- Email: [email protected]
Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate
Accepted Manuscript Title: Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate Author: Xiao-Yue Zhang Li-Han Zi Xu-Meng Ge Yong-Hao Li Chen-Guang Liu Feng-Wu Bai PII: DOI: Reference:
S1359-5113(16)30518-9 http://dx.doi.org/doi:10.1016/j.procbio.2016.12.027 PRBI 10906
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
5-10-2016 29-11-2016 26-12-2016
Please cite this article as: Zhang Xiao-Yue, Zi Li-Han, Ge Xu-Meng, Li Yong-Hao, Liu Chen-Guang, Bai Feng-Wu.Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.12.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate Xiao-Yue Zhang1, Li-Han Zi1*, Xu-Meng Ge3, Yong-Hao Li1, 2, Chen-Guang Liu2*, Feng-Wu Bai1, 2 1
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024,
China 2
State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology,
Shanghai Jiao Tong University, Shanghai 200240, China 3
Department of Food, Agricultural and Biological Engineering, The Ohio State
University/Ohio Agricultural Research and Development Center, Wooster, OH 44691-4096, USA
*
Corresponding authors: LH Zi Tel: +86-411-8470-6785, fax: +86-411-8470-6329, E-mail: [email protected]; CG Liu, Tel: +86-21-3420-5125, fax: +86-21-3420-5708, E-mail: [email protected]
1
Graphical abstract
2
Highlights •
Trichoderma reesei mutants were obtained with chemical and irradiation treatment.
•
Mutant T. reesei D-7 showed 57% higher filter paper activity (FPA) than wild type.
•
Soluble inducer was prepared by hydrolysis of corn starch with α-amylase.
•
Mixture of corn starch hydrolysate and cellulose achieved higher FPA.
3
Abstract: The high cost of cellulase production is one of the key barriers to commercialization of cellulose-based biofuels. Screening high cellulase-producing strains and exploring low-cost inducers of cellulase are efficient strategies to address this issue. In this study, Trichoderma reesei Rut-C30 mutants were obtained by treatment with Ethyl Methyl Sulfonate (EMS) and plasma-irradiation. Mutant T. reesei D-7 was selected as the most promising cellulase producer based on screening through phosphoric acid-swollen cellulose plates. The filter paper activity (FPA) of T. reesei D-7 was 57.55% higher than that of the wild-type strain. Corn starch hydrolysate (CSH) prepared through hydrolysis of corn starch by α-amylase were used as soluble inducer for cellulase production with T. reesei D-7. FPA of 4.53 IU/ml was achieved at an optimal initial concentration of CSH (40 g/l reducing sugar). Moreover, CSH and cellulose were proved to be the most effective soluble and insoluble inducers for cellulase production with T. reesei D-7. Mixture of CSH and cellulose further improved FPA up to 15.62 IU/ml, which was 3.45-fold or 1.52-fold as high as that achieved using CSH or cellulose as an inducer.
Keywords: Trichoderma reesei; Mutation; Cellulase; Corn starch hydrolysate; Soluble inducer
4
1. Introduction Cellulosic biomass, such as agricultural and forestry residues, is the most abundant renewable carbon source with low cost in the world. Production of biofuels from cellulosic biomass has been widely recognized as a sustainable strategy for reducing dependence on non-renewable fossil fuels resources and mitigating the devastating effects of climate change due to overconsumption of fossil fuels [1]. Conversion of cellulosic biomass to biofuels usually includes three main steps: pretreatment to reduce recalcitrance, enzymatic hydrolysis to release fermentable sugars, and fermentation to produce biofuels [2]. Enzymatic hydrolysis is conducted with cellulolytic enzymes including exoglucanases, endoglucanases and β-glucosidases, which break down cellulose into glucose with cellobiase and soluble gluco-oligomers as intermediates [1]. However, high cost of cellulase production remains one of the key barriers to commercialization of biofuels from cellulosic biomass [3]. A variety of microorganisms, including bacteria and fungi, can produce cellulase [4], while most of cellulases exploited for industrial application are produced by filamentous fungi, such as Trichoderma and Penicillium. Compared to other strains, Trichoderma reesei Rut-C30 is well-known for its high cellulase secretion capacity, thus is suitable to be used as a good starting point for further improvement of cellulase production performance [5]. Both random mutagenesis and rational genetic modifications have been studied for improving cellulase production from Trichoderma reesei. Compared to rational genetic modifications, random mutagenesis methods are simple and easy to operate, though they have limitations, such as lack of stability. Random mutagenesis can be conducted using chemical mutagens, such as nitrous guanidine (NTG) and Ethyl Methyl Sulfonate (EMS), or physical mutagens, such as Ultraviolet (UV) [6]. Plasma-irradiation has recently been known as an effective mutagenesis method to obtain high-productivity strains with stable performance 5
[7,8]. Furthermore, different mutagenesis can be combined to improve the efficiency of mutation. Medium composition, especially carbon and nitrogen sources, is another important factor that affects the cellulase activity and productivity. Carbon sources can act as both energy supplier and inducer for cellulase production, and can be classified into soluble and insoluble carbon sources based on their solubility in water [9]. Cellulase biosynthesis can be induced by soluble and insoluble substrates such as lactose, cellulose and is repressed in the presence of readily metabolisable carbon sources such as glucose and xylose [10]. Many previous studies have shown that high cellulase activity was usually achieved using insoluble carbon sources including nearly pure cellulose, such as Avicel and Solka Floc, and impure cellulose-containing substrates such as lignocellulosic materials [11,12]. Among insoluble substrates, pure cellulose has been recognized as the most effective inducer for cellulase production of T. reesei, which achieving the FPA of 22.7 IU/ml with total 70 g/l cellulose by fed-batch fermentation [11]. However, the cost of pure cellulose is too high for industrial production [13]. Soluble substrates, such as sophorose, lactose, cellobiose and sorbitol, have been used to induce cellulase production, while sophorose was proved as the most effective inducer that, with low concentrations, achieved 2500 and 233 times higher cellulase productivity than cellobiose and lactose, respectively [14]. However, the high price of sophorose limited its industrial application. Lactose has been widely used as an un-expensive soluble substrate that can obtain higher cellulase activity than those for cellobiose and sorbitol [15,16]. A common drawback of monosaccharide inducers is that they can be easily consumed by microorganisms, resulting in reduced efficiency of induction. This issue can be addressed by using soluble oligosaccharides that are difficult to be utilized by the developed strain. Currently, oligosaccharide inducers are prepared through hydrolysis of lignocellulose or 6
starch using inorganic acid such as hydrogen chloride and sulfuric acid [17,18]. However, detoxification process may be needed to remove inhibitors generated during hydrolysis process, which increases operating costs. Alternatively, oligosaccharides can be prepared through hydrolysis of corn starch with α-amylase, which is known as the first step for industrial production of glucose from corn starch. Enzymatic hydrolysis does not produce inhibitors, and requires only mildly operating conditions and low-cost of α-amylase. As a result, the corn starch hydrolysate (CSH) prepared by enzymatic hydrolysis of corn starch with α-amylase could be an excellent soluble inducer for cellulase production with T. reesei. To the authors’ knowledge, there is only one report on using wheat flour hydrolysate as an inducer for cellulase production, but without information about the cellulase activity [19]. In this study, the wild strain T. reesei Rut-C30 was treated by random mutagenesis with EMS followed by plasma-irradiation and mutant T. reesei D-7 was isolated by screening on phosphoric acid-swollen cellulose plate and evaluated by liquid cultivation. The selected mutant T. reesei D-7 was further investigated for cellulase production using CSH as soluble inducer prepared by enzymatic hydrolysis of corn starch. Moreover, cellulase production of T. reesei D-7 using CSH and cellulose as mixed inducer substrates was performed for the first time.
