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Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source
Accepted Manuscript Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source. Takashi Kogo, Yuki Yoshida, Keisuke Koganei, Hitoshi Matsumoto, Taisuke Watanabe, Jun Ogihara, Takafumi Kasumi PII: DOI: Reference:
S0960-8524(17)30147-5 http://dx.doi.org/10.1016/j.biortech.2017.01.075 BITE 17603
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
8 November 2016 20 January 2017 21 January 2017
Please cite this article as: Kogo, T., Yoshida, Y., Koganei, K., Matsumoto, H., Watanabe, T., Ogihara, J., Kasumi, T., Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source., Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.01.075
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Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.
Takashi Kogo, Yuki Yoshida, Keisuke Koganei, Hitoshi Matsumoto, Taisuke Watanabe, Jun Ogihara, and Takafumi Kasumi*
Applied Microbiology and Biotechnology Laboratory, Department of Chemistry and Lifescience, Nihon University (1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan)
*Corresponding author (Tel & Fax: +466-84-3943; E-mail: [email protected])
List of Abbreviations CMCase, carboxymethyl cellulase; pNP-Xase, p-nitrophenyl xylosidase; pNP-Lase, p-nitrophenyl lactosidase
Highlights Rice straw is a viable carbon source for fungal production of rice straw cellulases. T. reesei and H. insolens enzymes synergistically enhanced rice straw degradation. NH4OH-treated rice straw in the culture medium improved T. reesei enzyme activity.
Abstract Rice straw was evaluated as a carbon source for the fungi, Trichoderma reesei and Humicola insolens, to produce enzymes for rice straw hydrolysis. The enzyme activity of T. reesei and H. insolens cultivated in medium containing non-treated rice straw 1
were almost equivalent to the enzyme of T. reesei cultivated in Avicel medium, a form of refined cellulose. The enzyme activity of T. reesei cultivated in medium containing NH4OH-treated rice straw was 4-fold higher than enzyme from cultures grown in Avicel medium. In contrast, H. insolens enzyme from cultures grown in NH4OH-treated rice straw had significantly lower activity compared with non-treated rice straw or Avicel. The combined use of T. reesei and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. Our data suggest that rice straw is a promising low-cost carbon source for fungal enzyme production for rice straw hydrolysis.
Key words Biomass degrading enzymes, Trichoderma reesei, Humicola insolens, rice straw, synergistic effect.
Introduction Lignocellulose is probably the most abundant but under-utilized biomass resource in the world. The monosaccharides obtained from the degradation of lignocellulose into its component sugars can be converted to bio-ethanol, which is an important alternative to fossil fuels as concerns about oil, gas and coal shortages and their environmental impacts continue to grow (Sun and Cheng, 2002). In recent years, the potential of biomass for the production of chemicals and pharmaceuticals has also received increased attention. For example, butanol or erythritol obtained by fermentation can be converted to polymers such as bio-resins or plastic (Kiyoshi et al., 2015; Rahnama et al., 2014; Kobayashi et al., 2015). The increased interest in the potential of biomass stems from shortages in the raw materials on which naphtha plants depend, and a 2
world-wide shift towards the use of alternative energy sources (Yoshimura et al., 2012). The utilization of biomass is consistent with global sentiment driving a move away from a high-consumption fossil fuel dependent society to more eco-friendly communities powered by renewable bioresources. In Japan, thirteen million tons of rice straw are produced annually. Although 30% is utilized as livestock feed or manure, the remaining 70% is discarded (Nagashima,
2010). The polysaccharide composition of rice straw is similar to that of other herbaceous plants such as sugarcane or corn stover (Templeton et al., 2009; SN, 2008; Sun and Cheng, 2002). It seems no great difference in cost among these biomasses; actually no authentic comparative data are shown, probably because that they depend on how much amount is reliably available in each local area. Anyway, the rice is one of the typical and important crop in this country, and therefore rice straw concomitantly produced is much handy biomass. Hence, this hugely under-utilized resource is an attractive proposition from the perspective of reduced cost, reliable supply, and minimal impact on cropping and food production, all of which are of vital importance for biomass utilization. In addition, rice straw recycling has the potential to improve the prosperity of regional communities based on a local-production for local-consumption system (Singh and Bishnoi, 2012). Rice straw is mainly composed of polysaccharides such as cellulose (40%), hemicellulose (35%), lignin (10%), and silica (5%) (Kahar, 2013). These polymers are crosslinked to form a rigid three-dimensional structure that hinders rapid degradation into component sugars required for fermentation (Park et al., 2010). The development of efficient, practical and low cost approaches to rice straw degradation are essential to fully realize the potential of rice straw as a bioresource. In this regard, the fungus genus Trichoderma, includes strains that produce large amounts of cellulolytic 3
enzymes. Among them, T. reesei (ATCC-66589), which is a genetically engineered type of T. reesei (QM9414), is one of the most promising for cellulase production. Depending on T. reesei culture conditions, the maximal amount of extracellular protein produced is estimated to be 60-100g per liter of culture (Park et al., 2010). One of the characteristics of T. reesei is that more than 90% of the enzyme protein is composed of cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II), which are both essential for rice straw degradation. However, xylanase is another essential enzyme that is not necessarily present in sufficient quantities in T. reesei enzyme preparations (Sweeney and Xu, 2012). Hence, the growth and/or enzyme composition of T. reesei have been extensively studied from the view point of efficient hydrolysis of lignocellulosic rice straw (Ogasawara and Shida, 2012), augmented with chemical (alkali or acid), mechanical, or biological (white or brown rotting fungi) pre-treatment to promote enzymatic degradation (Alvira et al., 2010). The carbon source supplied to microorganisms is crucial for enzyme production. In this regard, Avicel is a form of refined cellulose that is currently the preferred substrate for T. reesei to produce inducible cellulosic enzymes. However, although Avicel is widely used in culture media, it is prohibitively expensive as a carbon source for large-scale biomass projects. Previously, we reported that the fungus, Humicola insolens, produces biomass degrading enzymes in different amounts depending on the carbon source in the medium (Matsumoto et al., 2014). If T. reesei is similar, using rice straw instead of Avicel might induce enzyme constituents more suitable for rice straw hydrolysis, and concomitantly bring about a significant cost reduction. In addition, since H. insolens enzyme preparations are rich in xylanase, combining enzyme preparations from T. reesei and H.
4
insolens may result in a synergistic and improved efficiency in rice straw decomposition. In this study, we investigated the induction, constituents and functions of rice straw degradation enzymes from T. reesei and H. insolens grown using rice straw as the carbon source. In addition, the synergistic effects of mixed T. reesei/H. insolens enzyme preparations were evaluated as a more efficient approach to rice straw degradation.
2. Materials and methods
2.1 Fungal strains
The T. reesei (ATCC-66589) and H. insolens (ATCC-26908) strains were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The strains were maintained in 10% glycerol at -80 °C.
2.1.1. Culture conditions
T. reesei and H. insolens were propagated on potato dextrose agar (PDA) medium, and yeast extract starch soluble agar (YpSsA) medium, respectively. For the preparation of enzyme, T. reesei was pre-cultured in 50 mL of medium consisting of 3% glucose, 0.6% (NH4)2SO4, and 0.6% corn steep liquor at 30 °C for 3 days at 200 rpm. The pre-culture was transferred into 100 mL of medium containing 0.9% Avicel or 2% rice straw (0.9% Avicel carbon equivalent), 0.18% (NH4)2SO4 and 0.18% corn steep liquor, and cultured at 30 °C for 7 days at 200 rpm. 5
H. insolens was pre-cultured in 50 mL of medium composed of 5% glucose and 0.3% ExSBM at 30 °C for 5 days at 200 rpm. The pre-culture was transferred into 100 mL of medium containing 2% Avicel or 4.4% rice straw (2% Avicel carbon equivalent) and 2% SBM, and cultured at 30 °C for 7 days at 200 rpm. After centrifugation at 12,000 rpm for 10 min, T. reesei and H. insolens supernatants were used for the measurement of enzymatic activity.
