Studies on microbial prospecting for exobiopolymeric flocculants

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 30 o C and in presence of CaCl2 (5mM). Thermal gravimetric (TGA) and rheological analysis of the bioflocculant demonstrated it to b e thermostable (100 o C, 5min) with temperature dependent viscosity and elasticity changes. The purified flocculant had a high molecular weight (~2.6×10 6 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 (2845 o C) 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. List of Abbreviations ARDRA Amplified ribosomal DNA restriction analysis BLAST Basic Local Alignment Search Tool CLSM Confocal laser scanning micrograph CPC Cetylpyridinium chloride DNA Deoxyribonucleic acid dNTP 2‟-deoxynucleoside-5‟-triphosphate EDTA Ethylenediaminetetraacetic acid EPS Extracellular polymeric substances FIB Flocculant isolation broth FISH Fluorescent in situ hybridization FTIR Fourier transform infrared spectroscopy GPC Gas permeation chromatography GSH L-glutamyl-L-cysteinyl glycine GTP Glutathione-S-transferases HPLC High performance liquid chromatography KBF Klebsiella bioflocculants LB Luria Bertani MRD Maximum recovery diluents NCC Nebauer cell counter NTG N-methyl-N'-nitro-N-nitrosoguanidine NTU Nephelometric Turbidity Units OD Optical Density PCR Polymerase chain reaction rDNA Ribosomal deoxyribonucleic acid SEM Scanning electron micrography SOD Superoxide dismutase TAL Thin Agar Layer TFA Trifluoroacetic acid TGA Thermogravimetric analysis TLC Thin layer chromatography UDP Uridine diphosphate UV Ultraviolet VDW van der Waals forces List of Tables Tables Page No. 2.1 Microbial Bioflocculants, their dose and flocculating activity 28 3.1 Analytical Conditions for analysis of metals in solution by AAS 61 4.1 Bacterial isolates from industrial units with more than 50 % of flocculant activity 64 4.2 Morphological and biochemical tests of the bacterial isolate RD4 66 4.3 Distance matrix based on nucleotide sequence homology 67 4.4 Aligned sequence data of Klebsiella terrigena RD4 67 4.5 Growth Kinetics of Klebsiella terrigena and bioflocculant production and its activity 70 4.6 Comparision of flocculating efficiency of bioflocculant with other flocculants 75 4.7 Compositional analysis of the bioflocculant 82 4.8 Effect of different carbon sources on flocculant activity and yield of bioflocculant produced by Klebsiella terrigena 91 4.9 Compositional analysis of the auxotrophic mutant KTM1 100 4.10 Selective removal of S. typhimurium ATCC 23564 from mixed cultures in tap water by purified Klebsiella terrigena biopolymer 103 4.11 Optimization of dose of purified Klebsiella terrigena biopolymer for flocculating S. typhimurium ATCC 23564 spiked in tap water 104 4.12 Comparison of flocculation efficiency between bioflocculant and alum for removal of Salmonella sp. from poultry wastewater 107 4.13 Comparison of removal of Cryptosporidium parvum oocysts by bioflocculant and alum 110 4.14 Optimization of medium pH for the removal of Cryptosporidium parvum oocysts by bioflocculant 111