Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a′ using response surface methodology

Abstract

The novel exopolysaccharide bioflocculant HBF-3 is produced by Halomonas sp. V3a′, which is a mutant strain of the deep-sea bacterium Halomonas sp. V3a. Response surface methodology (RSM) was employed to optimize the production medium for increasing HBF-3 production. Using a Plackett–Burman experimental design to aid in the first step of optimization, edible glucose, MgSO₄·7H₂O, and NH₄Cl were found to be significant factors affecting HBF-3 production. To determine the optimal concentration of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the critical components were obtained as follows: edible glucose, 16.14 g/l; MgSO₄·7H₂O, 2.73 g/l; and NH₄Cl, 1.97 g/l. HBF-3 production obtained by using the optimized medium was 4.52 g/l, which was in close agreement with the predicted value of 4.55 g/l. By scaling up fermentation from flask to fermenter, HBF-3 production was further increased to 5.58 g/l.

Introduction

Microbial-produced bioflocculants have received increased scientific and technical attention because they are biodegradable and nontoxic and their degradation intermediates are not secondary pollutants (Salehizadeh et al., 2000, Salehizadeh and Shojaosadati, 2001). Bioflocculants are mainly composed of protein, glycoprotein, polysaccharide, and nucleic acid (Labille et al., 2005). Many microorganisms have been shown to produce bioflocculants e.g., Aspergillus sojae (Nakamura et al., 1976), Paecilomyces sp. (Hiroaki and Kiyoshi, 1985), Rhodococcus erythropolis (Kurane et al., 1994), Serratia ficaria (Gong et al., 2008), Bacillus mucilaginosus (Lian et al., 2008). The high-cost and low yield of bioflocculant production, however, are the major factors limiting the development of bioflocculants for commercial use in wastewater treatment. To overcome these production limitations, mutational methods to obtain more efficient strains and a search for low-cost feedstocks have been active areas of research (Wang et al., 2007).

Response surface methodology (RSM) is a powerful statistical tool that has been successfully applied to optimize fermentation process (e.g., biomass cultivation (Lhomme and Roux, 1991)), spore generation (Chen et al., 2005), enzymes production (Park et al., 2002, Gangadharan et al., 2008), metabolite secretion (Wang and Wan, 2008, Bajaj et al., 2009). Compared with conventional methods, which are time-consuming because they require a large number of experiments to determine the optimal content of each factor one at a time, RSM can distinguish interaction effects from the effects of individual components. This offers essential information for executing optimization processes while simultaneously incorporating multiple responses.

In this study, a novel exopolysaccharide bioflocculant, named HBF-3, is produced by Halomonas sp. V3a′ which is a mutant of the deep-sea bacterium Halomonas sp. V3a (CCTCC M205050). Its average molecular weight, determined by gel permeation chromatography (GPC), is about 2.35 × 10⁵ Da. In addition, the crude preparation of HBF-3 contains 15.66% (w/w) rhamnose, 6.76% glucuronate, 0.77% trehalose, 2.73% glucose, 1.12% mannose and 5.30% sulfate groups. Promising results accumulated from preliminary research demonstrate that HBF-3 is efficient to flocculate inorganic solid suspensions, dyes solution, heavy metal ions and other synthetic suspensions in several types of wastewaters. To enhance the production of HBF-3 using RSM, a Plackett–Burman (PB) experimental design (Plackett and Burman, 1946) was performed to screen for components of the production medium that had significant effects on HBF-3 production. A central composite design (CCD) was then employed to optimize the PB-identified factors to further increase HBF-3 yields. Finally, batch fermentation was scaled up in 10-l fermenters based on the optimized medium.