Study On Isolation And Identification Of A Flocculant- Producing Bacterium And Its Flocculation Properties

With the advancement of technology and the development of economics, the aquaculture has been rapidly developed in China during the last decades. However, the rapid development of aquaculture has also been accompanied by the occurrence of some serious problems, such as the accumulation of large amounts of aquatic feed, food residues, wastes of aquaculture animals and other residues present in aquaculture water due to the application of large amount of aquatic feeds, which seriously affect aqua farming water quality. Coagulation of flocculants and their metabolites and the flocculation mechanism of flocculant-producing bacterial species are the coagulation and condensation process during which the flocculant-producing bacterium itself or its metabolites or the combinations of both are utilized to carry out coagulation and condensation of the flocs in aquaculture water. through several mechanisms including an individual or combined/cooperative utilization of adsorption bridging, charge neutralization/adsorption, flocculation mechanism network. This process is similar to the flocculation mechanism of Bio-Floc, in which the microorganisms or the flocculants they produced are used to form flocs from the organic matters, protozoa, algae, fecal bait present in aquaculture waters.Currently, the studies on screening and isolation of flocculant-producing bacteria from the biological floc in aquaculture water to obtain bioflocculant are rarely available. Thus, to carry out the related studies on flocculant-producing bacteria in bioflocs will be helpful to understand the characteristics of stimulating the growth of flocculant-producing bacteria and to analyze the compositions of its flocculators, aiming for providing certain references for further application of flocculant-producing bacteria in biolflocs.In this study, a total of 73 single colonies were isolated from the biological floc. Through preliminary screening, 19 strains that possess >60% of flocculation activity were obtained. After repeated screening and subculture for several generations, a flocculant-producing bacterial strain had good and stable flocculating activity named G201441 was obtained. Its flocculating activity was stabilized at around 90%. Through morphological observation, API system identification and molecular identification(Gen Bank accession number KP747687) was identified as a strain of Bacillus thuringiensis(Bacillus thuringiensis).Preliminary study was conducted on optimization of the fermentation conditions for this flocculant-producing floc bacterial strain G201441. Single factor experiment was conducted to find out the optimal sources of carbon, nitrogen, and inorganic ions for its fermentation medium for this strain as the follows: the carbon, nitrogen, and inorganic ions for its medium were beef extract, peptone and Na Cl. Orthogonal design method was applied to optimize the amounts of medium components for this strain. The amounts of optimal medium components were beef extract(3g/L), peptone(8g/L), and Na Cl(5g/L). With this optimized medium and under the optimum culture conditions, the flocculation activity fermentation broth of this strain in fermentation broth was up to 93.97%.Through the single factor test experiment, the optimal culture conditions for this flocculants-producing strain G201441 were determined as follows: initial p H of the fermentation broth, 8.0; the culture temperature, 30 ℃; shaking speed, 150 r / min; determining the yield floc bacteria G201441 the optimal flocculation conditions the key factors influencing the optimal flocculation conditions for this bacterial G201441 strain were determined as follows: kaolin suspension p H value, 7.0, the fermentation liquid dosage, 8%(volume fraction V / V) and coagulant, Ca2 + etc.The flocculation and coagulation activities of the supernatant of flocculantproducing bacterial strain G201441, untreated fermentation broth and the cell suspension were 93.80%, 69.62% and 0.9%, respectively. Thus, floc flocculation substances produced by this strain was mainly distributed in the fermentation liquid. Flocculant was visually observed as a white solid with a loose texture. When being visualized under a scanning electron microscope, its micro-morphology displayed as elongated filaments.The chemical, physical methods were applied to analyze the compositions. The flocculation substances produced by strain G201441 were heated in a water bath at 100℃ for 10-60 min. The qualitative analysis indicated that the flocculation active substances were not only stable, but also could reach more than 86% flocculation activity. There were multiple-colored reactions of carbohydrates. The crude components of flocculation substances were found to be mainly polysaccharide. Qualitative analysis revealed that flocculation substance accounted for 34.5%, and 8.6% of which contained the small amount of protein. At both 230 nm and 280 nm of UV spectroscopy, the typical absorption peaks of-COOH groups with conjugated double bonds and proteins were found. In the infrared spectroscopic analysis of polysaccharide, the-OH, C-H, C-O and C-O-C functional groups were present in flocculation substances. In summary, the crude material components of flocculation substances are mainly polysaccharides.