Impact of Vermicompost Manure on Microbial Population in Carp Rearing Pond

Effect of vermicompost, poultry manure, cow dung and pig manure used to monitor on microbial population in carp rearing pond water of Indian carp fviz. Catla catla, Labeo rohita and Cirrhinus mrigala. The fingerlings stocked @ 30 fish per pond in the ratio of 3 : 4 : 3. The bacterial strains isolated in ponds water treated with different manures were seven gram negative (A. hydrophilla, E. coli, E. aerogens, Shigella sp., K. oxytoca, P. aeruginosa, P. fluorescens) and three gram positive (M. luteus, S. aureus and Streptococcus sp.). The average counts of heterotrophic pathogenic bacteria in poultry manure founded to be maximum in decreasing order followed by pig manure, cow dung, vermicompost, vermicompost and control, respectively. However, bacteria, E. aerogens, P. fluorescens, P. aeruginosa, Shigella sp., K. oxytoca and Streptococcus sp. found absent in vermicompost treatments. All the three species gained maximum growth in vermicompost followed by cow dung > poultry manure and pig manure.


Introduction
More than half of the world population depends upon fish as a source of animal protein. Fish flesh contains all the essential amino acid and minerals i.e. iodine, phosphorus, potassium, iron, copper and vitamin A and D in desirable concentrations 1 . It serves as valuable ingredient to a healthy diet because of its low carbohydrate and unsaturated fat contents. It is often recommended by doctors to heart patients since it is an excellent source of Omega 3 fatty acid. So the inclusion of fish in our diet can make a valuable contribution to any diet that contains mainly of cereals, starchy roots and sugar for the healthy growth 2 . Freshwater fish in Indian ponds commonly suffer from bacterial diseases such as various kinds of skin ulcerations, albinoderma, erythroderma, furunculosis, and vertical-scale disease, primarily caused by Aeromonas sp. and Pseudomonas sp. Among the various practices, depending upon the variable inputs, semi-intensive carp culture practices in rural aquaculture involve utilization of various organic manures for plankton production. These manures are either directly utilized by the fish or they enrich the aquatic ecosystem with autotrophic (plankton) and heterotrophic microbial communities. Nearly half of the fish currently consumed as food worldwide are raised in fish pond rather than caught in the wild. In most of the situations, cultured fish remain healthy even in the continuous presence of pathogens. However, when environmental stresses occur and the balance shifts in favor of the disease, the characteristic pathogens flourish. Due to the outbreak of disease in aquaculture industry, use of antibiotic has led to the development of drug-resistant strains resulting in reduction of natural defense mechanism in the aqua cultural animals. To overcome these short comings, we have come to develop a newer technology through the use of vermicompost as pond fertilizer, which not only keeps the watery environment congenial for growth, but also lower the incidence of developing the pathogenic organism. Vermicompost is a product of vermi-biotechnology that is frequently used in agro-ecosystems as organic manure. The advantage of use of vermicompost as organic manure is the quick availability of nutrients in 'ready-to-uptake' forms 3 . So far, the information regarding efficacy of vermicompost as manure in aquaculture pond is scanty. In view of the above points in the present investigation we evaluated the effect of different manures on the bacterial population of treated fish ponds.

Estimation of Microbial Population from Manures Treated Fish Ponds
For microbial analysis, water and sediment samples were collected from the treated ponds after 30 days in sterilized glass bottles and processed within 6 hrs of collection. The analysis of bacterial samples was done in the laboratory.

Culture of Microbes
The water and soil samples enumerated in nutrient agar by serial dilution of the sample followed by the conventional spread plate method. These samples spreaded over the nutrient agar (NA) medium under aseptic conditions.

Tertiary Test for the Confirmation of Microbe
The confirmation test of these bacteria done with the help of selective media used for culturing that particular bacterium. Specific media provide nutrients that enhance the growth and predominance of a particular bacterial species and prevent the growth of other bacterial species. Growth of bacteria on specific medium taken as a confirmation of the identification of isolated bacterium.

