Improvement Of A Deep-sea Halomonas Sp.V3a By Mutation In Production Of Polysaccharide Bioflocculant And Product

Microbial flocculants are newly developed natural macromolecular organic substances produced by microorganisms, which have the ability to flocculate the suspensions and separate solid from the mixture. Compared with traditional inorganic and organic flocculants, they have the characteristics of biodegradability, high performance, environment friendly and so on. Microbial flocculants can be used in wastewater treatment , food processing and bio-pharmacy field. So bioflocculants have drawn more and more attentions and become front line of flocculants investigation.A spontaneous mutant strain was obtained from fermentation broth of Halomonas sp.V3a which was isolated from deep-sea sediment with high capability of producing bioflocculant. Fermentation condition optimization showed that the optimal production conditions of the mutant: cultivating in 50ml 2.0% NaCl medium in a 250ml flask with two-stage temperature control (first cultured at 24℃for 15h and then cultured at 28℃for the rest fermentation time ) at 160r/min and 28℃for about 25-28h.. After optimization of fermentation condition, the culture could achieve a flocculating rate as high as 99.86% for Kaolin suspension at a dosage of 2ml/L.The Halomonas sp.V3a (parent strain) grew slowly and the fermentation cycle was as long as 10d on it's optimal production condition (18℃, 75r/min). The results showed that the spontaneous mutation strain has much more advantage in bioflocculant production compared with its parent strain.. Bioflocculant produced by Halomonas sp.V3a-3# is HBF-3 which was obtained after purification.The result showed crude HBF-3 contained neutral sugar 20.56%, uronic acids 7.15%, amino sugarl.06% and tatal sugar 29.8%. UV Spectral Analysis showed that HBF-3 didn't contain any protein or nucleic acid. The molecular weight was determined about 232,589 Da by GPC Column-Ultrahydrogel Linear in HPLC system and the refractive index detector was used as the the detector. HBF-3 had a typical infrared spectrogram of EPSs and the characteristic absorption peak was 3294, 2951, 2953, 1646, 1074, 862cm^-1. TLC analysis of HBF-3 showed one single point on silica gel plate. HPLC analysis showed that HBF-3 was consist of glucose 2.73%,fucose 0.77%,mannose 1.12%,rhamnose 15.66% and glucuronic acid 6.76%.It contained the highest rhamnose content among the previously reported bioflolcculants.The result showed that HBF-3 showed a high stability against heat and extreme pH. Slight derease in activity was observed as pH changed from 1 to 12. In addition, Ca²⁺ was found to be unnecessary to flocculate Kaolin clay. The result of application in various wastewater indicated that it had a wide flo-spectrum no matter cell, algae or suspended solids. Furthermore, the potential of HBF-3 in removal of trace amounts metal ions and emulsification of oil was also investigated.