Development of a flat sheet woven fabric membrane fermenter for xylanase production by Thermomyces lanuginosus

Abstract

Fermentation processes are vital for the production of numerous bioproducts. Fermentation being the mass culture of micro – organisms for the production of some desired product, is an extensive field, with immense prospects for study and improvement. Enzyme production is of significance as these proteins are biological catalysts, finding niches in numerous industries, xylanase for example is utilized in the pulp and paper, animal feed, biofuel and food production processes. During enzyme production, a critical step is biomass separation, whereby the valuable product, the enzyme, is removed from the broth or micro – biological culture before it is denatured. This is typically achieved via centrifugation. The aim of this study was to develop and evaluate a submerged membrane fermenter system with the specific outcome of increasing the rate of production of xylanase, from the thermophilic fungal species Thermomyces lanuginous DSM 5826. Preliminary shake flask experiments were performed to determine the optimal production conditions, followed by partial characterization of the enzyme. A bioreactor was then fabricated to include a flat sheet membrane module, with outlets for permeate and broth withdrawal and inlets for feed and sterile air input. Experiments were conducted to determine the optimal dilution rate for maximum volumetric productivity. Results from the shake flask experiments indicated that the best conditions for xylanase production, yielding xylanase activity of 5118.60 ± 42.76 U.mL-1 was using nutrient medium containing beechwood xylan (1.5 % w/v), yeast extract (1.5 % w/v), potassium dihydrogen phosphate (0.5 % w/v), adjusted to a pH of 6.5 and inoculated with 1.0 mL of spore solution, rotating in a shaking incubator set to 150 rpm at 50 °C. Apart from analysis of the effect of the carbon source on xylanase activity, coarse corn cobs were used in the shake flask experiments as a cost saving initiative. The pH optima was determined to be 6.5 while the temperature optima of the enzyme was 70 °C. SDS PAGE analysis revealed that the molecular weight of the enzyme was between 25 and 35 kDa and qualitative analysis via a zymogram revealed clear zones of hydrolysis on a xylan infused agarose gel. During short run membrane fermenter experiments the percentage increase in enzyme activity between the batch operation (610.58 ± 34.54 U.mL-1) and semi – continuous operation (981.73 ± 55.54 U.mL-1) with beechwood xylan nutrient replenishment was 60.78 %. The maximum volumetric productivity achieved with beechwood supplementation after 192 hours in semi – continuous operation (5.32 ± 0.30 U.mL-1.hr-1) was 2.1 times greater than that of batch operation (2.54 ± 0.14 U.mL-1.hr-1) which equates to an increase of 110.28 % in productivity measured at its peak. The increase in total activity between batch (610 576.92 U) and beechwood xylan medium supplemented semi – continuous mode (1 184 937.50 U) resulted in a 94.07 % increase. During long run experimental periods, the increase in production of xylanase between the batch (873.26 ± 61.78 U.mL-1) and the xylan medium membrane system (1522.41 ± 107.65 U.mL-1) was determined to be 74.34 % while an overall average increase in productivity between the batch and xylan fed membrane system was 43.25%. The total enzyme activity with in membrane mode with beechwood xylan nutrient medium feed was 160 % greater than the batch process offering a 2.6 – fold increase. Experiments where de – ionized water was alternated with beechwood xylan nutrient medium had no significant impact on the productivity or enzyme activity. The optimal dilution rate for maximum volumetric productivity as determined to be 0.0033 hr-1. The results are indicative of the potential viability of such a design, yielding the desired outcome of a membrane integrated system to significantly increase the production of enzymes during fermentation.