Evaluation of extracellular enzyme-producing autochthonous gut bacteria in walking catfish, Clarias batrachus (L.)

  • Atrayee Dey Entomology Research Unit, Department of Zoology, The University of Burdwan, West Bengal 713104, India
  • Koushik Ghosh Aquaculture Laboratory, Department of Zoology, The University of Burdwan, West Bengal 713104, India
  • Niladri Hazra Entomology Research Unit, Department of Zoology, The University of Burdwan, West Bengal 713104, India
Keywords: Autochthonous bacteria, Bacillus, Clarias batrachus, extracellular enzymes, gut, 16S rRNA


The present study was carried out to screen autochthonous gut bacteria in freshwater air breathing walking catfish, Clarias batrachus Linnaeus. Altogether, 100 extracellular enzyme-producing bacteria were isolated from the foregut (FG) and hindgut (HG) regions. Data were presented as log viable counts g-1 gut (LVC). The occurrence of heterotrophic bacterial population was higher in the FG region (LVC = 8.25) than the HG (LVC= 7.3). Similarly, proteolytic, amylolytic and lipolytic bacteria in FG outnumbered (LVC=7.25, 6.77 and 5.23 respectively) the HG (LVC=6.38, 5.58 and 4.04 respectively). However, occurrence of cellulolytic bacteria in both, FG and HG was less (LVC=2.1 and 1.34 respectively) in comparison to the other extracellular enzyme-producing bacteria. Out of the 100 bacterial isolates, 22 isolates were primarily selected through qualitative assay of extracellular enzyme activities. Among them, 3 promising isolates were chosen as potent extracellular enzyme producers on the basis of cumulative scores (≥11) of the qualitative assay and quantitative enzyme assay. Maximum protease activity was revealed by the strain FG10 (201±2.40U), while FG43 exhibited maximum amylase (208.3±10.8U) and lipase (4.73±0.05U) activities. Among the strains isolated from the HG, the highest protease (188.3±1.2U), amylase (97.6±0.46U) and lipase (3.7±0.11U) activities were recorded with the strain HG01. 


Asfie M, Yoshijima T and Sugita H (2003) Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method. Fisheries Science 69: 21-26.

Askarian F, Zhou Z, Olsen RE, Sperstad S and Ringø E (2012) Culturable autochthonous gut bacteria in Atlantic salmon (Salmo salar L.) fed diets with or without chitin. Characterization by 16S rRNA gene sequencing, ability to produce enzymes and in vitro growth inhibition of four fish pathogens. Aquaculture 326-329: 1-8. DOI: doi:10.1016/j. aquaculture.2011.10.016

Bairagi A, Sarkar Ghosh K, Sen SK and Ray AK (2002) Enzyme producing bacterial flora isolated from fish digestive tracts. Aquaculture International 10: 109-121.

Banerjee G, Dan SK, Nandi A, Ghosh P and Ray AK (2015) Autochthonous gut bacteria in two Indian air-breathing fish, climbing perch (Anabas testudineus) and walking catfish (Clarias batrachus) : mode of association, identification, and enzyme producing ability. Polish Journal of Microbiology 64(4): 361-368.

Banerjee G, Nandi A, Dan SK, Ghosh P and Ray AK (2016) Mode of association, enzyme producing ability and identification of autochthonous bacteria in the gastrointestinal tract of two Indian air-breathing fish murrel (Channa punctatus) and stinging catfish (Heteropneustes fossilis). Proceedings of the Zoological Society, Kolkata. DOI: 10.1007/s12595-016-0167-x

Banerjee G, Ray AK, Askarian F and Ringø E (2013) Characterisation and identification of enzyme-producing autochthonous bacteria from the gastrointestinal tracts of two Indian air-breathing fish. Beneficial Microbes 4(3): 277-284. DOI: 10.3920/BM2012.0051

Banerjee S, Mukherjee A, Dutta D and Ghosh K (2015) Evaluation of chitinolytic gut bacteria in some carps and optimization of culture conditions for chitinase production by the selected bacteria. Journal of Microbiology, Biotechnology and Food Sciences 5(1): 12-19. DOI: 0.15414/jmbfs

Bernfeld P (1955). Amylases, α and ß Meth. Enzymology 1: 149-158.

