Application of SCD Probiotics Technology in Aquaculture

Sustainable Community Development, LLC ("SCD"), based in Kansas City, Missouri, USA, is a company specializing in natural microbial-based products and services for human health, agriculture industry, industrial waste management, and environmental sustainability. Through 10 years of research and development, SCD has selected 14 different microbial strains for production of various probiotic products. These include lactic acid bacteria such as Lactobacillus spp. and Streptococcus sp.; yeast such as Saccharomyces sp.; photosynthetic bacteria such as Rhodopseudomonas spp.; and other beneficial bacteria such as Bacillus sp.

In essence, SCD Probiotics Inside is a technology consortium of lactic acid bacteria, phototrophic bacteria, nutritional yeast, and other beneficial microorganisms. SCD’s consortium culture synergistically works to inhibit the growth of pathogenic harmful bacteria through competitive exclusion. Competitive exclusion is when two species compete for a single source of food. The microbe with the more efficient absorption system will acquire most of the food, grow faster, reproduce faster, and eventually displace the microbe that absorbs food slower and thus cannot grow as fast. Average pH of SCD products ranges from 3.2 - 3.5. Some products with SCD Probiotics Inside microorganisms consortium can include for instance, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. fermentum, L. plantarum, Rhodoseudomonas palustris, Saccharomyces cerevisiae, Streptococcus thermophilus, etc The above-mentioned microorganisms have been known to be associated with human foods and are ubiquitously used to manufacture dietary supplement probiotic products for human, animal, and aquaculture health.

While the traditional meaning of the word “probiotic” is applied to human and animal digestive microorganisms, SCD is on the cutting edge of developing technology to apply the concept of “probiotics” to many fields globaly including waste water treatment, odor control, environmental bioremediation, agriculture, pest control, mold remediation, industrial and home agriculture, aquaculture, gardening, prevention of skin diseases, turf grass, composting and other fields.

SCD Essential Probiotics™, ProBio Balance™ Plus and Certification

Information about SCD Essential Probiotics and ProBio Balance Plus can be found at www.SCDProbiotics.com. In general, both products are manufactured under high and strict quality control standard. In brief, SCD Essential Probiotics™ is recommended for human dietary supplement to enhance and maintain beneficial microorganisms in human and animal (both livestock and aquatic) digestive system. ProBio Balance™ Plus is an enhanced phototrophic bacteria and therefore is more suitable for agricultural and environment applications.

SCD Essential Probiotics complies with the Food Grade current Good Manufacturing Practices (cGMP). These guidelines are enforced in the United States by the Food and Drug Administration. All SCD products are manufactured under the cGMP program. GMP Requirements are guidelines that provide a system of processes, procedures, and documentation to ensure the product produced has the identity, strength, composition, quality, and purity that it is represented to possess. As a result, SCD Essential Probiotics are safe for both human and animal consumption. Products are manufactured in a food grade facility utilizing food grade equipment, raw materials, and utensils. All SCD products are manufactured under high and strict quality control standards.

Organic Materials Review Institute (OMRI) provides certifiers, growers, manufacturers and suppliers an independent review of products intended for use in certified organic production, handling, and processing (Organic Materials Review Institute, 2006). OMRI’s services are directed to all aspects of the organic industry with a primary focus on the decision makers who deal with the compliance status of generic meterials and brand name products. With the OMRI Generic Materials List and OMRI Products List, OMRI provides guidance on the suitability of material inputs under the USDA National Organic Program standards (Organic Materials Review Institute, 2006). Both SCD Essential Probiotics and ProBio Balance Plus are listed with OMRI as “safe for use in organic production.” Go to www.omri.org for more information and to verify SCD’s listing.

SCD’s compliance with the above-mentioned regulatory agencies illustrates the safety and quality of the products manufactured as well as routine testing that has been conducted over the past two years. Both SCD Essential Probiotics and ProBio Balance Plus have been routinely tested both in house and by third party independent laboratories for the incidence of pathogenic activity, heavy metals, and mycotoxins. A nutritional analysis has also been conducted to show the safety of the product for human consumption. Certificate of analysis of both SCD Essential Probiotics and ProBio Balance Plus are enclosed.

Both SCD Essential Probiotics and ProBio Balance Plus are recommended for use in aquaculture application. They can be used to manufacture secondary products for specific applications during shrimp grow out. The secondary products are as below.

