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Efficient saccharification of ammonia soaked rice straw by combination of Clostridium thermocellum cellulosome and Thermoanaerobacter brockii β-glucosidase
Bioresource Technology 107 (2012) 352–357
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Efficient saccharification of ammonia soaked rice straw by combination of Clostridium thermocellum cellulosome and Thermoanaerobacter brockii b-glucosidase Rattiya Waeonukul a,b,c,1, Akihiko Kosugi a,⇑,1, Chakrit Tachaapaikoon c, Patthra Pason c, Khanok Ratanakhanokchai b, Panida Prawitwong a, Lan Deng a, Masayoshi Saito a, Yutaka Mori a a b c
Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 303-8686, Japan School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok, Thailand Pilot Plant Development and Training Institute (PDTI), King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok, Thailand
a r t i c l e
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Article history: Received 14 November 2011 Received in revised form 22 December 2011 Accepted 23 December 2011 Available online 2 January 2012 Keywords: Cellulosome b-Glucosidase Clostridium thermocellum Thermoanaerobacter brockii Enzyme loading
a b s t r a c t Clostridium thermocellum is known to produce the cellulosomes with efficient plant cell wall degradation ability. To bring out the maximum cellulolytic ability of the cellulosomes, it is necessary to eliminate the end product inhibition by cellobiose. Combinations of b-glucosidases from thermophilic anaerobic bacteria and Aspergillus niger and C. thermocellum S14 cellulosomes were evaluated for optimization of cellulose degradation. b-Glucosidase (CglT) from Thermoanaerobacter brockii, in combination with cellulosomes, exhibited remarkable saccharification ability for microcrystalline cellulose. When rice straw, soaked in 28% aqueous ammonia for 7 days at 60 °C, was hydrolyzed by an enzyme loading combination of 2 mg cellulosome and 10 units CglT per g glucan, 91% of glucan was hydrolyzed to glucose, indicating roughly1/10 the enzyme load of a Trichoderma reesei cellulase (Celluclast 1.5L) and Novozyme-188 combination is enough for the combination of C. thermocellum S14 cellulosomes and CglT to achieve the same level of saccharification of rice straw. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose, consisting of the three major polymers, i.e. cellulose, hemicellulose and lignin, is expected to be utilized as an abundant renewable resource. However, the plant cell wall is difficult to hydrolyze because the cellulose is surrounded by a lignin seal that has a covalent association with hemicellulose, and some parts of cellulose have a tightly packed crystalline structure (Aspinall, 1980). Thus, the rate-limiting step in lignocellulose conversion to useful materials such as bioethanol is hydrolysis of the cellulose and hemicellulose polymers to sugars. Many microorganisms capable of producing cellulose and hemicellulose-degrading enzymes have been reported and characterized (Lynd et al., 2002). Two enzyme systems for the degradation of lignocellulose have been demonstrated in microorganisms. In many aerobic fungi and bacteria, endoglucanase, exoglucanase and ancillary enzymes are individually secreted and act synergistically to degrade lignocellulose. The best studied of these enzymes are the glycosyl hydrolases of Trichoderma reesei (Dashtban et al., 2009). On the other hand, several anaerobic microorganisms have evolved distinct enzyme systems, which involve the formation of a ⇑ Corresponding author. Tel./fax: +81 29 838 6623. 1
E-mail address: [email protected] (A. Kosugi). These authors contributed equally to this work.
0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.126
large, extracellular enzyme complex called the cellulosome (Lynd et al., 2002). Clostridium thermocellum, an anaerobic, thermophilic, and spore-forming bacterium, is the most potent cellulose-degrading bacterium known to produce cellulosomes (2–3.5 MDa) (Bayer et al., 2008; Demain et al., 2005). Recent genome sequencing efforts have identified more than 70 dockerin containing proteins in the genome of C. thermocellum ATCC27405 (Demain et al., 2005). Thus, the cellulosome of C. thermocellum provides for a surprisingly large variety of enzymes and obviously attractive enzymatic properties for the degradation of complex plant biomass. In a previous study, we successfully isolated the C. thermocellum S14 strain that produced cellulosomes with strong lignocellulose degrading ability from bagasse paper sludge in Thailand (Tachaapaikoon et al., 2011). The cellulosomes prepared from C. thermocellum S14 showed not only higher activity against microcrystalline cellulose and hemicellulose, but also for alkaline treated rice straw than the cellulosomes from the best-studied type strain, C. thermocellum ATCC27405. In order to optimize the cellulolytic abilities of cellulosomes, it is necessary to eliminate cellulosome inhibition by the major end product, cellobiose. Kadam and Demain (1989) reported that the addition of cloned C. thermocellum b-glucosidase (BglB) to its cell-free extract promoted the hydrolysis of microcrystalline cellulose. Likewise, enhancement of microcrystalline cellulose
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degradation by a combination of C. thermocellum cellulosome and commercial b-glucosidase (Novozyme-188) from Aspergillus niger has been reported (Lamed et al., 1991). However, the improvement of cellulose hydrolysis by addition of BglB and Novozyme-188 to the cellulosome preparations was not enough for efficient saccharification of cellulosic biomass. In this paper, we tested b-glucosidases from various thermophilic microorganisms and found that Thermoanaerobacter brockii b-glucosidase (CglT) remarkably improved the cellulose hydrolysis activity of the cellulosome prepared from C. thermocellum S14. The combination of T. brockii b-glucosidase and C. thermocellum S14 cellulosome effectively hydrolyzed the rice straw soaked in aqueous ammonia with the required enzyme loading of 1/10 the amount required with a combination of T. reesei cellulase and A. niger b-glucosidase for the same saccharification level, showing this combination provides a powerful saccharification system as a substitute fungal cellulase for natural lignocellulosic material. 2. Methods 2.1. Organisms, media, and growth conditions The hyper cellulolytic strain C. thermocellum S14 has been deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE P-627. Thermoanaerobacter pseudethanolicus ATCC33223, T. brockii ATCC33075 and C. thermocellum ATCC27405 were obtained from the American Type Culture Collection (Manassas, Virginia, USA). Thermoanaerobacter thermohydrosulfuricus YM3 (Mori, 1990) and Thermoanaerobacterium thermosaccharolyticum NOI-1 (Chimtong et al., 2011) were originally isolated from soils and are stored in our laboratory. C. thermocellum S14 was grown on BM7CO media (Tachaapaikoon et al., 2011). The medium was supplemented with10 g/L microcristalline cellulose powder (Sigmacell type-20; Sigma–Aldrich, St. Louis, USA). T. brockii was grown in modified DSMZ 122 medium (Qiang et al., 2009). The medium was adjusted to pH 7.0 and supplemented with 5 g/L cellobiose as the carbon source. All BM7CO and DSMZ 122 media were degassed in boiling water and bubbled with high purity carbon dioxide gas. Escherichia coli DH5a (TAKARA BIO, Shiga, Japan), BL21 (DE3), and plasmids pET19b (Merck KGaA, Darmstadt, Germany) served as the cloning host, expression host, and vector, respectively. E. coli cells were grown at 37 °C in Luria–Bertani (LB) medium containing ampicillin (50 lg/mL). The sake-brewing yeast Saccharomyces cerevisiae Kyokai no. 7 (K7) was obtained from the National Research Institute of Brewing (NRIB). In preculture, the yeast was grown aerobically in static culture at 30 °C on complete medium (YPD) containing 20 g peptone, 10 g yeast extract (Difco Laboratories, Detroit, MI, USA), and 20 g glucose per liter. 2.2. Preparation of cellulosomal enzymes from C. thermocellum S14 and crude enzymes from culture supernatants of thermophilic anaerobic bacteria Cellulosomes were prepared from cell-free broths using C. thermocellum S14 grown culture in BM7CO medium supplemented with 1% (w/v) microcrystalline cellulose for 4 days at 60 °C and recovered by the affinity digestion method (Morag et al., 1992). Each of the crude b-glucosidases was prepared from cell free broths using T. pseudethanolicus, T. brockii, T. thermohydrosulfuricus YM3 and T. thermosaccharolyticum NOI, respectively. After the culture was grown at 60 °C for 2 days with gentle shaking, the culture supernatant was obtained by centrifugation at 8000 rpm for 10 min at 4 °C. Ammonia sulfate precipitation was carried out at 80% saturation against the culture supernatant at 4 °C, overnight,