Determination of Colony Forming Unit (cfu/ml) of Isolated Microbes
Colony Forming Unit (cfu/ml) used to identify the number of viable microorganisms in a fixed amount of liquid. The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony. Thus, the number of colonies should give the number of bacteria that can grow under the incubation conditions employed. The bacterial sample diluted by factors of 10 and plated on agar. After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies determined. A plate having 30-300 colonies chosen because this range is considered statistically significant. Determination of cfu of bacteria involved the serial dilution method. On the following day number of colonies on each NA plates was counted and colony forming unit (cfu) recorded by using formula:-CFU/ ml = No. of colonies × Dilution factor.

Results
Ten bacterial strains isolated and identified from different organic manures treated fish ponds. Seven gram negative (A. hydrophila, E. coli, E. aerogens, P. fluorescens, P. aeruginosa, Shigella sp., K. oxytoca) and three grams positive (M. luteus, S. aureus, S. sp.) bacterial strains identified ( Table  1). The pathogenic heterotrophic A. hydrophila, E. coli, M. luteus, S. aureus bacteria presented in treatment control, pig manure, poultry manure, cow dung, vermicompost @ 10,000 and vermicompost @ 15,000 kg/ha/yr. The bacteria E. aerogens, P. fluorescens, P. aeruginosa, Shigella sp., K. oxytoca and Streptococcus sp. found absent in vermicompost @ 10,000 and vermicompost @ 15,000 kg/ha/yr. Only ten species of bacteria were identified and characterized under the study condition. The following species of bacteria were isolated and identified from water and sediment samples are presented as:-

Aeromonas hydrophila
Aeromonas hydrophila Gram negative, single short rod shaped aerobic bacteria. This was positive for catalase and oxidase. The results of secondary tests revealed that this bacterium was positive for glucose fermentation, nitrate reduction, glucose acid, maltose, mannitol, glycerol, fructose, galactose, sucrose, lactose, starch, and grew well at 37°C. However, the bacterium found negative for urease, Simmon citrate, inositol, TSI and H 2 S production. The bacterium showed good growth in Vogus-Proskauer medium at 37°C. Based on these tests, identification score assigned to this isolate by the PIBWin Programme was 0.99954 (Table 2).

Escherichia coli
Escherichia coli Gram negative, aerobic, fermentive, rod shaped and observed positive for catalase, glucose fermentation, nitrate, glucose acid, maltose, mannitol, glycerol, inositol and growth at 37°C. The bacterium showed negative results for urease, starch and voges-proskauer test. The identification score assigned to the bacterium was 0.99987 (Table 2).

Enterobacter aerogens
Enterobacter aerogens Gram negative, aerobic, rod shaped and positive for catalase, glucose fermentation, nitrate, glucose acid, maltose, mannitol, glycerol, inositol, and showed growth at 37°C. Negative results for urease, starch and Voges-proskauer tests observed. Based on above mentioned tests the identification score assigned to this bacterium by PIBW in was 0.95528 (Table 2).

Klebseilla oxytoca
The results of primary tests of this isolate revealed that this bacterium gram negative, aerobic, rod shaped, catalase positive. The results of secondary tests revealed that the bacterium positive for sucrose, mannitol, galactose, inositol, adonitol, maltose, fructose, lactose, sorbitol, indole, starch, D (+) xylose, Erhlich indole, urea and glycerol. However, the bacterium negative for arginine dehydrogenase, Simmon citrate, nitrate reduction and motility test. The bacterium showed good growth in methyl red and did not grow in Vogus-Proskauer broth at 37°C. Based on these tests, identification score assigned to this isolate by the PIBWin Programme was 0.96612 (Table 3).

