Optimization Of Culture Conditions For Bioflocculant HBF-3 Production By Halomonas Sp. V3a' And Scaling-up Fermentation

Microbial flocculants (MBFs), secreted by some microorganisms during their growth and metabolisms, are kinds of extracellular macromolecular biopolymers. Because of their wide flocculating spectra, high flocculating activities, harmlessness and lack of secondary pollution, MBFs have been attracting much more attentions than conventional flocculants. However, studies in recent years are mainly localized in screening bioflocculant-producing microorganisms and investigate the characterizations of their respective bioflocculants. While acquiring microorganisms which can secret high active bioflocculant, exploring their optimal culture media and conditions, and seeking low-cost substrates will be of most importance for further studies and practical application of bioflocculants.In this study, Halomonas sp. V3a', a mutant strain of Halomonas sp. V3a, can produce an exopolysaccharide bioflocculant which is designated HBF-3. Fermentation medium for HBF-3 production optimized using response surface methodology includes (g/L) edible glucose, 16.14; MgSO₄·7H₂O, 2.73; NH₄Cl, 1.97; yeast extract, 0.6; NaCl, 24; KH₂PO₄, 2; and K₂HPO₄, 5. And the optimal culture conditions are initial pH valuses, 7.5-8.0; inoculum concentration, 3% (v/v); culture temperature, 28℃; and rotary speed of flask-shakers, 160 rpm. The yield of HBF-3 were increased to be 4.52 g/L which was about one times more than its yield based on the previous unoptimized medium and culture conditions.To further increase the production of HBF-3, fed batch fermentation was carried out in 250-mL flasks based on optimized culture medium and conditions. The results showed that when fresh medium containing 0.5% (v/v) edible glucose was fed after Halomonas sp. V3a' was cultured for 25-28 h, the bioflocculant production was increased to be 4.94-5.10 g/L. Aeration rate and agitation speed was investigated using one-factor-at-one-time method in 10-L fermentors, and the optimal values of them were 1.5 vvm and 75 rpm, respectively with HBF-3 yield of 5.58 g/L. Scaling up fermentations were investigated in 50-L fermentors. Unexpectedly, HBF-3 yield was not increased but decreased. However, it could keep at the level of 5.41 g/L.Some reagents, such as Sorbic acid, sorbic alcohol, potassium sorbate, Methyl 4-methyl benzoate, sodium benzoate and methanol, were involved in the preservation of fermentative broth. The results showed that methanol played a much better role in preserving the fermentative broth. The flocculating activities decreased slightly when the fermentative broth added into methanol was incubated at room temperature for 10 days.