Beveridge MCM, Sikdar PK, Frerichs GN and Millar S (1991) The ingestion of bacteria in suspension by the common carp Cyprinus carpio L. Journal of Fish Biology 39: 825-831. DOI: DOI: 10.1111/j.1095-8649.1991.tb04412.x

Bier M (1955) Methods in enzymology, In: Colowick SP, Kaplan NO (eds). Academic Press, New York. pp. 627-642.

Clements KD (1997) Fermentation and gastrointestinal microorganisms in fishes. In: Mackie RI, White BA (eds.) Gastrointestinal Microbiology Vol I. Gastrointestinal Ecosystems and Fermentations. Chapman & Hall, New York. pp 156-198.

Cruz PM, Ibản˜ez AL, Hermosillo OAM and Saad HCR (2012) Use of probiotics in Aquaculture: Review article. ISRN Microbiology 2012: 1-14. DOI: 10.5402/2012/916845

Das KM and Tripathi SD (1991) Studies on the digestive enzymes of grass carp Ctenopharyngodon idella (Val). Aquaculture 92: 21-32. DOI: 10.1016/0044-8486(91)90005-R

Das P, Mandal S, Khan A, Manna SK and Ghosh K (2014) Distribution of extracellular enzyme-producing bacteria in the digestive tracts of 4 brackish water fish species. Turkish Journal of Zoology 38: 79-88. DOI: 10.3906/zoo-1205-3

Debnath S (2011) Clarias batrachus, the medicinal fish: An excellent candidate for aquaculture & employment generation. International Conference on Asia Agriculture and Animal IPCBEE 13:32-37, IACSIT Press, Singapore.

Dutta D and Ghosh K (2015) Screening of extracellular enzyme-producing and pathogen inhibitory gut bacteria as putative probiotics in mrigal, Cirrhinus mrigala (Hamilton, 1822). International Journal of Fisheries and Aquatic Studies 2(4): 310-318.

Ganguly S and Prasad A (2012) Microflora in fish digestive tract plays significant role in digestion and metabolism. Reviews in Fish Biology and Fisheries 22: 11-16. DOI: 10.1007/s11160-011-9214-x

Ganguly S, Paul I, and Mukhopadhayay SK (2010) Immunostimulant, probiotic and probiotic-their applications and effectiveness in aquaculture. Israeli Journal of Aquaculture — Bamidgeh 62(3): 130-138.

Ghosh K, Roy M, Kar N and Ringø E (2010) Gastrointestinal bacteria in rohu, Labeo rohita (Actinopterygii: Cypriniformes: Cyprinidae): Scanning electron microscopy and bacteriological study. Acta Ichthyologica et Piscatoria 40: 129-135.

Gildberg A, Mikkelsen H, Sandaker E, and Ringø E (1997) Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua). Hydrobiologia 352: 279-285.

Hasegawa M, Kishino H and Yano T (1985) Dating the human-ape split by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22: 160-174.

Jacob MB and Gerstein MJ (1960) Handbook of Microbiology. D Van Nostrand Co. Inc. Princeton, New Jersey.

Kar N and Ghosh K (2008) Enzyme Producing Bacteria in the Gastrointestinal Tracts of Labeo rohita (Hamilton) and Channa punctatus (Bloch). Turkish Journal of Fisheries and Aquatic Sciences 8: 115-120.

Kinnear PR and Gray CD (2000) SPSS for Windows made simple. Psychology Press, Sussex.

Lowry OH, Rosebrough NJ, Farr AL and Randell RJ (1951) Protein measurement with folin reagent. Journal of Biological Chemistry 193: 265-273.

