Continuous feeding of spent ammonium sulphite liquor improves the production and saccharification performance of cellulase by Penicillium oxalicum
Introduction
With the booming demand for renewable fuels and chemicals, and the weakening market competitiveness of traditional pulp and paper enterprises gradually, some researchers proposed the concept of pulp biorefinery (Axegård, 2005, Janssen et al., 2008). By transforming into biorefinery plants, traditional pulp and paper enterprises can use short fibers and hemicellulose to produce biofuels and value-added chemicals in addition to the pulping process, which enables the enterprises to improve raw material utilization and reduce environmental pollution. However, the bioconversion of lignocellulosic materials from pulping is limited by the cost of cellulase which degrades cellulosics to fermentable sugars (Phitsuwan et al., 2013). If some wastes generated in the industrial pulping process can be used for on-site cellulase production, the feedstock cost for producing cellulase can be reduced which can effectively improve the economic competitiveness of biorefinery.
Spent ammonium sulphite liquor (SASL) is the main effluent from the ammonium sulphite pulping process, and contains significant amounts of dissolved organic matter including mono- and oligo-saccharides released from cellulose and hemicellulose. These oligosaccharides were reported to induce the expression of lignocellulose-degrading enzymes in fungi (Znameroski et al., 2012). Furthermore, the residual ammonium in SASL can be utilized as nitrogen source for microorganisms. Thus, SASL has the potential to be applied in cellulase fermentation as an alternative for some raw materials.
Submerged fermentation is the main method used in industrial production of cellulase. In general, the concentration of cellulosic materials serving as both carbon source and cellulase inducer in the medium is positively correlated with the yield of cellulase (Hendy et al., 1982, Xue et al., 2012). However, since cellulosic materials have strong water absorption capacity, adding high concentration of them in the medium would increase the viscosity of fermentation broth and affect oxygen-transfer efficiency, water activity, etc. Fed-batch fermentation can overcome this problem to a certain extent and effectively improve the yield and productivity in cellulase production (dos Reis et al., 2013, Ma et al., 2013). Nevertheless, insoluble cellulosic materials can only be fed intermittently at low amounts, making it difficult in operation and increasing the risk of bacterial contamination.
In this study, fed-batch fermentation of cellulase by P. oxalicum through continuously feeding of SASL was explored. The effects of feeding rate on cellulase production were studied and a multi-stage feeding strategy was developed. Further, the hydrolysis performances of crude cellulases produced with or without SASL feeding were compared, and proteomic analysis was conducted to reveal the underlying mechanism for the differences.
Section snippets
Microorganism
The previously reported cellulase high-producing recombinant stain P. oxalicum RE-10 was using in this work. RE-10 was constructed by genetically modifying three key regulators (gpdA(p)-clrB-ptra; Δbgl2::hph; ΔcreA::bar) to significantly improve the production of cellulolytic enzymes (Yao et al., 2015).
Medium and culture conditions
The P. oxalicum RE-10 spores kept in 30% (v/v) glycerinum vials were inoculated on wheat bran extract slant and cultured for 5 days at 30 °C (Han et al., 2017a, Han et al., 2017b), followed by
Impact of SASL on cellulase production and cell growth
SASL used in this study contains certain amounts of soluble oligomers from lignocellulose (76.47 g/L arabinoxylan and 35.01 g/L β-glucan), monosaccharides (6.48 g/L glucose, 14.48 g/L xylose and 3.19 g/L arabinose) and 8.13 g/L ammonium. We therefore speculated that addition of SASL to the medium might increase cellulase production by P. oxalicum RE-10. The hypothesis was first tested in shake flask by adding SASL at the beginning of cultivation. As shown in Fig. 1a, the adding of 2%, 4% and 6% (v/v)
Conclusion
In this study, cellulase was produced by P. oxalicum with continuous feeding of SASL and then used for the saccharification of ammonium sulfite pulp and SASL to produce glucose. The rate-varying feeding strategy significantly increased the FPase from 12.69 U/mL in batch fermentation without SASL feeding to 17.66 U/mL in 7.5-L fermenter. In addition, the cellulase preparation with SASL feeding had higher specific lignocellulolytic activities and higher cellulose hydrolysis efficiency than that
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China (Grant No. 31370086 and 31670079), Major Projects of Science and Technology in Shandong Province (2015ZDXX0403B02), the Key Research and Development Project of Shandong Province (2016GSF121026) and the Fundamental Research Funds of Shandong University.
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