Contains details on biofloc, bioremediation and probiotics

1. USE OF
MICROBES IN
AQUACULTURE
BY,
AMRUTHA J NAIR
CHANDANA B L

2. • Microorganisms have major roles in pond culture,
particularly in the:
- Productivity
- Nutrient cycling
- Nutrition
- Water quality
- Disease control
- Environmental effluent management.

3. PROBIOTICS

4. • Live microbial feed supplement which beneficially
affects the host animal by improving its intestinal
microbial balance
• Benefits include improving the feed value, enzymatic
contribution to digestion, inhibition of pathogenic
microorganism, anti-mutagenic and anti-carcinogenic
activity, growth promoting factor and increase
immune response

6. ACTION OF PROBIOTICS
• Very important role to play in the degradation of organic matter
• Water quality would improve by reducing the disease (including
Vibrio sp., Aeromonas sp. and viruses) incidences, enhancing
zooplankton numbers, reducing odours and ultimately
enhancing aquacultural production.
• Free amino acids and glucose are also released providing food
sources for the beneficial microorganisms.

7. • Inorganic forms of nitrogen, such as ammonia, nitrate
and nitrite are also reduced.
• By improving total water quality and FCR, the
overall health and immunity of the shrimp will be
improved

8. SELECTION OF PROBIOTICS
• General selection criteria are mainly determined by bio
safety considerations,
a. Methods of production and processing.
b. Method of administration of the probiotic and
c. The location in the body where the microorganisms
are expected to be active

9.  CRITERIA FOR SELECTION
• General aspects
- Origin.
- Biosafety.
• Physiological aspects
- Activity and viability.
-Resistance to pH, gastric juice, bile and pancreatic fluid.
-Functional and physiological aspects
-Adherence to intestinal epithelium or tissue.

10. -Antagonism to pathogens, antimicrobial activity.
-Stimulation of immune response.
-Selective stimulation of beneficial microbes and
suppression of harmful ones.
-Beneficial systemic efforts

11. • Methods to select probiotic bacteria for use in the
aquaculture might include the following steps.
1. Collection of background information
2. Acquisition of putative probiotics
3. Screening of putative probiotics
4. Evaluation of pathogenicity and survival test
5. In-vivo evaluation
6. Effects in rearing condition

12. TYPES OF PROBIOTICS
• Probiotics are mainly of two types
a) gut probiotics which can be blended with feed and
administrated orally to enhance the useful microbial
flora of the gut
b) water probiotics which can proliferate in water
medium and exclude the pathogenic bacteria by
consuming all available nutrients. Thus, the pathogenic
bacteria are eliminated through starvation

13. PROBIOTICS CONSIDERED FOR USE IN
AQUACULTURE
• Lactobacillus sp., the lactic acid producing bacteria and many
other probiotics such as Aeromonas hydrophila, A. media,
Altermonas sp, Bacillus subtilis, Carnobacterium inhibens,
Debaryomyces hansenii, Enterococcus faecium, Lactobacillus
helveticus, L. plantarum, L. rhamnosus, Micrococcus luteus,
Pseudomonas fluorescens, Roseobacter sp., Streptococcus
thermopilus, Saccharomyces cerevisiae, S. exiguous, Vibrio
alginolyticus, V. fluvialis, Tetraselmis suecica and Weissella
helenica

16. METHODS OF APPPLICATION
• DRY FORMS
- probiotics that come in packets can be given with feed
or applied to water and have to be brewed at farm site
before application
- contains a packet of dry powder and a packet of
enzyme catalyst
- brewing has to be done in clean disinfected water at
27–32°C for 16 to 18 hours with continuous aeration

17. • LIQUID FORMS
- live and ready to act
- directly added to hatchery tanks or blended with
farm feeds
- can be applied any time of the day in indoor
hatchery tanks, while it should be applied either in the
morning or in the evening in outdoor tanks
- gives positive result in lesser time compared to dry
and spore form bacteria

18. BENEFITS
• Production of inhibitory compounds
• Competition for adhesion sites
• Competition for nutrients
• Source of nutrients and enzymatic contribution to
digestion
• Enhancement of immune response
• Influence on water quality
• Interaction with phytoplankton
• Antiviral activity

19. CONSTRAINTS
• Probiotic strains used in the feed may not be available to the
host animal because of leaching.
• Standardization of proper dosage is required.
• Many bacterial strains may not survive during feed
preparation.
• They lose their viability when exposed to the high temperature
and pressure of an extruder.
• High organic loads in the bottom sediments may reduce the
efficiency of the probiotics used.
• Depending on the water sediment status, exact dosage needs to
be calculated for each set of conditions

20. SAFETY CONCERNS
• Determination of strain resistance to a wide variety of common
classes of antibiotics such as tetracyclines, quinolones and
macrolides and subsequent confirmation of non-transmission of
drug resistance genes or virulence plasmids
• End-product formulation into consideration because this can
induce adverse effects in some subjects or negate the positive
effects altogether
• Modern molecular techniques should be applied to ensure that
the species of probiotics used in aquaculture are correctly
identified, for quality assurance as well as safety