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and a crude enzyme pellet was obtained by centrifugation (10,000 rpm at 4 °C). Crude enzymes were dissolved in MilliQ-filtered water and applied to a desalting column (Econo-Pac 10DG; Bio-Rad Laboratories, Hercules, CA, USA). 2.3. Preparation of recombinant b-glucosidases (rBglB and rCglT) from C. thermocellum and T. brockii Preparation of chromosomal and plasmid DNA, and transformation were carried out by standard procedures or according to supplier protocols (Qiagen, Frederick, MD, USA). To clone bglB (Gräbnitz et al., 1989) and cglT (Breves et al., 1997) genes, oligonucleotide primers were designed, respectively. The two primers containing artificial BamHI and Bpu1102 recognition sites (underlined) were used to amplify bglB (50 -CGCGGATCCGGCGGTAGATATCAAGAAA-30 for sense primer, and 50 -ATTGCTCAGCTTCCACGTTGTTTATTTTG-30 for antisense primer) and cglT (50 -CGCGGATCCGGCAAAATTTCCAAGAGAT-30 for sense primer, and 50 -ATTGCTCAGCATCTTCGATACCATCATC-30 for antisense primer) fragments by PCR using C. thermocellum and T. brockii genomic DNA as template, respectively. PCR was performed with Ex Taq polymerase (TAKARA BIO, Shiga, Japan) under standard conditions according to the manufacturer’s instructions. The amplified fragments were inserted between the BamHI and Bpu1102 sites of pET19b to generate pET19BglB and pET19CglT.rBglBand rCglT were purified by nickel affinity column chromatography (Ni-NTA agarose resins) and desalted using a desalting column. 2.4. Enzyme and protein assays The assays for the crude and recombinant b-glucosidase were performed at 60 °C in 0.1 M sodium acetate buffer (pH 6.0) with 5 mM CaCl2 under static conditions for 10 min (Tachaapaikoon et al., 2011). Determination of b-glucosidase activity was based on measurement of the release of p-nitrophenol from p-nitrophenyl b-D-glucoside (pNPG). One unit of enzyme releases 1 lmol equivalent of p-nitrophenol per min. Optimum temperature for the crude and recombinant enzymes was determined at temperatures from 40 to 80 °C at pH 6.0 (pH 5.0 and 6.0 for Novozyme-188). Optimum pH range profile was determined using different pH conditions [sodium acetate buffer (pH 4.0–6.0), sodium phosphate buffer (pH 7.0) or Tris–HCl buffer (pH 8.0–9.0)] at 50 or 60 °C. For thermostability assessment, crude and recombinant enzymes, and Novozyme-188 were incubated in 0.1 M sodium acetate buffer (pH 6.0) at 60 °C for 24 h. The remaining activity was measured at 50 °C for Novozyme-188, and at 60 °C for crude and recombinant enzymes using pNPG as the substrate. Glucose inhibition of b-glucosidase activity was measured by adding glucose at different concentrations (0–1 M) to the standard reaction mixture with pNPG as the substrate. The concentration of glucose required to inhibit 50% of the initial b-glucosidase activity under the assay condition was determined. All protein concentrations were determined with the Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as the standard. 2.5. Pretreatment of rice straw by aqueous ammonia soaking Rice straw was purchased from Miyahara store (Nagano, Japan). Rice straw was ground with a 0.5-mm mesh screen (ZM-100; Retsch, Haan, Germany) and 20 g of the substrate was added to a 500 mL autoclavable bottle with 200 mL of 7%, 14%, or 28% (w/v) aqueous ammonia (Wako Pure Chemical, Osaka, Japan). The bottle was incubated for 7 days at 30 or 60 °C. After pretreatment, the wet solid was washed with deionized water until neutral and separated into two portions. One portion was oven dried at 70 °C for 3 days to measure moisture content and was subjected to composition
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analysis. The other, used in carrying out the enzymatic digestibility test, was stored at 4 °C in the refrigerator. 2.6. Compositional analysis of natural and ammonia soaked rice straw The chemical composition of oven-dried natural and ammonia soaked rice straw was analyzed following NREL Chemical Analysis and Testing Standard Procedure. Each sample was subjected to 72% sulfuric acid hydrolysis at 30 °C for 60 min, followed by 3% sulfuric acid hydrolysis at 121 °C for 60 min. The autoclaved hydrolysis solution was neutralized to pH 6.0 with calcium carbonate and vacuum filtered through a filtering crucible. Mono- and oligosaccharide components were quantified by high performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) with a refractive index (Shimadzu RID-10A) detector on a Bio-Rad Aminex HPX87P column (Bio-Rad Laboratories, Hercules, CA, USA) operated at 80 °C with MilliQ-filtered water (Millipore) at a flow rate of 0.6 mL/min. Acid insoluble lignin (Klason lignin) content was defined as the weight of the filter cake (oven-dried at 70 °C to constant weight). 2.7. Microcrystalline cellulose hydrolysis by combination of cellulosome and b-glucosidase Commercial A. niger b-glucosidase (Novozyme-188) was purchased from Sigma–Aldrich. Its b-glucosidase activity was 383 units/mL against pNPG as substrate and the protein content was 203 mg/mL (1 unit: 0.53 mg protein). Microcrystalline cellulose hydrolysis experiments were performed in Hungate tubes containing 10% (w/v) microcrystalline cellulose in the presence of 5 mL, 100 mM sodium acetate buffer (pH 6.0) containing 5 mM CaCl2 and 5 mM dithiothreitol (DTT). The reactions were initiated by mixing 2 mg of cellulosome and 10 units of each b-glucosidase, as determined by the pNPG assay, per gram cellulose. One unit of rCglT and rBglB were approximately 0.09 mg/mL and 0.2 mg/mL as protein concentration, respectively. The serum bottles were placed on an air-bath shaker (TAITEC, Saitama, Japan) at 60 °C and 130 rpm. Samples were periodically removed (0, 24, 48, 72, 96 and 120 h), centrifuged at 12,000 rpm for 10 min and the supernatants were analyzed for oligosaccharides and glucose by HPLC. Since cellobiose and glucose were exclusively detected, digestibility (%) was calculated by the total amount of released cellobiose and glucose per amount of cellulose used in terms of glucose equivalent. 2.8. Enzymatic hydrolysis of ammonia soaked rice straw Washed rice straw from the ammonia soaked pretreatment was enzymatically hydrolyzed under different enzyme loading concentrations of prepared cellulosome and rCglT per gram of glucose obtained by acid-hydrolysis. Enzymatic digestibility experiments were performed at 60 °C with shaking (130 rpm) in 30 mL serum bottles containing 1% (w/v) glucan (based on content of dried pretreated rice straw) in the presence of 100 mM sodium acetate buffer (pH 6.0) containing 5 mM CaCl2 and 5 mM DTT with 10 mL total working volume. The reactions were initiated by mixing of the cellulosome (2 mg/g-glucan) and 10 units (0.9 mg/g-glucan) of rCglT. Commercial fungal cellulase (Celluclast 1.5L) derived from T. reesei was purchased from Sigma–Aldrich. T. reesei cellulase protein concentration was estimated as 140 mg/mL. Three different enzyme loadings were examined in this study: T. reesei cellulase at 2, 10, or 20 mg/g-glucan; and supplemented with Novozyme-188 at 5.3 mg (10 units) or 10.6 mg (20 units)/g-glucan. The hydrolysis of T. reesei cellulase was carried out in 100 mM sodium acetate buffer (pH 5.0) at 50 °C with shaking (130 rpm). As glucose was exclusively detected in the reaction mixtures containing b-glucosidase,
glucan digestibility (%) was expressed as the amount of glucose generated by enzymatic hydrolysis per amount of glucose obtained by acid-hydrolysis in the rice straw used. When cellulosomes alone were used, the amount of released cellobiose was counted in the calculation of glucan digestibility. 2.9. Fermentation tests Slurries of rice straw hydrolysates were prepared using 4% (w/v) glucan (based on content of dried pretreated rice straw), cellulosome (4 mg/g-glucan) and rCglT (20 units or 1.8 mg/g-glucan) in 50 mM sodium acetate buffer (pH 6.0) containing 5 mM CaCl2 with 10 mL total working volume. The saccharification slurry was supplemented with yeast extract and yeast nitrogen base with amino acids (Difco Laboratories, Detroit, MI, USA) at a final concentration of 1 g/L and 5 g/L. Synthetic medium without saccharification slurry (containing the same concentration of glucose, yeast extract and yeast nitrogen base) was used as a reference fermentation test. Precultured S. cerevisiae strain K7 on YPD medium was inoculated into each medium at 10% (v/v) and incubated at 30 °C. Samples were analyzed for ethanol using a gas chromatograph (model GC-2014: Shimadzu) with a flame-ionization detector (FID). 