2.2. Preparation of rice straw
Rice straw was harvested in Ibaraki Prefecture, Japan on Oct. 10th, 2013 and kept in a cool and dry room to avoid deterioration and rotting. After washing with water and air-drying, the straw was cut into approximately 5 cm lengths, ground in a milling machine (P-15, Fritsch Japan Co., Ltd., Yokohama, Japan), and passed through size 40 mesh sieves. The milled straw was used as a carbon source or an enzyme substrate after treatment for 72 h at 4 °C with 1.25% NH4OH or 5% NH4OH, respectively, followed by neutralization with 2N (NH4)2SO4. Milled straw swollen with water was used as substrate for enzyme assays.
2.3. Chemical reagents
p-nitrophenyl β-D-lactopyranoside (pNP-L), p-nitrophenyl β-D-xylopyranoside (pNP-X) and beech wood xylan were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Carboxymethyl cellulose (CMC) was purchased from nacalai tesque (Kyoto, Japan). All other reagents used were general commercial supplies from WAKO chemicals (Osaka, Japan). ExSBM (extruded soybean meal) and SBM 6
(soybean meal) were supplied courtesy of J-Oil Mills, CO., LTD (Osaka, Japan).
2.4. Enzyme activity and protein content assays
Xylanolytic and CMC hydrolyzing activities were assayed using a reaction mixture consisting of 0.8% beech wood xylan or CMC, McIlvaine buffer (pH 5.0 for T. reesei enzyme, pH 6.0 for H. insolens enzyme), and culture supernatant as the enzyme preparation. After incubation at 50 °C for 10 min, the release of reducing sugar was assayed by the DNS method (Miller, 1958). One unit of activity was defined as the amount of enzyme that released 1 µmol of reducing sugar per minute. pNP-L and pNP-X degrading activities were assayed using a reaction mixture composed of 100 µL 4 mM pNP-L or pNP-X, 100 µL 200 mM McIlvaine buffer (pH 5.0 or 6.0), and 200 µL culture supernatant in a total volume of 400 µL. After incubation at 50 °C for 10 min, 400 µL 1M sodium carbonate was added to the reaction mixture which was then colorimetrically assayed at 415 nm using a spectrophotometer (iMark Microplate Reader, Bio-Rad Laboratories Inc., Hercules, CA, USA) (Wood and Bhat, 1988). One unit of activity was defined as the amount of enzyme that liberates 1 µmol of p-nitrophenol per minute. The protein content of enzyme preparations was measured by the Lowry-Folin method against a bovine serum albumin standard (Lowry et al., 1951). Rice straw hydrolyzing activity was assayed in a reaction mixture composed of 800 µL 5% NH4OH-treated or non-treated rice straw as substrate, 100 µL of McIlvaine buffer (pH 5.0 or 6.0), and 100 µL of culture supernatant in a 1.5 mL tube. Equal amounts of enzyme protein were assayed. The reaction mixture was incubated at 50 °C for 180 min and the reducing sugar was assayed with the DNS method (Miller, 1958). 7
One unit of rice straw hydrolyzing activity was defined as the amount of enzyme that liberated 1 µmol of reducing sugar per minute. Apparent optimum pH was determined in a range of pH 2-8 using glycine buffer (pH 2-3), MacIlcvane buffer (pH 3-6), and phosphate buffer (pH 6-8), and apparent optimum temperature was determined in a range of 30-70 °C.
2.5. SDS-Polyacrylamide electrophoresis
Supernatant from a 7-day culture of T. reesei or H. insolens was subjected to SDS-PAGE on a NuPAGE Novex 4 to 12% Bis-Tris gel (Invitrogen, Thornton, Australia). Samples were prepared each containing 10 µL of supernatant (approximately 10 mg/L of protein) and 10 µL of loading buffer [0.5 M Tris-HCl, pH 6.8, 50% (v/v) glycerol, 10% SDS, 5% -mercaptoethanol, and 0.05% bromophenol blue]. The samples were boiled for 3 min, centrifuged for 30 sec, and cooled to room temperature before being loaded onto the gel. Electrophoresis was carried out at 75 V for 90 min using Tris-glycine running buffer containing 1.44% Glycine (Bio-Rad, Sydney, Australia), 0.3% Tris-Base and 0.1% SDS, (Peterson et al., 2011). After electrophoresis, the gel was soaked in fixing solution (100mL methanol + 100mL acetic acid + 100 mL distilled water) for 40 min, and washed with washing solution (100 mL methanol + 100 mL distilled water) for 10 min and subsequently with distilled water for 10 min. Following dipping in sensitizing solution (10 mg sodium thiosulfate dissolved in 50 mL distilled water) for 1 min, the gel was rinsed 3 times with distilled water, and soaked in developing solution (50 mg silver nitrate dissolved in 50 mL distilled water) for 20 min. The stained gel was rinsed 3 times with distilled water, and 8
transferred into stopping solution (10 mL acetic acid + 10 mL acetic acid) to stop the excess developing.
2.6. Scanning electron microscopy
The non-treatment rice straw and 1.25% NH4OH-treated rice straw were immersed in 2% glutaraldehyde, washed three times with 0.1 M cacodylate buffer (pH 7.0), and re-suspended in 1% osmium solution. The solution was dehydrated for 10 min each with 50%, 70%, 80%, 90%, and 99.8% acetone, and twice with 100% acetone for 30 min. The rice straw residue was then soaked for 30 min in a mixture of 100% acetone and isoamylacetone (1:1), followed by 90 min in 100% isoamylacetone, and stored at 4 °C before use. The final preparation was dried using a critical point drier (HCP-2, Hitachi High-Technologies Cooperation, Tokyo, Japan) and observed with a scanning electron microscope (S-3500, Hitachi High-Technologies Cooperation) following vacuum deposition.
2.7. Scale-up using a jar fermenter
The effect of scaling up the culture volume on enzyme production was examined using a 2 L jar fermenter (TBR-2-3, Sakura Seiki Bioreactor, Tokyo, Japan). The pre-culture obtained as described above was inoculated (10% (v/v)) into 1 L of medium composed of 2% (w/v) of 1.25% NH4OH-treated rice straw (w/v) and 0.4% corn steep liquor, and cultivated for 3 days at 30 °C at 300 rpm. The culture was supplemented with additional rice straw (3% w/v) and cultivation was continued for a further 7 days under the same conditions. The space velocity of aeration was 1 vvm, 9
and the agitation rate was 300 rpm.
3. Results and Discussion
Our data suggest that rice straw is a promising carbon source for the cultivation of T. reesei and H. insolens to produce enzymes for the decomposition of rice straw. The relatively low cost of rice straw compared with alternative refined cellulosic carbon sources such as Avicel and others, addresses one of the most crucial problems for the practical use of enzymes for digesting lignocellulosic materials.
