Studies on Microbial Prospecting for Exobiopolymeric Flocculants

Abstract

There are currently few effective bioflocculants as alternatives to chemical flocculants used extensively in solid-liquid separation processes. The present study was carried out with an objective of bioprospecting efficient and novel flocculant producing bacterial strains. A potential strain (isolated from activated sludge) exhibiting high flocculating activity, metal and pathogen removal ability was identified as Klebsiella terrigena. The bioflocculant purified from culture supernatants exhibited flocculating ability over a wide range of colloid particles (0.5 to 100μm) at low dosage, its flocculating ability being better than or equivalent to both natural and synthetic flocculants. Highest flocculating activity was observed in the pH range of 6-8, temperature 30oC and in presence of CaCl2 (5mM). Thermal gravimetric (TGA) and rheological analysis of the bioflocculant demonstrated it to be thermostable (100oC, 5min) with temperature dependent viscosity and elasticity changes. The purified flocculant had a high molecular weight (~2.6×106 Da) as determined by gel permeation chromatography; the ultrastructural pattern of purified bioflocculant was visualized by SEM as a porous structure with randomly distributed small pores interconnected with channels. HPLC of hydrolyzed flocculant could establish it to be a polysaccharide comprising of D-Glc, D-Man, D-Gal and D-GlcA monomers with linkage pattern resembling galactoglucans. FTIR of the biopolymer showed abundance of hydroxyl, carboxyl, and methoxyl groups as well as uronic acid residues. To elucidate the role of cellular mechanisms in flocculation, mutants defective in glucose utilization were generated and their flocculant analyzed for functionality as well as composition. High Performance liquid Chromatograms of flocculants obtained from mutant indicated a complete absence of galactose residues and a loss of flocculating activity, suggesting the crucial role of galactose for functionality of the flocculant. The effect of Water activity (aW) and oxidative stress were studied to understand their involvement in flocculant production by K.terrigena. Though growth was observed at oxidative stress and low water activity, the flocculant production could not be ascribed to protective mechanism by the cells. The purified flocculant could remove Salmonella significantly (80.3%) both under simulated conditions and contaminated poultry waste water. Fluorescent in situ hybridization (FISH) with genus specific Sal3 probe of the flocculated samples confirmed complete removal of Salmonella enmeshed in the bioflocculant matrix. The flocculant could remove (62.3%) Cryptosporidium oocysts as well from tap water at low dosage of 2mg/L, incorporation of CaCl2 (5mM) enhanced removal efficacy. The purified bioflocculant was also capable of removing Cadmium, Copper, Zinc and Lead within a wide range of temperature (28-45oC) and pH (5-8). Metal removal however varied with both the type and concentration of the metal (57-84%) and was completed within 2 hours of contact time. Overall the result of the current study suggests the flocculant to be of considerable potential, suitable for further commercial applications.