Enhanced cellulase production by Trichoderma viride ...

Bioresource Technology 133 (2013) 175–182

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Enhanced cellulase production by Trichoderma viride in a rotating fibrous bed bioreactor Tian-Qing Lan a, Dong Wei a,⇑, Shang-Tian Yang a,b,⇑, Xiaoguang Liu c a

State Key Laboratory of Pulp and Paper Engineering, School of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Rd., Guangzhou 510640, PR China William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA c Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL 35487, USA b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Cellulase production by Trichoderma

viride in bioreactors was studied. " Immobilization in fibrous bed

enhanced cellulase production from sugarcane bagasse. " Better reactor operation and control were achieved with improved biofilm morphology. " Higher saccharification yield can be attributed to the higher FPase activity. " Proteomics analysis showed differential expressions of cellulases (CBH and EG).

a r t i c l e

i n f o

Article history: Received 25 September 2012 Received in revised form 17 January 2013 Accepted 19 January 2013 Available online 31 January 2013 Keywords: Cellulase Sugarcane bagasse Trichoderma viride Fibrous-bed bioreactor Proteome

a b s t r a c t Filamentous fungi are widely used to produce cellulase, but how the fermentation conditions affect their production is not well known. In this study, cellulase production by Trichoderma viride in submerged fermentations with free cells in a stirred-tank reactor (STR) and immobilized cells in a rotating fibrous-bed bioreactor (RFBB) were investigated. Compared to free-cell fermentation, immobilized-cell fermentation gave 35.5% higher FPase activity and 69.7% higher saccharification yield of sugarcane bagasse (SCB). The secretory proteins in the fermentation broths were analyzed with two-dimensional gel electrophoresis (2-DE) and MALDI-TOF–TOF mass spectrometry, which identified 24 protein spots with differential expression levels. Among them, cellobiohydrolase CBH II and endoglucanase EG II were highly expressed and secreted in the immobilized-cell fermentation, while the free-cell fermentation produced more CBH I and EG IV. These results showed that immobilized-cell fermentation with T. viride in the RFBB was advantageous for cellulase production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Lignocellulases are widely used in food, animal feed, textile, pulp and paper industries, and especially in the production of bioethanol. Lignocellulases mainly include cellulase, hemicellulase ⇑ Corresponding authors at: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA. Tel.: +1 614 2926611; fax: +1 614 2923769 (S.-T. Yang), tel./fax: +86 20 87113849 (D. Wei). E-mail addresses: [email protected] (D. Wei), [email protected] (S.-T. Yang). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.088

and ligninase. Cellulase is a group of enzymes, which can be classified into several types based on the reactions they catalyze, including endoglucanase (EG) or carboxymethyl cellulase (CMCase), exoglucanase (such as FPase) or cellobiohydrolase (CBH), cellobiase or b-glucosidase. The synergistic action of these enzymes plays an important role in the hydrolysis of cellulose. Hemicellulase consists of xylanase, xylosidase, mannanase, mannosidase and other enzymes, with xylanase as the most significant one. The high cost and low efficiency of hydrolyzing lignocellulosic biomass to fermentable sugars negatively impact the economics of bioethanol and other biobased fuels and chemicals (Adsul et al.,

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2007; Xia and Shen, 2004). How to reduce the production cost of cellulase and increase its efficiency in cellulose hydrolysis has long been a challenging problem. It is thus desirable to develop novel fermentation processes for economical production of highly efficient cellulase for biorefinery. Current biorefineries require low-cost, high-efficient cellulase, which is commercially produced by using mainly filamentous fungi, such as Aspergillus niger and Trichoderma reesei, in submerged fermentation. However, the process usually suffers from poor mass transfer due to difficulty in controlling the fungal morphology during fermentation (Domingues et al., 2000). Extensive research has thus focused on the immobilization of fungal spores and mycelia on solid supports such as cotton towel, polyester fabrics, polyurethane foam, loofah sponge, and alginate beads for morphology control (Akihiro et al., 2007; Talabardon and Yang, 2005; Thongchul and Yang, 2006; Wang et al., 2010; Xia and Shen, 2004). Cell immobilization also facilitates the reuse of cells, which can increase reactor productivity due to increased cell density and also simplify product recovery. However, cell immobilization by entrapment in porous particles or matrices could impose mass transfer limitation and reduce reactor productivity. Recently, a rotating fibrous-bed bioreactor (RFBB) was developed to immobilize fungal mycelia, which attached and formed a biofilm on a rotating surface along with the agitation shaft in a stirred-tank bioreactor (Tay and Yang, 2002; Xu and Yang, 2007). The RFBB was successfully used to produce lactic acid by Rhizopus oryzae and mycophenolic acid by Penicillium brevicompactum in repeated batch and fed-batch fermentations, with greatly increased product titers, productivities and operational stability because of the improved oxygen transfer to and product secretion from the biofilm and better pH and DO controls in the virtually cell-free fermentation broth, which also had a reduced viscosity and facilitated down-stream processing. Fungal cells immobilized on a solid surface or forming a biofilm have also been shown to possess higher productivities for the production of secretary proteins compared to suspended cell clumps and pellets (Talabardon and Yang, 2005). With these advantages, the RFBB can be beneficially used to produce industrial enzymes such as cellulase, which has not been investigated before, however. The objective of this study was thus to investigate the feasibility and potential advantages of producing cellulase from SCB and wheat bran by Trichoderma viride immobilized in the RFBB. T. viride is a cellulase-overproducing filamentous fungus and has been extensively applied in industry. However, its use in an immobilized culture for cellulase production has not been well studied before. In this work, the fermentation kinetics of T. viride as biofilm in the RFBB and as freely suspended mycelia and clumps in conventional stirred-tank bioreactors were studied. The production of cellulase and xylanase in these fermentations was compared for their activities and abilities to hydrolyze SCB to reducing sugars. In addition, the secretory proteins of T. viride in these fermentations were analyzed with two-dimensional electrophoresis (2-DE) and MAL-

