Isolation and Characterization of Bacteria from the Gut of Blue Gourami (Trichogaster tricopters) and its Role on Growth

Five variant groups were isolated from the gut of Blue gourami Trichogaster tricopterus through serial dilution, identified by using biochemical tests and bacteria were Bacillus sp., (G1) Pseudomonas sp., (G2) Enterobacter sp., (G3) Aeromonas sp., (G4) and Escherichia sp., (G5). Based on enzymatic productivity (Amylase, Cellulase, Lipase, and Protease) and antimicrobial activity Bacillus sp., Enterobacter sp., and Aeromonas sp., and mass multiplied using nutrient broth. Four different feeds such as feed I (Control), II (1ml Bacillus sp.,) III (1ml each of Bacillus sp. and Enterobacter sp.) and IV (1ml each of Bacillus sp., Enterobacter sp. and Aeromonas sp.,) were prepared. Feed utilization parameters of blue gourami Trichogaster tricopterus were studied after 21 days of rearing and all feed utilization parameters were higher in feed IV.


INTRODUCTION
Ornamental fish rearing has emerged as the second most popular hobby next to photography due to its aesthetic beauty (Aly et al., 2008). The trade-in ornamental fish and aquarium supplies are multi-million dollar industries that span the globe, with a retail value of approximately US$ 500 million. Besides this, it also provides employment and revenue generation for the country (Balacazar, 2006). The major problem facing aquaculturists is the uncontrollable mortalities associated with disease and related disorders. Since the economic importance of aquarium fish is not less than that of the food fish. Ornamental fishes are susceptible to bacteria, viral, fungi, protozoa, and parasitic organisms and cause loss to the produce. Among the various pathogens affecting the cultured fish species, bacteria cause severe damage. Control of the bacterial disease is made possible by using drugs and antibiotics. The traditional use of antibiotics as growth promoters in aquaculture has been challenged because of the potential development of antibiotic-resistant bacteria. The use of vaccines is laborious, costly and highly stressful to the animals. Since these methods have certain limitations, alternative, productive methods must be examined to reduce the incidence of the pathogen in ornamental fish culture. The bacterial community in the gut of aquatic animals is much more crowded compared to terrestrial animals, as water serves an ideal medium for bacterial growth. The microbial network in the gastrointestinal tract of fish is very complex and plays a vital role in fish nutrition and disease prevention. The composition of the community of microbes in the fish gut is not constant and may change with nutritional status, age, surrounding water and other environmental conditions (Banerjee and Ray, 2017). However, the microbial balance in the gastrointestinal tract is crucial in response to host metabolism, disease prevention, and physiology. The microbial composition in the gut of vertebrates, including fish largely depends on the nutritional status of the host. Protease producing bacterial communities is dominant flora in carnivorous animals, which helps to degrade complex proteins to simple amino acids. Similarly, amylase and cellulose producing bacterial communities are reported to be highest in herbivorous animals (Ray et al. 2012). In addition to host nutrition, the gastrointestinal microbiota serves a variety of other beneficial functions in the host such as preventing the colonization of infectious agents, energy homeostasis and maintenance of healthy mucosal immunity. The normal microflora in the intestinal tract of the fish includes P s e u d o m o n a s s p p . A e r o m o n o u s s p p . , Enterobacteriaceae spp., Micrococcus spp., Escherichia spp, and Bacillus spp and these bacteria play a vital role in fish nutrition and disease prevention. Bairagi et al. (2002) have reported the existence of several enzymes producing bacterial strains, isolated from different freshwater fishes having different feeding habits. The study related to the isolation and characterization of bacteria from the gut of Blue Gourami Trichogaster tricopterus and its role in growth is entirely absent. So present research was undertaken.

MATERIALS AND METHODS Fish collection
For the present study, Blue gourami, Trichogaster tricopterus were collected from Aqua Garden, Madurai, Tamil Nadu, India and transported to the laboratory in polyethylene bags filled with aerated water. Fishes were acclimated in glass aquaria (60 ‫׳‬ 45 ‫׳‬ 45 cm) for15 days at 28 ± 2°C. During acclimation fishes were fed with trainee feed containing fish meal, groundnut oil cake, wheat flour and rice bran in the form of dry Pellets.

Isolation of gut bacteria
After transportation to the laboratory the gut content of Blue gourami was collected, consequently diluted and 10 -6 was selected for the separation of bacteria. The diluted sample was plated over sterilized nutrient agar medium and incubated at 37°C for 24 hours. (Bergy's Manual of Determinative Bacteriology, 1994). Nutrient Agar was used for the present study, different incubation temperature was used to obtain a wider range of isolation and the incubation time ranges from 24 hours, depending on the incubation temperature, groups were enumerated and separated.