3.1. Rice straw as a carbon source - effect on enzyme activity.
Generally, rice straw must be pre-treated to be used as a carbon source to efficiently nourish microorganisms. For that purpose, acid treatment with H2SO4 or HCl, or alkali treatment with NaOH, have been used in the pre-treatment process (Sarkar and Aikat, 2012; Alvira et al., 2010; Galbe and Zacchi, 2007; Kim et al., 2013). However, these methods sometimes result in a reduced yield due to excess decomposition of constitutional sugars, or the production of organic compounds that inhibit fermentation (Sun and Cheng, 2002). Hence, we selected NH4OH as the pre-treatment agent in this study because it is a less hazardous, weak alkali solution known to effectively loosen plant cell walls (Uppugundla et al., 2015). With the exception of CMCase, enzyme activities in T. reesei rice straw cultures were considerably higher than in Avicel cultures (Fig. 1A). The 1.25% NH4OH-treated rice straw had a much higher enzymatic activity compared with non-treated rice straw,
10
especially rice straw hydrolyzing activity. It suggests that cellulolytic enzymes other than CMCase were preferably induced during cultivation with alkali-peeled rice straw. While, the xylan and rice straw hydrolyzing activities of H. insolens enzyme cultivated with non-treated rice straw were comparable with enzyme from Avicel culture. However, when cultured in 1.25% NH4OH-treated rice straw, enzyme activity was significantly lower (Fig. 2A). pNP-L and CMCase activities of H. insolens cultured with non-treated rice straw were less than one half of those enzymes cultured with Avicel. Hence, significant part of rice straw hydrolyzing activity is considered to rely on xylanase activity, which may be insufficiently induced with alkali-peeled rice straw. Although xylan, pNP-X and pNP-L hydrolyzing activities of T. reesei enzyme were significantly higher in rice straw cultures compared with Avicel culture (Fig. 1A and Fig. 2A), CMCase activity did not substantially vary according to the carbon source. Hence, the greater rice straw hydrolyzing activity is possibly due to the elevation of activities of such enzymes as xylanase, pNP-Xase and pNP-Lase. Difference in rice straw hydrolytic activity between two fungal enzyme preparation may reflect the difference in enzyme composition induced by alkali-treated or non alkali-treated biomass. Thus, the alkali-treated rice straw is excellent carbon source for enzyme production of T. reesei on rice straw hydrolysis, in contrast, non alkali-treated rice straw is passable carbon source for enzyme production of H. insolens on rice straw hydrolysis.
3.2. SDS-PAGE profiles of enzymes
3.2.1 T. reesei enzyme
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Fig. 1B shows SDS-PAGE profile of the enzymes obtained from T. reesei in media containing 1.25% NH4OH-treated or non-treated rice straw, and Avicel. There were few clear differences between the SDS-PAGE profiles of the three enzyme preparations from T. reesei cultures. SDS-PAGE followed by silver staining revealed more prominent and well defined 23 kD and 32 kD bands in the rice straw enzyme preparations (lanes 2 and 3) compared with the Avicel enzyme preparation (lane 4). The 23 kD and 32 kD bands in the rice straw enzyme preparations may represent endoglucanase V (EG V) and xylanase III (XYN III), respectively. Some minor proteins other than cellulases and hemicellulases are known to work co-operatively to decompose rigid-structured biomass (Xiong, 2002). It is possible that these minor fractions visible on the SDS-PAGE gels are components of the enzyme assessed in this study, and are involved in the enzymatic degradation of rice straw. The 125 kD band is likely to be β-xylosidase (BXL) (Semenova et al., 2009), although this requires verification by immunoblotting. In the cellulase group between 45 kD and 66 kD (Chokhawala et al., 2015; kondo et al., 2012), a 57 kD band presumed to be cellobiohydrolase I (CBH I) is seen more distinctly in the rice straw enzyme preparations compared with the Avicel enzyme. T. reesei has been reported to produce xylanolytic enzymes such as xylanase I (XYN I), xylanase II (XYN II), and XYN III (Törrönen et al., 1992; Xu et al., 2000; Ogasawara et al., 2006) in a size range of 19, 21 and 32 kD, respectively. On the SDS-PAGE silver stained gels proteins in this size range are more prominent in the rice straw enzyme compared with the Avicel enzyme. Overall, these data suggest that rice straw is a preferable carbon source for T. reesei to produce rice straw hydrolyzing enzymes.
3.3.2 H. insolens enzyme 12
Analysis of silver stained SDS-PAGE gels of H. insolens enzyme revealed more prominent and well defined 32 and 37 kD bands in preparations from non-treated rice straw cultures compared with 1.25% NH4OH-treated rice straw culture (Fig. 2B). In addition, bands around 45-66 kD were observed that were faintly seen in 1.25% NH4OH-treated rice straw culture. The SDS-PAGE profile of enzyme of H. insolens grown with non-treated rice straw and Avicel were similar, but considerably different from 1.25% NH4OH-treated rice straw enzyme. Many bands visible in the enzyme preparations from non-treated rice straw and Avicel, whereas relatively few bands are seen in enzyme preparations from NH4OH-treated rice straw. The H. insolens enzyme activity profile was clearly different from that of T. reesei. H. insolens alters enzyme composition depending on the carbon source (Matsumoto et al., 2014). When supplied with NH4OH-treated rice straw, H. insolens enzyme activities were strikingly low compared with enzymes obtained from non-treated rice straw or Avicel. It is possible that H. insolens growth is inhibited by an excess of inorganic nitrogen from (NH4)2SO4 generated after neutralization. Although the overall enzyme activity of H. insolens is lower than T. reesei, many more bands are detected by SDS-PAGE in enzyme preparations obtained from H. insolens grown with non-treated rice straw compared with T. reesei. The band at 124 kD in enzyme obtained from H. insolens grown in non-treated straw and Avicel cultures is presumed to be H. insolens β-glucosidase 3A (HiBgl 3A) and H. insolens β-glucosidase 3B (HiBgl 3B), which are H. insolens specific proteins (Xia et al., 2016). Although indistinct on silver stained gels, the cellulosic enzymes such as CBH I, cellobiohydrolase II (CBH II), endoglucanase I (EG I), endoglucanase II (EG II), endoglucanase III (EG III) and EG V (Schiilein et al., 1997) may be present in H. insolens enzyme. In addition, band at 37 kD may represent 13
Xyl43A (β-xylosidase) (Mustaira et al., 2016). The verification of the presence of these proteins awaits further analysis by immunoblotting.
3.4 Conditions for optimal enzyme activity
The pH and temperature dependence of relative enzyme activities are shown in Fig. 3A and Fig. 3B, respectively. Both CMC and xylan hydrolyzing activity showed typical bell-shaped curves. The optimal pH and temperature for T. reesei enzyme activity were 4.0-5.0 and 60 °C, and 5.0-6.0 and 60 °C for H. insolens. The optimal pH for T. reesei and H. insolens enzymes rice straw degrading activity were 4.0 and 5.0, respectively, which was similar to CMCase and xylanase (data not shown). However, the 50 °C optimum temperature for T. reesei and H. insolens enzymes rice straw hydrolyzing activity was slightly lower compared with CMCase and xylanase. This difference could be related to the extended incubation time required for rice straw degradation compared with synthetic substrates; 10 min for CMC and xylan hydrolyzing activity, and 180 min for rice straw degrading activity.
3.5. Enzyme activity in scaled up culture using a jar fermenter
Fig.4 shows the time course enzyme activity of T. reesei (Fig. 4A) and H. insolens (Fig. 4B) during cultivation in a jar fermenter. For T. reesei, enzyme activity was detectable from day 3, and increased rapidly thereafter. The increase in CMCase activity was not as great as the other enzymes. After 7 days of cultivation, the xylan, pNP-L and pNP-X hydrolyzing activities were 7-fold, 29- fold, and 30 fold higher, respectively, than those obtained from small scale cultivation in a 500 mL shake flask. 14
For H. insolens, enzyme activity was detectable from day 1, and increased exponentially over 7 days. However, compared with T. reesei, the increases in enzyme activity were not as great as small scale cultivation; 2.5-fold for xylanase, 20-fold for pNP-X hydrolyzing activity, 10-fold for pNP-L hydrolyzing activity.