SCD ProBio Liquid 2™ (“PL2”): This is a probiotic culture for water treatment that has been acclimated with pond water at the farm site. It is a proprietary liquid probiotic material which has been specially acclimated at the farm to the unique water quality conditions of the farm site. In addition, it is used for treating the soil during preparation and treating the water quality during filling and rearing.

SCD ProBio Solid™ (“PBS”): This is a solid probiotic material for treating the soil during preparation and for treating the organic matter in the pond during rearing.

SCD BioFeed™ (“SBF”): This is a probiotic feed technology for increasing the immunity and improving feed conversion of the shrimp during rearing. This is made using the shrimp feed supplied by the farm.

Theory

Probiotics, modes of action, colonization and SCD Probiotics for Aquaculture

Definition

Fuller (1989) defined probiotics as a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Later, the term was defined as microbial dietary adjuvants that are administered in such a way, as to enter the gastrointestinal tract and to be kept alive, to beneficially affect the host physiology by modulating the mucosal and systemic immunity, as well as improving microbial balance by preventing the colonization of undesirable bacteria in the intestinal tract (Gatesoupe, 1999; Naidu et al. 1999). Later, Verschuere et al. (2000) gave a wider definition of probiotics as follows: a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient. It is important however to point out that bacteria that are delivering essential nutrients to the cultured species without exerting an active function in the host or in its environment should not be considered as probiotic. Environmental conditions as well as chance influence microbial community development, thus opening the opportunities for application of probiotics as biological control or conditioning agents since primary colonization of culture water could prevent the growing of bacteria accidentally present in the water that could occasionally be pathogenic for the cultured organisms. When the host or its environment has a well stable bacterial community, the application of the selected probiotic bacteria often needs to be made on a regular schedule mode in order to achieve the desired positive effects.

Modes of Action

There have been postulated several modes of action of probiotics in the host, like competition for nutrients, modulation of non-specific immune responses, production of antimicrobial compounds, competition for site attachment among others that have been observed during in vitro experiments; however it needs to be accepted that the efficiency of a selected probiotic in vitro may significantly change when administered to the host because it is influenced by more complex factors such as the selective ingestion (Balcazur et al., 2006), and the death in the intestinal tract (Vine et al., 2006) caused by the failure of the probiotic to maintain its in vitro physiology under circumstances of a more complex microbial interactions and/or nutritional environment (Tinh et al., 2007). In general, there is a sense of the lack of correlation between in vitro and in vivo experiments in the latest reviews on probiotic use in aquaculture (Balcazar et al., 2006; Vine et al., 2006; Tinh et al., 2007). The main claimed mechanisms are: competitive exclusion, digestion enhancement, immune response enhancement, water quality improvement and antiviral effects.

A. Competitive Exclusion
Bacterial antagonism is a common phenomenon in nature; therefore, microbial interactions play a major role in the equilibrium between competing beneficial and potentially pathogenic microorganisms. However, the composition of microbial communities can be altered by husbandry practices and environmental conditions that stimulate the proliferation of selected bacterial species. It is well known that the microbiota in the gastrointestinal tract of aquatic animals can be modified, for example by ingestion of other microorganisms; therefore, microbial manipulation constitutes a viable tool to reduce or eliminate the incidence of opportunist pathogens

There are selected microorganisms able to produce substances that can inhibit or kill other potential pathogenic bacteria like antibiotics, antibacterial substances, siderophores, bacteriolytic enzymes, proteases and protease inhibitor, lactic acid and other organic compounds like bacteriocins and hydrogen peroxide (Gatesoupe, 2008). One of the most well known bacteriocins is nisin, which is a ribosomally synthesized antimicrobial peptide produced by certain strains of Lactococcus lactis which has been proved to act against human multidrug resistant pathogens such Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and others (Gatesoupe, 2008).

B. Digestion Enhancement
Some researches have suggested that microorganisms have a beneficial effect in the digestive processes of aquatic animals, especially by supplying fatty acids and vitamins. In addition, some bacteria may participate in the digestion processes of bivalves by producing extracellular enzymes, such as proteases, lipases, as well as providing necessary growth factors (Balcazur et al., 2006; Vine et al., 2006). Similar observations have been reported for the microbial flora of adult penaeid shrimp (Penaeus chinensis), where a complement of enzymes for digestion and synthesize compounds that are assimilated by the animal (Wang et al., 2000). Microbiota may serve as a supplementary source of food and microbial activity in the tract digestive may be a source of vitamins or essential amino acids (Wang et al., 2000).