2. Material and Methods 2.1 Microorganisms and culture media The cellulase producing fungi T. reesei Rut-C30 was a gift from Agricultural Research Service Culture Collection (NRRL 11460), USA. The T. reesei Rut-C30 and its mutants were maintained on Malt Extract Agar medium (MEA medium, malt extract 30 g/l and agar 15 g/l) plates and sub-cultured every one week. Selection medium (SM) contained (g/l): phosphoric acid-swollen cellulose (PASC) 10, 7
sorbose 4, Triton X-100 1, KH2PO4 2.0, MgSO4·7H2O 0.3, CaCl2·2H2O 0.4, (NH4)2SO4 1.4, agar 15 [20,21]. Inoculum medium contained Basal Medium (BM) described by Mandels and Weber [22] with modification (g/l): lactose 10; peptone 1; (NH4)2SO4 1.4; KH2PO4 2.0; MgSO4·7H2O 0.3; CaCl2·2H2O 0.4; FeSO4·7H2O 0.005; MnSO4·H2O 0.0017; ZnSO4·7H2O 0.0014; CoCl2·2H2O 0.002; Urea 0.3. The fermentation medium was the same as cultivation medium except that 40 g/l cellulose (microcrystalline cellulose, Sangon Company, Shanghai, China), 30 g/l soy flour and 20 g/l wheat bran were used instead of lactose and peptone. The initial pH was adjusted to 4.8 by 0.2 mol/l Na2HPO4-citric acid (pH 5.0) without control during cultivation. The medium was autoclaved at 121ºC for 15 min.
2.2 Cellulase production in shake flasks Batch cultivation experiments were carried out in 250 ml Erlenmeyer flasks. For inoculum preparation, one ml of a spore suspension (108 spores/ml) was inoculated into 50 ml of inoculum medium in the flasks which were cultured at 28ºC, 150 rpm for 2 days. The seed was inoculated into 50 ml fermentation medium in 250 ml Erlenmeyer flasks at an inoculum ratio of 8% (v/v), at 28ºC with shaking at 150 rpm for 7 days. The samples were removed daily and were centrifuged at 4000 rpm for 5 min. The supernatant was analyzed for extra cellular enzyme activities and soluble proteins.
2.3 Mutagenesis and screening of mutants The strain was grown on MEA plates for 7 days at 28ºC for spore development. Spores were then harvested and washed with 0.1% Tween-80 solution and the spore concentration was diluted to 108 spores/ml. Random mutagenesis was first conducted using ethyl methyl sulfonate (EMS) treatment according to the method described by Jiang et al. [20] and followed by plasma-irradiation treatment. 50 μl EMS was added to 10 ml of the spore 8
suspension and it was kept at room temperature for 24 h, then followed by plasma-irradiation at 24 V, 1.7 A and gap 3 mm for 5 min corresponding to a lethal rate of 78.38%. 100 μl the treated spore suspension was subsequently spread on the screening plates and cultured at 28ºC for 4-7 days. The mutants were selected on the basis of clearance zones appearing on SM medium. The final selected mutants were then inoculated in MEA medium for 7 days and the cellulase activity of mutants were assessed in shake flasks to select the most promising enzyme producing mutant.
2.4 Corn starch hydrolysate (CSH) preparation Corn starch hydrolysate (CSH) was prepared using α-amylase hydrolysis of corn starch at 40% (w/v) dry solid with 5 IU/g corn starch at 93-95ºC under constant stirring condition [23]. The hydrolysate was sampled at 30, 60 and 90 min, respectively. After cooling, liquefied CSH were recovered by filtration. Total reducing sugar in the liquefied CSH were 300 g/l determined by dinitrosalicylic acid (DNS) method [24], which contained 24 g/l glucose determined by biological sensor(SBA-40, Institute of Biological, Shandong Academy of Sciences, Jinan, China).
2.5 Analytical methods The supernatants of samples were suitably diluted before analyzed extra cellular enzyme activities and soluble protein. Filter paper activity (FPA) was used to estimate total cellulase activity according to Ghose [25] by incubating 0.5 ml enzyme with 1.0 ml citrate buffer (0.2 M, pH 4.8) containing one Whatman No.1 filter paper strip (50 mg, 1×6 cm). The reaction mixture was incubated at 50ºC for 60 min. Endoglucanase activity (CMCase) was determined by incubating 0.5 ml enzyme with 1.0 ml of 1% (w/v) carboxymethylcellulose (CMC) dissolved in citrate buffer and the reaction mixture was incubated at 50ºC for 15 min. 9
β-glucosidase activity was carried out in the total reaction mixture of 1.5 ml containing 0.5 ml enzyme and 1.0 ml of 15 mM cellobiose solution in citrate buffer and the mixture was incubated at 50ºC for 10 min. The reducing sugar was determined by DNS method and glucose was determined by biological sensor. One unit of enzyme activity was defined as the amount of enzyme required for liberating 1 mmol/l of glucose per ml per minute under the assay conditions. Concentrations of secreted proteins were measured using Bradford reagent (Sangon Biotech, China). All experimental results are presented as mean and standard deviations of triplicated independent experiments with standard deviations at significance p0.05.
3.1 Results and discussion 3.1 Mutagenesis and screening of mutants After mutagenesis, 60 positive colonies were initially selected on the basis of forming clear zones of hydrolysis of phosphoric acid-swollen cellulose in the selection medium (SM). Then the colonies were transferred again to SM and 20 mutants were screened according to the size of clearance zone surrounding the colony. These potential mutants were further assessed for cellulase production in shake flasks. The FPA of mutants and the wild strain was shown in Fig. 1, 13 mutants had 1.42-57.55% higher FPA than wild strain T. reesei Rut-C30. D-7 demonstrated the highest FPA of 8.35 IU/ml, which was higher than the mutant of Rut-C30 (8 IU/ml) by diethyl sulfate and UV mutagenesis using 50 g/l cellulose as inducer [11]. Moreover, D-7 presented larger clear zone than Rut-C30 and mutants D-8 and D-9 in the second round of screening (Fig. 2). In addition, it should be noted that the conidia (green) of mutant D-7 was decreased compared with other three strains in SM medium, which is consistent to higher cellulase secretion [26]. Moreover, the stability of cellulase production was evaluated with serial sub-culture in MEA medium for 8 times. The FPA in supernatant of 10
fermentation broth showed little changed, indicating a stable productive capacity of mutant T. reesei D-7 for cellulase production.
Fig. 1 and Fig. 2
The mutant T. reesei D-7 was further evaluated for extracellular production of cellulase, CMCase, and β-glucosidase in MEA medium for 7 days (Table 1). CMCase of T. reesei D-7 achieved 128.07 IU/ml, increasing by 50.81% compared with wild strain T. reesei Rut-C30 (84.92 IU/ml). The β-glucosidase of D-7 and Rut-C30 were obtained at 0.71 IU/ml and 0.51 IU/ml, respectively, which showed the low level for both mutant and the wild strain. This was corresponding with previous study that T. reesei could produce cellobiohydrolases (CBH) and endo-glucanases (EG) components of cellulase in large quantities but the amount of β-glucosidase was insufficient [27]. The D-7 could secret high mount of CMCase but low level of β-glucosidase because of screening scheme by using phosphoric acid-swollen cellulose (PASC) that preferred the strain with CMCase activity. The soluble protein content of crude enzymes from T. reesei D-7 was 3.39 mg/ml, 2.49-fold higher than wild strain, which reflected that the enhancement of cellulase production by mutant strain was owing to the increased of secretion of the enzyme.
