- Open Access
Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials
© Fujii et al; licensee BioMed Central Ltd. 2009
- Received: 1 May 2009
- Accepted: 1 October 2009
- Published: 1 October 2009
Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi.
We compared A. cellulolyticus and T. reesei cellulase activity against the three lignocellulosic materials: eucalyptus, Douglas fir and rice straw. Saccharification analysis using the supernatant from each culture demonstrated that the enzyme mixture derived from A. cellulolyticus exhibited 2-fold and 16-fold increases in Filter Paper enzyme and β-glucosidase specific activities, respectively, compared with that derived from T. reesei. In addition, culture supernatant from A. cellulolyticus produced glucose more rapidly from the lignocellulosic materials. Meanwhile, culture supernatant derived from T. reesei exhibited a 2-fold higher xylan-hydrolyzing activity and produced more xylose from eucalyptus (72% yield) and rice straw (43% yield). Although the commercial enzymes Acremonium cellulase (derived from A. cellulolyticus, Meiji Seika Co.) demonstrated a slightly lower cellulase specific activity than Accellerase 1000 (derived from T. reesei, Genencor), the glucose yield (over 65%) from lignocellulosic materials by Acremonium cellulase was higher than that of Accellerase 1000 (less than 60%). In addition, the mannan-hydrolyzing activity of Acremonium cellulase was 16-fold higher than that of Accellerase 1000, and the conversion of mannan to mannobiose and mannose by Acremonium cellulase was more efficient.
We investigated the hydrolysis of lignocellulosic materials by cellulase derived from two types of filamentous fungi. We found that glucan-hydrolyzing activity of the culture supernatant from A. cellulolyticus was superior to that from T. reesei, while the xylan-hydrolyzing activity was superior for the cellulase from T. reesei. Moreover, Acremonium cellulase exhibited a greater glucan and mannan-hydrolyzing activity than Accellerase 1000.
- Rice Straw
- Cellulase Activity
- Lignocellulosic Material
Lignocellulosic biomass represents a promising starting material for the production of bioethanol fuel, as it contains a large quantity of sugars in the form of cellulose and hemicellulose. Ethanol fuel production from lignocellulosic biomass is advantageous as it does not lead to competition for food resources . For ethanol fuel production from lignocellulosic materials, cellulose and hemicellulose must firstly be hydrolyzed to fermentable sugars. Sulfuric acid and cellulolytic enzymes are the major hydrolyzers of cellulose and hemicellulose identified to date . However, when sulfuric acid is used for the hydrolysis of lignocellulosic materials, it is necessary to remove the residual sulfuric acid from the hydrolyzing solution prior to yeast fermentation. Furthermore, sulfuric acid produces toxic compounds that inhibit fermentation [2–4]. Therefore, enzymatic saccharification of lignocellulosic materials that does not require the use of acidic compounds represents an important improvement in the generation of fermentable sugars during the bioethanol production process. The development of efficient pretreatment methods that do not require the use of chemicals before enzymatic hydrolysis, such as milling treatment, have also been eagerly investigated [5, 6]. Cellulases, a group of enzymes that hydrolyze crystalline cellulose to smaller oligosaccharides and subsequently glucose, and hemicellulases that hydrolyze hemicellulose to monomeric sugars, have been used for the enzymatic saccharification of lignocellulosic materials.
Filamentous fungal strains, also sometimes termed wood-degrading organisms, secrete a large quantity of cellulase and hemicellulase [7–10]. Cellulase produced by fungi comprises three major enzyme components: 1) endoglucanases that randomly hydrolyze internal glycosidic linkages; 2) cellobiohydrolases that produce cellobiose from cellulose chain ends; and 3) β-glucosidases that convert cellobiose to glucose . Xylan-hydrolyzing enzymes including xylanase and β-xylosidase, and mannan-hydrolyzing enzymes such as mannanase and β-mannosidase are examples of hemicellulases produced by fungi . Of the cellulases and hemicellulases produced by fungi, the enzymes derived from Trichoderma reesei represent the best characterized, and are often used for enzymatic saccharification of lignocellulosic materials . The genome of T. reesei QM6a has been sequenced and the sequence information is readily available . The cellulase derived from T. reesei demonstrates a relatively weak β-glucosidase activity, and the reaction from cellobiose to glucose has been shown to be slow . Yamanobe et al. isolated enzymes from the filamentous fungus strain Acremonium cellulolyticus Y-94, which produces high levels of cellulase . These enzymes demonstrated a significantly higher β-glucosidase activity than the cellulase derived from T. reesei. However, the cellulase and hemicellulase produced by A. cellulolyticus have not been as well characterized as those produced by T. reesei. A number of cellulase hyperproducing mutants have also been obtained from A. cellulolyticus Y-94 and T. reesei QM6a following treatment with mutagens including ultraviolet (UV) and various chemical compounds [12, 16–18]. Understanding the advantages and disadvantages of each of the enzymes derived from A. cellulolyticus and T. reesei may prove essential for the improvement of their hydrolyzing performance during bioethanol production processes.