DI-TOF–TOF mass spectrometry to identify the major types of cellulase produced under different fermentation conditions, which also provided a proteomic map for the secretory proteins of T. viride. Finally, the mycelial morphology in the RFBB was examined and the effects of different morphologies resulting from immobilization are discussed in this paper. 2. Methods 2.1. Culture and medium Fresh SCB (Jiangmen Sugarcane Chemical Engineering Co. Ltd., Guangdong, China) and wheat bran (Jiangsu Sanniu Flour Co. Ltd., Jiangsu, China) were dried at 70 °C for 24 h, and then treated with micro-milling (BFM-6BII Model, Jinan Billionpowder Tec & Eng Co., Ltd, Shandong, China) to reduce the particle size to 100 lm and used as the carbon source in fermentation. Wheat bran contained mainly cellulose (40% w/w), lignin (3%), protein, and minerals, and was used to induce cellulase production. The SCB contained (wt%) 41.6% cellulose, 26.4% hemicellulose, and 19.8% lignin. The SCB treated with 2% NaOH at 80 °C for 1 h contained 50.8% cellulose, 30.8% hemicelluloses, and 12.6% lignin. Unless otherwise noted, T. viride QM 9414 used in this study was cultured in the medium containing (g/L) dried SCB powder, 10; wheat bran, 10; urea, 0.3; KH2PO4, 3.0; MgSO47H2O, 0.5; CaCl2, 0.5 and trace element solution, 1 mL. The trace element solution consisted of (g/ L): FeSO47H2O, 7.5; ZnSO47H2O, 2.0; MnCl24H2O, 3.0; and CoCl2, 3.0. The initial pH of the medium was 5.02. To prepare the spores for the fermentation study, T. viride was cultured on solid medium with 2% agar in petri dishes at 28 °C for 6 days. Spores were then harvested, suspended in sterile distilled water, and used to inoculate the fermentation described below. 2.2. Free-cell fermentation in STR Batch fermentation was carried out in a 7-L stirred-tank reactor (STR) containing 5 L of the medium. After autoclaving at 121 °C for 20 min twice, the bioreactor was inoculated with 10 mL of a spore suspension (108/mL) and then operated at 28 °C, with aeration at 0.5–1.0 vvm and agitation at 120 rpm for 16 days. The medium pH was self-maintained at 4–6 without pH control. Air enriched with oxygen was sparged to bioreactor to maintain the dissolved oxygen (DO) at >10%. Broth samples were taken every other day, centrifuged at 13,000 rpm for 10 min, and the supernatants were collected and stored at 70 °C for further analysis. 2.3. Immobilized-cell fermentation in RFBB The rotating fibrous-bed bioreactor (RFBB) was modified from a 7-L bioreactor (see Fig. 1). Briefly, a perforated stainless steel

Fig. 1. Schematic of the rotating fibrous bed bioreactor (RFBB) and photos showing the biofilm in the RFBB and free cells and mycelial clumps in the stirred tank bioreactor (STR).


cylinder (9 cm in diameter, 15 cm in height) affixed with a polypropylene cloth (thickness: 0.4 mm) for cell immobilization was mounted onto the stirring shaft of the bioreactor. Cotton cloth was also tested but was not chosen for cell immobilization because cotton cloth would be degraded by the cellulase secreted by T. viride. The bioreactor containing 5 L of the medium was autoclaved at 121 °C for 20 min twice. After cooling, the bioreactor was inoculated with 10 mL of spore suspension (108/mL) and then operated at 28 °C with aeration at 0.5–1.0 vvm. The rotational speed of the RFB was initially at 60 rpm and then increased to 90 rpm when SCB, wheat bran and mycelia had all been adsorbed onto the polypropylene cloth. Oxygen-enriched air was sparged to keep DO at >10% during the fermentation. Broth samples were taken every other day and centrifuged at 13,000 rpm for 10 min to collect the supernatant, which was stored at 70 °C for further analysis. 2.4. Saccharification of SCB Unless otherwise noted, the saccharification of SCB was carried out in a citrate buffer (50 mM, pH 4.8) by mixing 1.0 mL of the buffer with 50 mg of SCB and 0.5 mL of the fermentation broth supernatant. The mixture was incubated at 50 °C with agitation at 140 rpm for 48 h. For comparison, commercial cellulase (ACCELLERASEÒ 1500, Genencor, NY, USA) and concentrated fermentation broth were also studied at the same enzyme loading of 8 FPU/g SCB (0.4 FPU enzyme in 1.5 mL buffer containing 50 mg of SCB). No wheat bran was added in these experiments. The saccharification yield was calculated based on the amount of the reducing sugars (RS) obtained in the hydrolysate divided by the total carbohydrate present in SCB and times 0.9 (to correct the increased weight from hydrolysis), as shown in the following equation:

Saccharification yieldð%Þ ¼ RSðgÞ 0:9 100=total carbohydrateðgÞ: The carbohydrate content of dried SCB was 67.97% (w/w). All samples, enzyme blank and substrate blank were tested in duplicate. 2.5. Two-dimensional electrophoresis (2-DE) The broth supernatant was ultrafiltered to concentrate proteins and eliminate salts using a 10 kD membrane (Millipore, USA). Then, acetone (20 °C) was slowly added into the concentrates. The volume ratio of acetone to the concentrate was 4:1. The mixture was kept at 20 °C overnight to precipitate proteins, and then was centrifuged at 12,000 rpm for 20 min. The precipitates were rinsed with the ice-cold acetone of 20 °C three times. The protein pellet was air-dried, resuspended in lysis buffer (7 M urea, 2 M thiourea, 4% chaps, 1% DTT), and vortexed for 1 h at room temperature. 100 lg of proteins were loaded onto pH 3–6 IPG strips (24 cm, linear). Isoelectrofocusing (IEF) and SDS–PAGE were carried out following the well established procedures described elsewhere (Bah et al., 2010). For each protein sample, three 2-DE gels were prepared. Two gels were silver-stained for image analysis and one was stained with Coomassie Brilliant Blue for mass spectrometric analysis. 2.6. Proteomics analysis The silver-stained gels were scanned using a PowerLook1100 scanner (UMAX, Taiwan), and protein spots images were analyzed using GE HealthCare Software (Amersham Biosciences, Sweden). The protein spots with at least 2-fold differences in absolute abundance and reproducible changes were considered to be

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differentially secreted proteins. These protein spots on the corresponding Coomassie-stained gels were excised and digested with trypsin using a Spot Handling Workstation (Amersham Biosciences, Sweden). The digested peptide masses were measured using a MALDI-TOF–TOF mass spectrometer (ABI4700 System, USA). Data were processed via the Data Explorer software and proteins were identified by searching against a comprehensive non-redundant sequence database (NCBInr: National Center for Biotechnology Information non-redundant protein sequence database) using the MASCOT search engine (http://www.matrixscience.com/cgi/ search_form.pl?FORMVER=2&SEARCH=MIS). 2.7. Analytical methods Cellulase activities (CMCase, FPase, cellobiase) were assayed with glucose as the standard. For FPase, the assay mixture comprising 0.5 mL fermentation broth sample, 1.0 mL citric acid/citrate buffer (50 mM, pH 4.8) and Whatman No. 1 filter paper strip (1.0 6.0 cm2) was incubated at 50 °C for 60 min. CMCase activity was determined by incubating the mixture of 0.5 mL 2% (w/v) CMC solution in citric acid/citrate buffer (50 mM, pH 4.8) and 0.5 mL fermentation broth sample at 50 °C for 30 min. To assay cellobiase activity, the mixture of 1.0 mL fermentation broth sample and 1.0 mL fresh cellobiose solution (15 mM) in citric acid/citrate buffer (50 mM, pH 4.8) was incubated at 50 °C for 30 min. One unit of CMCase or FPase activity was defined as the amount of enzyme that released 1 lmol of glucose equivalent per min per mL fermentation supernatant. One unit of cellobiase activity was defined as the amount of enzyme that released 2 lmol of glucose equivalent per min per mL fermentation supernatant. Xylanase activity was determined with xylose as the standard following the method described by Ponce-Noyola and Torre (2001). One unit of xylanase activity was defined as the amount of enzyme that released 1 lmol of xylose equivalent per min per mL fermentation supernatant. CMC, xylan, and cellobiose were purchased from Sigma–Aldrich Chemical Co. Protein concentration was measured by the Bradford method according to the instruction of Protein Assay Dye Reagent Concentrate (Rio-Rad, USA). Glucose concentration was determined using SBA-40D Glucose-Lactate Analyzer (The Science Academy of Shandong, Jinan, China). The reducing sugar was determined by the commonly used dinitrosalicylic acid (DNS) method. All assays were performed in duplicate. Sugars present in the hydrolysate were analyzed with HPLC with ZORBAX 843300-908 carbohydrate column (4.6 150 mm, 5 lm, Agilent) at 45 °C using 0.005 N H2SO4 as mobile phase at 0.6 ml/min and the samples were detected using a refractive index detector (Waters 2414, USA) maintained at 45 °C. Authentic chromatographic grade cellobiose, glucose and xylose were used as standards for identification and quantification of the sugars in the hydrolysates. 3. Results and discussion 3.1. Free-cell fermentation in STR Batch fermentation with free cells was carried out in a stirredtank reactor (STR) for 16 days. Fig. 2 shows the kinetics, including DO, pH, protein and reducing sugar (RS) concentrations, and enzyme activities in the fermentation broth. The DO decreased rapidly to 20% by day 4, continued to decline to 12% at day 11, and then gradually went up to 20% afterwards (Fig. 2a). The pH values during fermentation showed a similar trend and varied between 4.29 and 5.15, which was within the range of 4–6 suitable for T. viride to produce cellulase (Voragen et al., 1988). So pH was not controlled in this study. Throughout the fermentation, the


100

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0.18

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0.12 0.06 0

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8 10 Time (day)

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Fig. 2. Kinetics of free-cell fermentation of T. viride grown on sugarcane bagasse and wheat bran for the production of cellulase (CMCase, FPase, and cellobiase) and xylanase. RS: reducing sugars.