Table 1
3.2 Effect of nitrogen sources on cellulase production by mutant T. reesei D-7 Four different nitrogen sources including soy flour, yeast extract, corn steep liquor and peptone were tested for their effects on cellulase production of mutant T. reesei D-7 (Fig. 3). Yeast extract achieved maximum FPA of 10.27 IU/ml on day-6 which was 1.34, 1.42 and 11
1.58-fold higher than those obtained by using soy flour, corn steep liquor and peptone (7.68, 7.22 and 6.48 IU/ml), respectively, on day-7. Actually, on day-5, yeast extract has already achieved a FPA of 8.89 IU/ml, which was much (more than 2.39-fold) higher than those obtained by using other nitrogen sources. Therefore, yeast extract was chosen as a suitable nitrogen source for cellulase production of T. reesei D-7.
Fig. 3
3.3 Cellulase production using CSH as inducer In order to detect the effect of CSH prepared with different reaction time on cellulase production of mutant T. reesei D-7, the hydrolysates were sampled at 30, 60, and 90 min, which containing 300, 309 and 321 g/l of total reducing sugar, and 24, 28 and 30 g/l of glucose, respectively. However, there was no significant difference of FPA by using hydrolysates sampled at different time. Therefore, the hydrolysis time 30 min was selected for cost reduction. The cellulase production experiment was carried out at different total reducing sugar ranging from 10 to 50 g/l with yeast extract as nitrogen source. The result showed that the highest FPA increased with the reducing sugar concentration increased from 10 to 40 g/l and reached maximum 4.53 IU/ml at 40 g/l (Fig. 4A). The maximum FPA decreased slightly when the concentration of the total reducing sugar further increased to 50 g/l. Furthermore, the reducing sugar was quickly consumed at low initial concentration (10-30 g/l) and the residual sugar declined to roughly 1 g/l at the 3th or 4th days, which may result in the low cellulase production (Fig. 4B). Conversely, the reducing sugars were consumed at the 6th day when 40 or 50 g/l CSH were added and the FPA reached the highest level afterward (the 7th day). The delay of achieving maximum FPA was consistent with previous study [28]. 12
Fig. 4
Previous studies have shown that wheat starch hydrolysate prepared by acid hydrolysis rendered effective inducing ability, but the starch itself was a poor inducer for cellulase production by T. reesei [29]. The acid hydrolysis of starch resulted in the formation of reversion products from glucose, such as sophorose, which have been considered as the real inducers of cellulase production in Trichoderma species. Moreover, our study also showed that large amount of disaccharide, trisaccharide, etc. were formed after enzymatic hydrolysis of corn starch (data not shown), which may render inductive ability of cellulase biosynthesis of T. reesei.
3.4 Cellulase production using different soluble and insoluble inducers Various soluble and insoluble substrates (40 g/l) were tested as carbon sources for their effect on cellulase production of mutant T. reesei D-7. As depicted in Table 2, among the soluble substrates tested, CSH induced the highest FPA. Meanwhile, lactose has been widely used as soluble carbon source for cellulase production [15]. In present study the FPA obtained 2.79 IU/ml with 40 g/l lactose, which showed higher inductive ability than other tested soluble substrates including isomalto-oligosaccharide, whey powder, sorbose and alginic acid. However, CSH and lactose induced less cellulase production than cellulose which achieved a FPA of 10.27 IU/ml. Similar results were obtained in previous study that pure cellulose had been generally considered as the best inducer for producing a well-balanced cellulase system with high yields of enzymes [13]. Impure cellulose including corn stalk and wheat straw pretreated by sulfuric acid was also proved to be efficient inducers for cellulase production of T. reesei D-7 achieving FPA of 4.84 and 4.17 IU/ml, respectively. Inulin, CMC-Na and 13
low-cost kelp showed a lower inductive ability. Moreover, the peaking time for FPA was around the 7th day for different substrates. To our knowledge, kelp and its extract alginic acid were firstly used as inducers for cellulase synthesis.
Table 2
3.5 Cellulase production using CSH and cellulose as inducers CSH and cellulose were proved as the best soluble and insoluble inducers for cellulase production of T. reesei D-7 among tested carbon sources (Table 2). As soluble substrates could decrease the viscosity of fermentation broth, while high enzymatic activity would be achieved with insoluble inducers, combination of CSH and cellulose was further used as inducer for enhanced cellulase secretion with decreased amount of cellulose. The maximum FPA was all achieved at the 7th day. As shown in Fig. 5, FPA was enhanced with supplementation of cellulose amount at low concentrations of CSH (10 g/l), and then declined as CSH increased from 20 to 40 g/l. By using the mixture of 20 g/l CSH and 30 g/l cellulose, the maximum FPA of 15.62 IU/ml was achieved, which was 3.45-fold and 1.52-fold higher than those obtained via addition of 40 g/l CSH and 40 g/l cellulose as inducers, respectively. The CMCase and β-glucosidase of individual and mixture carbon sources were further analyzed (Table 3). The results showed that CMCase increased with the increase of FPA, while β-glucosidases maintained at a low level for all substrates tested, because the cocktail of cellulase produced by T. reesei featured insufficient of β-glucosidases [27].
Fig. 5 and Table 3
4. Conclusions 14
Treatment of T. reesei Rut-C30 with EMS followed by plasma-irradiation was proved to be an efficient method to obtain high-cellulase producing mutants. The FPA of mutant T. reesei D-7 was 1.58-fold as high as that of the wild strain. CSH prepared by hydrolysis of corn starch with α-amylase effectively induced cellulase production in T. reesei D-7. Supplementation with 40 g/l of CSH achieved an FPA of 4.53 IU/ml. Moreover, the mixture of prepared CSH (20 g/l) and cellulose (30 g/l) was used as inducer for cellulase production by T. reesei D-7, achieving an FPA of 15.62 IU/ml. This study did not only obtain a good-performing mutant with high cellulase productivity, but also presented a feasible pathway to explore more cheap and accessible inducers.
Conflict of interest The authors have no conflict of interest to declare.
Acknowledgements This work was supported by the Natural Sciences Foundation of China (21406029, 21406030 and 21276038).