3.6. Synergistic effect of mixed enzyme preparations on rice straw hydrolysis
Synergy between T. reesei cellulase and β-glucosidase from Taiwanese fungi (Ng et al., 2011), Aspergillus niger and T. reesei (van den Brink et al., 2014), or xylanase and glycoside hydrolases (Goncalves et al., 2015) has been reported in biomass degradation studies. The synergistic effect of mixed T. reesei and H. insolens enzyme preparations on saccharification of rice straw is presented in Fig. 5. The ratios were calculated from the released reducing sugar based on the estimated total sugar content of rice straw. As shown by the black bars, there was a significant synergistic effect of the combined enzyme preparations compared with the sums of the ratios obtained by using the individual enzyme preparations (gray bars) (Kai et al., 2010). To our knowledge, this is the first time that this phenomenon has been reported. Hydrolysis ratios of T. reesei and H. insolens were 70.3% and 23.5%, respectively, within 72h. However, the 75%: 25% (v/v) mixture of T. reesei and H. insolens enzymes produced a 79.8% hydrolysis ratio, which was approximately 10% greater than the T. reesei enzyme alone. While, weight loss in dry weight base of the rice straw was 44.1% after enzyme hydrolysis for 72 h using the 75%: 25% (T : H ; v/v) mixture. This value is close to the vale (49.1%) obtained from the result using commercial enzyme preparation, Celluclast 1.5L (Bussamra et al. 2015). When only 25% of H. insolens enzyme was substituted with the T. reesei enzyme, the ratio reached as high as 68.2%, 15
which was comparable to the level of 100% T. reesei enzyme. As mentioned before, T. reesei mainly produces cellulolytic enzymes such as CBH II, CBH III, or EG, and also produces xylanolytic enzymes such as XYN I and XYN II. Further, XYNs activities are known to enhance when cultivated with carbon sources rich in hemicellulose like rice straw and bagasse. Whereas, BG or BXL that can hydrolyze short chain cello-oligosaccharides into glucose is not always sufficient, especially as to T. reesei 66589 (PC-3-7) (Treebupachatsaku et al., 2015). In case of H. insolens, most of enzymes produced from the wild strain are still unclear. Hence, the results here postulate that xylanase and/or β-glucosidase of H. insolens complement the rice straw hydrolyzing activity of T. reesei, although the activity itself is rather low. Further, it suggests the possibility that H. insolens produce a peculiar protein like swollenin that helps rice straw hydrolysis, considering its wide variety of enzymes. Co-culture system has been reported to be much efficient for on-site production of enzymes (G-Correa et al., 1999, Kolasa et al., 2014). It involves to simplify the pre-treatment process of the materials and subsequent saccharification process, in addition to improve the yields of saccharification and ethanol production. From these viewpoints, SSF (simultaneous saccharification fermentation) system, SPS (simultaneous pretreatment fermentation) system, or SBS (simultaneous bio-delignification saccharification) system have recently been proposed (Vincent et al., 2014; Ma and Ruan, 2015; Dhiman et al., 2015). In case of SPS system, for example, approximately as much as 90 % of corn stover sugary constituent is reported to be hydrolyzed. Although not known it is applicable to our case, T. reesei and H. insolens, we are now trying co-culture system. Hopefully, these two fungi have quite similar culture conditions, and they do not interfere enzyme productivity each other (data will be shown in near future). 16
3.7. Structural change of rice straw after NH4OH-treatment and enzyme decomposition
Rice straw consists of cellulose, xylan, lignin and many other components (Xiong, 2004). T. reesei produces carbohydrate active enzymes in response to cellulose, hemicellulose, or their component sugars in the immediate environment (Singh and Bishnoi, 2012). Hence, enzyme preparations obtained from rice straw culture are expected to preferably hydrolyze rice straw. Lignin overlaying cellulose and xylan bundles is partly degraded by alkali-treatment, and the exposed cellulose and xylan effectively function as enzyme inducing carbohydrate compounds. After 5 % NH4OH-treatment the xylan and cell wall bundled with lignin peeled away to expose the inner cellulose fibril. When provided with non-treated rice straw as the enzyme substrate, the rigid cellulosic construction remained even after 3 hours of enzyme hydrolysis (data not shown). In contrast, when provided 5% NH4OH-treated rice straw as substrate, the cellulosic surface dissociated into microfibril tufts. The differences between T. reesei and H. insolens enzymes activities and constituents may be related to the surface structure of the rice straw used in the culture medium. Cellulose fibrils exposed by peeling the xylan layer with NH4OH could facilitate the induction of cellulosic enzyme production by T. reesei, whereas it could be less preferable for the induction of xylanolytic enzyme production by H. insolens.
4. Conclusions
We demonstrated that rice straw is a promising carbon source for T. reesei and H. insolens to produce rice straw degrading enzymes, and the combined use of T. reesei 17
and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. However, the enzyme activities were generally lower than commercially available enzymes (Kai et al., 2010). We are currently investigating innovative new culture techniques and conditions to improve the hydrolyzing activity of fugal enzyme, especially of H. insolens enzyme to levels comparable with commercial enzyme preparations.
Acknowledgements
We thank Dr. Ken Tokuyasu and Dr. Masakazu Ike of the National Food Research Institute, National Agricultural and Food Research Organization, for kind offer of rice straw samples. This study was partly supported by a grant from the Biomass Utilization Project of Ministry of Agriculture, Forestry, and Fisheries of Japan (BEC-C070).
Conflict of Interest The authors declare that they have no conflicts of interest.
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Figure Legends
Fig. 1. Enzyme activity and SDS-PAGE analysis of enzyme from T. reesei cultivated with three different carbon sources. A; Hydrolyzing activity of T. reesei enzyme grown on rice straw and Avicel. *; P < 0.05, the data were obtained from three independent experiments. Statistical analysis was conducted with the Stundent’s t-test. B; Representative silver stain of a SDS-PAGE gel of T. reesei enzyme. Lane 1; molecular mass makers, lane 2; 1.25% NH4OH-treated rice straw as the carbon source, lane 3; non-treated rice straw as the carbon source, lane 4; Avicel as the carbon source. In measuring rice straw hydrolyzing activity, 5% NH4 OH-treated rice straw was used as substrate.
Fig. 2. Enzyme activity and SDS-PAGE analysis of H. insolens enzyme cultivated with three different carbon sources. A; Hydrolyzing activity of H. insolens from grown on rice straw and Avicel. *; p < 0.05, the data were obtained from three independent experiments and statistical analysis was conducted with the Stundent’s t-test. B; Representative silver stain of a SDS-PAGE gel of H. insolens enzymes. Lane 1; 26
molecular mass makers, lane 2; 1.25% NH4OH-treated rice straw as the carbon source, lane 3; non-treated rice straw as the carbon source, lane 4; Avicel as the carbon source. In measuring rice straw hydrolyzing activity, 5% NH4OH-treated rice straw was used as substrate.
Fig. 3. Optimal pH and temperature for the activity of T. reesei and H. insolens enzymes. A; Optimal pH. B; Optimal temperature. Activities are represented relative to the highest value. The data were obtained from three independent experiments.
Fig. 4. Time course of cellulase and xylanase activities as a function of time for the culture of (A) T. reesei and (B) H. insolens. The data were obtained from three independent experiments.
Fig. 5. Synergistic effect of mixed T. reesei and H. insolens enzyme preparations on saccharification of rice straw. The data were obtained from three independent experiments. T and H represent enzymes from T. reesei and H. insolens, respectively. Figures added after T and H represent the percentage (v/v) of each enzyme included in the mixed preparation; black bar, actual measured value; gray bar, presumed value from combined enzyme preparation. Statistical analysis was conducted with the Stundent’s t-test. *; P < 0.05, **; P < 0.05
Supplementary figure (S-Fig A, B) Scanning electron microphotographs of rice straw. A; Non-treated rice straw used as a substrate. (magnification x600), B; 5 % NH4OH-treated rice straw (magnification x550).
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Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.
