5/24/2021 Selecting A Bacterial Formula To Compete with Vibrio spp.

Selecting A Bacterial Formula To Compete with Vibrio spp.

by Valerie Anne Edwards, Pres. Alken-Murray Corporation

Update 11/28/2006

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:

Facultative anaerobe: A bacterium capable of growing under either aerobic or

anaerobic conditions, in the presence of an inorganic ion i.e. SO4, NO3.
Chemoautotroph: An organism (only bacteria) that obtains its energy from the direct
oxidation of chemical compounds (e.g., NH3, NO2-, S2, and Fe2+), without using
sunlight, to yield energy and uses only organic compounds as a source of carbon.
This metabolic mode also requires energy for CO2 reduction, like photosynthesis, but
no lipid-mediated processes are involved.

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.

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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:

Two (2) sources of organic carbon: maltodextrin and milk whey concentrate.
Two (2) sources of nitrogen: yeast extract and amino-acids
Two (2) sources of phosphate (MSP and DSP)
Additional supplements; minerals, vitamins (including Biotin), trace elements, Yucca
schidigera

*This research note was added to this document 11/28/2006 by Valerie Anne Edwards,
President Alken-Murray Corporation.

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NUTRIENTS UTILIZED BY VIBRIO spp.

Key Bacterial
Species/Strains Hydrogen Petroleum
Citrate Sucrose Salt Fats
sulfide Hydrocarbons

Bacillus subtilis
Yes Yes Yes Yes
AMV 359 & 405

Bacillus
licheniformis Yes Yes Yes Yes
AMV 134S & 406

Bacillus
amyloliquefaciens
843, AMP 100, AMP Yes Yes Yes
104, AMV 119S &
AMV 923

Bacillus
sulfidophilus
AMH 100, AMH 108 Yes Yes Yes Yes Yes
& AMH 118

Bacillus pumilus
AMH 109, AMH 115, Yes Yes Yes Yes
& AMV 121S

Bacillus
megaterium Yes Yes Yes Yes
112 & AMC 300

Bacillus
thuringiensis 679 Yes Yes Yes Yes

Petroleum-
degrading Yes Yes Yes Yes Yes
vegetative, gram-
negative strains*

Pseudomonas Yes Yes

putida 412

Enterobacter Yes Yes Yes

cloacae 411

Paracoccus.
denitrificans Yes Yes Yes Yes
367

Paracoccus.
pantotrophus Yes Yes Yes Yes
512

Alken Clear-Flo Products Containing Selected Species/Strains

Key Bacterial
Species/Strains CF 1002 CF 1005 CF 1006 CF 1007 Enz-Odor 6

Bacillus subtilis
Yes Yes Yes Yes
AMV 359 & 405

Bacillus
licheniformis Yes Yes Yes Yes
AMV 134S &406

Bacillus Yes Yes Yes

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amyloliquefaciens
843, AMP 100, AMP
104, AMV 119S &
AMV 923

Bacillus pumilus
AMH 109, AMH 115 Yes
& AMV 121S

Bacillus
megaterium Yes Yes Yes
112 & AMC 300
Bacillus
sulfidophilus
AMH 100, AMH 108 Yes Yes Yes
& AMH 118

Bacillus
Yes Yes
thuringiensis 679

Pseudomonas
Yes Yes Yes
putida 412

Enterobacter Yes Yes

cloacae 411

Paracoccus.
denitrificans Yes Yes
367

Paracoccus.
pantotrophus Yes Yes
512

Cold-Temperature Yes Yes Yes

Catalyst strains

Petroleum-
degrading Yes
vegetative, gram-
negative 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
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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
molasse 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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1. Aquafarmer Information Sheet: Prevention of Black Gill Disease in Marine Shrimp,

published by the Center for Tropical and Subtropical Aquaculture, No. 126, July 1997.
2. The Fisheries and Oceans, Canada, SeaLane Diseases of Shellfish. Introduction to
Bioluminescence, Shrimp Diseases, an article published by the Center for Tropical
and Subtropical Aquaculture,The Oceanic Institute, No. 121, May 1996
3. Genetic diversity of related vibriophages isolated from marine environments around
Florida and Hawaii, USA, by Kellogg, C.A., Rose, J.B., Jiang, S. C., Thurmond, J. M.,
and Paul J.H., published by Inter-Research, Marine Ecology Series 1995.
4. FDA/CFSAN website
5. Biological Science, An Ecological Approach (BSCS Green Version) 3rd edition, Rand
McNally & Company (1973)
6. Biology of the Procaryotes, J.W. Lengeler, Gerhart Drews, & Hans G. Schlegel ,
Blackwell Science (1999)
7. Manual of Clinical Microbiology, Patrick R. Murray, Ellen Jo Baron, Michael A. Pfaller,
Fred C. Tenover, Robert H. Yolken, American Society of Microbiology, 7th Edition
(1999)
8. Microbiological Methods for Monitoring the Environment: Water and Wastes, Bordner
& Winter (1978)
9. Bacteria in their Natural Environments, M.M. Fletcher and G.D. Floodgate etc..(1985)
10. Microbiology, 5th ed., M.J. Pelczar, Jr, E.C.S. Chan, NR. Krieg (1986)
11. Microbial Growth The Marine Fish and Invert Reef Aquarium, 7th ed., Albert J. Thiel
(1990) Aardvark Press
12. Biological Indicators of Freshwater Pollution And Environmental Management, J.M.
Hellawell (1989)
13. Aquaculture and Water Quality, D.E. Brune & J.R. Tomasso (1991)
14. Prawn Culture, Scientific and Practical Approach, C.K. Jung, W.G. Co (1988)
15. Principles and Practices of Pond Aquaculture, J. A. Lannan, R.O. Smitherman, G.
Tchobanoglous (1986)
16. Scallop Farming, D. Hardy (1991)
17. Marine Shrimp Culture: Principles and Practice, A.W. Fast, L.J. Lester (1992)
18. Fish Aquaculture, Technology and Experiments, C. Meske (1985)
19. Fish Medicine, M. Stoskopf (1993)
20. Water Quality in Ponds for Aquaculture, CF Boyde (1990)
21. Control of Luminous Vibrio Species in Penaeid Aquaculture Ponds.
Aquaculture, D. J. W. Moriarty (1998), Microbial Interactions in Aquaculture, May
1998:164 (1-4): 351-358.
22. E-mail and fax communication from Adam Ogilvie.
23. Studies on the chemical control of luminous bacteria Vibrio harveyi and V.splendidus
isolated from diseased Penaeus monodon larvae and rearing water , Baticados, MCL,
Lavilla-Pitogo, CR, Cruz-Lacierda, ER, de la Pena, LD, Sunaz, NA (1990), DisAquat
Org 9: 133-139

See Laurence Evans' article on Probiotics (including CF 1006) in Shrimp Hatcheries

Clear-Flo || Even-Flo || Demulsifier || Polymers || Chemicals || American SH Cats

Dry Product Index Liquid Product Index Nutrient Index
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