21. BIOREMEDIATION

22. • Bioremediation is the process of using microorganisms
to transform hazardous compounds in a contaminated
environment to non-hazardous end products.
• Naturally occurring or laboratory cultivated
• Biotreatment, bioreclamation and biorestoration

23. RELATIVE DEGRADABILITY
• Simple hydrocarbons and petroleum fuels
- degradability decreases as molecular weight and
degree of branching increase
• Aromatic hydrocarbons
- one or two ring compounds degrade readily, higher
molecular weight compounds less readily
• Alcohols, esters
• Nitrobenzenes and ethers degrade slowly

24. • Chlorinated hydrocarbons
- decreasing degradability within increasing chlorine
substitution – highly chlorinated compounds like
PCBs and chlorinated solvents do not appreciably
degrade aerobically
• Pesticides are not readily degraded

25. BIOSTIMULATION
• Strategy by which the naturally occurring microorganisms are
provided with environmental conditions that favour their
growth and reproduction
• The nutrients, when artificially added to the pond, favours the
microorganisms which multiply in millions numbers, and in
return, the organic wastes and toxic substances are rapidly
broken down by them, into a less harmful state
• Can be achieved by providing an artificial substrate which will
encourage the natural occurring bacteria to grow and multiply
by forming biofilms

26. BIOAUGMENTATION
• Bioaugmentation involves either the addition
of commercially prepared bacteria strains or
the addition of a blend of isolated enzymes or
the combination of both the bacteria strains
and the enzymes (mostly preferred), to
increase the natural degradation carried out by
the indigenous bacterial population

27. REQUIREMENTS
• Carbon source
• Electron donor
• Electron acceptor
• Nutrients
• Optimum pH
• Optimum temperature
• Adequate mass transfer
• Organism retention time

28. • Microorganisms destroy organic contaminants in the
course of using the chemicals for their own growth and
reproduction
• Cells catalyze oxidation of organic chemicals (electron
donors), causing transfer of electrons from organic
chemicals to some electron acceptor
• In aerobic oxidation, acceptor is oxygen
• In anaerobic, acceptor is (with decreasing efficiency):
- Nitrate, manganese, iron, sulfate, C02 and organics
• Also need essential nutrients such as nitrogen,
phosphorus (macronutrients), Fe, Mg, Ca, Co, Ni
(micronutrients) and vitamins

29. ORGANIC DETRITUS
• Dissolved and suspended organic matter contains mainly
carbon chains and is abundantly available to microbes and
algae
• Bacillus subtilis, B. licheniformes, B. cereus, B. coagulans and
species Phenibacillus polymyxa are good examples of bacteria
suitable for bioremediation of organic detritus
• Mass produced, mixed with sand or clay ad broadcasted to be
deposited at the pond bottom
• These bacteria produce a variety of enzymes that break down
proteins and starch to small molecules, which are then taken up
as energy sources by other organisms

30. NITROGENOUS COMPOUNDS
• Ammonia oxidizers are placed under five genera
Nitrosomonas, Nitrosovibrio, Nitrosococcus, Nitrolobus
and Nitrospira
• Derives energy by oxidizing ammonia and ammonium
to nitrate
• Nitrification not only produces nitrate but also alters pH
towards the acidic range, facilitating the availability of
soluble materials
• CO2 acts as carbon source

31. HYDROGEN SULPHIDE
• Organic loading can stimulate H2S production and reduction in
the diversity of benthic fauna.
• Hydrogen sulphide is soluble in water and has been suggested
as the cause of gill damage and other ailments in fish.
• Unionized H2S is extremely toxic to fish that may occur in
natural waters as well as in aquaculture farms
• Purple and green sulphur bacteria that grow at the anaerobic
portion of the sediment-water interface that break H2S at pond
bottom have been widely used in aquaculture to maintain a
favorable environment
• Chromatiaceae and Chlorobiaceae

32. • Can be mass cultured and can be applied
• Being autotrophic and photosynthetic, mass culture is less
expensive
• The common examples of photosynthetic bacteria of importance
in aquaculture are Rhodospirillum, Rhodopseudomonas,
Chromatium, Thiocystis, Thiospirillum, Thiocapsa, Lamprocystis,
Thiodictyon, Thiopedia, Amoebobacter, Chlorobium,
Prosthecochloris, Pelodictyon and Clathrochloris.

33. ADVANTAGES
• It works on a variety of organic and inorganic
compounds
• Can be done either on-site or off-site, easy to
implement and maintain
• Low-cost compared to other treatment methods
• Environmentally-friendly and aesthetically pleasing
• Reduces the amount of wastes to be land filled

34. DISADVANTAGES
• It may take several years to remediate and depends on
climatic conditions
• Restricted to sites with contamination near the roots
• Harvested plants may be classified as hazardous
waste
• Consumption of contaminated plants may be harmful
• There may be harmful effects on the food chain

35. BIOFLOC
TECHNOLOGY

36. • “Bio floc systems" is recent technology which seeks
to solve the problems of water pollution and improve
the use of water resources, in addition to recycling the
nutrients found in the water by a community of
heterotrophic bacteria.
• In aquaculture, the “biofloc” system acts like a
retention trap for the nutrients in the pond, and
reduces maintenance cost.