C. Immune Response Enhancement
Several studies have documented the ability of a number of probiotics to modulate non specific immune responses in aquatic animals. In the case of fish, most of the published experiments with probiotics have demonstrated an increase in resistance during bacterial infections (Balcázar, 2006). Only recent studies have demonstrated the incidence of probiotics in immune system functions. Gatesoup (2008) reported that feeding with Gram-positive as well as Gram negative selected probiotics, caused and increase in cellular parameters such as the number of erythrocytes, lymphocytes and macrophages and enhanced lysozyme activity. Villamil et al. (2002) evaluated the immunomodulatory effects of several isolates of LAB from terrestrial origin, finding that Lactococcus lactis caused the major increases in immune functions of turbot (S. maximus) like chemiluminiscence and no production of head kidney macrophages. Later, Villamil et al. (2003b) showed that, in the case of LAB, not only the whole cell was able to induce an increase in the immune responses, but some extra cellular products such as nisin were able to increase chemiluminiscence and nitric oxide production in a dose and time dependant manner in turbot (Scophthalmus maximus).

In shrimp, Balcázar et al. (2003) found that a mixture administration of Bacillus and Vibrio sp. to Litopenaeus vannamei caused an increase in resistance against Vibrio harveyi and white spot syndrome that was correlated with an increase of the phagocytosis and antibacterial activity. Chiu et al. (2007) reported that the white shrimp, Litopenaeus vannamei, treated with food supplement with Lactobacillus plantarum significantly increased phenoloxidase (PO) activity, respiratory burst, superoxide dismutase (SOD) activity and clearance efficiency of Vibrio alginolyticus, as well as prophenoloxidase (proPO), and peroxinectin (PE) mRNA transcription.

D. Water Quality Improvement
It has been postulated that the primary principle for acceleration of organic matter decomposition by probiotics (particularly particulate matter that settles to create black sludge such as in prawn pond) is a function of C:N ratio management by beneficial heterotrophic bacteria (Gatesoup, 1999; Avnimelech and Ritvo, 2003). Inoculation with probiotics enhances the domination of heterotrophic bacteria in the environment. It has been proposed that bacteria selected as probiotics from the genus Bacillus, are known to convert organic mater to CO2, in contrast to Gram negative bacteria that would convert organic mater in bacterial biomass or slime (Gatesoup, 2008). The phototrophic bacteria are known to be very robust and versatile for their nutrient utilization. They are known to utilize any commonly found toxic substances, e.g. ammonia, nitrites, hydrogen sulfide, etc.) in a waste system (Noparatnaraporn et al., 1987; Kantachote et al., 2005). Principle chemical reactions are carbon decomposition via both respiration and fermentation, nitrification/de-nitrifciation (such as ammonia to nitrate) and sulfate reduction (sulfite to sulfate) (Avnimelech and Ritvo, 2003).

During the production cycle, high levels of gram-positive bacteria, particular heterotrophic bacteria, can be used to minimize the buildup of dissolved and suspended organic carbon. The end result is reduction in final black sludge buildup after harvest. It has been reported that use of Bacillus sp. improved water quality, reduced black sludge, improved survival and growth rates and increased the health status of juvenile Penaeus monodon and reduced the pathogenic vibrios (Gatesoup, 2008).

E. Antiviral Effects
Some bacteria used as candidate probiotics have antiviral effects. Although the exact mechanism by which these bacteria do this is not known, laboratory tests indicate that the inactivation of viruses can occur by chemical and biological substances, such as extracts from marine algae and extracellular agents of bacteria. The production of antagonistic compounds may also be active against virus as documented by Balcazur et al. (2006) who reported antiviral activity from Pseudomonas sp., Vibrios sp., Aeromonas sp., obtained from salmon hatcheries against infectious hematopoietic necrosis virus (IHNV) with more than 50% plaque reduction. Balcazur et al. (2006) reported the isolation of a strain of Pseudoalteromonas undina, which exerted antiviral effects by increasing survival in prawn (Penaeus sp.) and sea bream (Sparus aurata) experimentally infected with Sima-aji Neuro Necrosis Virus (SJNNV), Baculo-like viruses and Irido virus. Gatesoup (2008) reported a significant antiviral activity in two Vibrio strains isolated from a shrimp hatchery, especially against IHNV and Oncorhynchusmasou virus (OMV). Further studies should be conducted in order to establish if there is a direct antiviral activity or if the survival augmentation responded to other more complex factors.