concentration of reducing sugars remained at a low level of 0.025 g/L (Fig. 2b), indicating that all sugar released from the saccharification of SCB by cellulase in the broth was almost instantaneously consumed by cells. Meanwhile, protein production continued and reached the maximum concentration of 0.19 g/L on the 14th day, and then started to decline (Fig. 2b). Likewise, the production of cellulase and hemicellulase, including CMCase, FPase, cellobiase and xylanase, followed a similar trend, reaching maximum activities on day 12–14 (Fig. 2c). More CMCase (0.67 IU/mL) and xylanase (0.67 IU/mL) were produced than FPase (0.23 IU/mL) and cellobiase (0.26 IU/mL) in the fermentation. The fermentation was stopped at day 16 when these enzymes were no longer produced. It should be noted that throughout the fermentation, the broth was turbid because of the suspended SCB and wheat bran particles and mycelia. Besides numerous loose pellets and small mycelial clumps in suspension, large mycelial clumps (chunks) also formed and attached everywhere on the surfaces of the pH and DO probes and the wall of the reactor glass vessel (see Fig. 1), making it difficult to sample because of poor mixing. Cells stopped producing enzymes probably because of poor mixing and oxygen limitation inside the large mycelial clumps. 3.2. Immobilized-cell fermentation in RFBB Fig. 3 shows the kinetics of immobilized-cell fermentation in the RFBB. In general, the fermentation showed similar trends in DO, pH, protein and reducing sugar concentrations, and enzyme production to those in the free-cell fermentation. However, the immobilized-cell fermentation continued to produce protein and enzymes for 20 days, reaching a higher protein concentration of

3.3. Saccharification of SCB The saccharification of SCB by enzymes present in the fermentation broth was evaluated and the results as the saccharification yields are compared in Fig. 4. As expected, higher saccharification yields were obtained with the broths containing more enzymes produced towards the end of the fermentation. A saccharification yield of 29.4% was the highest obtained from the free-cell fermentation broth obtained at 12th day, whereas a much higher yield of 49.9% was obtained with the immobilized-cell fermentation broth 100

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0.29 g/L (Fig. 3b). Also, the pH varied more widely, from 4.16 to 5.62 (Fig. 3a), compared to free-cell fermentation, suggesting different metabolites might have been consumed and secreted by T. viride under different fermentation conditions. Compared to the free-cell fermentation, more FPase (0.31 IU/mL) and xylanase (0.76 IU/mL) but slightly lower cellobiase (0.22 IU/mL) and CMCase (0.61 IU/mL) were obtained in the RFBB fermentation (Fig. 3c). It should be noted that there was no suspended mycelia in the RFBB as all cells were attached to the rotating fibrous matrix forming a homogeneous biofilm (see Fig. 1). Also, only during the first several days the fermentation broth was turbid but became clear after 10 days when all SCB and wheat bran particles were also attached to the rotating fibrous matrix. The cell-free broth facilitated mass transfer and allowed the cells in the RFBB to continue to grow and produce enzymes for an extended period without losing productivity. Since all mycelia, SCB and wheat bran particles were attached onto the fibrous bed, the RFBB was relatively easy to operate without any problems such as fouling of pH and DO probes and clogging of the sampling port by the large mycelial clumps observed in the free-cell fermentation.

0.30 Protein

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CMCase FPase

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Fig. 3. Kinetics of immobilized-cell fermentation of T. viride grown on sugarcane bagasse and wheat bran for the production of cellulase (CMCase, FPase, and cellobiase) and xylanase. RS: reducing sugars.

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Saccharification yield (%)

50 Immobilized-cell fermentation 40

Free-cell fermentation

30 20 10 0

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Time (day) Fig. 4. Comparison of the saccharification of SCB using the broths from free-cell and immobilized-cell fermentations.

Table 1 Activities of various lignocullulases in the fermentation broths and sugar contents in the SCB hydrolysates treated with the corresponding broths. Free-cell fermentation

Immobilized-cell fermentation

0.62 ± 0.04 0.23 ± 0.02 0.25 ± 0.02 0.67 ± 0.04

0.60 ± 0.05 0.31 ± 0.02 0.22 ± 0.01 0.62 ± 0.05

Sugar content of hydrolysate (g/L) Glucose 4.08 ± 0.32 Xylose 1.52 ± 0.11 Cellobiose 0.78 ± 0.07 Total reducing sugars 7.4 ± 0.5 Saccharification yield (%) 29.4 ± 2.1

7.67 ± 0.61 2.58 ± 0.21 1.26 ± 0.10 12.6 ± 0.3 49.9 ± 1.3

Enzyme activities (IU/mL) CMCase FPase Cellobiase Xylanase

Fermentation broths were obtained at the end of the free-cell fermentation (12th day) and the immobilized-cell fermentation (22nd day). The averages and standard errors from two replicate runs are reported here.

