Purification and characterization of cellulose produced by Acetobacter xylinum
Cellulose was produced through batch-fermentation using a strain of Acetobacter xylinum, one of the attractive alternative sources of cellulose (Sinskey et al., 1986). From the preliminary and factorial experiments, the important factors in cellulose production were found to be the sources of nitrogen and carbohydrates and their concentrations and pH. Fructose alone and in combination with sucrose at 5.0% (w/v) total concentration gave the best yield among the sugars used. Nitrogen was found to be essential but excessive amounts did not enhance cellulose production. The optimum pH for cellulose synthesis using sucrose as the sole carbon source appeared to be 4.5. The optimum conditions for cellulose production were determined through Response Surface Methodology (RSM). A three-level, seven-factor design adopted from Box and Behnken (1960) was used with the following factors as input variables: nitrogen from an organic or inorganic source, fructose or sucrose as carbon sources, pH, inoculum concentration and temperature. Results of this experiment indicated the need to conduct another optimization experiment using only four factors (sucrose or fructose concentrations, pH and temperature). A central composite rotatable second order design by Cochran and Cox (1957) was used. The model for the response, cellulose yield, was highly significant and with nonsignificant lack of fit. The stationary point was a maximum and its critical values (or optimum fermentation conditions) were: fructose concentration, 24.8 g/l; sucrose concentration, 76.5 g/l; pH, 4.49 and temperature, 29.3$\sp\circ$C. The experimental yield obtained using these optimum conditions was 12.67 $\pm$ 0.14 g/l which was very close to the predicted value. This verification experiment showed that the model was a good predictor of cellulose yield. Simple and effective isolation and purification methods were developed. Mechanical treatment and solvent extraction using NaOH or SDS (sodium dodecyl sulfate) were found to be effective in removing most of the bacterial cells and other impurities present in the bacterial cellulose. Using any of these isolation procedures, a product with at most 2.8% protein and at least 75% yield. The water holding capacity (WHC) of bacterial cellulose using the centrifugation method was very high (about 30 g H$\sb2$O/g solids). Drying reduced its WHC but its level was better than a widely used commercial brand.
BeMiller, Purdue University.
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