F. Colonization
Colonization of the gastrointestinal tract of animals by probiotics is possible only after birth, and before the definitive installation of a very competitive indigenous microbiota. After this installation, only the addition of high doses of probiotic provokes its artificial and temporary dominance. In mature animals, the population of probiotic organisms in the gastrointestinal tract shows a sharp decrease within days after the intake had stopped (Fuller, 1992). According to Gatesoup (2008), a microorganism is able to colonize the gastrointestinal tract when it can persist there for a long time, by possessing a multiplication rate that is higher than its expulsion rate. For example, Vibrio sp. normally colonize the hepatopancreas of juvenile white shrimp; however, this normal microflora can artificially become dominated by Bacillus sp. (up to 50% of the total) if it is added to the water for 20 days (Gatesoup, 2008).

The process of colonization is characterized by attraction of bacteria to the mucosal surface, followed by association within the mucous gel or attachment to epithelial cells. Adhesion and colonization of the mucosal surfaces are possible protective mechanisms against pathogens through competition for binding sites and nutrients, or immune modulation (Balcazar et al., 2006).

Factors known to influence the colonization of microorganisms can be grouped as follows: (i) Host-related factors: body temperature, redox potential levels, enzymes, and genetic resistance. For example, bacteria may enter through the mouth, either with water or food particles, and pass down the alimentary tract, at which point some of them are retained as part of a resident microflora. Others are destroyed by the digestive process or pass through the gut, and are eliminated via the faeces. In addition, bacterial growth may be inhibited by any antimicrobial compound produced by the host. (ii) Microbe-related factors: effects of antagonistic microorganims, proteases, bacteriocins, lysozymes, hydrogen peroxide, formation of ammonia, diacetyl, and alteration of pH values by the production of organic acids (Balcazar et al., 2006). For example, lactic acid bacteria are known to produce compounds such as bacteriocins that inhibit the growth of other microorganisms.

In summary, the known benefits of probiotics in aquaculture farming include direct effects to the shrimp and the environment of the shrimp. Research has shown immunity enhancement, disease control, feed conversion improvement effects on the shrimp through microbial colonization of the digestive tract. Environmental effects include water quality improvement, reduction of water exchange, and reduction of sludge accumulation. All of these effects combined have a synergist impact on the financial performance of the farm. It is expected that probiotics will be used in prawn culture to replace the use of antibiotics and will lead the prawn industry to future organic farming.

SCD Probiotics for Aquaculture

Environmental Enhancement
SCD Probiotics Inside cultures contain naturally occurring beneficial heterotrophic bacteria, e.g. bacilli and lactobacilli bacteria. They primarily obtain their nutrients from organic sources. Due to the fact that they are naturally occurring microorganisms and the highly advanced SCD Probiotics Inside technology process, they survive and proliferate in the environment. Further, they are known for their fast growing properties. The primary principle for acceleration of organic breakdown reduction (particularly particulate matter that settles to create black sludge such as in shrimp pond) is a function of C:N ratio management by beneficial heterotrophic bacteria (Gatesoup, 1999; Avnimelech and Ritvo, 2003). Inoculation of SCD Probiotics cultures enhances the domination of heterotrophic bacteria in the environment. SCD Probiotic cultures are unique due to the addition of the phototrophic bacteria and the technology culturing process, which allows these diverse organisms to thrive in a synergistic consortium. The phototrophic bacteria are very robust and versatile for their nutrient utilization. They are known to utilize any commonly found toxic substances, e.g. ammonia, nitrites, hydrogen sulfide, etc.) in a waste system (Noparatnaraporn et al., 1987; Kantachote et al., 2005). Purple Non-Sulfur Bacteria (PNSB), is a family of purple-red photosynthetic microbes (normally found in deep soil, in pond mud and on leaves of wild plants). Four genera are most frequently isolated: Rhodopseudomonas, Rhodobacter, Rhodomicrobium, and Rhodospirillum (Lindquist, 2001). PNSB can use various substrates as sources of organic carbon and energy with ammonium and/or nitrate as a source of nitrogen and may use sulfide or thiosulphate as an electron donor under photosynthetic conditions (Imhoff and Trüper, 1989). Because of these attributes, they have the potential for treating various sources of wastewater and removing odor (Kim, 2004; Do et al., 2003). They have been used for treatment of many types of wastes: concentrated latex wastewater (Choorit et al. 2002), aquarium wastewater (Nagadomi et al. 1999), and agricultural waste (Hiraishi et al. 1989). For swine wastewater treatment, only anaerobic lagoons are used and many researchers have described a relationship between decreased odor intensity and total suspended solid in the anaerobic lagoons and phototrophic purple bacteria looming on them (Chen et al. 2003, Do et al. 2003). The reports indicated that the reduction in biological oxygen demand (BOD) and chemical oxygen demand (COD) ranged 46 to 91%. Swine dry manure treated with EM-wash and drinking water contained 23.9% crude protein and significant quantities of essential amino acids. Consistent results were found from dairy plant, of which removal of BOD, COD and total volatile solids (TVS) averaged from 85%, 82%, and 84%, respectively.