obtained at 22nd day. Consequently, more glucose, xylose, and cellobiose were released from SCB treated with the immobilized-cell fermentation broth (see Table 1). Interestingly, both free-cell and immobilized-cell fermentation broths contained similar amounts of CMCase, but the latter had a 35% higher FPase activity, which probably was responsible for the increased cellulose hydrolysis efficiency and glucose yield. However, the immobilized-cell fermentation broth had 8% lower xylanase activity, but still produced more xylose, which could be attributed to the synergy of cellulases and xylanases in the hydrolysis process (Murashima et al., 2003). The higher FPase activity caused more degradation of cellulose, and therefore, more hemicellulose including xylan was exposed

to xylanase to produce more xylose. Clearly, the fermentation broth produced in the RFBB contained enzymes that were more efficient to hydrolyze SCB. It should be noted that the immobilized-cell fermentation broth from days 0–14 contained less enzymes and gave lower saccharification yields compared to the free-cell fermentation broths from the same period. This seemingly slower enzymes production in the RFBB can be attributed to enzyme adsorption to the SCB and wheat bran particles. The adsorbed enzymes were released into the broth upon the saccharification of SCB and wheat bran later in the fermentation, however. This could explain the rapid increase in the FPase activity in the immobilized-cell fermentation broth after 14 days and consequently the sharp increase in the saccharification yield from 32.0% using the fermentation broth obtained at day 20 to 49.9% with the broth obtained at day 22. In contrast, enzymes produced in the free-cell fermentation were mostly present in the broth as almost all SCB and wheat bran particles were engulfed by the mycelia clumps (see SEM pictures in Fig. S1a in Supplemental materials). It is noted that the 49.9% saccharification yield from the immobilized-cell fermentation broth was obtained without any thermal or chemical pretreatments, which usually can greatly improve the sugar yield (Adsul et al., 2005; Kuo and Lee, 2009; Rabelo et al., 2008; Zhao et al., 2009). A much higher saccharification yield of 82.5% was obtained from the SCB pretreated with 2% (w/v) NaOH at 80 °C for 1 h and with a higher enzyme loading of 8 FPU/g substrate. Table 2 compares SCB yields from various pretreatments and enzyme sources reported in the literature. Adsul et al. (2005) reported that the saccharification yield increased twofold to 94.6% when the SCB was pretreated with steam explosion and bleaching as compared to simple milling and alkali treatment. Similarly, increasing the lime pretreatment time from 12 h to 36 h also increased saccharification yield from 38.1% to 78.5% (Rabelo et al., 2008). In addition to the pretreatment conditions, the saccharification yield is also greatly affected by the solid loading (substrate consistency) and enzyme loading (Jorgensen et al., 2007). In this study, the saccharification yield of 82.5% obtained with the immobilized-cell fermentation broth was higher than that (62.1%) using the commercial cellulase (Genencor ACCELLERASE 1500) under the same saccharification conditions (50 °C, pH 4.8, 48 h) and was comparable to those with other enzymes reported in the literature (Table 2). Considering the relatively high solid loading of 3.3% and mild pretreatment conditions used in this study, the enzymes produced by T. viride in the RFBB fermentation were effective for SCB saccharification and are promising for industrial application. 3.4. Effects of mycelial morphology To evaluate the cell morphology and its possible effects on the fermentation, mycelial pellets and clumps at the end of the freecell fermentation (12th day) and the biofilm in the RFBB at the

Table 2 Comparison of saccharification yields of sugarcane bagasse treated with cellulases from various sources. Enzyme source

Pretreatments

Solid loading (%)

Enzyme loading (FPU/g SCB)

Saccharification Yield (%)

References

Unknown Unknown P. janthinellum

Ammonia at 120 oC 10% NaOH and 10% peracetic acid Steam explosion and bleaching Milling and alkali N-methylmorpholine-N-oxide at 130 oC Lime at 70 oC for 12 h to 36 h Micro-milling Micro-milling and alkali pretreatmenta Micro-milling and alkali pretreatmenta

5.2 2.0 1.0

Unknown 15 Unknown

Kurakake et al., 2001 Zhao et al., 2009 Adsul et al., 2005

1.0 0.33 3.3 3.3 3.3

5 3.42 3.1 8.0 8.0

48.1 92.0 48.5 94.6 80.7 38.1–78.5 49.9 82.5 62.1

A. niger T. reesei + A. niger T. viride Genencor ACCELLERASE 1500 a

2% NaOH at 80 °C for 1 h.