Highlights Rice straw is a viable carbon source for fungal production of rice straw cellulases. T. reesei and H. insolens enzymes synergistically enhanced rice straw degradation. NH4OH-treated rice straw in the culture medium improved T. reesei enzyme activity.
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S0960-8524(17)30147-5 http://dx.doi.org/10.1016/j.biortech.2017.01.075 BITE 17603
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
8 November 2016 20 January 2017 21 January 2017
Please cite this article as: Kogo, T., Yoshida, Y., Koganei, K., Matsumoto, H., Watanabe, T., Ogihara, J., Kasumi, T., Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source., Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.01.075
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Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.
Takashi Kogo, Yuki Yoshida, Keisuke Koganei, Hitoshi Matsumoto, Taisuke Watanabe, Jun Ogihara, and Takafumi Kasumi*
Applied Microbiology and Biotechnology Laboratory, Department of Chemistry and Lifescience, Nihon University (1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan)
*Corresponding author (Tel & Fax: +466-84-3943; E-mail: [email protected])
List of Abbreviations CMCase, carboxymethyl cellulase; pNP-Xase, p-nitrophenyl xylosidase; pNP-Lase, p-nitrophenyl lactosidase
Highlights Rice straw is a viable carbon source for fungal production of rice straw cellulases. T. reesei and H. insolens enzymes synergistically enhanced rice straw degradation. NH4OH-treated rice straw in the culture medium improved T. reesei enzyme activity.
Abstract Rice straw was evaluated as a carbon source for the fungi, Trichoderma reesei and Humicola insolens, to produce enzymes for rice straw hydrolysis. The enzyme activity of T. reesei and H. insolens cultivated in medium containing non-treated rice straw 1
were almost equivalent to the enzyme of T. reesei cultivated in Avicel medium, a form of refined cellulose. The enzyme activity of T. reesei cultivated in medium containing NH4OH-treated rice straw was 4-fold higher than enzyme from cultures grown in Avicel medium. In contrast, H. insolens enzyme from cultures grown in NH4OH-treated rice straw had significantly lower activity compared with non-treated rice straw or Avicel. The combined use of T. reesei and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. Our data suggest that rice straw is a promising low-cost carbon source for fungal enzyme production for rice straw hydrolysis.
Key words Biomass degrading enzymes, Trichoderma reesei, Humicola insolens, rice straw, synergistic effect.
Introduction Lignocellulose is probably the most abundant but under-utilized biomass resource in the world. The monosaccharides obtained from the degradation of lignocellulose into its component sugars can be converted to bio-ethanol, which is an important alternative to fossil fuels as concerns about oil, gas and coal shortages and their environmental impacts continue to grow (Sun and Cheng, 2002). In recent years, the potential of biomass for the production of chemicals and pharmaceuticals has also received increased attention. For example, butanol or erythritol obtained by fermentation can be converted to polymers such as bio-resins or plastic (Kiyoshi et al., 2015; Rahnama et al., 2014; Kobayashi et al., 2015). The increased interest in the potential of biomass stems from shortages in the raw materials on which naphtha plants depend, and a 2
world-wide shift towards the use of alternative energy sources (Yoshimura et al., 2012). The utilization of biomass is consistent with global sentiment driving a move away from a high-consumption fossil fuel dependent society to more eco-friendly communities powered by renewable bioresources. In Japan, thirteen million tons of rice straw are produced annually. Although 30% is utilized as livestock feed or manure, the remaining 70% is discarded (Nagashima,
2010). The polysaccharide composition of rice straw is similar to that of other herbaceous plants such as sugarcane or corn stover (Templeton et al., 2009; SN, 2008; Sun and Cheng, 2002). It seems no great difference in cost among these biomasses; actually no authentic comparative data are shown, probably because that they depend on how much amount is reliably available in each local area. Anyway, the rice is one of the typical and important crop in this country, and therefore rice straw concomitantly produced is much handy biomass. Hence, this hugely under-utilized resource is an attractive proposition from the perspective of reduced cost, reliable supply, and minimal impact on cropping and food production, all of which are of vital importance for biomass utilization. In addition, rice straw recycling has the potential to improve the prosperity of regional communities based on a local-production for local-consumption system (Singh and Bishnoi, 2012). Rice straw is mainly composed of polysaccharides such as cellulose (40%), hemicellulose (35%), lignin (10%), and silica (5%) (Kahar, 2013). These polymers are crosslinked to form a rigid three-dimensional structure that hinders rapid degradation into component sugars required for fermentation (Park et al., 2010). The development of efficient, practical and low cost approaches to rice straw degradation are essential to fully realize the potential of rice straw as a bioresource. In this regard, the fungus genus Trichoderma, includes strains that produce large amounts of cellulolytic 3
enzymes. Among them, T. reesei (ATCC-66589), which is a genetically engineered type of T. reesei (QM9414), is one of the most promising for cellulase production. Depending on T. reesei culture conditions, the maximal amount of extracellular protein produced is estimated to be 60-100g per liter of culture (Park et al., 2010). One of the characteristics of T. reesei is that more than 90% of the enzyme protein is composed of cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II), which are both essential for rice straw degradation. However, xylanase is another essential enzyme that is not necessarily present in sufficient quantities in T. reesei enzyme preparations (Sweeney and Xu, 2012). Hence, the growth and/or enzyme composition of T. reesei have been extensively studied from the view point of efficient hydrolysis of lignocellulosic rice straw (Ogasawara and Shida, 2012), augmented with chemical (alkali or acid), mechanical, or biological (white or brown rotting fungi) pre-treatment to promote enzymatic degradation (Alvira et al., 2010). The carbon source supplied to microorganisms is crucial for enzyme production. In this regard, Avicel is a form of refined cellulose that is currently the preferred substrate for T. reesei to produce inducible cellulosic enzymes. However, although Avicel is widely used in culture media, it is prohibitively expensive as a carbon source for large-scale biomass projects. Previously, we reported that the fungus, Humicola insolens, produces biomass degrading enzymes in different amounts depending on the carbon source in the medium (Matsumoto et al., 2014). If T. reesei is similar, using rice straw instead of Avicel might induce enzyme constituents more suitable for rice straw hydrolysis, and concomitantly bring about a significant cost reduction. In addition, since H. insolens enzyme preparations are rich in xylanase, combining enzyme preparations from T. reesei and H.
4
insolens may result in a synergistic and improved efficiency in rice straw decomposition. In this study, we investigated the induction, constituents and functions of rice straw degradation enzymes from T. reesei and H. insolens grown using rice straw as the carbon source. In addition, the synergistic effects of mixed T. reesei/H. insolens enzyme preparations were evaluated as a more efficient approach to rice straw degradation.
2. Materials and methods
2.1 Fungal strains
The T. reesei (ATCC-66589) and H. insolens (ATCC-26908) strains were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The strains were maintained in 10% glycerol at -80 °C.
2.1.1. Culture conditions
T. reesei and H. insolens were propagated on potato dextrose agar (PDA) medium, and yeast extract starch soluble agar (YpSsA) medium, respectively. For the preparation of enzyme, T. reesei was pre-cultured in 50 mL of medium consisting of 3% glucose, 0.6% (NH4)2SO4, and 0.6% corn steep liquor at 30 °C for 3 days at 200 rpm. The pre-culture was transferred into 100 mL of medium containing 0.9% Avicel or 2% rice straw (0.9% Avicel carbon equivalent), 0.18% (NH4)2SO4 and 0.18% corn steep liquor, and cultured at 30 °C for 7 days at 200 rpm. 5
H. insolens was pre-cultured in 50 mL of medium composed of 5% glucose and 0.3% ExSBM at 30 °C for 5 days at 200 rpm. The pre-culture was transferred into 100 mL of medium containing 2% Avicel or 4.4% rice straw (2% Avicel carbon equivalent) and 2% SBM, and cultured at 30 °C for 7 days at 200 rpm. After centrifugation at 12,000 rpm for 10 min, T. reesei and H. insolens supernatants were used for the measurement of enzymatic activity.