37. • The term “biofloc” applies to a compound made out of
- 60 to 70% of organic matter, which includes a
heterogeneous mixture of microorganisms (fungus, algae,
protozoans, and rotifers)
- 30 – 40% of inorganic matter such as colloids, organic
polymers, and dead cells.
• They can reach a size up to 1000 µm, irregular shape, full
of pores, and allow the pass of fluids

38. Structure of Biofloc

39. BIOFLOC DEVELOPMENT
• The principle of this technique is the generation of
nitrogen cycle by maintaining higher C: N ratio
through stimulating heterotrophic microbial growth
• Higher C : N is maintained through the addition of
carbohydrate source (molasses)
• 20 units of carbon is required to assimilate one
nitrogen unit

40. • Dense microorganisms develop and function both as
bioreactor controlling water quality and protein food
source.
• The non-consumed nitrogen by the organisms in the
culture can be used to produce microbe protein,
instead of generating toxic compounds
• It is very important to note that this process reduces
the total amount of dissolved oxygen available for the
organisms.
• Density between 10 and 15 mL must be there, so the
system can keep functioning properly.

41. Bio floc system operation in pond

42. BENEFITS OF BIOFLOC CULTURE
• Eco-friendly culture system.
• It reduces environmental impact.
• Improves land and water use efficiency
• Limited or zero water exchange
• Higher productivity (It enhances survival rate, growth
performance, feed conversion in the culture systems of fish).

43. • Higher biosecurity.
• Reduces water pollution and the risk of introduction and
spread of pathogens
• Cost-effective feed production.
• It reduces utilization of protein rich feed and cost of standard
feed.
• It reduces the pressure on capture fisheries i.e., use of cheaper
food fish and trash fish for fish feed formulation.

44. DISADVANTAGES OF BIOFLOC
CULTURE
• Increased energy requirement for mixing and aeration
• Reduced response time because water respiration rates are
elevated
• Start-up period required
• Alkalinity supplementation required
• Increased pollution potential from nitrate accumulation
• Inconsistent and seasonal performance for sunlight-exposed
systems

45. IMMUNOSTIMULANTS

46. • Immunostimulant “IS“ is a natural occurring
compound that modulates the immune system by
increasing the host's resistance against diseases
• Comprise a group of biological and synthetic
compounds that enhance the non-specific cellular and
humoral defence mechanism

48. DISADVANTAGES OF
IMMUNOSTIMULANTS
• High cost.
• Limited efficiency upon parentally administration
• Are not effective against all diseases.
• Over doses of IS induce immunosuppression .
• In few cases, IS failed to render enhanced protection or
increase in immunity .
• Immunostimulants are used with success in aquaculture
against many pathogens, but the ability to improve innate
resistance to columnaris disease has not been studied

52. GLUCAN
• Most popular immunostimulants
• Derived from yeast cell wall
• It increase protection against V. anguillarum and V.
salmonicida.
• It induce significantly protection against
Furunculosis

53. LIPOPOLYSACCHARIDE
• Cell wall component of Gram-negative bacteria
• LPS stimulate B cell proliferation and enhance
macrophage phagocytic activity

54. FREUND’S COMPLETE ADJUVAENT
(FCA)
• Mineral oil adjuvent containing killed Mycobacterium
butyricum
• FCA enhances immune responses and increase the
efficacy of vaccination in fish

55. REFERENCES
• Priyadarshini Pandiyan, Deivasigamani Balaraman, Rajasekar Thirunavukkarasu, Edward Gnana
Jothi George, Kumaran Subaramaniyan, Sakthivel Manikkam, Balamurugan Sadayappan.
Probiotics in aquaculture. Drug Invention Today 5 (2013) 55 – 59
• Maloy Kumar Sahu · .N. S. Swarnakumar · K. Sivakumar · T. Thangaradjou · L. Kannan.
Probiotics in aquaculture: importance and future perspectives. Indian J. Microbiol. (September
2008) 48:299–308
• PatriciaMart´ınez Cruz, Ana L. Ib´a˜nez, Oscar A. MonroyHermosillo, and Hugo C. Ram´ırez
Saad. Use of Probiotics in Aquaculture. 2012
• Swapna P. Antony and Rosamma Philip. Probiotics in aquaculture
• M. Vidali. Bioremediation. An overview. Pure Appl. Chem., Vol. 73, No. 7, pp. 1163–1172, 2001.
• Babak Pakdaman Sardrood, Ebrahim Mohammadi Goltapeh, and Ajit Varma. An Introduction to
Bioremediation. 10.1007/978-3-642-33811-3_1.2013
• Ph. Lakshmi Chanu & Sagar C. Mandal. Concepts of Bioremediation and its Application in
Aquaculture
• Artin Hatzikioseyian. Principles of bioremediation processes. Trends in Bioremediation and
Phytoremediation, 2010: 23-54 ISBN: 978-81-308-0424-8
• S. Musyoka, I. Fernandez. Types and mechanisms of bioremediation in aquaculture wastes;
Review . International Journal of Advanced Scientific and Technical Research .2016:5.,6