Kuo and Lee, 2009 Rabelo et al., 2008 This study This study

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end of the immobilized-cell fermentation (22nd day) were examined with scanning electron microscopy (see Supplemental materials). In the free-cell fermentation, most SCB and wheat bran particles were covered by a thick and dense layer of mycelia, and the majority of the SCB and wheat bran particles inside the clumps were not degraded even at the end of the fermentation (see Fig. S1a in Supplemental materials), indicating mass transfer limitation in these compact pellets. In contrast, the biofilm was made of relatively porous layers of mycelia with large pores throughout, which should facilitate oxygen and nutrients transfer to cells and thus allow extended growth period and more efficient enzymes production even though the biofilm had grown to a thickness of 4.0 mm by day 22 when the fermentation was terminated. The biofilm in the RFBB had four layers from the inner fibrous matrix to the biofilm surface, including two distinguishable middle layers – one with smaller SCB and wheat bran particles near the fibrous matrix and the other with larger SCB and wheat bran particles near the surface of the biofilm (see Fig. S1b in Supplemental materials), suggesting that the SCB and wheat bran particles in the interior of the biofilm had been degraded extensively by the fungal cells. It is also possible that soon after spores had germinated and grown into mycelia, smaller SCB and wheat bran particles were adsorbed onto the biofilm attached on the polypropylene cloth before the larger SCB and wheat bran particles were attracted to the more developed biofilm. It is thus clear that the large-size mycelia clumps and pellets with compact mycelia in the free-cell fermentation not only hampered mass transfer but also prevented the ingrowth of cells to SCB and wheat bran particles inside, resulting in inferior production of FPase as compared to the immobilized-cell fermentation. In addition to the mycelial clumps with various sizes of up to >1 cm, there were numerous freely dispersed mycelia fragments in the broth, which greatly increased the broth viscosity in the free-cell fermentation. The high-viscosity broth and shear damage to the suspended mycelia in the stirred tank reactor might have also adversely affected cell growth, which were alleviated when mycelia formed biofilm in the RFBB operated under a relatively low shear environment. In addition, the biofilm morphology in the RFBB could also promote the production and secretion of proteins and metabolites (Talabardon and Yang, 2005; Tay and Yang, 2002; Xu and Yang, 2007). 3.5. Comparative proteomics analysis of secretory proteins To further investigate the effects of fermentation mode on the production of cellulase and hemicellulase, the proteins secreted by T. viride in free-cell (12th day) and immobilized-cell (22nd day) fermentations were analyzed with 2-DE and MALDI-TOF– TOF mass spectrometry. The preliminary investigation by using IPG strips with a pH range of 3–10 showed that the pI values of most proteins were from 3 to 6. So the IPG strips with a pH range

of 3–6 were chosen for the better resolution of the protein spots. The extracellular proteins in the fermentation broths on the 2-DE maps were analyzed. There were about 1000 (956 ± 29 from freecell fermentation and 1011 ± 51from immobilized-cell fermentation) detectable protein spots on the 2-DE maps (see Fig. S2 in Supplemental materials). Among them, 30 distinct protein spots with the most significant differences (>2-fold difference) in absolute abundance and reproducible changes (p < 0.05) were identified to be differentially secreted proteins. Twenty-four of these protein spots, 6 (tagged in Fig. S1a) were more abundant in the free-cell fermentation broth and 18 (tagged in Fig. S1b) in the immobilized-cell fermentation broth, were identified successfully using the MS database (www.matrixscience.com) from various fungi including T. viride, Hypocrea jecorina and T. reesei (H. jecorina’s anamorph). Table 3 lists the identified proteins, including 2 exoglucanases (cellobiohydrolase: CBH I, CBH II), 2 endoglucanases (EG II, EG IV), swollenin, and a-amylase. It is noted that 15 of the 18 protein spots tagged in the immobilized-cell fermentation were identified as CBH II, whereas 4 of the 6 tagged proteins in the free-cell fermentation were CBH I. Clearly, different exoglucanases or cellobiohydrolases were produced by T. viride under different fermentation conditions. The CBH II from T. viride was reported to have about 2fold higher activity and play a more important role in hydrolyzing cellulose than CBH I (Mach et al., 1995). This finding is consistent with the higher FPase activity and higher SCB hydrolysis yield found with the immobilized-cell fermentation broth. It is common to see many CBHs spots on the 2-DE maps as T. viride is an excellent cellulase-producing fungus (Li et al., 2010). Isabelle et al. (2008) found that both Cel6A/CBH II and Cel7A/CBH I isoforms were present, with Cel7A occupying a high percentage of the total protein spot volume (57.1%) in the 2-DE gel when lactose medium was used to grow T. viride. Sun et al. (2008) also reported that CBHs dominated over 37% of the total extracellular proteins of T. reesei based on 2-DE analysis. However, it should be noted that the theoretical molecular mass and pI values for the identified CBHs were not consistent with their positions on the 2-D gels (see Supplemental materials Tables S1 and S2), which can be attributed to the heterogeneous post-translational modifications and protein degradation in 2-DE sample preparation (Mach et al., 1995; Vinzant et al., 2001). There are five known endoglucanases, which randomly cleave the internal bonds at amorphous sites in cellulose (Isabelle et al., 2008). Two of them, EG II and EG IV, were found to have different expression levels between the free-cell and immobilized-cell fermentations. More EG II, which is the major endoglucanase (Saloheimo et al., 1997), was present in the broth of the immobilized-cell fermentation, whereas more EG IV was produced in the free-cell fermentation. Swollenin does not have any hydrolytic activity but can swell cotton fibers, thus making the substrate more accessible to cellulases

Table 3 Proteins with differential expression levels in the fermentation broths identified by MALDI-TOF–TOF mass spectrometry. Protein

Accession number

Theoretical mol. mass (Da)

Proteins with higher expression levels in free-cell fermentation broth CBH I gi|809286 47.1 gi|223874 52.8 gi|50400675 54.6 EG IV gi|21263647 35.9 Swollenin gi|8052455 53.1 Proteins with higher expression levels in immobilized-cell fermentation broth CBH II gi|6137484 39.2 EG II gi|121794 44.9 Swollenin gi|8052455 53.1 a-amylase gi|197631747 51.0 a

Theoretical pI value

Spot number on 2-DEa

4.3 4.3 4.8 5.3 4.8

6, 7 11 13 10 9

4.8 5.0 4.8 4.7

1, 2, 3, 6, 10, 14, 22, 24, 25, 26, 27, 28, 31, 37, 38 4 36 9

Identified protein spots on the 2-DE gel maps (see Fig. S1 in Supplemental materials) with scores greater than required scores and p < 0.05.