In brief, while certain groups of SCD Probiotics products are capable of shifting microbial ecology balance, where organic compounds are converted into mineral nutrients, other co-existing microbial groups are capable of utilizing either those mineral nutrients or by-products from other water living organisms. Principle chemical reactions are carbon decomposition via both respiration and fermentation, nitrification/de-nitrifciation (such as ammonia to nitrate) and sulfate reduction (sulfite to sulfate) (Avnimelech and Ritvo, 2003).

Byproducts such as organic acids and enzymes generated through fermentation with SCD Probiotics cultures will also enable break down of wastes and solids to reduce turbidity of the water. The production of vitamins, anti-oxidants and various bio-active micronutrients by SCD Probiotics further stimulate the growth of biology, thereby increasing the diversity index and increase the overall capacity of biology to consume and decompose organic matter. These mechanisms are critical for water quality improvement, sludge reduction and therefore can be used to improve financial results in aquaculture applications as well as to minimize negative impacts to the environment.

Impacts on Host Animal

It has been recognized for years that the maintenance of vigorously fermenting lactobacilli bacteria producing a low pH in the human, animal and aquatic organism intestine can help in protecting the intestine against colonization by harmful bacteria or viruses (Gatesoup, 1999; Avnimelech and Ritvo, 2003). In addition, the improvement of environment and neutralization of toxic compounds lowers stress, which in improves natural resistance and immune responses to pathogens or diseases. The production of nutritive substances by SCD Probiotics in the digestive system provides further improved feed conversion, assimilation of nutrients and boosting of the immune system. Nutritive substances produced by SCD Probiotics include vitamins, antioxidants, assimilable minerals, amino acids and enzymes. These beneficial bio-active substances are the building blocks of health cell generation in all plants, animals, humans, fish and shrimp.

Special beneficial bacteria in SCD Probiotic cultures are known to generate a second generation immunostimulant such as Bio-Glucan which plays a major role in stimulating the natural immune system of the target organisms. For example there are numerous studies showing immunity enhancement against Vibrio spp and White Spot Virus (Noparatnaraporn et al., 1987; Gatesoup, 1999; Kobayashi and Kobayashi, 2000).

SCD Probiotics Technology Benefit Analysis

The successful application of SCD Probiotics Technology is expected to have the following impact on production costs at the farm:

Decrease the pumping cost in the intensive ponds by improving water quality and decreasing the frequency and quantum of water exchange as well as black sludge after harvest.
Decrease the aeration cost in the intensive and hyper-intensive ponds through microbial action and more efficient degradation of organic material, thereby reducing bottom sedimentation.
Improve feed conversion. A 2.4% improvement has been seen in trials
Improve survival rate. An 8.1% increase has been achieved in trials.
Direct 1% reduction in feed cost, not including improved feed conversion.
Reduce the duration of the cycle to achieve target weight.
Increased average weight and corresponding market value.
Increased shrimp quality and corresponding market value.
Increased growth rate. As much as a 37.3% increase as compared to control has been achieved in trials.
Increase in average weight. As much as a 27.5% increase as compared to control has been achieved in trials.

Due to a number of critical unknown variables particularly cost required for feed, pond water exchange and pond aeration, it is premature to estimate the return in amount of dollar spent on SCD Probiotics Technology, even though previous trial data has shown increase in improvement of production performance (kg/ha) by 38.4%.

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