Characterization of gut bacteria
The preponderant groups on the nutrient agar medium were selected and identified based on the cellular morphology, microscopic and  (10 -6 Cells) were mass multiplied by inoculating into the nutrient broth.

Experimental Feed Preparation:
The raw materials such as fish meal, groundnut oil cake, wheat flour, and tapioca were used for preparing the feed. After knowing the protein content by Micro -Kjeldahl method (Jayaraman,1992) (Table 1), one control(without bacteria), three experimental feeds (Ali, 1980) were prepared by using different isolated bacteria. The components used for feed preparation were dried, powdered and sieved through 425-micron sieve. The ingredients were weighed and mixed thoroughly with 130 -150 ml of distilled water.
The mixed feedstuff was put in an autoclave for 15 min at 100°C and cooled. After cooling, fish oil, sunflower oil, supplevite -mix, sodium chloride, sodium benzoate, and different isolated (1ml of Bacillus sp.,1ml of Bacillus sp.,+Enterobactor sp., and 1ml of Bacillus sp.,+ Enterobacter sp., and Aeromonas sp.,) bacteria were mixed with the feed. And then it was extruded with the help of Pelletizer. The pellets were dried at room temperature. This formulated feed was kept in an airtight container in -20°C until used to prevent contamination ( Table 2).

In vivo experimental design and growth study
For growth study, uniform size of Blue gourami Trichogaster tricopterus (3.66 ± 0.36 g) were selected and were introduced in rectangular glass tanks (45 cm L ‫׳‬ 22 cm B ‫׳‬ 22cm H) having a capacity of 18 liters. Five fishes were introduced in each tank and triplicates were maintained. During rearing, the fishes were fed on the adlibitum diet of the prepared feed twice a day for 1 hour each from 9 -10 am and 4 -5 pm. The unfed were collected after one hour of feeding

RESULTS AND DISCUSSION
The organisms isolated from the intestinal content were identified Bacillus sp, Enterobacter sp, Aeromonas sp., using biochemical tests as given in table 3. The selected intestinal bacteria were Bacillus sp., Enterobacter sp., Aeromonas sp., based on the Biochemical tests, Enzyme Production (Amylase, Cellulase, Lipase, and Protease) ( Table 4). Based on the test the selected   producing bacterial strains in the digestive tract of 4 brackish water fish species. Antibacterial Activity (Table 5), and enumeration of bacteria are given in Table  6. Based on antibacterial activity Bacillus sp., Enterobacter sp., Aeromonas sp., (G1), (G3), (G4) has shown higher inhibition against pathogens Staphylococcus aureus, Shigella sonnei, Enterococcus faecalis, Pseudomonas aeruginosa and Klebsilla pneumoniae. Zapata et al. (2013) reported that Lactic acid bacterial strains isolated from Nile Tilapia intestine showed the high ability to inhibit the growth of freshwater fish pathogen particularly Vibrio sp. and Mycobacterium sp.  Table 7. Condition factor (K)   of blue gourami Trichogaster tricopterus was estimated for comparative purposes to assess the feed. The average initial condition factor is 1.15 ± 0.13 and the final condition factor increased in feed IV (1.55 ± 0.18) and in all others, the final condition factor was decreased. Sivakumar et al., (2016) reported that the average initial condition factor of yellow molly was 1.84 and the final condition factor increased in feed V (2.65) when fed with Escherichia fergusonii. Suganya et al. (2018) also reported that the final condition factor was higher in feed containing 4ml of Pseudomonas sp. in the feed of Zebrafish. Feed utilization parameters of blue gourami Trichogaster tricopterus is presented in Table 8. Feed consumption of Blue gourami was higher in feed IV (5,4 ± 0.13) containing every 1 ml each of Bacillus sp., Enterobacter sp., Aeromonas sp. and lower in feed I (control) (3.1 ± 0.41). Rajan and Revathi (2011) reported that the feed consumption of Platy Xiphophorus maculates was higher in Ex. Feed V containing 10 4 cells of Bacillus subtilis. Bisht et al., (2012) reported that the feed consumption in common carp was higher (95%) in diet D3 and lower (85%) in diet D1. Wang et al. (2015) reported that the feed intake was higher in juvenile Pseudobagrus ussuriensis. Feed Conversion Efficiency of Blue gourami was higher in feed IV (0.06 ± 0.01). But in the case of gold fish, the feed conversion efficiency gradually decreased from lower to a higher quantity of