| The method of selecting an ideal bacterial formula to compete with a specific organism begins with a thorough understanding of the target organism, including its nutrient and environmental requirements. As with algae, if one can reduce the availability of the required nutrients, the result is cleaner, healthier water for the shrimp or fish, and a reduced number of undesirable bacterial species. | ||||||
| Vibrio spp. are gram-negative, facultatively anaerobic chemoautotrophs that are common in marine environments. | ||||||
| Kenneth Todar, University of Wisconsin-Madison, Department of Bacteriology, says of Vibrio spp., in his Bacteriology 303 course, "They have structural and metabolic properties that overlap with both the enterics and the pseudomonads. In Bergey's Manual, Vibrionaceae is a family on the level with Enterobacteriaceae. Vibrios are facultative like enterics, but they have polar flagella, are oxidase-positive, and dissimilate sugars in the same manner as the pseudomonads. In aquatic habitats they overlap with the Pseudomonadaceae in their ecology, although pseudomonads favor fresh water and Vibrios prefer salt water." | ||||||
Definitions:
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| Vibrio spp. are of concern to humans because certain species, including Vibrio cholerae can cause serious human disease if infected shellfish and oysters are consumed raw. [4] [7] Vibrio spp. are of concern to shrimp and prawn farmers because various species can cause Penaeid bacterial septicemia (also known as Penaeid vibriosis, Luminescent vibriosis, Red-leg disease, "sien dun" in Thai which translates to black splint, Sea gull syndrome or "Sindrome gaviota" in Latin America), resulting in death of up to ninety percent of their crops, with the most significant impact on post-larvae and juvenile shrimp. Outbreaks of severe Vibrio spp. contamination in shrimp farms have frequently occurred in conjunction with an excess of hydrogen sulfide in the sludge (1) and an overgrowth of Cyanobacteria spp.[3[ Vibrio harveyi, is the species of luminescent Vibrio spp. most commonly implicated in pathogenic outbreaks at shrimp and prawn farms around the world. [23] | ||||||
| Recent collapses of the shrimp industry in Vietnam, India, Bangladesh, the Philippines and China have been partly attributed to an increase in the incident of Vibriosis. The industry has also been seriously affected by the emergence of a number of pathogenic viruses notably the Yellowhead baculovirus; however the ability of Alken Clear-Flo® to reduce stress in shrimp through the control of pollutants and the inhibition of Vibriosis will enable the shrimp to withstand viral attacks with a greater expectation of survival. The Fisheries and Oceans, Canada, SeaLane Diseases of Shellfish agrees that maintaining water quality has been proven to decrease the incidence of disease. | ||||||
| Vibrio spp. ferment glucose, test positive on the oxidase test, and grow well in non-selective culture media. [7] Most of the species, especially the pathogenic strains, require sodium chloride, so they should not grow well in fresh water. Electrolytes stimulate their growth, so a nicely balanced shrimp or fish feed will encourage their proliferation. Vibrio spp. prefer a pH from 7 to 9, as they are acid sensitive. Their growth is severely inhibited in an acid environment, as is the growth of Cyanobacteria, another species unwelcome in shrimp and fish farms. Most ponds with a problem with Vibrio spp. have reported a concurrent problem with hydrogen sulfide buildup in the sludge. Vibrio spp. feeds on various forms of sulfur, which includes hydrogen sulfide. The Standard Methods test for identifying pathogenic Vibrio spp. is a two stage procedure. First the bacteria is grown on an alkaline peptone agar. Then it is subcultured on a Thiosulphate, citrate, bile salt, sucrose agar. [4] Examining the nutrients required for Vibrio spp. proliferation, citrate, sucrose, sulfur and salt stand out for the selection of a product to compete with Vibrio spp. | ||||||
Some shrimp farmers have reported a degree of success in limiting the pathogenic strains of Vibrio spp. by using molasses instead of table sugar (sucrose) in their feed., as a means of boosting the carbon to nitrogen nutrient balance, so that the heterotrophic bacteria population increases in the pond, digesting a higher proportion of excess ammonia. Although there is some disagreement in the industry, most environmental engineers notice optimum bacterial utilization of organic carbon pollution, when the nutrient balance matches C= 100, N = 6, and P = 1.2, with a ratio of C:N = 17:1 :and N:P = 5. Shrimp farmers can then manipulate the selective utilization of specific nutrients in a pond by altering the nutrient ratio. For example, when ammonia levels are high, addition of an easily digested carbon source, like molasses, to produce a ratio of C:N = 12 will force the heterotrophic cultures to utilize a higher level of ammonia, present in pond water, to balance the molasses, since molasses is easier to digest than the organic carbon pollutants naturally present (feces, leftover feed, petroleum and fertilizer runoff). Environmental engineers and Alken-Murray have noticed that when heterotrophic bacteria digest petroleum hydrocarbons, they require and utilize a