2.2. Preparation of rice straw
Rice straw was harvested in Ibaraki Prefecture, Japan on Oct. 10th, 2013 and kept in a cool and dry room to avoid deterioration and rotting. After washing with water and air-drying, the straw was cut into approximately 5 cm lengths, ground in a milling machine (P-15, Fritsch Japan Co., Ltd., Yokohama, Japan), and passed through size 40 mesh sieves. The milled straw was used as a carbon source or an enzyme substrate after treatment for 72 h at 4 °C with 1.25% NH4OH or 5% NH4OH, respectively, followed by neutralization with 2N (NH4)2SO4. Milled straw swollen with water was used as substrate for enzyme assays.
2.3. Chemical reagents
p-nitrophenyl β-D-lactopyranoside (pNP-L), p-nitrophenyl β-D-xylopyranoside (pNP-X) and beech wood xylan were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Carboxymethyl cellulose (CMC) was purchased from nacalai tesque (Kyoto, Japan). All other reagents used were general commercial supplies from WAKO chemicals (Osaka, Japan). ExSBM (extruded soybean meal) and SBM 6
(soybean meal) were supplied courtesy of J-Oil Mills, CO., LTD (Osaka, Japan).
2.4. Enzyme activity and protein content assays
Xylanolytic and CMC hydrolyzing activities were assayed using a reaction mixture consisting of 0.8% beech wood xylan or CMC, McIlvaine buffer (pH 5.0 for T. reesei enzyme, pH 6.0 for H. insolens enzyme), and culture supernatant as the enzyme preparation. After incubation at 50 °C for 10 min, the release of reducing sugar was assayed by the DNS method (Miller, 1958). One unit of activity was defined as the amount of enzyme that released 1 µmol of reducing sugar per minute. pNP-L and pNP-X degrading activities were assayed using a reaction mixture composed of 100 µL 4 mM pNP-L or pNP-X, 100 µL 200 mM McIlvaine buffer (pH 5.0 or 6.0), and 200 µL culture supernatant in a total volume of 400 µL. After incubation at 50 °C for 10 min, 400 µL 1M sodium carbonate was added to the reaction mixture which was then colorimetrically assayed at 415 nm using a spectrophotometer (iMark Microplate Reader, Bio-Rad Laboratories Inc., Hercules, CA, USA) (Wood and Bhat, 1988). One unit of activity was defined as the amount of enzyme that liberates 1 µmol of p-nitrophenol per minute. The protein content of enzyme preparations was measured by the Lowry-Folin method against a bovine serum albumin standard (Lowry et al., 1951). Rice straw hydrolyzing activity was assayed in a reaction mixture composed of 800 µL 5% NH4OH-treated or non-treated rice straw as substrate, 100 µL of McIlvaine buffer (pH 5.0 or 6.0), and 100 µL of culture supernatant in a 1.5 mL tube. Equal amounts of enzyme protein were assayed. The reaction mixture was incubated at 50 °C for 180 min and the reducing sugar was assayed with the DNS method (Miller, 1958). 7
One unit of rice straw hydrolyzing activity was defined as the amount of enzyme that liberated 1 µmol of reducing sugar per minute. Apparent optimum pH was determined in a range of pH 2-8 using glycine buffer (pH 2-3), MacIlcvane buffer (pH 3-6), and phosphate buffer (pH 6-8), and apparent optimum temperature was determined in a range of 30-70 °C.
2.5. SDS-Polyacrylamide electrophoresis
Supernatant from a 7-day culture of T. reesei or H. insolens was subjected to SDS-PAGE on a NuPAGE Novex 4 to 12% Bis-Tris gel (Invitrogen, Thornton, Australia). Samples were prepared each containing 10 µL of supernatant (approximately 10 mg/L of protein) and 10 µL of loading buffer [0.5 M Tris-HCl, pH 6.8, 50% (v/v) glycerol, 10% SDS, 5% -mercaptoethanol, and 0.05% bromophenol blue]. The samples were boiled for 3 min, centrifuged for 30 sec, and cooled to room temperature before being loaded onto the gel. Electrophoresis was carried out at 75 V for 90 min using Tris-glycine running buffer containing 1.44% Glycine (Bio-Rad, Sydney, Australia), 0.3% Tris-Base and 0.1% SDS, (Peterson et al., 2011). After electrophoresis, the gel was soaked in fixing solution (100mL methanol + 100mL acetic acid + 100 mL distilled water) for 40 min, and washed with washing solution (100 mL methanol + 100 mL distilled water) for 10 min and subsequently with distilled water for 10 min. Following dipping in sensitizing solution (10 mg sodium thiosulfate dissolved in 50 mL distilled water) for 1 min, the gel was rinsed 3 times with distilled water, and soaked in developing solution (50 mg silver nitrate dissolved in 50 mL distilled water) for 20 min. The stained gel was rinsed 3 times with distilled water, and 8
transferred into stopping solution (10 mL acetic acid + 10 mL acetic acid) to stop the excess developing.
2.6. Scanning electron microscopy
The non-treatment rice straw and 1.25% NH4OH-treated rice straw were immersed in 2% glutaraldehyde, washed three times with 0.1 M cacodylate buffer (pH 7.0), and re-suspended in 1% osmium solution. The solution was dehydrated for 10 min each with 50%, 70%, 80%, 90%, and 99.8% acetone, and twice with 100% acetone for 30 min. The rice straw residue was then soaked for 30 min in a mixture of 100% acetone and isoamylacetone (1:1), followed by 90 min in 100% isoamylacetone, and stored at 4 °C before use. The final preparation was dried using a critical point drier (HCP-2, Hitachi High-Technologies Cooperation, Tokyo, Japan) and observed with a scanning electron microscope (S-3500, Hitachi High-Technologies Cooperation) following vacuum deposition.
2.7. Scale-up using a jar fermenter
The effect of scaling up the culture volume on enzyme production was examined using a 2 L jar fermenter (TBR-2-3, Sakura Seiki Bioreactor, Tokyo, Japan). The pre-culture obtained as described above was inoculated (10% (v/v)) into 1 L of medium composed of 2% (w/v) of 1.25% NH4OH-treated rice straw (w/v) and 0.4% corn steep liquor, and cultivated for 3 days at 30 °C at 300 rpm. The culture was supplemented with additional rice straw (3% w/v) and cultivation was continued for a further 7 days under the same conditions. The space velocity of aeration was 1 vvm, 9
and the agitation rate was 300 rpm.
3. Results and Discussion
Our data suggest that rice straw is a promising carbon source for the cultivation of T. reesei and H. insolens to produce enzymes for the decomposition of rice straw. The relatively low cost of rice straw compared with alternative refined cellulosic carbon sources such as Avicel and others, addresses one of the most crucial problems for the practical use of enzymes for digesting lignocellulosic materials.
