Bacillus strains that Aquaintech Inc. works with produce a range of enzymes at high levels under a wide range of environmental conditions.  They are Generally Recognizes as Safe (GRAS) by the USFDA-which is the reason why they are not subject to regulation (provided no claims are made regarding curing or preventing disease).  Widely used in billions of fish and shrimp annually, they are the standard against which other products are measured.  Bacillus species are also used in the waste management industry including sewage treatment plants where their versatility is essential for the conversion of waste into a benign form, bacterial biomass.  


Spore containing Bacillus species.


As discussed previously, caveat emptor applies here.  Let the buyer beware.  The use of bacteria for improving water quality and the overall production environment is a slowly evolving paradigm that has the potential for being an important element of sustainable production.  Not all bacteria being sold are suitable for ensuring that one can gain the maximum possible benefit from their use without resorting to very costly application protocols and approaches that are no guarantees.  It unfortunately also has the potential for being a huge waste of money that harms the crop instead of benefiting it especially when mixtures are employed with no understanding about how they interact.






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