higher level of ammonia-nitrogen than for digestion of simple carbohydrates, sewage,etc. The ideal balance for petroleum remediation is C=100,N=10, P= 2, so although the N:P ratio remains 5:1, the C:N ratio changes from 17:1 to 10:1. The results of diet change or carbon supplementation have not been reliable, but the use of unsulfured molasses was reported to produce better results than sulfured molasses. Since molasses comes from the same cane sugar source as sucrose, it is possible that using another carbohydrate, one capable of exerting selective pressure on the microbial population, would provide more consistent results. Various human probiotic formulas include maltodextrin for this purpose, since enteric pathogens, such as the luminescent Vibrio harveyi, are unable to break down the complex maltodextrin, while a solid population of probiotic species will be selectively encouraged to grow and reproduce, increasing their biomass. | ||||||
*Alken-Murray advises adding maltodextrin to aquaculture feeds amended with Alken Clear-Flo 1002, since maltodextrin serves as a preferred organic carbon source for 10 of the 12 Bacillus strains present in Alken Clear-Flo 1002, Alken-Murray also includes maltodextrin in Alken Clear-Flo 1015 to enhance performance of this specialty product, designed to manage toxic effects of Cyanobacteria blooms. Alken-Murray will also soon be introducing Alken Bio-Nutrient 10, a prebiotic nutrient blend, designed to selectively encourage growth of floc-forming, heterotrophic probiotic bacteria, while discouraging pathogenic and filamentous bacteria.. Key ingredients are as follows:
*This research note was added to this document 11/28/2006 by Valerie Anne Edwards, President Alken-Murray Corporation. | ||||||
AMV 359 & 405 |
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AMV 134S & 406 |
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843, AMP 100, AMP 104, AMV 119S & AMV 923 |
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AMH 100, AMH 108 & AMH 118 |
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| Bacillus pumilus AMH 109, AMH 115, & AMV 121S |
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112 & AMC 300 |
Yes | |||||
| Petroleum-degrading vegetative, gram-negative strains* | ||||||
367 |
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512 |
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AMV 359 & 405 |
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AMV 134S &406 |
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843, AMP 100, AMP 104, AMV 119S & AMV 923 |
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| Bacillus pumilus AMH 109, AMH 115 & AMV 121S |
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112 & AMC 300 |
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AMH 100, AMH 108 & AMH 118 |
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367 |
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512 |
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| Cold-Temperature Catalyst strains | |||||
| *Petroleum hydrocarbon degrading strains belong to genii: Pseudomonas & Marinobacter. | |||||
| When both economy and competition for nutrients required by Vibrio spp. are considered, the ideal blend is Alken Clear-Flo® 1006. For optimum results, apply at 50% of the dosages prescribed on the dosage chart for Clear-Flo® 1003 - 1008. | |||||
| Alken Clear-Flo® 1006.can be applied directly to a functioning fish or shrimp pond to generally clean the water and eliminate the formation of sludge while reducing the proliferation of Vibrio spp.. If applied to sludge, contaminated with hydrogen sulfide and Vibrio spp., under anaerobic conditions is desired, nitrate formed from nitrification of ammonia should provide an alternate electron acceptor. The ideal nutrient BALANCE of "Total Soluble Carbon to Total Soluble Nitrogen to Total Soluble Phosphorus" ratio = 100/6/1.2, provides optimum growth and digestion rates for heterotrophic microbes (those that digest organic carbon-based foods, such as milk, sugar, starch, etc.). As long as the balance of nutrients remains close to this level, the biomass population will adjust, first with those strains best able to benefit from the food present AND the environmental conditions growing and reproducing best. If one primary nutrient, again soluble carbon, nitrogen or phosphate, is SO over-abundent that the pond is again completely out of balance, the environmental engineer or pond owner will consider other options, including the use of autotrophic bacteria (bacteria that gain energy from specific chemical reactions, while using carbon dioxide as their only carbon source). The most common over-abundent nutrient in aquaculture ponds and other water-bodies is Nitrogen, with ammonia showing up the most, first resulting from fecal bacteria, such as E.coli, using the "urease" enzyme to convert urea into ammonia, but then protein-degrading bacteria convert protein into amino-acids and finally into ammonia. Alken-Murray offers a pair of autotrophic nitrifier species of bacteria, isolated from soil, Nitrosomonas europaea, strain 978, and Nitrobacter winogradkyi, strain 391, in Alken Clear-Flo 1100-50x, which oxidize excess ammonia, first to nitrite, and finally to nitrate. Links from this product information bulletin to our Nitrification Tutorial, the QC-2 lab test and the product MSDS should be useful to clients. Our "QC-53 - Ammonia Oxidation Measurement" , the protocol used to verify performance of the nitrifiers we market, is posted to the Alken-Murray website, and can be accessed by clicking the link provided on the product technical information bulletin for Alken Clear-Flo 1100-50x. Also available by link from that product technical bulletin, is our "QC2", which is a simpler test procedure that is used to verify survival of nitrifiers, following transport or prolonged storage. | |||||