Pseudomonas fluorescens
Pseudomonas fluorescens aerobic, non fermentive, Gram negative, rod shaped and positive for catalase, oxidase, Simmon citrate, xylose, glucose, fructose, arginine dihydrolase, 10% glucose, lactose, glycerol. The bacterium showed growth at 37°C P. fluorescens negative for urease, nitrate reducation, malonate, maltose, glucose fermentation, starch hydrolysis, Ehrlich indole, adonitol, glucose acid, sucrose, sorbitol, lactose and cellobiose. Based on these tests, identification score assigned to this bacterium by the PIBWin Programme was 0.99565 (Table 2).      Table 3). The bacterium showed positive growth at 37°C. Based on the abovementioned tests an identification score of 0.99573 was assigned to the bacterium. Growth on Antibiotic assay medium C confirmed the identification of bacterium.

Micrococcus luteus
Micrococcus luteus bacterium Gram positive, arobic and positive for catalase, oxidase, glucose, fructose, urease, Simmon citrate and Vogus-Proskauer. However, the bacterium was negative for maltose, mannitol, adonitol, xylose, lactose, nitrate. insuline and galactose. The bacterium showed good growth in Vogus-Proskauer medium but did not grow in methyl red at 37°C. Based on these tests, identification score assigned to this isolate by the PIBWin Programme was 0.99620 (Table 4).

Staphylococcus aureus
Staphylococcus aureus bacterium Gram positive, arobic and positive for lactose, maltose, mannitole, sucrose, sorbitol, glucose, fructose, Vogus-Proskauer, glycerol, galactose and arginine dihydrolase. However, the bacterium was negative for starch, Ehrlich Indole, Simmon citrate and inositol. The bacterium showed good growth in Vogus-Proskauer medium. Based on these tests, identification score assigned to this isolate by the PIBWin Programme was 0.90954 (Table 4).

Discussion
In the present investigation seven-gram negative and three gram positive heterotrophic pathogenic bacterial strains isolated from different manure treated fish ponds. The number of gram negative bacterial isolates correlated with studies of various scientists reported that manures treated water samples micro flora of aquatic animals consists mainly of gram-negative aerobic, obligate anaerobic and facultative anaerobic bacteria, the composition of which may change with environmental stresses 4 , Diet 5 , and fish age 6 . The isolated bacterial strains belonged to Aeromonas sp., Pseudomonas sp., Shigella sp. and members of the family Entero bacteriaceae. These bacteria dominate microflora of freshwater species 7 13 . The control treatment, however, significantly reduced the population of total heterotrophic pathogenic bacteria in both water and sediment, compared to the manure treatments. The water quality was also influenced by the management conditions. Similar results observed by Blackburn and Henriksen 14 ; Jana and Barat 15 ; Mei et al. 16 ; Yao and Zhaoyang 17 . In an earlier experiment, a direct significant (P≤0.05) observed between the weight gain of Indian major carps and the amount of zooplankton present in tanks under different doses of organic manuring, Jha et al. 18 . These results were corroborating with the finding of Jana and Chakrabarti 19 , Ludwig 20 . All aquaculture production systems must provide a suitable environment to promote the growth of aquatic crops. Although application of organic manure does not directly cause bacterial diseases in fish, the significantly greater abundance of heterotrophic pathogenic bacteria in the water and sediments of the manured treatments vermicompost @ 15,000 kg/ha/yr, vermicompost @ 10,000 kg/ha/yr, cow dung @ 10,000 kg/ha/yr, poultry manure @ 6,000 kg/ha/yr and pig manure @ 4,000 kg/ha/yr could lead to diseases. Should fish resistance to disease be low, the possibility of occurrence of bacterial disease is higher in these treatments. Therefore, proper pond management should be observed to prevent any chance of bacterial disease. Though it has been established that high fish yield in culture systems can be achieved by higher abundance of plankton through organic manuring, practical alternatives to pond manuring are needed because manuring may reduce water quality. Intensive stocking of Indian major carp ponds in India requires a standard water quality to be maintained throughout, so that fish growth is not adversely affected. In view of the financial constraints of marginal farmers who cannot afford modern aeration or waste-treatment equipments, raising of Indian major carp larvae in ponds fed exogenously with zooplankton is of considerable significance because not only would such feeding support high rates of survival and production, it would also maintain greater abundance of zooplankton in the system and better water quality with lower concentrations of heterotrophic pathogenic bacteria in the system.