Reference [1] A. Gupta, J.P. Verma, Sustainable bio-ethanol production from agro-residues: A review, Renew. Sustain. Energ. Review. 41(2015) 550-567. [2] V. Parisutham, T.H. Kim, S.K. Lee, Feasibilities of consolidated bioprocessing microbes: From pretreatment to biofuel production, Bioresour. Technol. 161(2014) 431-440. [3] D. Klein-Marcuschamer, P. Oleskowicz-Popiel, B.A. Simmons, H.W. Blanch, The challenge of enzyme cost in the production of lignocellulosic biofuels, Biotechnol. Bioeng. 109(2012) 1083-1087. 15
[4] T. Hasunuma, F. Okazaki, N. Okai, K.Y. Hara, J. Ishii, A. Kondo, A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology, Bioresour. Technol. 135(2013) 513-522. [5] R. Peterson, H. Nevalainen, Trichoderma reesei RUT-C30-thirty years of strain improvement, Microbiol. 158(2012) 58-68. [6] Y.H.P. Zhang, M.E. Himmel, J.R. Mielenz, Outlook for cellulase improvement: Screening and selection strategies, Biotechnol. Adv. 24(2006) 452-481. [7] X. Zhang, X. Zhang, H. Li, L. Wang, C. Zhang, X. Xing, C. Bao, Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool, Appl. Microbiol. Biotechnol. 98(2014) 5387-5396. [8] R. Yao, M. Li, S. Deng, H. Hu, W. Huai, F. Li, Mutagenesis of Trichoderma viride by ultraviolet and plasma, Plasma Sci. Technol. 14(2012) 353-365. [9] S.K. Tangnu, H.W. Blanch, C.R. Wilke, Enhanced production of cellulase, hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30), Biotechnol. Bioeng. 23(1981) 1837-1849. [10] L.R. Lynd, P.J. Weimer, W.H. van Zyl, I.S. Pretorius, Microbial cellulose utilization: fundamentals and biotechnology, Microbiol. Mol. Biolog. Review. 66(2002) 506-577. [11] L.J. Ma, C. Li, Z.H. Yang, W.D. Jia, D.Y. Zhang, S.L. Chen, Kinetic studies on batch cultivation of Trichoderma reesei and application to enhance cellulase production by fed-batch fermentation, J. Biotechnol. 166(2013) 192-197. [12] A. Culbertson, M. Jin, L.C. Sousa, B.E. Dale, V. Balan, In-house cellulase production from AFEX™ pretreated corn stover using Trichoderma reesei RUT C-30, RSC Adv. 3(2013) 25960-25969. [13] L. Olssona, T.M.I.E. Christensen, K.P. Hansen, E.A. Palmqvist, Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei 16
Rut C-30, Enzyme Microb. Technol. 33(2003) 612-619. [14] M. Mandels, F.W. Parrish, E.T. Reese, Sophorose as an inducer of cellulase in Trichoderma viride, J Bacteriol. 83(1962) 400-408. [15] X. Fang, S. Yano, H. Ionue, S. Sawayama, Lactose enhances cellulase production by the filamentous fungus Acremonium cellulolyticus, J Biosci. Bioeng. 106(2008) 115-120. [16] M. Nogawa, M. Goto, H. Okada, Y. Morikawa, L-sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei, Cur. Genet. 38(2001) 329-334. [17] C.M. Lo, Q. Zhang, N.V. Callow, L. Ju, Cellulase production by continuous culture of Trichoderma reesei Rut-C30 using acid hydrolysate prepared to retain more oligosaccharides for induction, Bioresour. Technol. 101(2010) 717-723. [18] Y.H.P. Zhang, L.R. Lynd, Cellodextrin preparation by mixed-acid hydrolysis and chromatographic separation, Anal. Biochem. 322(2003) 225-232. [19] M. Wayman, S. Chen, Cellulase production be Trichoderma reesei using whole wheat flour as a carbon source, Enzyme Microb. Technol. 14(1992) 825-831. [20] X. Jiang, A. Geng, N. He, Q. Li, New isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production, J Biosci. Bioeng. 111(2011) 121-127. [21] M.R. Tansey, Agar-diffusion assay of cellulolytic ability of thermophilic fungi, Arch. Microbiol. 77(1971) 1-11. [22] M. Mandels, J. Weber, Production of cellulase, Adv. Chem. Series 95(1969) 391-414. [23] L.H. Zi, C.G. Liu, C.B. Xin, F.W. Bai, Stillage backset and its impact on ethanol fermentation by the flocculating yeast, Process Biochem. 48(2013) 753-758. [24] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31(1959) 426-428. [25] T.K. Ghose, Measurement of cellulase activities. Pur. Appl. Chem. 59(1987) 257-268. 17
[26] V. Novy, M. Schmid, M. Eibinger, Z. Petrasek, B. Nidetzky, The micromorphology of Trichoderma reesei analyzed in cultivations on lactose and solid lignocellulosic substrate, and its relationship with cellulase production, Biotechnol. Biofuels 9(2016) 169. [27] B. Wang, L. Xia, High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei, Bioresour. Technol. 102(2011) 4568-4572. [28] Y.H. Li, C.G. Liu, F.W. Bai, X.Q. Zhao, Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture, Bioresour. Technol. 216(2016) 503-510. [29] S. Chen, M. Wayman, Novel inducers derived from starch for cellulase production by Trichoderma reesei, Process Biochem. 27(1992) 327-334.
18
Table 1 Enzyme activity and soluble protein content of T. reesei Rut-C30 and its mutant T. reesei D-7
Organisms
CMCase,
β-glucosidase,
Protein,
U/ml
IU/ml
mg/ml
FPA, IU/ml
T. reesei Rut-C30
5.30 ± 0.13
84.92 ± 8.67
0.51 ± 0.02
1.36 ± 0.04
Mutant T. reesei D-7
8.35 ± 0.15
128.07 ± 9.84
0.71 ± 0.04
3.39 ± 0.28
19
Table 2 Effect of soluble and insoluble carbon sources on FPA of T. reesei D-7
Soluble
Insoluble
Carbon source
Maximum FPA, IU/ml
Time, d
CSH
4.53 ± 0.13
7
Lactose
2.79 ± 0.19
7
Isomalto-oligosaccharide
2.47 ± 0.21
6
Sorbose
2.41 ± 0.15
7
Alginic acid
1.81 ± 0.18
8
Whey powder
1.55 ± 0.17
7
Cellulose
10.27 ± 0.11
7
Pretreated corn stalk
4.84 ± 0.09
7
Pretreated wheat straw
4.17 ± 0.12
7
Inulin
2.38 ± 0.16
6
Kelp
1.86 ± 0.19
6
CMC-Na
1.43 ± 0.13
7
20
Table 3 Enzyme activity of T. reesei D-7 with CSH and/or cellulose as inducers β-glucosidase,
Cellulose, CSH, g/l
FPA, IU/ml
CMCase, IU/ml
g/l
IU/ml
20
0
1.12 ± 0.08
23.96 ± 0.57
0.83 ± 0.04
40
0
4.53 ± 0.13
55.84 ± 0.33
1.04 ± 0.10
20
30
15.62 ± 0.13
195.75 ± 11.32
0.95 ± 0.04
0
30
8.35 ± 0.22
129.98 ± 12.63
0.73 ± 0.07
0
40
10.27 ± 0.11
183.45 ± 13.49
0.69 ± 0.06
21
Figure legends Fig. 1. Cellulase production of the mutants and wild type strain T. reesei Rut-C30 in shake flask fermentation for 7 days. Mutants were selected from the second screening in phosphoric acid-swollen cellulose plates Fig. 2. Colonies of the mutants (T. reesei D-7, D-8, D-9) and T. reesei Rut-C30 formed on the screening plate containing 1% phosphoric-acid-swollen cellulose Fig. 3. Effect of nitrogen sources (30 g/l) on cellulase production of T. reesei D-7 with 40 g/L cellulose as carbon source Fig. 4. Time-course profiles of cellulase production (A) and reducing sugar (B) for T. reesei D-7 taking CSH as carbon source and yeast extract (30 g/l) as nitrogen source Fig. 5. Effect of initial CSH and cellulose content on cellulase production of T. reesei D-7
22
Fig. 1. Cellulase production of the mutants and wild type strain T. reesei Rut-C30 in shake flask fermentation for 7 days. Mutants were selected from the second screening in phosphoric acid-swollen cellulose plates
23
Fig. 2. Colonies of the mutants (T. reesei D-7, D-8, D-9) and T. reesei Rut-C30 formed on the screening plate containing 1% phosphoric-acid-swollen cellulose
24
Fig. 3. Effect of nitrogen sources (30 g/l) on cellulase production of T. reesei D-7 with 40 g/L cellulose as carbon source
25
Fig. 4. Time-course profiles of cellulase production (A) and reducing sugar (B) for T. reesei D-7 taking CSH as carbon source and yeast extract (30 g/l) as nitrogen source
26
Fig. 5. Effect of initial CSH and cellulose content on cellulase production of T. reesei D-7
27
S1359-5113(16)30518-9 http://dx.doi.org/doi:10.1016/j.procbio.2016.12.027 PRBI 10906
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
5-10-2016 29-11-2016 26-12-2016
Please cite this article as: Zhang Xiao-Yue, Zi Li-Han, Ge Xu-Meng, Li Yong-Hao, Liu Chen-Guang, Bai Feng-Wu.Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.12.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of Trichoderma reesei mutants by combined mutagenesis and induction of cellulase by low-cost corn starch hydrolysate Xiao-Yue Zhang1, Li-Han Zi1*, Xu-Meng Ge3, Yong-Hao Li1, 2, Chen-Guang Liu2*, Feng-Wu Bai1, 2 1
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024,
China 2
State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology,
Shanghai Jiao Tong University, Shanghai 200240, China 3
Department of Food, Agricultural and Biological Engineering, The Ohio State
University/Ohio Agricultural Research and Development Center, Wooster, OH 44691-4096, USA
*
Corresponding authors: LH Zi Tel: +86-411-8470-6785, fax: +86-411-8470-6329, E-mail: [email protected]; CG Liu, Tel: +86-21-3420-5125, fax: +86-21-3420-5708, E-mail: [email protected]
1
Graphical abstract
2
Highlights •
Trichoderma reesei mutants were obtained with chemical and irradiation treatment.