(Salohimo et al., 2002). It has been found in many cellulose-degrading fungi, including T. reesei (Salohimo et al., 2002), Aspergillus fumigatus (Chen et al., 2010) and Trichoderma pseudokoningii (Zhou et al., 2011), but not in T. viride in any previous study. The identified aamylase had a higher expression level in the immobilized-cell fermentation broth. It is well known that T. viride can secrete a-amylase, which belongs to the glycosyl hydrolase family 13 and cannot degrade cellulose. It is not clear why this enzyme was produced in the fermentation as there was no starch present in the medium. It is noted that cellobiase, hemicellulase, and lignin-degrading enzymes were not among the 24 protein spots identified in this study, suggesting no difference in their production/secretion levels between free-cell and immobilized-cell fermentations. T. viridi and other cellulase-producing Trichoderma spp. normally do not produce lignin-degrading enzymes. However, the native cellobiase, which has a reported pI value of 6.5 (Vinzant et al., 2001) might not be present on the 2-DE gel maps because of the range of IEF gel used in this study was only pH 3–6. Nevertheless, the comparative proteomic data from this study would contribute to the construction of a complete proteome or secretome for T. viride. A comprehensive analysis of secretome is desirable for the development of better lignocellulases, and has been extensively studied for several fungi, including T. reesei, Trichoderma harzianum, A. niger, Aspergillus nidulans, Phanerochaete chrysosporium, Neurospora crassa, and Postia placenta (Do Vale et al., 2012; Martinez et al., 2009; Phillips et al., 2011; Sayali et al., 2012; Sunil et al., 2011; Sunil et al., 2010). However, to date little has been done on T. viride’s secretome (Zhou et al., 2008). 4. Conclusions This study demonstrated the advantages of immobilizing mycelia in the rotating fibrous bed for cellulase production because of the easier reactor operation and better process control with improved biofilm morphology. The broth produced from the RFBB fermentation gave higher saccharification efficiency because of the higher FPase activity, which could be attributed to better exoglucanases and cellobiohydrolases produced by T. viride under the RFBB fermentation conditions as identified in the comparative proteomics analysis. This study also provided new insights on the differential secretome of T. viride under different fermentation conditions. Acknowledgements This work was supported in part by Open Funding of State Key Laboratory of Pulp and Paper Engineering (Grant No. 200937 and 2012TS03), South China University of Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.01. 088. References Adsul, M.G., Bastawde, K.B., Varma, A.J., Gokhale, D.V., 2007. Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production. Bioresource Technol. 98, 1467–1473. Adsul, M.G., Ghule, J.E., Shaikh, H., Singh, R., Bastawde, K.B., Gokhale, D.V., Varma, A.J., 2005. Enzymatic hydrolysis of delignified bagasse polysaccharides. Carbohyd. Polym. 62, 6–10. Akihiro, H., James, C.O., Hideki, A., Hideo, T., 2007. Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulase. J. Biosci. Bioeng. 103, 311–317.

