Characterization of a bioflocculant produced from the consortium of three marine bacteria of the genera Cobetia and Bacillus and its application for wastewater treatment Ugbenyen Anthony1,3, Vine Niall2, Simonis Jean1, Albert Basson3, Anthony Okoh4 1Department of Hydrology, University of Zululand, KwaDlangezwa, KZN, South Africa 2Department of Zoology and Entomology, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa. 3Department of Biochemistry and Microbiology, University of Zululand, South Africa. 4Applied and Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology. University of Fort Hare, Alice, South Africa Corresponding author: ugbenyenanthony@gmail.com Fig 1a Fig 1b Fig 2 Fig 1a and 1b: Images of flocculation process Fig 2 : mechanism of flocculation INTRODUCTION Flocculants, or flocculating agents are chemicals that promote flocculation by causing colloids and other suspended particles in liquids to aggregate, forming a floc (1). Thus they are widely applied in different industrial processes, including wastewater treatment, downstream processing, food and fermentation processes (2). Although chemical flocculants have been used widely due to their effective flocculating activity and low cost, some of these synthetic flocculant have threats to public health and increase environmental risks (3). In recent years, utilization of microbial flocculants have been anticipated due to their biodegradability and harmlessness of their degradative intermediates (4). Thus screening new microorganisms that could produce flocculants with excellent flocculating activity seem to be a feasible approach to solving the problem posed by synthetic flocculants (5). Marine bacteria are among the most economically and biotechnologically valuable prokaryotes. Thus, it is important that new groups of bacteria from unexplored or underexploited habitat like the marine environment be considered as sources of novel bioactive compound including bioflocculants. METHODS Bacteria and Culture Conditions The bacteria were isolated from sediment samples of Algoa Bay in the Eastern Cape Province of South Africa as part of the bioflocculant producing culture collection. The bacteria were maintained in 20% glycerol at -80oC (6). Extraction and Purification of Bioflocculant Isolation and purification of the bioflocculant was done according to the method described by previous reports (6) Flocculation Test of Bioflocculant Flocculating activity was measured as described by Kurane et al. (7) and Ugbenyen et al. (6) Analysis of purified bioflocculant Total protein content of bioflocculants was determined by the Lowry’s method using Bovine Serum Albumin (BSA) as a standard (8). Total sugar content of bioflocculant was determined by the phenol-sulphuric acid method using glucose as standard solution (9) and uronic acid was quantified by the carbazole method (10). FTIR analysis of the purified bioflocculant was done using a Fourier-transform infrared spectrophotometer (Perkin Elmer System 2000, England) over a wave number range of 4000 to 370 cm-1.Scanning electron microscopy (SEM) image of the purified bioflocculant was taken using JEOL (JSM-6390LV, Japan). Analysis of wastewaters River water, dairy wastewaters and brewery wastewaters were collected. COD (Chemical oxygen demand), turbidity and pH, of the river water and wastewaters were measured using a spectrophotometer (Spectroquant Pharo 100 M, EU), turbidimeter (HACH, USA) and pH meter respectively. Comparison with traditional flocculants was done by replacing the bioflocculant with alum and polyacrylamide Fig 4. FTIR Spectrum of the purified bioflocculant from the consortium of Cobetia sp. OAUIFE, Bacillus sp MAYA and Bacillus sp Gilbert. Table 2. A comparison of the removal of COD, turbidity and flocculating activity of the bioflocculants from consortium of Cobetia sp. OAUIFE, Bacillus sp MAYA and Bacillus sp. Gilbert with tradition flocculants. Flocculant used Wastewater Turbidity removal (% ) COD reduction (%) Flocculating activity (%) Bioflocculant Brewery 92 58.5 93.7   Dairy 72.5 99.8 82.2 River 96.6 70.6 96.4 Alum 87 98.0 75.1 71.4 98.6 67.0 33.1 70.0 57.4 Polyacrylamide 85.4 99.5 66.1 67.3 48.8 68.8 70.8 RESULTS a b CONCLUSION The bioflocculant produced from a mixed culture of Cobetia sp. OAUIFE, Bacillus sp. MAYA, and Bacillus sp. Gilbert was very effective in removing turbidity and COD from brewery wastewater, dairy wastewater, and river water. The high-flocculating activity of the bioflocculant could be due to the presence of functional groups such as hydroxyl, amino, and carboxyl groups which enhance flocculation. Microbial consortium in bioflocculant production could be a means of improving flocculant characteristics and efficiency. This bioflocculant could be an attractive substitute to the harmful chemical flocculants such as polyacrylamide and alum which have commonly been used in water and wastewater treatment. Fig 3a and 3b :SEM analysis of the purified bioflocculant produced by the consortium of Cobetia sp. OAUIFE, Bacillus sp MAYA and Bacillus sp Gilbert and SEM analysis of the purified bioflocculant from the consortium flocculating kaolin suspension Table 1: Percentage composition of purified bioflocculant produced by the consortium of Cobetia sp. OAUIFE, Bacillus sp MAYA and Bacillus sp. Gilbert REFERENCES Composite % (w/w) Protein 43.2 Neutral sugar 0.53 Uronic acid 9.02 Hubbard, A.T., 2004. CRC Press. p.4230. Salehizadeh, H., Shojaosadati, S.A., 2001. Biotechnol. Adv., 19: 371-385. Kowall, N.W., Pendleury, W.W., Kessler, J.B., 1989. Neurosci. 29: 329-337. Liu, W., Wang, K.,Li B., Yuan, H., Yang, J., 2010. Bioresour. Technol. 101: 1044 -1048. Daolun, L.F., Shihong, H. X., 2008. 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