•
Mutant T. reesei D-7 showed 57% higher filter paper activity (FPA) than wild type.
•
Soluble inducer was prepared by hydrolysis of corn starch with α-amylase.
•
Mixture of corn starch hydrolysate and cellulose achieved higher FPA.
3
Abstract: The high cost of cellulase production is one of the key barriers to commercialization of cellulose-based biofuels. Screening high cellulase-producing strains and exploring low-cost inducers of cellulase are efficient strategies to address this issue. In this study, Trichoderma reesei Rut-C30 mutants were obtained by treatment with Ethyl Methyl Sulfonate (EMS) and plasma-irradiation. Mutant T. reesei D-7 was selected as the most promising cellulase producer based on screening through phosphoric acid-swollen cellulose plates. The filter paper activity (FPA) of T. reesei D-7 was 57.55% higher than that of the wild-type strain. Corn starch hydrolysate (CSH) prepared through hydrolysis of corn starch by α-amylase were used as soluble inducer for cellulase production with T. reesei D-7. FPA of 4.53 IU/ml was achieved at an optimal initial concentration of CSH (40 g/l reducing sugar). Moreover, CSH and cellulose were proved to be the most effective soluble and insoluble inducers for cellulase production with T. reesei D-7. Mixture of CSH and cellulose further improved FPA up to 15.62 IU/ml, which was 3.45-fold or 1.52-fold as high as that achieved using CSH or cellulose as an inducer.
Keywords: Trichoderma reesei; Mutation; Cellulase; Corn starch hydrolysate; Soluble inducer
4
1. Introduction Cellulosic biomass, such as agricultural and forestry residues, is the most abundant renewable carbon source with low cost in the world. Production of biofuels from cellulosic biomass has been widely recognized as a sustainable strategy for reducing dependence on non-renewable fossil fuels resources and mitigating the devastating effects of climate change due to overconsumption of fossil fuels [1]. Conversion of cellulosic biomass to biofuels usually includes three main steps: pretreatment to reduce recalcitrance, enzymatic hydrolysis to release fermentable sugars, and fermentation to produce biofuels [2]. Enzymatic hydrolysis is conducted with cellulolytic enzymes including exoglucanases, endoglucanases and β-glucosidases, which break down cellulose into glucose with cellobiase and soluble gluco-oligomers as intermediates [1]. However, high cost of cellulase production remains one of the key barriers to commercialization of biofuels from cellulosic biomass [3]. A variety of microorganisms, including bacteria and fungi, can produce cellulase [4], while most of cellulases exploited for industrial application are produced by filamentous fungi, such as Trichoderma and Penicillium. Compared to other strains, Trichoderma reesei Rut-C30 is well-known for its high cellulase secretion capacity, thus is suitable to be used as a good starting point for further improvement of cellulase production performance [5]. Both random mutagenesis and rational genetic modifications have been studied for improving cellulase production from Trichoderma reesei. Compared to rational genetic modifications, random mutagenesis methods are simple and easy to operate, though they have limitations, such as lack of stability. Random mutagenesis can be conducted using chemical mutagens, such as nitrous guanidine (NTG) and Ethyl Methyl Sulfonate (EMS), or physical mutagens, such as Ultraviolet (UV) [6]. Plasma-irradiation has recently been known as an effective mutagenesis method to obtain high-productivity strains with stable performance 5
[7,8]. Furthermore, different mutagenesis can be combined to improve the efficiency of mutation. Medium composition, especially carbon and nitrogen sources, is another important factor that affects the cellulase activity and productivity. Carbon sources can act as both energy supplier and inducer for cellulase production, and can be classified into soluble and insoluble carbon sources based on their solubility in water [9]. Cellulase biosynthesis can be induced by soluble and insoluble substrates such as lactose, cellulose and is repressed in the presence of readily metabolisable carbon sources such as glucose and xylose [10]. Many previous studies have shown that high cellulase activity was usually achieved using insoluble carbon sources including nearly pure cellulose, such as Avicel and Solka Floc, and impure cellulose-containing substrates such as lignocellulosic materials [11,12]. Among insoluble substrates, pure cellulose has been recognized as the most effective inducer for cellulase production of T. reesei, which achieving the FPA of 22.7 IU/ml with total 70 g/l cellulose by fed-batch fermentation [11]. However, the cost of pure cellulose is too high for industrial production [13]. Soluble substrates, such as sophorose, lactose, cellobiose and sorbitol, have been used to induce cellulase production, while sophorose was proved as the most effective inducer that, with low concentrations, achieved 2500 and 233 times higher cellulase productivity than cellobiose and lactose, respectively [14]. However, the high price of sophorose limited its industrial application. Lactose has been widely used as an un-expensive soluble substrate that can obtain higher cellulase activity than those for cellobiose and sorbitol [15,16]. A common drawback of monosaccharide inducers is that they can be easily consumed by microorganisms, resulting in reduced efficiency of induction. This issue can be addressed by using soluble oligosaccharides that are difficult to be utilized by the developed strain. Currently, oligosaccharide inducers are prepared through hydrolysis of lignocellulose or 6
starch using inorganic acid such as hydrogen chloride and sulfuric acid [17,18]. However, detoxification process may be needed to remove inhibitors generated during hydrolysis process, which increases operating costs. Alternatively, oligosaccharides can be prepared through hydrolysis of corn starch with α-amylase, which is known as the first step for industrial production of glucose from corn starch. Enzymatic hydrolysis does not produce inhibitors, and requires only mildly operating conditions and low-cost of α-amylase. As a result, the corn starch hydrolysate (CSH) prepared by enzymatic hydrolysis of corn starch with α-amylase could be an excellent soluble inducer for cellulase production with T. reesei. To the authors’ knowledge, there is only one report on using wheat flour hydrolysate as an inducer for cellulase production, but without information about the cellulase activity [19]. In this study, the wild strain T. reesei Rut-C30 was treated by random mutagenesis with EMS followed by plasma-irradiation and mutant T. reesei D-7 was isolated by screening on phosphoric acid-swollen cellulose plate and evaluated by liquid cultivation. The selected mutant T. reesei D-7 was further investigated for cellulase production using CSH as soluble inducer prepared by enzymatic hydrolysis of corn starch. Moreover, cellulase production of T. reesei D-7 using CSH and cellulose as mixed inducer substrates was performed for the first time.