3. Results and discussion 3.1. Comparison of thermostability and glucose tolerance of bglucosidases from various thermophilic bacteria In order to evaluate the compatibility of b-glucosidases for use in combination with cellulosomes, major properties, including glucose tolerance and thermostability, of enzymes from various thermophilic bacteria and A. niger (Novozyme-188) were compared (Table 1). The crude enzyme of T. brockii demonstrated not only the highest thermostability, but also high glucose tolerance among the enzymes evaluated, which is in accord with the results with CglT reported by Breves et al. (1997). Interestingly, the crude enzyme of T. thermosaccharolyticum NOI, which contains an endocellulase-free multienzyme complex, indicated the highest glucose tolerance (650 mM); however, its thermostability was low compared to the enzymes of T. pseudethanolicus and T. brockii. Glucose tolerance and thermostability of Novozyme-188 were much lower than those of the thermophilic anaerobic bacteria. The recombinant b-glucosidase of C. thermocellum (rBglB) was inhibited by low glucose concentration and, strangely, showed low stability at 60 °C under which condition cellulosome is quite stable. The recombinant CglT (rCglT) had the same levels of thermostability and glucose tolerance as the crude enzyme and showed approximately 2-fold higher specific activity than rBglB. Based on the above-mentioned results, we chose CglT (or rCglT) as the cellulosome partner in the cellulose hydrolysis study. 3.2. Enhancement of cellulose hydrolysis ability by combination of C. thermocellum cellulosome and CglT In order to confirm whether CglT is a compatible b-glucosidase, microcrystalline cellulose hydrolysis ability was assessed using a combination of C. thermocellum S14 cellulosomes supplemented with 10 units of Novozyme-188, rBglB or rCglT. When a combination of cellulosomes and Novozyme-188 was tested against 10% (w/v) microcrystalline cellulose, no significant enhancement of hydrolysis activity was observed compared to that of cellulosomes alone (Fig. 1). Addition of rBglB to cellulosomes was observed to slightly increase the saccharification rate of cellulose compared to that of Novazyme-188 and cellulosomes alone. In contrast, when cellulosomes were combined with rCglT, the hydrolysis activity
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R. Waeonukul et al. / Bioresource Technology 107 (2012) 352–357 Table 1 Comparison of b-glucosidase activities from thermophilic anaerobic bacteria. Specific activity (U/mg)a
Thermal stability (%)b
Optimum temperature (°C)c
Optimum pHc
Glucose inhibition (mM)d
Crude enzymes T. pseudethanolicus ATCC33223 T. brockii ATCC33075 T. thermohydrosulfuricus YM3 T. thermosaccharolyticum NOI Novozyme-188
0.07 ± 0.01 0.15 ± 0.02 0.02 ± 0.01 0.35 ± 0.04 1.89 ± 0.02
90 97 50 40 25
60–70 60–75 60–70 60–70 50–60
6.0–7.0 6.0–7.0 6.0–7.0 6.0–7.0 5.0–6.0
320 ± 5.0 450 ± 8.0 300 ± 7.0 650 ± 3.2 60 ± 2.0
Recombinant enzymes rCglT (T. brockii) rBglB (C. thermocellum)
11.0 ± 0.05 5.1 ± 0.05
97 43
60–75 50–60
6.0–7.0 6.0–7.0
450 ± 10.2 95 ± 5.1
The values are the means of triplicate experiments ±standard deviation. a Specific activity; units of b-glucosidase per mg-protein. b Thermal stability is indicated as percentage of remaining b-glucosidase activity. c The range reported represents where >90% of the maximal activity was maintained. d Glucose inhibition was calculated as the glucose concentration required to inhibit 50% of initial b-glucosidase activity.
ment conditions employed. When the rice straw was treated with 7%, 14%, and 28% ammonia at 30 °C for 7 days, the lignin content of pretreated solids was drastically decreased and exhibited delignification rates of 49.2–57.0% compared to raw rice straw. Higher delignification rates were observed with pretreatment at 60 °C than at 30 °C. Under all pretreatment conditions, high glucan and xylan recovery rates were obtained.
Cellulose digestibility (%)
100 80 60 40
3.4. Hydrolysis of aqueous ammonia soaked rice straw using a combination of cellulosome and rCglT
20 0 0
24 48 72 96 120 Time (h)
Cellulosomes with rCglT (T. brockii) Cellulosomes with rBglB (C. thermocellum) Cellulosomes with Novozyme-188 Cellulosomes Fig. 1. Enhancement of cellulose degradation by C. thermocellum S14 cellulosomes in combination with b-glucosidases from different sources. Hydrolysis conditions: substrate, 10% (w/v) Sigmacell; enzymes, 2 mg cellulosomes with/without 10 units of each b-glucosidase per g cellulose; pH 6.0; 60 °C. Error bars represent ±SD (n = 3).
against microcrystalline cellulose increased dramatically (Fig. 1). Differences in the hydrolysis rates are ascribable to differences in the properties of b-glucosidases from A. niger, C. thermocellum and T. brockii (Table 1). In particular, the poor thermostability at 60 °C and low glucose tolerance of Novozyme-188 and rBglB presumably caused the low ability in their cooperative cellulose degradation with C. thermocellum cellulosomes. These results indicated that CglT is a distinctly excellent partner in combination with C. thermocellum cellulosomes.
3.3. Rice straw and pretreated rice straw compositions The chemical composition (percent dry weight) of rice straw is as follows: glucan 35.6 ± 0.1%, xylan 15.6 ± 0.1%, arabinan 4.7 ± 0.1%, galactan 3.4 ± 0.1%, and Klason lignin 20.7 ± 0.1% (containing 1.7% ash). The polysaccharide content (over 50%) together with the relatively low lignin content suggests that rice straw is the good lignocellulosic biomass for ethanol production. Rice straw was subjected to 7%, 14%, and 28% aqueous ammonia soaking treatment at 30° and 60 °C for 7 days, respectively. The chemical composition of rice straw obtained in the filtration cakes after aqueous ammonia soaking pretreatment is shown in Table 2. The percent recovery of solids (SR) was >65% under all pretreat-
When cellulosome alone was applied to the hydrolysis reaction of 28% aqueous ammonia soaked rice straw at 60 °C for 7 days, glucan digestibility was low (37%) (Fig. 2). In contrast to cellulosome alone, when an enzyme combination of 2 mg of cellulosome and 10 units of rCglT per g glucan was used to hydrolyze pretreated rice straw, efficient degradation rates were obtained; 91% glucan saccharification was achieved with rice straw soaked in 28% aqueous ammonia (60 °C), indicating that the presence of a suitable b-glucosidase, such as CglT, is necessary to enhance cellulosome performance. In achieving cost-effective enzymatic hydrolysis of lignocellulosic biomass, the need to reduce the enzyme loading is a major technical challenge. Improvement of pretreatment methods for lignocellulosic materials (Pallapolu et al., 2011), as well as supplementation with accessory enzymes (b-glucosidase, xylanase, and pectinase) (Hu et al., 2011) and ionic or nonionic additives (Zheng et al., 2008) have been assessed in an effort to reduce the enzyme loading. In general, minimum T. reesei cellulase loadings between 15 and 80 mg protein/g-glucan in combination with A. niger b-glucosidase are required for efficient hydrolysis (over 70% glucan conversion) of a broad range of pretreated lignocellulosic substrates (Pallapolu et al., 2011; Arantes and Saddler, 2011). When T. reesei cellulase and Novozyme-188 were used in hydrolysis tests for pretreated rice straw soaked at 60 °C for 7 days, the required total enzyme loading was estimated to be more than 10 times that needed to obtain the same saccharification rates observed using a combination of cellulosomes and rCglT (Fig. 3). These results clearly showed that the combination of cellulosomes and rCglT could not only achieve remarkably efficient saccharification with rice straw, but is also expected to result in a reduction of enzyme loading, leading to an economically feasible saccharification process. Arantes and Saddler (2011) reported that the enzyme loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates and suggested that the rate-limiting step during hydrolysis is the limited accessibility of the
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Table 2 Composition of aqueous ammonia soaked rice straw (g/100 g). Pretreatment conditions Temperature (°C)
Duration (days)
NH3 concentration (%, v/v)
Untreated 30
0 7 7 7 7 7 7
0 7 14 28 7 14 28
60
Klason lignina
Delignificationb (%, w/w)
Solid composition Glucan
Xylan
20.7 ± 0.1 7.7 ± 0.3 7.1 ± 0.4 6.1 ± 0.2 7.2 ± 0.2 5.5 ± 0.1 4.9 ± 0.1
0.0 62.8 65.7 70.5 65.2 73.4 76.3
35.6 ± 0.5 33.5 ± 0.2 32.6 ± 0.4 32.0 ± 0.6 32.7 ± 1.1 32.0 ± 0.5 31.1 ± 0.1
15.6 ± 0.6 12.1 ± 0.3 11.6 ± 0.2 11.3 ± 0.6 11.0 ± 0.2 11.6 ± 0.1 11.4 ± 0.3
7%, 14%, and 28% (v/v) aqueous ammonia concentrations were used. Pretreatments were carried out using a ratio of 1:10 of solid: liquid. The values are the means of triplicate experiments ±standard deviation. a Klason lignin contains acid insoluble inorganic materials. b Delignification indicates the rate of reduced weight of Klason lignin.
