Abstract
Present study was aimed at isolation of autochthonous chitinase-producing bacteria from the gastrointestinal tracts of 3 Indian Major Carps (Labeo rohita, Catla catla, Cirrhinus mrigala) and 3 exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio). Altogether, 119 bacteria were isolated from both the proximal and distal intestine and screened for chitinolytic activity. On the basis of chitin hydrolysis zone, 63 isolates were primarily selected for chitinase production, from which 34 potent strains were further studied for quantitative enzyme assay. Amongst them, the strains HMH1 and CMF2 exhibited potent chitinolytic activity and were identified as Bacillus pumilus (KF454036) and Bacillus flexus (KF454035), respectively by 16S rRNA partial gene sequence analysis. Optimization of various fermentation parameters (e.g., temperature, pH, inoculums size, surfactant, colloidal chitin concentration, incubation time, carbon sources, organic and inorganic nitrogen sources) were carried out in chitinase production medium. Incubation for 72 h at 350C and initial pH 7.5 revealed optimum chitinase productions by B. pumilus HMH1 in the media supplemented with colloidal chitin 0.1% (w/v), maltose 2% (w/v), ammonium sulphate 1.0% (w/v) and Tween-80 0.2% (v/v). However, B. flexus CMF2 required 48 h incubation at 35°C and initial pH 8.0 with colloidal chitin 0.15% (w/v), sucrose 1% (w/v), yeast extract 2.0% (w/v) and Tween-20 0.2% (v/v) supplementation for optimum yield. The results indicate that there is ample scope for further research to appraise fish gut microorganisms for chitinase production or as probiotics to improve feed efficiency in fish.
Highlights
Chitin (C8H13O5N)n has been estimated as the second most abundant biomass in the world after cellulose forming structural component of many fish food organisms, including fungi, crustaceans, coelenterates, protozoan and green algae (Rinaudo, 2006; Khoushab and Yamabhai, 2010)
Enumeration of gut microbial community in the 6 fish species studied revealed that autochthonous culturable heterotrophic and chitinase producing microorganisms were present in both PI and DI regions in all the fish species studied (Table 2)
Out of the 119 randomly selected isolates, 63 chitinase-producing bacteria were primarily selected by qualitative enzyme assay, which were further evaluated by quantitative enzyme assay
Summary
Chitin (C8H13O5N)n has been estimated as the second most abundant biomass in the world after cellulose forming structural component of many fish food organisms, including fungi, crustaceans, coelenterates, protozoan and green algae (Rinaudo, 2006; Khoushab and Yamabhai, 2010). The micro-environment of fish gut confers a favourable niche for the microorganisms (Kar and Ghosh, 2008), and the gut microbiota in fish is closely related to the food that they use to consume (Han et al, 2010). These distinct microbial communities may contribute uniquely to the nutrient cycling in the system (Ringø et al, 2012). Studies have indicated that fish feeding on chitin rich diets have higher chitinase activity (Danulat, 1986; Gutowska et al, 2004) Apart from such sporadic information, likely occurrence of chitinolytic bacteria in fish gut and their significance in feed utilization of the host species are inconclusive and contradictory. The present study aimed at (1) isolation and enumeration of chitinaseproducing gut microorganisms in 3 Indian Major Carps and 3 exotic carps, (2) identification of the most promising chitinase-producing micro-organisms by 16S rRNA partial gene sequence analysis, and (3) optimization of the various process parameters that influence chitinase production by the promising bacterial strains, Bacillus pumilus HMH1 (KF454036) and Bacillus flexus CMF2 (KF454035)
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