3.1. Rice straw as a carbon source - effect on enzyme activity.
Generally, rice straw must be pre-treated to be used as a carbon source to efficiently nourish microorganisms. For that purpose, acid treatment with H2SO4 or HCl, or alkali treatment with NaOH, have been used in the pre-treatment process (Sarkar and Aikat, 2012; Alvira et al., 2010; Galbe and Zacchi, 2007; Kim et al., 2013). However, these methods sometimes result in a reduced yield due to excess decomposition of constitutional sugars, or the production of organic compounds that inhibit fermentation (Sun and Cheng, 2002). Hence, we selected NH4OH as the pre-treatment agent in this study because it is a less hazardous, weak alkali solution known to effectively loosen plant cell walls (Uppugundla et al., 2015). With the exception of CMCase, enzyme activities in T. reesei rice straw cultures were considerably higher than in Avicel cultures (Fig. 1A). The 1.25% NH4OH-treated rice straw had a much higher enzymatic activity compared with non-treated rice straw,
10
especially rice straw hydrolyzing activity. It suggests that cellulolytic enzymes other than CMCase were preferably induced during cultivation with alkali-peeled rice straw. While, the xylan and rice straw hydrolyzing activities of H. insolens enzyme cultivated with non-treated rice straw were comparable with enzyme from Avicel culture. However, when cultured in 1.25% NH4OH-treated rice straw, enzyme activity was significantly lower (Fig. 2A). pNP-L and CMCase activities of H. insolens cultured with non-treated rice straw were less than one half of those enzymes cultured with Avicel. Hence, significant part of rice straw hydrolyzing activity is considered to rely on xylanase activity, which may be insufficiently induced with alkali-peeled rice straw. Although xylan, pNP-X and pNP-L hydrolyzing activities of T. reesei enzyme were significantly higher in rice straw cultures compared with Avicel culture (Fig. 1A and Fig. 2A), CMCase activity did not substantially vary according to the carbon source. Hence, the greater rice straw hydrolyzing activity is possibly due to the elevation of activities of such enzymes as xylanase, pNP-Xase and pNP-Lase. Difference in rice straw hydrolytic activity between two fungal enzyme preparation may reflect the difference in enzyme composition induced by alkali-treated or non alkali-treated biomass. Thus, the alkali-treated rice straw is excellent carbon source for enzyme production of T. reesei on rice straw hydrolysis, in contrast, non alkali-treated rice straw is passable carbon source for enzyme production of H. insolens on rice straw hydrolysis.
3.2. SDS-PAGE profiles of enzymes
3.2.1 T. reesei enzyme
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Fig. 1B shows SDS-PAGE profile of the enzymes obtained from T. reesei in media containing 1.25% NH4OH-treated or non-treated rice straw, and Avicel. There were few clear differences between the SDS-PAGE profiles of the three enzyme preparations from T. reesei cultures. SDS-PAGE followed by silver staining revealed more prominent and well defined 23 kD and 32 kD bands in the rice straw enzyme preparations (lanes 2 and 3) compared with the Avicel enzyme preparation (lane 4). The 23 kD and 32 kD bands in the rice straw enzyme preparations may represent endoglucanase V (EG V) and xylanase III (XYN III), respectively. Some minor proteins other than cellulases and hemicellulases are known to work co-operatively to decompose rigid-structured biomass (Xiong, 2002). It is possible that these minor fractions visible on the SDS-PAGE gels are components of the enzyme assessed in this study, and are involved in the enzymatic degradation of rice straw. The 125 kD band is likely to be β-xylosidase (BXL) (Semenova et al., 2009), although this requires verification by immunoblotting. In the cellulase group between 45 kD and 66 kD (Chokhawala et al., 2015; kondo et al., 2012), a 57 kD band presumed to be cellobiohydrolase I (CBH I) is seen more distinctly in the rice straw enzyme preparations compared with the Avicel enzyme. T. reesei has been reported to produce xylanolytic enzymes such as xylanase I (XYN I), xylanase II (XYN II), and XYN III (Törrönen et al., 1992; Xu et al., 2000; Ogasawara et al., 2006) in a size range of 19, 21 and 32 kD, respectively. On the SDS-PAGE silver stained gels proteins in this size range are more prominent in the rice straw enzyme compared with the Avicel enzyme. Overall, these data suggest that rice straw is a preferable carbon source for T. reesei to produce rice straw hydrolyzing enzymes.
3.3.2 H. insolens enzyme 12
Analysis of silver stained SDS-PAGE gels of H. insolens enzyme revealed more prominent and well defined 32 and 37 kD bands in preparations from non-treated rice straw cultures compared with 1.25% NH4OH-treated rice straw culture (Fig. 2B). In addition, bands around 45-66 kD were observed that were faintly seen in 1.25% NH4OH-treated rice straw culture. The SDS-PAGE profile of enzyme of H. insolens grown with non-treated rice straw and Avicel were similar, but considerably different from 1.25% NH4OH-treated rice straw enzyme. Many bands visible in the enzyme preparations from non-treated rice straw and Avicel, whereas relatively few bands are seen in enzyme preparations from NH4OH-treated rice straw. The H. insolens enzyme activity profile was clearly different from that of T. reesei. H. insolens alters enzyme composition depending on the carbon source (Matsumoto et al., 2014). When supplied with NH4OH-treated rice straw, H. insolens enzyme activities were strikingly low compared with enzymes obtained from non-treated rice straw or Avicel. It is possible that H. insolens growth is inhibited by an excess of inorganic nitrogen from (NH4)2SO4 generated after neutralization. Although the overall enzyme activity of H. insolens is lower than T. reesei, many more bands are detected by SDS-PAGE in enzyme preparations obtained from H. insolens grown with non-treated rice straw compared with T. reesei. The band at 124 kD in enzyme obtained from H. insolens grown in non-treated straw and Avicel cultures is presumed to be H. insolens β-glucosidase 3A (HiBgl 3A) and H. insolens β-glucosidase 3B (HiBgl 3B), which are H. insolens specific proteins (Xia et al., 2016). Although indistinct on silver stained gels, the cellulosic enzymes such as CBH I, cellobiohydrolase II (CBH II), endoglucanase I (EG I), endoglucanase II (EG II), endoglucanase III (EG III) and EG V (Schiilein et al., 1997) may be present in H. insolens enzyme. In addition, band at 37 kD may represent 13
Xyl43A (β-xylosidase) (Mustaira et al., 2016). The verification of the presence of these proteins awaits further analysis by immunoblotting.
3.4 Conditions for optimal enzyme activity
The pH and temperature dependence of relative enzyme activities are shown in Fig. 3A and Fig. 3B, respectively. Both CMC and xylan hydrolyzing activity showed typical bell-shaped curves. The optimal pH and temperature for T. reesei enzyme activity were 4.0-5.0 and 60 °C, and 5.0-6.0 and 60 °C for H. insolens. The optimal pH for T. reesei and H. insolens enzymes rice straw degrading activity were 4.0 and 5.0, respectively, which was similar to CMCase and xylanase (data not shown). However, the 50 °C optimum temperature for T. reesei and H. insolens enzymes rice straw hydrolyzing activity was slightly lower compared with CMCase and xylanase. This difference could be related to the extended incubation time required for rice straw degradation compared with synthetic substrates; 10 min for CMC and xylan hydrolyzing activity, and 180 min for rice straw degrading activity.
3.5. Enzyme activity in scaled up culture using a jar fermenter
Fig.4 shows the time course enzyme activity of T. reesei (Fig. 4A) and H. insolens (Fig. 4B) during cultivation in a jar fermenter. For T. reesei, enzyme activity was detectable from day 3, and increased rapidly thereafter. The increase in CMCase activity was not as great as the other enzymes. After 7 days of cultivation, the xylan, pNP-L and pNP-X hydrolyzing activities were 7-fold, 29- fold, and 30 fold higher, respectively, than those obtained from small scale cultivation in a 500 mL shake flask. 14
For H. insolens, enzyme activity was detectable from day 1, and increased exponentially over 7 days. However, compared with T. reesei, the increases in enzyme activity were not as great as small scale cultivation; 2.5-fold for xylanase, 20-fold for pNP-X hydrolyzing activity, 10-fold for pNP-L hydrolyzing activity.