80
28% ammonia (60 ˚C) 14% ammonia (60 ˚C)
60
28% ammonia (30 ˚C) 14% ammonia (30 ˚C)
40
28% ammonia (60 ˚C ) without rCglT Untreated rice straw
20
0
24 48 Times (h)
72
Fig. 2. Saccharification of aqueous ammonia soaked rice straw using a combination of cellulosomes and rCglT. Hydrolysis conditions: substrate, 1% (w/v) glucancontaining rice straw; enzymes, 2 mg cellulosomes with/without 10 units rCglT per g glucan; pH 6.0; 60 °C. Error bars represent ±SD (n = 3).
3.5. Ethanol fermentation of rice straw hydrolysate by enzyme combination
Glucan digestibility (%)
100
A fermentation test was carried out using the saccharification slurry derived from 28% aqueous ammonia soaked rice straw containing 4% (w/v) glucan and degraded by a combination of 4 mg
80 60 40 20 0 0
24 48 Times (h)
72
Cellulosomes (2 mg) with rCglT (0.9 mg; 10 units) T. reesei cellulase (20 mg) with Novozyme-188 (10.6 mg; 20 units) T. reesei cellulase (10 mg) with Novozyme-188 (5.3 mg; 10 units) T. reesei cellulase (2 mg) with Novozyme-188 (5.3 mg; 10 units) Fig. 3. Comparison of the saccharification ability of a cellulosome–rCglT combination and T. reesei cellulase (Celluclast 1.5L) supplemented with Novozyme-188. Rice straw (1% glucan) soaked in 28% aqueous ammonia at 60 °C for 7 days was used as the substrate. Hydrolysis conditions for T. reesei cellulase: substrate, 1% (w/v) glucan-containing rice straw; enzymes, 2 and 10 mg of Celluclast 1.5L with 10 units Novozyme-188 or 20 mg Celluclast 1.5L with 20 units Novozyme-188 per g glucan; pH 5.0; 50 °C. Error bars represent ±SD (n = 3).
enzymes to the cellulose chains due to the physical structure of the lignocellulosic substrate. The ammonia treatment has been
20
10
10
5
0
0
12
24
36
48
Ethanol concentration (g/L)
0
reported to increase the porosity of the lignocellulosic materials with the removal of crosslinks between xylan hemicelluloses and other components as well as to cause swelling leading to an increase in internal surface area (Sun and Cheng, 2002). Those effects should increase the accessibility of the cellulose for the enzymes to improve cellulose hydrolysis. On the other hand, the C. thermocellum cellulosomes exhibit strong hydrolytic ability for native crystalline cellulose compared to fungal cellulases (Demain et al., 2005) because of their highly ordered molecular structure (Mayer et al., 1987). Through the whole genome sequence and subunit protein analyses, it has been demonstrated that the enzymatic subunits of the C. thermocellum cellulosomes have a higher diversity of polysaccharide binding modules than that of the T. reesei enzymes (Demain et al., 2005), which supposedly facilitates binding of the cellulosomes to lignocellulosic substrates. These characteristics of the cellulosomes together with their diversified hemicellulolytic activities enable the cellulosomes to effectively hydrolyze the ammonia-soaked rice straw.
Glucose concentration (g/L)
Glucan digestibility (%)
100
0
Time (h) Ethanol production (synthetic medium) Glucose consumption (synthetic medium) Ethanol production (rice straw hydrolysate) Glucose consumption (rice straw hydrolysate) Fig. 4. Ethanol fermentation profile using ammonia soaked rice straw hydrolysates obtained by a combination of cellulosomes and rCglT. Error bars represent ±SD (n = 3).
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cellulosomes and 20 units rCglT per g glucan. The slurry was supplemented with nitrogen and finally prepared with an initial glucose concentration of 18.6 g/L, which corresponds to 89% glucan saccharification. Fermentation profiles of ethanol production and glucose consumption were obtained from cultures grown on the rice straw hydrolysate, as well as from cultures grown on synthetic medium (Fig. 4). Ethanol productivity of 9.2 g/L was achieved, which corresponds to approximately 95% of the theoretical yield, indicating that the saccharification slurry obtained by combination of the cellulosome and rCglT can be fermented without any inhibition. 4. Conclusions This study investigated the effectiveness of combinations of cellulosomes and compatible b-glucosidases in hydrolyzing rice straw. The combination with CglT drastically accelerated degradation of cellulose. When rice straw soaked in 28% aqueous ammonia was hydrolyzed by an enzyme loading of 2 mg cellulosome and 0.9 mg rCglT per g-glucan, 91% of glucan in rice straw was hydrolyzed, which means the required total enzyme loading is more than 10 times lower than that observed for a combination of fungal cellulases. These results showed that the combination has great potential as a cellulolytic system to be an alternative to fungal cellulases. Acknowledgements Waeonukul, R. was supported by the JIRCAS Visiting Research Fellowship Program 2008 and 2009. The authors would like to thank the Thailand Research Fund (TRF Grant) for financial support in Thailand. This work was conducted as part of a research project funded by a grant (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry, and Fisheries of Japan. References Arantes, V., Saddler, J.N., 2011. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnol. Biofuels 4, 3–16. Aspinall, G.O., 1980. Chemistry of cell-wall polysaccharides. In: Preiss, J. (Ed.), The Biochemistry of Plants. Academic Press Inc., New York, pp. 473–500. Bayer, E.A., Shoham, Y., Lamed, R., 2008. Cellulosome-enhanced conversion of biomass: on the road to bioethanol. In: Wall, J.D., Harwood, C.S., Demain, A.L. (Eds.), Bioenergy. ASM Press, Washington, DC, pp. 75–96.