2. Material and Methods 2.1 Microorganisms and culture media The cellulase producing fungi T. reesei Rut-C30 was a gift from Agricultural Research Service Culture Collection (NRRL 11460), USA. The T. reesei Rut-C30 and its mutants were maintained on Malt Extract Agar medium (MEA medium, malt extract 30 g/l and agar 15 g/l) plates and sub-cultured every one week. Selection medium (SM) contained (g/l): phosphoric acid-swollen cellulose (PASC) 10, 7
sorbose 4, Triton X-100 1, KH2PO4 2.0, MgSO4·7H2O 0.3, CaCl2·2H2O 0.4, (NH4)2SO4 1.4, agar 15 [20,21]. Inoculum medium contained Basal Medium (BM) described by Mandels and Weber [22] with modification (g/l): lactose 10; peptone 1; (NH4)2SO4 1.4; KH2PO4 2.0; MgSO4·7H2O 0.3; CaCl2·2H2O 0.4; FeSO4·7H2O 0.005; MnSO4·H2O 0.0017; ZnSO4·7H2O 0.0014; CoCl2·2H2O 0.002; Urea 0.3. The fermentation medium was the same as cultivation medium except that 40 g/l cellulose (microcrystalline cellulose, Sangon Company, Shanghai, China), 30 g/l soy flour and 20 g/l wheat bran were used instead of lactose and peptone. The initial pH was adjusted to 4.8 by 0.2 mol/l Na2HPO4-citric acid (pH 5.0) without control during cultivation. The medium was autoclaved at 121ºC for 15 min.
2.2 Cellulase production in shake flasks Batch cultivation experiments were carried out in 250 ml Erlenmeyer flasks. For inoculum preparation, one ml of a spore suspension (108 spores/ml) was inoculated into 50 ml of inoculum medium in the flasks which were cultured at 28ºC, 150 rpm for 2 days. The seed was inoculated into 50 ml fermentation medium in 250 ml Erlenmeyer flasks at an inoculum ratio of 8% (v/v), at 28ºC with shaking at 150 rpm for 7 days. The samples were removed daily and were centrifuged at 4000 rpm for 5 min. The supernatant was analyzed for extra cellular enzyme activities and soluble proteins.
2.3 Mutagenesis and screening of mutants The strain was grown on MEA plates for 7 days at 28ºC for spore development. Spores were then harvested and washed with 0.1% Tween-80 solution and the spore concentration was diluted to 108 spores/ml. Random mutagenesis was first conducted using ethyl methyl sulfonate (EMS) treatment according to the method described by Jiang et al. [20] and followed by plasma-irradiation treatment. 50 μl EMS was added to 10 ml of the spore 8
suspension and it was kept at room temperature for 24 h, then followed by plasma-irradiation at 24 V, 1.7 A and gap 3 mm for 5 min corresponding to a lethal rate of 78.38%. 100 μl the treated spore suspension was subsequently spread on the screening plates and cultured at 28ºC for 4-7 days. The mutants were selected on the basis of clearance zones appearing on SM medium. The final selected mutants were then inoculated in MEA medium for 7 days and the cellulase activity of mutants were assessed in shake flasks to select the most promising enzyme producing mutant.
2.4 Corn starch hydrolysate (CSH) preparation Corn starch hydrolysate (CSH) was prepared using α-amylase hydrolysis of corn starch at 40% (w/v) dry solid with 5 IU/g corn starch at 93-95ºC under constant stirring condition [23]. The hydrolysate was sampled at 30, 60 and 90 min, respectively. After cooling, liquefied CSH were recovered by filtration. Total reducing sugar in the liquefied CSH were 300 g/l determined by dinitrosalicylic acid (DNS) method [24], which contained 24 g/l glucose determined by biological sensor(SBA-40, Institute of Biological, Shandong Academy of Sciences, Jinan, China).
2.5 Analytical methods The supernatants of samples were suitably diluted before analyzed extra cellular enzyme activities and soluble protein. Filter paper activity (FPA) was used to estimate total cellulase activity according to Ghose [25] by incubating 0.5 ml enzyme with 1.0 ml citrate buffer (0.2 M, pH 4.8) containing one Whatman No.1 filter paper strip (50 mg, 1×6 cm). The reaction mixture was incubated at 50ºC for 60 min. Endoglucanase activity (CMCase) was determined by incubating 0.5 ml enzyme with 1.0 ml of 1% (w/v) carboxymethylcellulose (CMC) dissolved in citrate buffer and the reaction mixture was incubated at 50ºC for 15 min. 9
β-glucosidase activity was carried out in the total reaction mixture of 1.5 ml containing 0.5 ml enzyme and 1.0 ml of 15 mM cellobiose solution in citrate buffer and the mixture was incubated at 50ºC for 10 min. The reducing sugar was determined by DNS method and glucose was determined by biological sensor. One unit of enzyme activity was defined as the amount of enzyme required for liberating 1 mmol/l of glucose per ml per minute under the assay conditions. Concentrations of secreted proteins were measured using Bradford reagent (Sangon Biotech, China). All experimental results are presented as mean and standard deviations of triplicated independent experiments with standard deviations at significance p0.05.
3.1 Results and discussion 3.1 Mutagenesis and screening of mutants After mutagenesis, 60 positive colonies were initially selected on the basis of forming clear zones of hydrolysis of phosphoric acid-swollen cellulose in the selection medium (SM). Then the colonies were transferred again to SM and 20 mutants were screened according to the size of clearance zone surrounding the colony. These potential mutants were further assessed for cellulase production in shake flasks. The FPA of mutants and the wild strain was shown in Fig. 1, 13 mutants had 1.42-57.55% higher FPA than wild strain T. reesei Rut-C30. D-7 demonstrated the highest FPA of 8.35 IU/ml, which was higher than the mutant of Rut-C30 (8 IU/ml) by diethyl sulfate and UV mutagenesis using 50 g/l cellulose as inducer [11]. Moreover, D-7 presented larger clear zone than Rut-C30 and mutants D-8 and D-9 in the second round of screening (Fig. 2). In addition, it should be noted that the conidia (green) of mutant D-7 was decreased compared with other three strains in SM medium, which is consistent to higher cellulase secretion [26]. Moreover, the stability of cellulase production was evaluated with serial sub-culture in MEA medium for 8 times. The FPA in supernatant of 10
fermentation broth showed little changed, indicating a stable productive capacity of mutant T. reesei D-7 for cellulase production.
Fig. 1 and Fig. 2
The mutant T. reesei D-7 was further evaluated for extracellular production of cellulase, CMCase, and β-glucosidase in MEA medium for 7 days (Table 1). CMCase of T. reesei D-7 achieved 128.07 IU/ml, increasing by 50.81% compared with wild strain T. reesei Rut-C30 (84.92 IU/ml). The β-glucosidase of D-7 and Rut-C30 were obtained at 0.71 IU/ml and 0.51 IU/ml, respectively, which showed the low level for both mutant and the wild strain. This was corresponding with previous study that T. reesei could produce cellobiohydrolases (CBH) and endo-glucanases (EG) components of cellulase in large quantities but the amount of β-glucosidase was insufficient [27]. The D-7 could secret high mount of CMCase but low level of β-glucosidase because of screening scheme by using phosphoric acid-swollen cellulose (PASC) that preferred the strain with CMCase activity. The soluble protein content of crude enzymes from T. reesei D-7 was 3.39 mg/ml, 2.49-fold higher than wild strain, which reflected that the enhancement of cellulase production by mutant strain was owing to the increased of secretion of the enzyme.