3.6. Synergistic effect of mixed enzyme preparations on rice straw hydrolysis
Synergy between T. reesei cellulase and β-glucosidase from Taiwanese fungi (Ng et al., 2011), Aspergillus niger and T. reesei (van den Brink et al., 2014), or xylanase and glycoside hydrolases (Goncalves et al., 2015) has been reported in biomass degradation studies. The synergistic effect of mixed T. reesei and H. insolens enzyme preparations on saccharification of rice straw is presented in Fig. 5. The ratios were calculated from the released reducing sugar based on the estimated total sugar content of rice straw. As shown by the black bars, there was a significant synergistic effect of the combined enzyme preparations compared with the sums of the ratios obtained by using the individual enzyme preparations (gray bars) (Kai et al., 2010). To our knowledge, this is the first time that this phenomenon has been reported. Hydrolysis ratios of T. reesei and H. insolens were 70.3% and 23.5%, respectively, within 72h. However, the 75%: 25% (v/v) mixture of T. reesei and H. insolens enzymes produced a 79.8% hydrolysis ratio, which was approximately 10% greater than the T. reesei enzyme alone. While, weight loss in dry weight base of the rice straw was 44.1% after enzyme hydrolysis for 72 h using the 75%: 25% (T : H ; v/v) mixture. This value is close to the vale (49.1%) obtained from the result using commercial enzyme preparation, Celluclast 1.5L (Bussamra et al. 2015). When only 25% of H. insolens enzyme was substituted with the T. reesei enzyme, the ratio reached as high as 68.2%, 15
which was comparable to the level of 100% T. reesei enzyme. As mentioned before, T. reesei mainly produces cellulolytic enzymes such as CBH II, CBH III, or EG, and also produces xylanolytic enzymes such as XYN I and XYN II. Further, XYNs activities are known to enhance when cultivated with carbon sources rich in hemicellulose like rice straw and bagasse. Whereas, BG or BXL that can hydrolyze short chain cello-oligosaccharides into glucose is not always sufficient, especially as to T. reesei 66589 (PC-3-7) (Treebupachatsaku et al., 2015). In case of H. insolens, most of enzymes produced from the wild strain are still unclear. Hence, the results here postulate that xylanase and/or β-glucosidase of H. insolens complement the rice straw hydrolyzing activity of T. reesei, although the activity itself is rather low. Further, it suggests the possibility that H. insolens produce a peculiar protein like swollenin that helps rice straw hydrolysis, considering its wide variety of enzymes. Co-culture system has been reported to be much efficient for on-site production of enzymes (G-Correa et al., 1999, Kolasa et al., 2014). It involves to simplify the pre-treatment process of the materials and subsequent saccharification process, in addition to improve the yields of saccharification and ethanol production. From these viewpoints, SSF (simultaneous saccharification fermentation) system, SPS (simultaneous pretreatment fermentation) system, or SBS (simultaneous bio-delignification saccharification) system have recently been proposed (Vincent et al., 2014; Ma and Ruan, 2015; Dhiman et al., 2015). In case of SPS system, for example, approximately as much as 90 % of corn stover sugary constituent is reported to be hydrolyzed. Although not known it is applicable to our case, T. reesei and H. insolens, we are now trying co-culture system. Hopefully, these two fungi have quite similar culture conditions, and they do not interfere enzyme productivity each other (data will be shown in near future). 16
3.7. Structural change of rice straw after NH4OH-treatment and enzyme decomposition
Rice straw consists of cellulose, xylan, lignin and many other components (Xiong, 2004). T. reesei produces carbohydrate active enzymes in response to cellulose, hemicellulose, or their component sugars in the immediate environment (Singh and Bishnoi, 2012). Hence, enzyme preparations obtained from rice straw culture are expected to preferably hydrolyze rice straw. Lignin overlaying cellulose and xylan bundles is partly degraded by alkali-treatment, and the exposed cellulose and xylan effectively function as enzyme inducing carbohydrate compounds. After 5 % NH4OH-treatment the xylan and cell wall bundled with lignin peeled away to expose the inner cellulose fibril. When provided with non-treated rice straw as the enzyme substrate, the rigid cellulosic construction remained even after 3 hours of enzyme hydrolysis (data not shown). In contrast, when provided 5% NH4OH-treated rice straw as substrate, the cellulosic surface dissociated into microfibril tufts. The differences between T. reesei and H. insolens enzymes activities and constituents may be related to the surface structure of the rice straw used in the culture medium. Cellulose fibrils exposed by peeling the xylan layer with NH4OH could facilitate the induction of cellulosic enzyme production by T. reesei, whereas it could be less preferable for the induction of xylanolytic enzyme production by H. insolens.
4. Conclusions
We demonstrated that rice straw is a promising carbon source for T. reesei and H. insolens to produce rice straw degrading enzymes, and the combined use of T. reesei 17
and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. However, the enzyme activities were generally lower than commercially available enzymes (Kai et al., 2010). We are currently investigating innovative new culture techniques and conditions to improve the hydrolyzing activity of fugal enzyme, especially of H. insolens enzyme to levels comparable with commercial enzyme preparations.
Acknowledgements
We thank Dr. Ken Tokuyasu and Dr. Masakazu Ike of the National Food Research Institute, National Agricultural and Food Research Organization, for kind offer of rice straw samples. This study was partly supported by a grant from the Biomass Utilization Project of Ministry of Agriculture, Forestry, and Fisheries of Japan (BEC-C070).
Conflict of Interest The authors declare that they have no conflicts of interest.
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Figure Legends
Fig. 1. Enzyme activity and SDS-PAGE analysis of enzyme from T. reesei cultivated with three different carbon sources. A; Hydrolyzing activity of T. reesei enzyme grown on rice straw and Avicel. *; P < 0.05, the data were obtained from three independent experiments. Statistical analysis was conducted with the Stundent’s t-test. B; Representative silver stain of a SDS-PAGE gel of T. reesei enzyme. Lane 1; molecular mass makers, lane 2; 1.25% NH4OH-treated rice straw as the carbon source, lane 3; non-treated rice straw as the carbon source, lane 4; Avicel as the carbon source. In measuring rice straw hydrolyzing activity, 5% NH4 OH-treated rice straw was used as substrate.
Fig. 2. Enzyme activity and SDS-PAGE analysis of H. insolens enzyme cultivated with three different carbon sources. A; Hydrolyzing activity of H. insolens from grown on rice straw and Avicel. *; p < 0.05, the data were obtained from three independent experiments and statistical analysis was conducted with the Stundent’s t-test. B; Representative silver stain of a SDS-PAGE gel of H. insolens enzymes. Lane 1; 26
molecular mass makers, lane 2; 1.25% NH4OH-treated rice straw as the carbon source, lane 3; non-treated rice straw as the carbon source, lane 4; Avicel as the carbon source. In measuring rice straw hydrolyzing activity, 5% NH4OH-treated rice straw was used as substrate.
Fig. 3. Optimal pH and temperature for the activity of T. reesei and H. insolens enzymes. A; Optimal pH. B; Optimal temperature. Activities are represented relative to the highest value. The data were obtained from three independent experiments.
Fig. 4. Time course of cellulase and xylanase activities as a function of time for the culture of (A) T. reesei and (B) H. insolens. The data were obtained from three independent experiments.
Fig. 5. Synergistic effect of mixed T. reesei and H. insolens enzyme preparations on saccharification of rice straw. The data were obtained from three independent experiments. T and H represent enzymes from T. reesei and H. insolens, respectively. Figures added after T and H represent the percentage (v/v) of each enzyme included in the mixed preparation; black bar, actual measured value; gray bar, presumed value from combined enzyme preparation. Statistical analysis was conducted with the Stundent’s t-test. *; P < 0.05, **; P < 0.05
Supplementary figure (S-Fig A, B) Scanning electron microphotographs of rice straw. A; Non-treated rice straw used as a substrate. (magnification x600), B; 5 % NH4OH-treated rice straw (magnification x550).
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Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.
Highlights Rice straw is a viable carbon source for fungal production of rice straw cellulases. T. reesei and H. insolens enzymes synergistically enhanced rice straw degradation. NH4OH-treated rice straw in the culture medium improved T. reesei enzyme activity.
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