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Breves, R., Bronnenmeier, K., Wild, N., Lottspeich, F., Staudenbauer, W.L., Hofemeister, J., 1997. Genes encoding two different b-glucosidases of Thermoanaerobacter brockii are clustered in a common operon. Appl. Environ. Microbiol. 10, 3902–3910. Chimtong, S., Tachaapaikoon, C., Pason, P., Kyu, K.L., Kosugi, A., Mori, Y., Ratanakhanokchai, K., 2011. Isolation and characterization of endocellulasefree multienzyme complex from newly isolated Thermoanaerobacterium thermosaccharolyticum strain NOI-1. J. Microbiol. Biotechnol. 21, 284–292. Dashtban, M., Schraft, H., Qin, W., 2009. Fungal bioconversion of lignocellulosic residues; opportunities & perspectives. Int. J. Biol. Sci. 5, 578–595. Demain, A.L., Newcomb, M., Wu, J.H.D., 2005. Cellulases, clostridia, and ethanol. Microbiol. Mol. Biol. Rev. 69, 124–154. Gräbnitz, F., Rücknagel, K.P., Seiss, M., Staudenbauer, W.L., 1989. Nucleotide sequence of the Clostridium thermocellum bglB gene encoding thermostable bglucosidase B: homology to fungal b-glucosidases. Mol. Gen. Genet. 217, 70–76. Hu, J., Arantes, V., Saddler, J.N., 2011. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect? Biotechnol. Biofuels 4, 36–49. Kadam, S.K., Demain, A.L., 1989. Addition of cloned beta-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex. Biochem. Biophys. Res. Commun. 161, 706–711. Lamed, R., Kenig, R., Morag, E., Calzada, J.F., Micheo, F.D., Bayer, E.A., 1991. Efficient cellulose solubilization by a combined cellulosome-b-glucosidase system. Appl. Biochem. Biotechnol. 27, 173–183. Lynd, L.R., Weimer, PJ., van Zyl, W.H., Pretorius, I.S., 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66, 506– 577. Mayer, F., Coughlan, M.P., Mori, Y., Ljungdahl, L.G., 1987. Macromolecular organization of the cellulolytic enzyme complex of Clostridium thermocellum as revealed by electron microscopy. Appl. Environ. Microbiol. 53, 2785–2792. Morag, E., Bayer, E.A., Lamed, R., 1992. Affinity digestion for the near-total recovery of purified cellulosome from Clostridium thermocellum. Enzyme Microbial Technol. 14, 289–292. Mori, Y., 1990. Charactarization of a symbiotic coculture of Clostridium themohydrosulfuricum YM3 and Clostridium thermocellum YM4. Appl. Environ. Microbiol. 56, 37–42. Pallapolu, V.R., Lee, Y.Y., Garlock, R.J., Balan, V., Dale, B.E., Kim, Y., Mosier, N.S., Ladisch, M.R., Falls, M., Holtzapple, M.T., Sierra-Ramirez, R., Shi, J., Ebrik, M.A., Redmond, T., Yang, B., Wyman, C.E., Donohoe, B.S., Vinzant, T.B., Elander, R.T., Hames, B., Thomas, S., Warner, R.E., 2011. Effects of enzyme loading and bglucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies. Bioresour. Technol. doi:10.1016/ j.biortech.2011.03.085. Qiang, He., Lokken, P.M., Chen, Si., Zhou, J., 2009. Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus. Bioresour. Technol. 100, 5955–5965. Sun, Ye., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83, 1–11. Tachaapaikoon, C., Kosugi, A., Pason, P., Waeonukul, R., Ratanakhanokchai, R., Kyu, K.L., Arai, T., Murata, Y., Mori, Y., 2011. Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain. Biodegradation. doi:10.1007/s10532-011-9486-9. Zheng, Y., Pan, Z., Zhang, R., Wang, D., Jenkins, B., 2008. Non-ionic surfactants and non-catalytic protein treatment on enzymatic hydrolysis of pretreated Creeping Wild Ryegrass. Appl. Biochem. Biotechnol. 146, 231–248.
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Efficient saccharification of ammonia soaked rice straw by combination of Clostridium thermocellum cellulosome and Thermoanaerobacter brockii b-glucosidase Rattiya Waeonukul a,b,c,1, Akihiko Kosugi a,⇑,1, Chakrit Tachaapaikoon c, Patthra Pason c, Khanok Ratanakhanokchai b, Panida Prawitwong a, Lan Deng a, Masayoshi Saito a, Yutaka Mori a a b c
Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 303-8686, Japan School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok, Thailand Pilot Plant Development and Training Institute (PDTI), King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok, Thailand
a r t i c l e
i n f o
Article history: Received 14 November 2011 Received in revised form 22 December 2011 Accepted 23 December 2011 Available online 2 January 2012 Keywords: Cellulosome b-Glucosidase Clostridium thermocellum Thermoanaerobacter brockii Enzyme loading
a b s t r a c t Clostridium thermocellum is known to produce the cellulosomes with efficient plant cell wall degradation ability. To bring out the maximum cellulolytic ability of the cellulosomes, it is necessary to eliminate the end product inhibition by cellobiose. Combinations of b-glucosidases from thermophilic anaerobic bacteria and Aspergillus niger and C. thermocellum S14 cellulosomes were evaluated for optimization of cellulose degradation. b-Glucosidase (CglT) from Thermoanaerobacter brockii, in combination with cellulosomes, exhibited remarkable saccharification ability for microcrystalline cellulose. When rice straw, soaked in 28% aqueous ammonia for 7 days at 60 °C, was hydrolyzed by an enzyme loading combination of 2 mg cellulosome and 10 units CglT per g glucan, 91% of glucan was hydrolyzed to glucose, indicating roughly1/10 the enzyme load of a Trichoderma reesei cellulase (Celluclast 1.5L) and Novozyme-188 combination is enough for the combination of C. thermocellum S14 cellulosomes and CglT to achieve the same level of saccharification of rice straw. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose, consisting of the three major polymers, i.e. cellulose, hemicellulose and lignin, is expected to be utilized as an abundant renewable resource. However, the plant cell wall is difficult to hydrolyze because the cellulose is surrounded by a lignin seal that has a covalent association with hemicellulose, and some parts of cellulose have a tightly packed crystalline structure (Aspinall, 1980). Thus, the rate-limiting step in lignocellulose conversion to useful materials such as bioethanol is hydrolysis of the cellulose and hemicellulose polymers to sugars. Many microorganisms capable of producing cellulose and hemicellulose-degrading enzymes have been reported and characterized (Lynd et al., 2002). Two enzyme systems for the degradation of lignocellulose have been demonstrated in microorganisms. In many aerobic fungi and bacteria, endoglucanase, exoglucanase and ancillary enzymes are individually secreted and act synergistically to degrade lignocellulose. The best studied of these enzymes are the glycosyl hydrolases of Trichoderma reesei (Dashtban et al., 2009). On the other hand, several anaerobic microorganisms have evolved distinct enzyme systems, which involve the formation of a ⇑ Corresponding author. Tel./fax: +81 29 838 6623. 1
E-mail address: [email protected] (A. Kosugi). These authors contributed equally to this work.
0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.126
large, extracellular enzyme complex called the cellulosome (Lynd et al., 2002). Clostridium thermocellum, an anaerobic, thermophilic, and spore-forming bacterium, is the most potent cellulose-degrading bacterium known to produce cellulosomes (2–3.5 MDa) (Bayer et al., 2008; Demain et al., 2005). Recent genome sequencing efforts have identified more than 70 dockerin containing proteins in the genome of C. thermocellum ATCC27405 (Demain et al., 2005). Thus, the cellulosome of C. thermocellum provides for a surprisingly large variety of enzymes and obviously attractive enzymatic properties for the degradation of complex plant biomass. In a previous study, we successfully isolated the C. thermocellum S14 strain that produced cellulosomes with strong lignocellulose degrading ability from bagasse paper sludge in Thailand (Tachaapaikoon et al., 2011). The cellulosomes prepared from C. thermocellum S14 showed not only higher activity against microcrystalline cellulose and hemicellulose, but also for alkaline treated rice straw than the cellulosomes from the best-studied type strain, C. thermocellum ATCC27405. In order to optimize the cellulolytic abilities of cellulosomes, it is necessary to eliminate cellulosome inhibition by the major end product, cellobiose. Kadam and Demain (1989) reported that the addition of cloned C. thermocellum b-glucosidase (BglB) to its cell-free extract promoted the hydrolysis of microcrystalline cellulose. Likewise, enhancement of microcrystalline cellulose