181

Bah, A.M., Sun, H., Chen, F., Zhou, J., Dai, H., Zhang, G., Wu, F., 2010. Comparative proteomic analysis of Typha angustifolia leaf under chromium, cadmium and lead stress. J. Hazard. Mater. 184, 191–203. Chen, X., Ishida, N., Todaka, N., Nakamura, R., Maruyama, J., Takahashi, H., Kitamoto, K., 2010. Promotion of efficient saccharification of crystalline cellulose by Aspergillus fumigatus Swo1. Appl. Environ. Microbiol. 76, 2556–2561. Do Vale, L.H.F., Gomez-Mendoza, D.P., Kim, Min-Sik, Pandey, A., Ricart, C.A.O., Filho, E.X.F., Sousa, M.V., 2012. Secretome analysis of the fungus Trichoderma harzianum grown on cellulose. Proteomics 12, 2716–2728. Domingues, F.C., Queiroz, J.A., Cabral, J.M.S., Fonseca, L.P., 2000. The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30. Enzyme Microb. Technol. 26, 394–401. Isabelle, H.G., Antoine, M., Alain, D., Gwénaël, J., Daniel, M., Sabrina, L., Hughes, M., Jean-Claude, S., Frédéric, M., Marcel, A., 2008. Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol. Biofuels 1, 18. Jorgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction of lignocellulose at high-solids concentrations. Biotechnol. Bioeng. 96, 862–870. Kuo, C.H., Lee, C.K., 2009. Enhanced enzymatic hydrolysis of sugarcane bagasse by N-methylmorpholine-N-oxide pretreatment. Bioresource Technol. 100, 866– 871. Kurakake, M., Kisaka, W., Ouchi, K., Komaki, T., 2001. Pretreatment with ammonia water for enzymatic hydrolysis of corn husk, bagasse, and switchgrass. Appl. Biochem. Biotechnol. 90, 251–259. Li, X.H., Yang, H.J., Roy, B., Park, E.Y., Jiang, L.J., Wang, D., Miao, Y.G., 2010. Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet. Microbiol. Res. 165, 190–198. Mach, R.L., Seiboth, B., Myasnikov, A., 1995. The bgl 1 gene of Trichoderma reesei QM9414 encodes an extracellular, cellulose-inducible glucosidase involved in cellulase induction by sophorose. Mol. Microbiol. 16, 687–697. Martinez, D., Challacombe, J., Morgenstern, I., Hibbett, D., Schmoll, M., Kubicek, C.P., Ferreira, P., Ruiz-Duenas, F.J., Martinez, A.T., Kersten, P., Hammel, K.E., Vanden Wymelenberg, A., Gaskell, J., Lindquist, E., Sabat, G., Bondurant, S.S., Larrondo, L.F., Canessa, P., Vicuna, R., Yadav, J., Doddapaneni, H., Subramanian, V., Pisabarro, A.G., Lavín, J.L., Oguiza, J.A., Master, E., Henrissat, B., Coutinho, P.M., Harris, P., Magnuson, J.K., Baker, S.E., Bruno, K., Kenealy, W., Hoegger, P.J., Kües, U., Ramaiya, P., Lucas, S., Salamov, A., Shapiro, H., Tu, H., Chee, C.L., Misra, M., Xie, G., Teter, S., Yaver, D., James, T., Mokrejs, M., Pospisek, M., Grigoriev, I.V., Brettin, T., Rokhsar, D., Berka, R., Cullen, D., 2009. Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc. Natl. Acad. Sci. USA 106, 1954– 1959. Murashima, K., Kosugi, A., Doi, R.H., 2003. Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation. J. Bacteriol. 185, 1518–1524. Phillips, C.M., Iavarone, A.T., Marletta, M.A., 2011. Quantitative proteomic approach for cellulose degradation by Neurospora crassa. J. Proteome Res. 10 (9), 4177– 4185. Ponce-Noyola, T., Torre, M.D.L., 2001. Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture. Bioresource Technol. 78, 285–291. Rabelo, S.C., Filho, R.M., Costa, A.C., 2008. A comparison between lime and alkaline hydrogen peroxide pretreatments of sugarcane bagasse for ethanol production. Appl. Biochem. Biotechnol. 148, 45–58. Saloheimo, M., Nakari-SetäLä, T., Tenkanen, M., Penttilä, M., 1997. CDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast. Eur. J. Biochem. 249, 584–591. Salohimo, M., Paloheimo, M., Hakola, S., Pere, J., Swanson, B., Nyyssönen, E., Bhatia, A., Ward, M., Penttilä, M., 2002. Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur. J. Biochem. 269, 4202–4211. Sayali, S., Anamika, R., Patricia, A.C., Steven, D.H., Rolf, P., Andrew, J.M., 2012. A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover. Biotechnol. Biofuels 5, 52. Sun, W.C., Cheng, C.H., Lee, W.C., 2008. Protein expression and enzymatic activity of cellulases produced by Trichoderma reesei Rut C-30 on rice straw. Process Biochem. 43, 1083–1087. Sunil, S.A., An, A.L., Arulmani, M., Peter, P., Siu, K.S., 2010. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes. J. Proteome Res. 9, 3932–3940. Sunil, S.A., Anita, R., Lim, T.C., Lynette, T., Sunil, S., Siu, K.S., 2011. Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei. J. Proteome Res. 10, 4579–4596. Talabardon, M., Yang, S.T., 2005. Production of GFP and glucoamylase by recombinant Aspergillus niger: effects of fermentation conditions on fungal morphology and protein secretion. Biotechnol. Prog. 21, 1389–1400. Tay, A., Yang, S.T., 2002. Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor. Biotechnol. Bioeng. 80, 1–12. Thongchul, N., Yang, S.T., 2006. Controlling biofilm growth and lactic acid production by Rhizopus oryzae in a rotating fibrous bed bioreactor: effects of dissolved oxygen, rotational speed, and urea concentration. J. Chinese Inst. Chem. Eng. 37, 49–61. Vinzant, T.B., Adney, W.S., Decker, S.R., Baker, J.O., Kinter, M.T., Sherman, N.E., Fox, J.W., Himmel, M.E., 2001. Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation. Appl. Biochem. Biotechnol. 91–93, 99–107.

182


Voragen, A.G., Beldman, J.G., Rombouts, F.N., 1988. Cellulases of mutant strain of Trichoderma viride QM9414. Methods Enzymol. 160, 243–251. Wang, Z., Wang, Y., Yang, S.T., Wang, R., Ren, H., 2010. A novel honeycomb matrix for cell immobilization to enhance lactic acid production by Rhizopus oryzae. Bioresource Technol. 101, 5557–5564. Xia, L.M., Shen, X.L., 2004. High-yield cellulase production by Trichoderma reseei ZU02 on corn cob residue. Bioresource Technol. 91, 259–262. Xu, Z.N., Yang, S.T., 2007. Production of mycophenolic acid by Penicillium brevicompactum immobilized in a rotating fibrous-bed bioreactor. Enz. Microb. Technol. 40, 623–628.

Zhao, X., Peng, F., Cheng, K., Liu, D., 2009. Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali-peracetic acid pretreatment. Enzyme Microb. Technol. 44, 17–23. Zhou, J., Wang, Y.H., Chu, J., Zhuang, Y.P., Zhang, S.L., Yin, P., 2008. Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100–14. Bioresource Technol. 99, 6826–6833. Zhou, Q.X., Lv, X.X., Zhang, X., Meng, X.F., Chen, G.J., Liu, W.F., 2011. Evaluation of swollenin from Trichoderma pseudokoningii as a potential synergistic factor in the enzymatic hydrolysis of cellulose with low cellulase loadings. World J. Microb. Biotechnol. 27, 1905–1910.