Table 1
3.2 Effect of nitrogen sources on cellulase production by mutant T. reesei D-7 Four different nitrogen sources including soy flour, yeast extract, corn steep liquor and peptone were tested for their effects on cellulase production of mutant T. reesei D-7 (Fig. 3). Yeast extract achieved maximum FPA of 10.27 IU/ml on day-6 which was 1.34, 1.42 and 11
1.58-fold higher than those obtained by using soy flour, corn steep liquor and peptone (7.68, 7.22 and 6.48 IU/ml), respectively, on day-7. Actually, on day-5, yeast extract has already achieved a FPA of 8.89 IU/ml, which was much (more than 2.39-fold) higher than those obtained by using other nitrogen sources. Therefore, yeast extract was chosen as a suitable nitrogen source for cellulase production of T. reesei D-7.
Fig. 3
3.3 Cellulase production using CSH as inducer In order to detect the effect of CSH prepared with different reaction time on cellulase production of mutant T. reesei D-7, the hydrolysates were sampled at 30, 60, and 90 min, which containing 300, 309 and 321 g/l of total reducing sugar, and 24, 28 and 30 g/l of glucose, respectively. However, there was no significant difference of FPA by using hydrolysates sampled at different time. Therefore, the hydrolysis time 30 min was selected for cost reduction. The cellulase production experiment was carried out at different total reducing sugar ranging from 10 to 50 g/l with yeast extract as nitrogen source. The result showed that the highest FPA increased with the reducing sugar concentration increased from 10 to 40 g/l and reached maximum 4.53 IU/ml at 40 g/l (Fig. 4A). The maximum FPA decreased slightly when the concentration of the total reducing sugar further increased to 50 g/l. Furthermore, the reducing sugar was quickly consumed at low initial concentration (10-30 g/l) and the residual sugar declined to roughly 1 g/l at the 3th or 4th days, which may result in the low cellulase production (Fig. 4B). Conversely, the reducing sugars were consumed at the 6th day when 40 or 50 g/l CSH were added and the FPA reached the highest level afterward (the 7th day). The delay of achieving maximum FPA was consistent with previous study [28]. 12
Fig. 4
Previous studies have shown that wheat starch hydrolysate prepared by acid hydrolysis rendered effective inducing ability, but the starch itself was a poor inducer for cellulase production by T. reesei [29]. The acid hydrolysis of starch resulted in the formation of reversion products from glucose, such as sophorose, which have been considered as the real inducers of cellulase production in Trichoderma species. Moreover, our study also showed that large amount of disaccharide, trisaccharide, etc. were formed after enzymatic hydrolysis of corn starch (data not shown), which may render inductive ability of cellulase biosynthesis of T. reesei.
3.4 Cellulase production using different soluble and insoluble inducers Various soluble and insoluble substrates (40 g/l) were tested as carbon sources for their effect on cellulase production of mutant T. reesei D-7. As depicted in Table 2, among the soluble substrates tested, CSH induced the highest FPA. Meanwhile, lactose has been widely used as soluble carbon source for cellulase production [15]. In present study the FPA obtained 2.79 IU/ml with 40 g/l lactose, which showed higher inductive ability than other tested soluble substrates including isomalto-oligosaccharide, whey powder, sorbose and alginic acid. However, CSH and lactose induced less cellulase production than cellulose which achieved a FPA of 10.27 IU/ml. Similar results were obtained in previous study that pure cellulose had been generally considered as the best inducer for producing a well-balanced cellulase system with high yields of enzymes [13]. Impure cellulose including corn stalk and wheat straw pretreated by sulfuric acid was also proved to be efficient inducers for cellulase production of T. reesei D-7 achieving FPA of 4.84 and 4.17 IU/ml, respectively. Inulin, CMC-Na and 13
low-cost kelp showed a lower inductive ability. Moreover, the peaking time for FPA was around the 7th day for different substrates. To our knowledge, kelp and its extract alginic acid were firstly used as inducers for cellulase synthesis.
Table 2
3.5 Cellulase production using CSH and cellulose as inducers CSH and cellulose were proved as the best soluble and insoluble inducers for cellulase production of T. reesei D-7 among tested carbon sources (Table 2). As soluble substrates could decrease the viscosity of fermentation broth, while high enzymatic activity would be achieved with insoluble inducers, combination of CSH and cellulose was further used as inducer for enhanced cellulase secretion with decreased amount of cellulose. The maximum FPA was all achieved at the 7th day. As shown in Fig. 5, FPA was enhanced with supplementation of cellulose amount at low concentrations of CSH (10 g/l), and then declined as CSH increased from 20 to 40 g/l. By using the mixture of 20 g/l CSH and 30 g/l cellulose, the maximum FPA of 15.62 IU/ml was achieved, which was 3.45-fold and 1.52-fold higher than those obtained via addition of 40 g/l CSH and 40 g/l cellulose as inducers, respectively. The CMCase and β-glucosidase of individual and mixture carbon sources were further analyzed (Table 3). The results showed that CMCase increased with the increase of FPA, while β-glucosidases maintained at a low level for all substrates tested, because the cocktail of cellulase produced by T. reesei featured insufficient of β-glucosidases [27].
Fig. 5 and Table 3
4. Conclusions 14
Treatment of T. reesei Rut-C30 with EMS followed by plasma-irradiation was proved to be an efficient method to obtain high-cellulase producing mutants. The FPA of mutant T. reesei D-7 was 1.58-fold as high as that of the wild strain. CSH prepared by hydrolysis of corn starch with α-amylase effectively induced cellulase production in T. reesei D-7. Supplementation with 40 g/l of CSH achieved an FPA of 4.53 IU/ml. Moreover, the mixture of prepared CSH (20 g/l) and cellulose (30 g/l) was used as inducer for cellulase production by T. reesei D-7, achieving an FPA of 15.62 IU/ml. This study did not only obtain a good-performing mutant with high cellulase productivity, but also presented a feasible pathway to explore more cheap and accessible inducers.
Conflict of interest The authors have no conflict of interest to declare.
Acknowledgements This work was supported by the Natural Sciences Foundation of China (21406029, 21406030 and 21276038).