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degradation by a combination of C. thermocellum cellulosome and commercial b-glucosidase (Novozyme-188) from Aspergillus niger has been reported (Lamed et al., 1991). However, the improvement of cellulose hydrolysis by addition of BglB and Novozyme-188 to the cellulosome preparations was not enough for efficient saccharification of cellulosic biomass. In this paper, we tested b-glucosidases from various thermophilic microorganisms and found that Thermoanaerobacter brockii b-glucosidase (CglT) remarkably improved the cellulose hydrolysis activity of the cellulosome prepared from C. thermocellum S14. The combination of T. brockii b-glucosidase and C. thermocellum S14 cellulosome effectively hydrolyzed the rice straw soaked in aqueous ammonia with the required enzyme loading of 1/10 the amount required with a combination of T. reesei cellulase and A. niger b-glucosidase for the same saccharification level, showing this combination provides a powerful saccharification system as a substitute fungal cellulase for natural lignocellulosic material. 2. Methods 2.1. Organisms, media, and growth conditions The hyper cellulolytic strain C. thermocellum S14 has been deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE P-627. Thermoanaerobacter pseudethanolicus ATCC33223, T. brockii ATCC33075 and C. thermocellum ATCC27405 were obtained from the American Type Culture Collection (Manassas, Virginia, USA). Thermoanaerobacter thermohydrosulfuricus YM3 (Mori, 1990) and Thermoanaerobacterium thermosaccharolyticum NOI-1 (Chimtong et al., 2011) were originally isolated from soils and are stored in our laboratory. C. thermocellum S14 was grown on BM7CO media (Tachaapaikoon et al., 2011). The medium was supplemented with10 g/L microcristalline cellulose powder (Sigmacell type-20; Sigma–Aldrich, St. Louis, USA). T. brockii was grown in modified DSMZ 122 medium (Qiang et al., 2009). The medium was adjusted to pH 7.0 and supplemented with 5 g/L cellobiose as the carbon source. All BM7CO and DSMZ 122 media were degassed in boiling water and bubbled with high purity carbon dioxide gas. Escherichia coli DH5a (TAKARA BIO, Shiga, Japan), BL21 (DE3), and plasmids pET19b (Merck KGaA, Darmstadt, Germany) served as the cloning host, expression host, and vector, respectively. E. coli cells were grown at 37 °C in Luria–Bertani (LB) medium containing ampicillin (50 lg/mL). The sake-brewing yeast Saccharomyces cerevisiae Kyokai no. 7 (K7) was obtained from the National Research Institute of Brewing (NRIB). In preculture, the yeast was grown aerobically in static culture at 30 °C on complete medium (YPD) containing 20 g peptone, 10 g yeast extract (Difco Laboratories, Detroit, MI, USA), and 20 g glucose per liter. 2.2. Preparation of cellulosomal enzymes from C. thermocellum S14 and crude enzymes from culture supernatants of thermophilic anaerobic bacteria Cellulosomes were prepared from cell-free broths using C. thermocellum S14 grown culture in BM7CO medium supplemented with 1% (w/v) microcrystalline cellulose for 4 days at 60 °C and recovered by the affinity digestion method (Morag et al., 1992). Each of the crude b-glucosidases was prepared from cell free broths using T. pseudethanolicus, T. brockii, T. thermohydrosulfuricus YM3 and T. thermosaccharolyticum NOI, respectively. After the culture was grown at 60 °C for 2 days with gentle shaking, the culture supernatant was obtained by centrifugation at 8000 rpm for 10 min at 4 °C. Ammonia sulfate precipitation was carried out at 80% saturation against the culture supernatant at 4 °C, overnight,
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and a crude enzyme pellet was obtained by centrifugation (10,000 rpm at 4 °C). Crude enzymes were dissolved in MilliQ-filtered water and applied to a desalting column (Econo-Pac 10DG; Bio-Rad Laboratories, Hercules, CA, USA). 2.3. Preparation of recombinant b-glucosidases (rBglB and rCglT) from C. thermocellum and T. brockii Preparation of chromosomal and plasmid DNA, and transformation were carried out by standard procedures or according to supplier protocols (Qiagen, Frederick, MD, USA). To clone bglB (Gräbnitz et al., 1989) and cglT (Breves et al., 1997) genes, oligonucleotide primers were designed, respectively. The two primers containing artificial BamHI and Bpu1102 recognition sites (underlined) were used to amplify bglB (50 -CGCGGATCCGGCGGTAGATATCAAGAAA-30 for sense primer, and 50 -ATTGCTCAGCTTCCACGTTGTTTATTTTG-30 for antisense primer) and cglT (50 -CGCGGATCCGGCAAAATTTCCAAGAGAT-30 for sense primer, and 50 -ATTGCTCAGCATCTTCGATACCATCATC-30 for antisense primer) fragments by PCR using C. thermocellum and T. brockii genomic DNA as template, respectively. PCR was performed with Ex Taq polymerase (TAKARA BIO, Shiga, Japan) under standard conditions according to the manufacturer’s instructions. The amplified fragments were inserted between the BamHI and Bpu1102 sites of pET19b to generate pET19BglB and pET19CglT.rBglBand rCglT were purified by nickel affinity column chromatography (Ni-NTA agarose resins) and desalted using a desalting column. 2.4. Enzyme and protein assays The assays for the crude and recombinant b-glucosidase were performed at 60 °C in 0.1 M sodium acetate buffer (pH 6.0) with 5 mM CaCl2 under static conditions for 10 min (Tachaapaikoon et al., 2011). Determination of b-glucosidase activity was based on measurement of the release of p-nitrophenol from p-nitrophenyl b-D-glucoside (pNPG). One unit of enzyme releases 1 lmol equivalent of p-nitrophenol per min. Optimum temperature for the crude and recombinant enzymes was determined at temperatures from 40 to 80 °C at pH 6.0 (pH 5.0 and 6.0 for Novozyme-188). Optimum pH range profile was determined using different pH conditions [sodium acetate buffer (pH 4.0–6.0), sodium phosphate buffer (pH 7.0) or Tris–HCl buffer (pH 8.0–9.0)] at 50 or 60 °C. For thermostability assessment, crude and recombinant enzymes, and Novozyme-188 were incubated in 0.1 M sodium acetate buffer (pH 6.0) at 60 °C for 24 h. The remaining activity was measured at 50 °C for Novozyme-188, and at 60 °C for crude and recombinant enzymes using pNPG as the substrate. Glucose inhibition of b-glucosidase activity was measured by adding glucose at different concentrations (0–1 M) to the standard reaction mixture with pNPG as the substrate. The concentration of glucose required to inhibit 50% of the initial b-glucosidase activity under the assay condition was determined. All protein concentrations were determined with the Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as the standard. 2.5. Pretreatment of rice straw by aqueous ammonia soaking Rice straw was purchased from Miyahara store (Nagano, Japan). Rice straw was ground with a 0.5-mm mesh screen (ZM-100; Retsch, Haan, Germany) and 20 g of the substrate was added to a 500 mL autoclavable bottle with 200 mL of 7%, 14%, or 28% (w/v) aqueous ammonia (Wako Pure Chemical, Osaka, Japan). The bottle was incubated for 7 days at 30 or 60 °C. After pretreatment, the wet solid was washed with deionized water until neutral and separated into two portions. One portion was oven dried at 70 °C for 3 days to measure moisture content and was subjected to composition
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analysis. The other, used in carrying out the enzymatic digestibility test, was stored at 4 °C in the refrigerator. 2.6. Compositional analysis of natural and ammonia soaked rice straw The chemical composition of oven-dried natural and ammonia soaked rice straw was analyzed following NREL Chemical Analysis and Testing Standard Procedure. Each sample was subjected to 72% sulfuric acid hydrolysis at 30 °C for 60 min, followed by 3% sulfuric acid hydrolysis at 121 °C for 60 min. The autoclaved hydrolysis solution was neutralized to pH 6.0 with calcium carbonate and vacuum filtered through a filtering crucible. Mono- and oligosaccharide components were quantified by high performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) with a refractive index (Shimadzu RID-10A) detector on a Bio-Rad Aminex HPX87P column (Bio-Rad Laboratories, Hercules, CA, USA) operated at 80 °C with MilliQ-filtered water (Millipore) at a flow rate of 0.6 mL/min. Acid insoluble lignin (Klason lignin) content was defined as the weight of the filter cake (oven-dried at 70 °C to constant weight). 