Optimized Solid-state Fermentation Of Aspergillus Niger For Production Of Feed-purpose β-glucanase And Xylanase Using Rice Residues From Mass Production Of Fungal Biocontrol Agents

Entomopathogenic hyphomycetes are important fungal biocontrol agents against insect pests, being well represented by the fungal species Beauveria Vuillemin, Metarhizium. An improved technology was developed in our laboratory for mass production of aerial conidia of fungal biocontrol agents, such as Beauveria and Metarhizium, on rice in the upright multi-tray conidiation chamber. This process could not only control the conidiation period easily, but also havn't any wastewater and exhaust gas produced. In spite of these advantages, this process would leave bulk rice residues after aerial conidia being harvested from culture media. In order to establish environment-friendly process for bulk production of fungal biocontrol agents, this study was sought the technique that produced the feed-purposeβ-glucanase and xylanase with rice residue and wheat bran. A series of experiments, including one-factor-at-a-time optimization and orthogonal experiments were performed to enhance the production yield of these two enzymes. Detailed results are summarized as follows:The optimal ratio of wheat bran to rice residue and the fermentation period The experiment was achieved by solid-state fermentation (SSF) of Aspergillus niger at 25℃during the process of 3 d, using the 11 mixtures of increase by 10% degrees of rice residues and wheat bran as substrate, adding 0.5% NH4NO3. The yields of P-glucanase and xylanase from the 11 treatments were measured after ending the fermentation course. After grinding rice residue, the same experiment was performed using milled rice instead of rice residue as substrate. The results revealed that the mixture of 30% milled rice and 70% wheat bran was selected as an acceptable substrate in the trials of 11 ratios of the two components, the differences of the enzyme yields were not significant between the mixture of 30% milled rice and 70% wheat bran and the trial of wheat bran. At the base of the acceptable substrate, the next experiment was performed by examining the production ofβ-glucanase and xylanase during the dynamic course from 42 h to 96 h, 6 h as an interval. The results indicated that the highest yields ofβ-glucanase and xylanase were simultaneity obtained at 54h during the fermentation course. Consequently, the further experiments were achieved using the mixture of 30% milled rice and 70% wheat bran as substrate and 54h as fermentation course.One-factor-at-a-time optimization of solid-state fermentation In order to further increase the yields ofβ-glucanase and xylanase, batch experiments of one-factor-at-a-time optimization were carried out, including water content, supplementary nitrogen, metal ion, Tween-80 and initial pH in the water. The results showed that the optimal water content was found to be 55.6%, which was the representation of the1:1.2 ratio of substrate to water. Among the nine additional nitrogen sources, the efficiency of inorganic nitrogen sources were found to be better than the organic nitrogen sources at the aspect of promoting enzyme production, and (NH₄)₂SO₄ as the most favorable nitrogen. The salt of KH₂PO₄ was determined to be the optimal for increasingβ-glucanase yield, whereas CaCl₂ was the best for xylanase. Given Tween-80 as the surfactant at the concentration range of 0.1%-0.5%, the treatments of 0.2% and 0.3% were more appropriate than other treatments.β-glucanase and xylanase exhibited their maximum activity at pH 6 and pH 4 or 5, respectively, and both of them showed remarkable yield at acidic conditions.Orthogonal experiments of solid-state fermentation Based on above results, factors found to significantly affect the enzyme yields in the mixture during SSF were the type of metal ion as trace nutrition (A), the content of (NH₄)₂SO₄ as supplementary nitrogen (B), and the concentration of Tween-80 (C) and pH in the water (D) used to stir the mixture for SSF. Combinations of the selected factors were further optimized for enzyme production in triple four-factor, three-level orthogonal experiments. Factor A was found to be the most important factor for production ofβ-glucanase, and followed by factor D, C and B, and the A₂B₁C₃D₂ as the best combination. Factor C was observed to be the most remarkable factor for xylanase, and followed by factor D, B and A, and the A₂B₂C₃D₂ as the optimal combination. This optimized culture conditions were confirmed with three repeated whole trials of 54-h SSF at 25℃. The results indicated there was no remarkable difference in the yields ofβ-glucanase and xylanase between the two combinations, which were greatly enhanced as compared to yield before optimization. The optimized combination of A₂B₂C₃D₂ caused the greatest improvement in all trials, and was the rice-bran mixture (initial pH, 5) supplemented with 0.1% KH₂PO₄, 1.0% (NH₄)₂SO₄ and 0.3% Tween-80.Conclusive remarks In summary, rice residues from mass production of fungal biocontrol agents against insect pests could be utilized effectively for production of feed-purposeβ-glucanase and xylanase. Compared to those before optimization, yields ofβ-glucanase and xylanase had considerable improvement under optimized conditions. The results highlighted an alternative approach to utilize the rice residues for the enzyme production at the reduced cost of 30% wheat bran and would facilitate to establish a rice-based, environment-friendly technology for bulk production of fungal biocontrol agents.