Reference [1] A. Gupta, J.P. Verma, Sustainable bio-ethanol production from agro-residues: A review, Renew. Sustain. Energ. Review. 41(2015) 550-567. [2] V. Parisutham, T.H. Kim, S.K. Lee, Feasibilities of consolidated bioprocessing microbes: From pretreatment to biofuel production, Bioresour. Technol. 161(2014) 431-440. [3] D. Klein-Marcuschamer, P. Oleskowicz-Popiel, B.A. Simmons, H.W. Blanch, The challenge of enzyme cost in the production of lignocellulosic biofuels, Biotechnol. Bioeng. 109(2012) 1083-1087. 15
[4] T. Hasunuma, F. Okazaki, N. Okai, K.Y. Hara, J. Ishii, A. Kondo, A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology, Bioresour. Technol. 135(2013) 513-522. [5] R. Peterson, H. Nevalainen, Trichoderma reesei RUT-C30-thirty years of strain improvement, Microbiol. 158(2012) 58-68. [6] Y.H.P. Zhang, M.E. Himmel, J.R. Mielenz, Outlook for cellulase improvement: Screening and selection strategies, Biotechnol. Adv. 24(2006) 452-481. [7] X. Zhang, X. Zhang, H. Li, L. Wang, C. Zhang, X. Xing, C. Bao, Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool, Appl. Microbiol. Biotechnol. 98(2014) 5387-5396. [8] R. Yao, M. Li, S. Deng, H. Hu, W. Huai, F. Li, Mutagenesis of Trichoderma viride by ultraviolet and plasma, Plasma Sci. Technol. 14(2012) 353-365. [9] S.K. Tangnu, H.W. Blanch, C.R. Wilke, Enhanced production of cellulase, hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30), Biotechnol. Bioeng. 23(1981) 1837-1849. [10] L.R. Lynd, P.J. Weimer, W.H. van Zyl, I.S. Pretorius, Microbial cellulose utilization: fundamentals and biotechnology, Microbiol. Mol. Biolog. Review. 66(2002) 506-577. [11] L.J. Ma, C. Li, Z.H. Yang, W.D. Jia, D.Y. Zhang, S.L. Chen, Kinetic studies on batch cultivation of Trichoderma reesei and application to enhance cellulase production by fed-batch fermentation, J. Biotechnol. 166(2013) 192-197. [12] A. Culbertson, M. Jin, L.C. Sousa, B.E. Dale, V. Balan, In-house cellulase production from AFEX™ pretreated corn stover using Trichoderma reesei RUT C-30, RSC Adv. 3(2013) 25960-25969. [13] L. Olssona, T.M.I.E. Christensen, K.P. Hansen, E.A. Palmqvist, Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei 16
Rut C-30, Enzyme Microb. Technol. 33(2003) 612-619. [14] M. Mandels, F.W. Parrish, E.T. Reese, Sophorose as an inducer of cellulase in Trichoderma viride, J Bacteriol. 83(1962) 400-408. [15] X. Fang, S. Yano, H. Ionue, S. Sawayama, Lactose enhances cellulase production by the filamentous fungus Acremonium cellulolyticus, J Biosci. Bioeng. 106(2008) 115-120. [16] M. Nogawa, M. Goto, H. Okada, Y. Morikawa, L-sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei, Cur. Genet. 38(2001) 329-334. [17] C.M. Lo, Q. Zhang, N.V. Callow, L. Ju, Cellulase production by continuous culture of Trichoderma reesei Rut-C30 using acid hydrolysate prepared to retain more oligosaccharides for induction, Bioresour. Technol. 101(2010) 717-723. [18] Y.H.P. Zhang, L.R. Lynd, Cellodextrin preparation by mixed-acid hydrolysis and chromatographic separation, Anal. Biochem. 322(2003) 225-232. [19] M. Wayman, S. Chen, Cellulase production be Trichoderma reesei using whole wheat flour as a carbon source, Enzyme Microb. Technol. 14(1992) 825-831. [20] X. Jiang, A. Geng, N. He, Q. Li, New isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production, J Biosci. Bioeng. 111(2011) 121-127. [21] M.R. Tansey, Agar-diffusion assay of cellulolytic ability of thermophilic fungi, Arch. Microbiol. 77(1971) 1-11. [22] M. Mandels, J. Weber, Production of cellulase, Adv. Chem. Series 95(1969) 391-414. [23] L.H. Zi, C.G. Liu, C.B. Xin, F.W. Bai, Stillage backset and its impact on ethanol fermentation by the flocculating yeast, Process Biochem. 48(2013) 753-758. [24] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31(1959) 426-428. [25] T.K. Ghose, Measurement of cellulase activities. Pur. Appl. Chem. 59(1987) 257-268. 17
[26] V. Novy, M. Schmid, M. Eibinger, Z. Petrasek, B. Nidetzky, The micromorphology of Trichoderma reesei analyzed in cultivations on lactose and solid lignocellulosic substrate, and its relationship with cellulase production, Biotechnol. Biofuels 9(2016) 169. [27] B. Wang, L. Xia, High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei, Bioresour. Technol. 102(2011) 4568-4572. [28] Y.H. Li, C.G. Liu, F.W. Bai, X.Q. Zhao, Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture, Bioresour. Technol. 216(2016) 503-510. [29] S. Chen, M. Wayman, Novel inducers derived from starch for cellulase production by Trichoderma reesei, Process Biochem. 27(1992) 327-334.
18
Table 1 Enzyme activity and soluble protein content of T. reesei Rut-C30 and its mutant T. reesei D-7
Organisms
CMCase,
β-glucosidase,
Protein,
U/ml
IU/ml
mg/ml
FPA, IU/ml
T. reesei Rut-C30
5.30 ± 0.13
84.92 ± 8.67
0.51 ± 0.02
1.36 ± 0.04
Mutant T. reesei D-7
8.35 ± 0.15
128.07 ± 9.84
0.71 ± 0.04
3.39 ± 0.28
19
Table 2 Effect of soluble and insoluble carbon sources on FPA of T. reesei D-7
Soluble
Insoluble
Carbon source
Maximum FPA, IU/ml
Time, d
CSH
4.53 ± 0.13
7
Lactose
2.79 ± 0.19
7
Isomalto-oligosaccharide
2.47 ± 0.21
6
Sorbose
2.41 ± 0.15
7
Alginic acid
1.81 ± 0.18
8
Whey powder
1.55 ± 0.17
7
Cellulose
10.27 ± 0.11
7
Pretreated corn stalk
4.84 ± 0.09
7
Pretreated wheat straw
4.17 ± 0.12
7
Inulin
2.38 ± 0.16
6
Kelp
1.86 ± 0.19
6
CMC-Na
1.43 ± 0.13
7
20
Table 3 Enzyme activity of T. reesei D-7 with CSH and/or cellulose as inducers β-glucosidase,
Cellulose, CSH, g/l
FPA, IU/ml
CMCase, IU/ml
g/l
IU/ml
20
0
1.12 ± 0.08
23.96 ± 0.57
0.83 ± 0.04
40
0
4.53 ± 0.13
55.84 ± 0.33
1.04 ± 0.10
20
30
15.62 ± 0.13
195.75 ± 11.32
0.95 ± 0.04
0
30
8.35 ± 0.22
129.98 ± 12.63
0.73 ± 0.07
0
40
10.27 ± 0.11
183.45 ± 13.49
0.69 ± 0.06
21
Figure legends Fig. 1. Cellulase production of the mutants and wild type strain T. reesei Rut-C30 in shake flask fermentation for 7 days. Mutants were selected from the second screening in phosphoric acid-swollen cellulose plates Fig. 2. Colonies of the mutants (T. reesei D-7, D-8, D-9) and T. reesei Rut-C30 formed on the screening plate containing 1% phosphoric-acid-swollen cellulose Fig. 3. Effect of nitrogen sources (30 g/l) on cellulase production of T. reesei D-7 with 40 g/L cellulose as carbon source Fig. 4. Time-course profiles of cellulase production (A) and reducing sugar (B) for T. reesei D-7 taking CSH as carbon source and yeast extract (30 g/l) as nitrogen source Fig. 5. Effect of initial CSH and cellulose content on cellulase production of T. reesei D-7
22
Fig. 1. Cellulase production of the mutants and wild type strain T. reesei Rut-C30 in shake flask fermentation for 7 days. Mutants were selected from the second screening in phosphoric acid-swollen cellulose plates
23
Fig. 2. Colonies of the mutants (T. reesei D-7, D-8, D-9) and T. reesei Rut-C30 formed on the screening plate containing 1% phosphoric-acid-swollen cellulose
24
Fig. 3. Effect of nitrogen sources (30 g/l) on cellulase production of T. reesei D-7 with 40 g/L cellulose as carbon source
25
Fig. 4. Time-course profiles of cellulase production (A) and reducing sugar (B) for T. reesei D-7 taking CSH as carbon source and yeast extract (30 g/l) as nitrogen source
26
Fig. 5. Effect of initial CSH and cellulose content on cellulase production of T. reesei D-7
27