2.7. Microcrystalline cellulose hydrolysis by combination of cellulosome and b-glucosidase Commercial A. niger b-glucosidase (Novozyme-188) was purchased from Sigma–Aldrich. Its b-glucosidase activity was 383 units/mL against pNPG as substrate and the protein content was 203 mg/mL (1 unit: 0.53 mg protein). Microcrystalline cellulose hydrolysis experiments were performed in Hungate tubes containing 10% (w/v) microcrystalline cellulose in the presence of 5 mL, 100 mM sodium acetate buffer (pH 6.0) containing 5 mM CaCl2 and 5 mM dithiothreitol (DTT). The reactions were initiated by mixing 2 mg of cellulosome and 10 units of each b-glucosidase, as determined by the pNPG assay, per gram cellulose. One unit of rCglT and rBglB were approximately 0.09 mg/mL and 0.2 mg/mL as protein concentration, respectively. The serum bottles were placed on an air-bath shaker (TAITEC, Saitama, Japan) at 60 °C and 130 rpm. Samples were periodically removed (0, 24, 48, 72, 96 and 120 h), centrifuged at 12,000 rpm for 10 min and the supernatants were analyzed for oligosaccharides and glucose by HPLC. Since cellobiose and glucose were exclusively detected, digestibility (%) was calculated by the total amount of released cellobiose and glucose per amount of cellulose used in terms of glucose equivalent. 2.8. Enzymatic hydrolysis of ammonia soaked rice straw Washed rice straw from the ammonia soaked pretreatment was enzymatically hydrolyzed under different enzyme loading concentrations of prepared cellulosome and rCglT per gram of glucose obtained by acid-hydrolysis. Enzymatic digestibility experiments were performed at 60 °C with shaking (130 rpm) in 30 mL serum bottles containing 1% (w/v) glucan (based on content of dried pretreated rice straw) in the presence of 100 mM sodium acetate buffer (pH 6.0) containing 5 mM CaCl2 and 5 mM DTT with 10 mL total working volume. The reactions were initiated by mixing of the cellulosome (2 mg/g-glucan) and 10 units (0.9 mg/g-glucan) of rCglT. Commercial fungal cellulase (Celluclast 1.5L) derived from T. reesei was purchased from Sigma–Aldrich. T. reesei cellulase protein concentration was estimated as 140 mg/mL. Three different enzyme loadings were examined in this study: T. reesei cellulase at 2, 10, or 20 mg/g-glucan; and supplemented with Novozyme-188 at 5.3 mg (10 units) or 10.6 mg (20 units)/g-glucan. The hydrolysis of T. reesei cellulase was carried out in 100 mM sodium acetate buffer (pH 5.0) at 50 °C with shaking (130 rpm). As glucose was exclusively detected in the reaction mixtures containing b-glucosidase,
glucan digestibility (%) was expressed as the amount of glucose generated by enzymatic hydrolysis per amount of glucose obtained by acid-hydrolysis in the rice straw used. When cellulosomes alone were used, the amount of released cellobiose was counted in the calculation of glucan digestibility. 2.9. Fermentation tests Slurries of rice straw hydrolysates were prepared using 4% (w/v) glucan (based on content of dried pretreated rice straw), cellulosome (4 mg/g-glucan) and rCglT (20 units or 1.8 mg/g-glucan) in 50 mM sodium acetate buffer (pH 6.0) containing 5 mM CaCl2 with 10 mL total working volume. The saccharification slurry was supplemented with yeast extract and yeast nitrogen base with amino acids (Difco Laboratories, Detroit, MI, USA) at a final concentration of 1 g/L and 5 g/L. Synthetic medium without saccharification slurry (containing the same concentration of glucose, yeast extract and yeast nitrogen base) was used as a reference fermentation test. Precultured S. cerevisiae strain K7 on YPD medium was inoculated into each medium at 10% (v/v) and incubated at 30 °C. Samples were analyzed for ethanol using a gas chromatograph (model GC-2014: Shimadzu) with a flame-ionization detector (FID). 3. Results and discussion 3.1. Comparison of thermostability and glucose tolerance of bglucosidases from various thermophilic bacteria In order to evaluate the compatibility of b-glucosidases for use in combination with cellulosomes, major properties, including glucose tolerance and thermostability, of enzymes from various thermophilic bacteria and A. niger (Novozyme-188) were compared (Table 1). The crude enzyme of T. brockii demonstrated not only the highest thermostability, but also high glucose tolerance among the enzymes evaluated, which is in accord with the results with CglT reported by Breves et al. (1997). Interestingly, the crude enzyme of T. thermosaccharolyticum NOI, which contains an endocellulase-free multienzyme complex, indicated the highest glucose tolerance (650 mM); however, its thermostability was low compared to the enzymes of T. pseudethanolicus and T. brockii. Glucose tolerance and thermostability of Novozyme-188 were much lower than those of the thermophilic anaerobic bacteria. The recombinant b-glucosidase of C. thermocellum (rBglB) was inhibited by low glucose concentration and, strangely, showed low stability at 60 °C under which condition cellulosome is quite stable. The recombinant CglT (rCglT) had the same levels of thermostability and glucose tolerance as the crude enzyme and showed approximately 2-fold higher specific activity than rBglB. Based on the above-mentioned results, we chose CglT (or rCglT) as the cellulosome partner in the cellulose hydrolysis study. 3.2. Enhancement of cellulose hydrolysis ability by combination of C. thermocellum cellulosome and CglT In order to confirm whether CglT is a compatible b-glucosidase, microcrystalline cellulose hydrolysis ability was assessed using a combination of C. thermocellum S14 cellulosomes supplemented with 10 units of Novozyme-188, rBglB or rCglT. When a combination of cellulosomes and Novozyme-188 was tested against 10% (w/v) microcrystalline cellulose, no significant enhancement of hydrolysis activity was observed compared to that of cellulosomes alone (Fig. 1). Addition of rBglB to cellulosomes was observed to slightly increase the saccharification rate of cellulose compared to that of Novazyme-188 and cellulosomes alone. In contrast, when cellulosomes were combined with rCglT, the hydrolysis activity
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R. Waeonukul et al. / Bioresource Technology 107 (2012) 352–357 Table 1 Comparison of b-glucosidase activities from thermophilic anaerobic bacteria. Specific activity (U/mg)a
Thermal stability (%)b
Optimum temperature (°C)c
Optimum pHc
Glucose inhibition (mM)d
Crude enzymes T. pseudethanolicus ATCC33223 T. brockii ATCC33075 T. thermohydrosulfuricus YM3 T. thermosaccharolyticum NOI Novozyme-188
0.07 ± 0.01 0.15 ± 0.02 0.02 ± 0.01 0.35 ± 0.04 1.89 ± 0.02
90 97 50 40 25
60–70 60–75 60–70 60–70 50–60
6.0–7.0 6.0–7.0 6.0–7.0 6.0–7.0 5.0–6.0
320 ± 5.0 450 ± 8.0 300 ± 7.0 650 ± 3.2 60 ± 2.0
Recombinant enzymes rCglT (T. brockii) rBglB (C. thermocellum)
11.0 ± 0.05 5.1 ± 0.05
97 43
60–75 50–60
6.0–7.0 6.0–7.0
450 ± 10.2 95 ± 5.1
The values are the means of triplicate experiments ±standard deviation. a Specific activity; units of b-glucosidase per mg-protein. b Thermal stability is indicated as percentage of remaining b-glucosidase activity. c The range reported represents where >90% of the maximal activity was maintained. d Glucose inhibition was calculated as the glucose concentration required to inhibit 50% of initial b-glucosidase activity.
ment conditions employed. When the rice straw was treated with 7%, 14%, and 28% ammonia at 30 °C for 7 days, the lignin content of pretreated solids was drastically decreased and exhibited delignification rates of 49.2–57.0% compared to raw rice straw. Higher delignification rates were observed with pretreatment at 60 °C than at 30 °C. Under all pretreatment conditions, high glucan and xylan recovery rates were obtained.
Cellulose digestibility (%)
100 80 60 40
3.4. Hydrolysis of aqueous ammonia soaked rice straw using a combination of cellulosome and rCglT
20 0 0
24 48 72 96 120 Time (h)
Cellulosomes with rCglT (T. brockii) Cellulosomes with rBglB (C. thermocellum) Cellulosomes with Novozyme-188 Cellulosomes Fig. 1. Enhancement of cellulose degradation by C. thermocellum S14 cellulosomes in combination with b-glucosidases from different sources. Hydrolysis conditions: substrate, 10% (w/v) Sigmacell; enzymes, 2 mg cellulosomes with/without 10 units of each b-glucosidase per g cellulose; pH 6.0; 60 °C. Error bars represent ±SD (n = 3).
against microcrystalline cellulose increased dramatically (Fig. 1). Differences in the hydrolysis rates are ascribable to differences in the properties of b-glucosidases from A. niger, C. thermocellum and T. brockii (Table 1). In particular, the poor thermostability at 60 °C and low glucose tolerance of Novozyme-188 and rBglB presumably caused the low ability in their cooperative cellulose degradation with C. thermocellum cellulosomes. These results indicated that CglT is a distinctly excellent partner in combination with C. thermocellum cellulosomes.