MacDonald NL, Stark JR and Austin B (1986) Bacterial microflora in the gastrointestinal tracts of Dover sole (Solea solea L.), with emphasis on the possible role of bacteria in the nutrition of the host. FEMS Microbiology Letters 35: 107-111. DOI: 10.1111/j.1574-6968.1986.tb01508.x

Mandal S and Ghosh K (2013) Isolation of tannase-producing microbiota from the gastrointestinal tracts of some freshwater fish. Journal of Applied Ichthyology 29: 145-153. DOI: 10.3750/AIP 2010.40.2.05

Merrifield DL, Harper GM, Dimitroglou A, Ringø E and Davies SJ (2010) Possible influence of probiotic adhesion to intestinal mucosa on the activity and morphology of rainbow trout (Oncorhynchus mykiss) enterocytes. Aquaculture Research 41: 1268-1272. DOI: 10.1111/j.1365-2109.2009.02397.x

Mukherjee A and Ghosh K (2014) Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp, Catla catla (Hamilton). Aquaculture Research 45: 1-13. DOI : 10.1111/are.12676

Ray AK, Ghosh K and Ringø E (2012) Enzyme-producing bacteria isolated from fish gut: A review. Aquaculture Nutrition 18: 465-492. DOI: 10.1111/j.1365-2095.2012.00943.x

Ray AK, Roy T, Mondal S and Ringø E (2010) Identification of gut-associated amylase, cellulase and protease producing bacteria in three species of Indian major carps. Aquaculture Research 41: 1462-1469. DOI: 10.1111/j.1365-2109.2009.02437.x

Ringø E, Olsen RE, Mayhew TM and Myklebust R (2003) Electron microscopy of the intestinal microflora of fish. Aquaculture 227: 395-415. DOI: 10.1016/j.aquaculture.2003.05.001

Ringø E, Strøm E and Tabachek JA (1995) Intestinal microflora of salmonids: a review. Aquaculture Research 26: 773-789. DOI: 10.1111/j.1365-2109.1995.tb00870.x

Sakhare VB and Chalak AD (2014) Food and feeding habits of Clarias batrachus (Linnaeus, 1758) from Ambajogai, Maharashtra, India. Journal of Fisheries 2(2): 148-150. DOI: dx.doi.org/10.17017/jfish.v2i2. 2014.33

Sangiliyandi G and Gunasekaran P (1996) Extracellular lipase producing Bacillus licheniformis from an oil mill refinery effluent. Indian Journal of Microbiology 36: 109-110.

Stickney RR (1975) Cellulase activity in the stomachs of freshwater fishes from Texas. Proceedings of the Annual Conference on Southeastern Association of Game Fish Community 29: 282-287.

Strøm E and Olafsen JA (1990) The indigenous microflora of wild-captured juvenile cod in net-pen rearing. In: Lesel R (ed) Microbiology of poecilotherms. Elsevier, Amsterdam, pp 181-185.

Sugita H, Shibuya K, Hanada H and Deguchi Y (1997) Antibacterial abilities of intestinal microflora of the river fish. Fisheries Science 63: 378-383.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-2739. DOI: 10.1093/molbev/msr121

Teather RM and Wood PJ (1982) Use of Congo-red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Applied Environmental Microbiology 43: 777-780.

Trust TJ and Sparrow RAH (1974) The bacterial flora in the alimentary tract of freshwater salmonid fishes. Canadian Journal of Microbiology 20: 1219-1228. DOI: 10.1139/m74-188

Verschuere L, Rombaut G, Sorgeloos P and Verstraete W (2000) Probiotic Bacteria as Biological Control Agents in Aquaculture. Microbiology and Molecular Biology Reviews 64(4): 655-671.

Walter HE (1984) Method with haemoglobin, caesin and azocoll as substrate. In: Methods of enzymatic analysis, Bergmeyer HU, Grassl M and Bergmeyer J, (eds). 3rd ed, Vol 5, Weinheim: Verlag Chemie, pp. 270-277.

Zar JH (1999) Biostatistical analysis. Pearson Education Singapore Pte. Ltd., New Delhi.

How to Cite
Dey, A., Ghosh, K., & Hazra, N. (2016). Evaluation of extracellular enzyme-producing autochthonous gut bacteria in walking catfish, Clarias batrachus (L.). Journal of Fisheries, 4(1), 345-352. https://doi.org/10.17017/j.fish.112