| Although there is minor disagreement among environmental engineers about the absolute ideal balance of nutrients to obtain maximum rates of bioremediation, most of them AGREE that heterotrophic bacteria digsting petroleum-hydrocarbons and some extremely difficult industrial chemicals, require a much higher level of nitrogen to balance the carbon level provided by these compounds. Thus, a Los Angeles pond experiencing severe parking lot runoff discovered that treatment with Alken Clear-Flo 1006 solved a double-problem better than anyone anticipated was possible. High levels of ammonia and phosphate arrived from a small poultry operation, followed by runoff from a large parking lot, where a church had decided to raise money by washing cars AND changing oil over a single weekend, counting on an expected deluge to wash away the evidence of their activity. Tests revealed higher COD and TPH than had been anticipated, but the balance ended up with"Total Carbon to Total Nitrogen to Total Phosphorus" = 100 to 9 to 2.5 pretty close to the level sought for bioremediation in land farming or in-situ soil spill cleanup of petroleum hydrocarbons (100 to 10 to 2 ). To try to rescue the fish and other pond residents, our distributor decided to continue treatment with aquatic products, but to amend dosages based on actual levels of pollution delivered during this deluge. Alken Clear-Flo 1006 talents that allow it to out-compete Vibrio spp. also make it an ideal treatment for this emergency pollution incident combining food Fats,Oils and Greases found in poultry manure runoff combined with petroleum oils from parking lot oil changes. Ever since 1991, distributors have reported to Alken-Murray researchers that shrimp farmers tend to wash down areas around their ponds with detergents and phenolic sanitizers, so most CF 1000 series products have included one strain of Pseudomonas putida that digests chemical detergents and another that digests phenols, so that today, these two strains are included in CF 1003, 1005, 1006, 1007, 1008 & 1015. Normal maintenance dosages of Alken Clear-Flo 1006 , were continued after a special three-week emergency cleanup treatment protocol. | |||||
| Uncontrolled and drastic pH shifts will stress the shrimp (or other species raised) , making them more susceptible to illness. pH can be stabilized with the addition of dolomitic Limestone, which will also tie up some excess phosphate, that cannot be used by the bacteria. Alken Clear-Flo 1002, 1005, 1008, 1008C, 1015 and 1015C all contain Limestone to assist maintenance of alkalinity during treatment, as is revealed on product MSDS, posted to the Alken-Murray website. | |||||
| If it is also possible to alter the environment, by lowering salinity and pH, and by substituting unsulphured molasses for sugar in the feed, the undesirable Vibrio spp. should be severely decreased. Reducing salt and pH would also decrease the desirability of a particular habitat for Vibrio spp., but this is seldom possible. Since the Alken-Murray treatments will improve overall water quality, as measured by an increased REDOX potential, the general immunity and health of the aquatic inhabitants should make them less susceptible to all diseases and parasites. | |||||
| One scientific support for the use of bacteria to control the presence of Vibrio spp. in large aquatic water bodies is D. J. W. Moriarty (1998) Control of Luminous Vibrio Species in Penaeid Aquaculture Ponds. Aquaculture. [21] Microbial Interactions in Aquaculture, May 1998 164 (1-4): 351-358. This paper discusses how the use of Bacillus spp. in high numbers (10,000 to 100,000) in prawn farms changed the bacterial species composition and improved prawn production while decreasing presence of luminous Vibrio spp.. We appreciate Sr. Sergio Escutia of Aquastrat S.A. de C.V. in Mexico informing us of the contents of this article. | |||||
| The Aquaculture Veterinary Services International, researcher Adam Ogilvie [22] , performed an independent study of two Alken-Murray custom probiotic formulations for the control of Vibrio angulliarum and V. fischeri and Yersinia ruckeri in Atlantic salmon raising. The custom blend currently named Alken Clear-Flo 1003 significantly inhibited Yersinia, reducing it by 89% in four days , but the formula named Alken Clear-Flo 1006, (created in the fall of 1996) totally inhibited all growth of various species of pathogenic, luminescent Vibrio, revealing a total inability of these strains to grow in water previously seeded with Alken Clear-Flo 1006 . These tests were run over a 7 day period of time in the fall of 2000 in Canada. | |||||
References:
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See Laurence Evans' article on Probiotics
(including CF 1006) in Shrimp Hatcheries