3.3. Rice straw and pretreated rice straw compositions The chemical composition (percent dry weight) of rice straw is as follows: glucan 35.6 ± 0.1%, xylan 15.6 ± 0.1%, arabinan 4.7 ± 0.1%, galactan 3.4 ± 0.1%, and Klason lignin 20.7 ± 0.1% (containing 1.7% ash). The polysaccharide content (over 50%) together with the relatively low lignin content suggests that rice straw is the good lignocellulosic biomass for ethanol production. Rice straw was subjected to 7%, 14%, and 28% aqueous ammonia soaking treatment at 30° and 60 °C for 7 days, respectively. The chemical composition of rice straw obtained in the filtration cakes after aqueous ammonia soaking pretreatment is shown in Table 2. The percent recovery of solids (SR) was >65% under all pretreat-
When cellulosome alone was applied to the hydrolysis reaction of 28% aqueous ammonia soaked rice straw at 60 °C for 7 days, glucan digestibility was low (37%) (Fig. 2). In contrast to cellulosome alone, when an enzyme combination of 2 mg of cellulosome and 10 units of rCglT per g glucan was used to hydrolyze pretreated rice straw, efficient degradation rates were obtained; 91% glucan saccharification was achieved with rice straw soaked in 28% aqueous ammonia (60 °C), indicating that the presence of a suitable b-glucosidase, such as CglT, is necessary to enhance cellulosome performance. In achieving cost-effective enzymatic hydrolysis of lignocellulosic biomass, the need to reduce the enzyme loading is a major technical challenge. Improvement of pretreatment methods for lignocellulosic materials (Pallapolu et al., 2011), as well as supplementation with accessory enzymes (b-glucosidase, xylanase, and pectinase) (Hu et al., 2011) and ionic or nonionic additives (Zheng et al., 2008) have been assessed in an effort to reduce the enzyme loading. In general, minimum T. reesei cellulase loadings between 15 and 80 mg protein/g-glucan in combination with A. niger b-glucosidase are required for efficient hydrolysis (over 70% glucan conversion) of a broad range of pretreated lignocellulosic substrates (Pallapolu et al., 2011; Arantes and Saddler, 2011). When T. reesei cellulase and Novozyme-188 were used in hydrolysis tests for pretreated rice straw soaked at 60 °C for 7 days, the required total enzyme loading was estimated to be more than 10 times that needed to obtain the same saccharification rates observed using a combination of cellulosomes and rCglT (Fig. 3). These results clearly showed that the combination of cellulosomes and rCglT could not only achieve remarkably efficient saccharification with rice straw, but is also expected to result in a reduction of enzyme loading, leading to an economically feasible saccharification process. Arantes and Saddler (2011) reported that the enzyme loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates and suggested that the rate-limiting step during hydrolysis is the limited accessibility of the
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Table 2 Composition of aqueous ammonia soaked rice straw (g/100 g). Pretreatment conditions Temperature (°C)
Duration (days)
NH3 concentration (%, v/v)
Untreated 30
0 7 7 7 7 7 7
0 7 14 28 7 14 28
60
Klason lignina
Delignificationb (%, w/w)
Solid composition Glucan
Xylan
20.7 ± 0.1 7.7 ± 0.3 7.1 ± 0.4 6.1 ± 0.2 7.2 ± 0.2 5.5 ± 0.1 4.9 ± 0.1
0.0 62.8 65.7 70.5 65.2 73.4 76.3
35.6 ± 0.5 33.5 ± 0.2 32.6 ± 0.4 32.0 ± 0.6 32.7 ± 1.1 32.0 ± 0.5 31.1 ± 0.1
15.6 ± 0.6 12.1 ± 0.3 11.6 ± 0.2 11.3 ± 0.6 11.0 ± 0.2 11.6 ± 0.1 11.4 ± 0.3
7%, 14%, and 28% (v/v) aqueous ammonia concentrations were used. Pretreatments were carried out using a ratio of 1:10 of solid: liquid. The values are the means of triplicate experiments ±standard deviation. a Klason lignin contains acid insoluble inorganic materials. b Delignification indicates the rate of reduced weight of Klason lignin.
80
28% ammonia (60 ˚C) 14% ammonia (60 ˚C)
60
28% ammonia (30 ˚C) 14% ammonia (30 ˚C)
40
28% ammonia (60 ˚C ) without rCglT Untreated rice straw
20
0
24 48 Times (h)
72
Fig. 2. Saccharification of aqueous ammonia soaked rice straw using a combination of cellulosomes and rCglT. Hydrolysis conditions: substrate, 1% (w/v) glucancontaining rice straw; enzymes, 2 mg cellulosomes with/without 10 units rCglT per g glucan; pH 6.0; 60 °C. Error bars represent ±SD (n = 3).
3.5. Ethanol fermentation of rice straw hydrolysate by enzyme combination
Glucan digestibility (%)
100
A fermentation test was carried out using the saccharification slurry derived from 28% aqueous ammonia soaked rice straw containing 4% (w/v) glucan and degraded by a combination of 4 mg
80 60 40 20 0 0
24 48 Times (h)
72
Cellulosomes (2 mg) with rCglT (0.9 mg; 10 units) T. reesei cellulase (20 mg) with Novozyme-188 (10.6 mg; 20 units) T. reesei cellulase (10 mg) with Novozyme-188 (5.3 mg; 10 units) T. reesei cellulase (2 mg) with Novozyme-188 (5.3 mg; 10 units) Fig. 3. Comparison of the saccharification ability of a cellulosome–rCglT combination and T. reesei cellulase (Celluclast 1.5L) supplemented with Novozyme-188. Rice straw (1% glucan) soaked in 28% aqueous ammonia at 60 °C for 7 days was used as the substrate. Hydrolysis conditions for T. reesei cellulase: substrate, 1% (w/v) glucan-containing rice straw; enzymes, 2 and 10 mg of Celluclast 1.5L with 10 units Novozyme-188 or 20 mg Celluclast 1.5L with 20 units Novozyme-188 per g glucan; pH 5.0; 50 °C. Error bars represent ±SD (n = 3).
enzymes to the cellulose chains due to the physical structure of the lignocellulosic substrate. The ammonia treatment has been
20
10
10
5
0
0
12
24
36
48
Ethanol concentration (g/L)
0
reported to increase the porosity of the lignocellulosic materials with the removal of crosslinks between xylan hemicelluloses and other components as well as to cause swelling leading to an increase in internal surface area (Sun and Cheng, 2002). Those effects should increase the accessibility of the cellulose for the enzymes to improve cellulose hydrolysis. On the other hand, the C. thermocellum cellulosomes exhibit strong hydrolytic ability for native crystalline cellulose compared to fungal cellulases (Demain et al., 2005) because of their highly ordered molecular structure (Mayer et al., 1987). Through the whole genome sequence and subunit protein analyses, it has been demonstrated that the enzymatic subunits of the C. thermocellum cellulosomes have a higher diversity of polysaccharide binding modules than that of the T. reesei enzymes (Demain et al., 2005), which supposedly facilitates binding of the cellulosomes to lignocellulosic substrates. These characteristics of the cellulosomes together with their diversified hemicellulolytic activities enable the cellulosomes to effectively hydrolyze the ammonia-soaked rice straw.
Glucose concentration (g/L)
Glucan digestibility (%)
100
0
Time (h) Ethanol production (synthetic medium) Glucose consumption (synthetic medium) Ethanol production (rice straw hydrolysate) Glucose consumption (rice straw hydrolysate) Fig. 4. Ethanol fermentation profile using ammonia soaked rice straw hydrolysates obtained by a combination of cellulosomes and rCglT. Error bars represent ±SD (n = 3).
R. Waeonukul et al. / Bioresource Technology 107 (2012) 352–357
cellulosomes and 20 units rCglT per g glucan. The slurry was supplemented with nitrogen and finally prepared with an initial glucose concentration of 18.6 g/L, which corresponds to 89% glucan saccharification. Fermentation profiles of ethanol production and glucose consumption were obtained from cultures grown on the rice straw hydrolysate, as well as from cultures grown on synthetic medium (Fig. 4). Ethanol productivity of 9.2 g/L was achieved, which corresponds to approximately 95% of the theoretical yield, indicating that the saccharification slurry obtained by combination of the cellulosome and rCglT can be fermented without any inhibition. 4. Conclusions This study investigated the effectiveness of combinations of cellulosomes and compatible b-glucosidases in hydrolyzing rice straw. The combination with CglT drastically accelerated degradation of cellulose. When rice straw soaked in 28% aqueous ammonia was hydrolyzed by an enzyme loading of 2 mg cellulosome and 0.9 mg rCglT per g-glucan, 91% of glucan in rice straw was hydrolyzed, which means the required total enzyme loading is more than 10 times lower than that observed for a combination of fungal cellulases. These results showed that the combination has great potential as a cellulolytic system to be an alternative to fungal cellulases. Acknowledgements Waeonukul, R. was supported by the JIRCAS Visiting Research Fellowship Program 2008 and 2009. The authors would like to thank the Thailand Research Fund (TRF Grant) for financial support in Thailand. This work was conducted as part of a research project funded by a grant (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry, and Fisheries of Japan. References Arantes, V., Saddler, J.N., 2011. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnol. Biofuels 4, 3–16. Aspinall, G.O., 1980. Chemistry of cell-wall polysaccharides. In: Preiss, J. (Ed.), The Biochemistry of Plants. Academic Press Inc., New York, pp. 473–500. Bayer, E.A., Shoham, Y., Lamed, R., 2008. Cellulosome-enhanced conversion of biomass: on the road to bioethanol. In: Wall, J.D., Harwood, C.S., Demain, A.L. (Eds.), Bioenergy. ASM Press, Washington, DC, pp. 75–96.
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