2.1. Experimental design: The experiment was conducted in 12 (twelve) fiberglass tanks for close observation of the biofloc technology. The experimental tanks were treated with the C/N=10:1 (denoted as T1), C/N=15:1 (denoted as T2) and C:N=20:1 (denoted as T3) to develop the biofloc. In addition, a control group was also maintained without any carbon addition. The heterotropic bacteria were developed in the flocs, which were collected and determined from each of the experimental groups. Afterward, macro and microbiological composition of the flocs were determined by laboratory analysis. Macroscopic organisms were determined by using light microscope and the potential bacterial composition was determined by microbiological plate assay. The floc samples were examined to observe the load of Enterococcus spp., Clostridium spp., Lactobacillus spp. and Salmonella spp.
2.2. Development of biofloc and rearing of prawn in the tanks The trials for biofloc development were carried out in the fiber glass tank in water chemistry laboratory of Fisheries and Marine Resource Technology (FMRT) Discipline, Khulna University and in the Molecular Biology Laboratory of the same Discipline from 02, August to 15, August, 2017. Biofloc volume was determined at the end of the tank trials and samples were determined for next one month for the bacterial composition of the flocs. For the determination of the bacterial composition, the freshwater prawn Macrobrachium rosenbergii were cultured with BFT treatment in the experimental tank and then the samples were taken in the Biochemistry and Molecular Genetics Laboratory of FMRT Discipline, Khulna University for culture and enumeration of bacterial species. Twelve tanks were set randomly in the water chemistry laboratory for developing bioflocs in prawn culture system. Before stocking the prawn juveniles were weighed and feed was given at 5% of their body weight. From the amount of feed, the proper amount of molasses was calculated to maintain proper C/N ratio (10, 15 and 20) in different treatments. After that, specific amount of feed and molasses were provided to the experimental tanks twice a day to adjust the feed nitrogen for targeted microbial growth. Besides, continuous aeration was provided in all the tanks for homogenous oxygen and nutrients distribution for flocs. The carbon nitrogen ratio was calculated according to the following equation.
2.3. Collection and Determination of floc Samples of floc were collected for three times from each of the experimental tank for each and every enumeration at every three days interval. So, nine samples were collected under each treatment. As a result thirty-six samples were collected from twelve experimental tanks under four treatment groups. The collected sample was carried immediately to the Molecular Biology Laboratory of FMRT Discipline, Khulna University. All the samples were instantly used for microbial analysis and also properly labeled and stored in -20oC temperature for further analysis. Total volume of floc per liter water was measured at first by using a specific cone. Then the proportion or percentage of different types of flocs such as protozoa, plankton, copepods, etc. in the collected sample was determined at first by observing them under a light microscope.
2.4. Enumeration of beneficial and harmful bacteria in the flocs Test tubes, petri dishes, conical flask, plastic tips and other glassware were sterilized by autoclaving at a temperature of 121°C and a pressure of 15 pound per square inch for 15 minutes. Petri dishes, tips box was subjected to dry heat sterilization at 155°C for 1 hour in electric oven [48] . Peptone water, Absolute alcohol, Ethyl alcohol (75%), distilled water and detergent powder were used as chemical and reagent during the study periods. Plate counting agar was used for standard plate count. For culture and normal counting of Salmonella spp., Broth Medium I (Tetrathionate Bile Brilliant Green Broth) was used. This media was specific or selective for harmful Salmonella spp. For culture and normal counting of E. faecalis, SF Broth Medium was used. Lactobacillus spp. bacteria were cultured in Lactobacillus MRS Agar medium. For culture and normal counting of Clostridium butyricum, selective Reinforced Clostridial Agar (M154) was used. Accurate amount (1 ml) of pooled sample was measured by pipette. Then the sample was taken into a sterile test tube containing 9 ml 0.1% sterile peptone water for vortex mixing. The mixture was homogenised for 2 min. This serial dilution was continued up to 10-5. Samples from each of the experimental tank were taken and prepared in the same way. The prepared samples of the six tanks were kept in separate test tube. At first the samples were cultured in the standard plate count agar media. Then the colonies of the total heterotrophic bacteria were counted by using colony counting equipment.
2.5. Statistical analysis The data were recorded during the experiment and collected data were stored, explored and analyzed using Microsoft Excel, SPSS and Microsoft Word program to present the results and discussion. The normality test of the data was done in SPSS at first. After verifying the normality of flocs volume and log-transformed bacterial data, one-way analysis of variance (one-way ANOVA) was performed to check the significant differences among the treatments. After detecting it (P<0.05), the Tukey test of means comparison was used in SPSS to compare the data of control and treated tanks.