The aim of this study was to further understand cellulases derived from A. cellulolyticus and T. reesei by analyzing the hydrolysis of lignocellulosic biomass. We determined the specific activity of cellulase and hemicellulase, and performed enzymatic saccharification of three lignocellulosic materials.
The cellulase hyperproducing strains used in this study were A. cellulolyticus CF-2612  and T. reesei CDU-11 . A. cellulolyticus CF-2612 was cultured in production medium in Erlenmeyer flasks , and the resulting culture supernatant was analyzed. The culture supernatant from T. reesei was kindly supplied by Kyowa Hakko Kougyo Co. (Tokyo, Japan). The commercial enzymes used in this study were Acremonium cellulase (AC, derived from A. cellulolyticus; Meiji Seika, Tokyo, Japan) and Accellerase 1000 (derived from T. reesei; Genencor, Rochester, NY, USA).
The soluble protein concentration was determined using the method of Lowry et al. . Filter-paper (FPase) activity was measured as described previously by Ghose , a method recommended by the Commission of Biotechnology, IUPAC. The activity of CMCase was assayed based on the method of Mandels et al. . Briefly, appropriately diluted supernatant and 0.5 ml of carboxymethylcellulose (CMC, 2% w/v) in citrate buffer (50 mM, pH 4.8) were mixed in equal volumes, and the enzyme reaction mixture was incubated at 50°C for 30 min. Avicelase activity was determined under similar conditions, with the exception that the enzyme reaction proceeded for 2 h in 1.0 ml of acetate buffer (0.1 M, pH 4.8), 10 mg of Avicel PH-101 (Fluka, Buchs, Switzerland) as the substrate and 1.0 ml of diluted enzyme solution. The reducing sugars released were analyzed via the dinitrosalicylic acid (DNS) assay . Xylanase activity was assayed in a 1.0-ml reaction mixture containing 1% (w/v) birchwood xylan (Sigma-Aldrich, St. Louis, MO, USA), 50 mM acetate buffer (pH 5.0) and appropriately diluted enzyme solutions. Following 30 min incubation at 45°C, the reducing sugar liberated was measured using the DNS assay. Mannanase activity was measured under similar conditions as xylanase activity, with the exception that 1% glucomannan (Megazyme, Wicklow, Ireland) served as the substrate. For the measurement of β-glucosidase activity, appropriately diluted enzyme solution and 10 mM p-nitrophenyl-β-D-glucopyranoside (Wako Pure Chemical Industries, Tokyo, Japan) were added to 100 mM citrate buffer, and the enzyme reaction mixture was incubated at 45°C for 10 min. Absorbance at 420 nm was then measured. β-Xylosidase and β-mannosidase activities were measured under similar conditions, with the exception that p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-β-D-mannopyranoside (Wako Pure Chemical Industries) were used as the substrates, respectively. For these experiments, one unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute.
Pretreatment of lignocellulosic materials
Eucalyptus and Douglas fir wood chips were kindly supplied by a pulp factory located close to our research center (Oji Paper Co., Tokyo, Japan). Rice straw was kindly supplied by Dr Tokuyasu (National Food Research Institute, Tsukuba, Japan). Ball-milling pretreatment of cellulosic materials was based on the methods described by Inoue et al. . The initial composition of eucalyptus (40.0% glucan and 10.4% xylan) and Douglas fir (51.9% glucan and 13.2% mannan) was determined as described previously . The initial composition of rice straw (37.0% glucan and 13.7% xylan) was determined by the method of Hideno et al. .
Enzymatic hydrolysis was performed using an enzyme constituting 22.5 or 90.0 mg protein per gram of dry substrate. The soluble protein concentration was determined using the method of Lowry et al. . The diluted enzyme solution (8 ml) in 50 mM acetate buffer (pH 5.0) was added to 2 g of pretreated material in a 15-ml tube. The reaction mixture was then incubated at 50°C for 72 h with mixing using a magnetic stirrer. The hydrolysate was centrifuged, and the supernatant analyzed. This experiment was performed in triplicate.
Analysis of substrate hydrolysates
Substrate hydrolysates were analyzed using a high-performance liquid chromatography (HPLC) system equipped with a RI-2031 Plus detector (Jasco, Tokyo, Japan). Glucose, xylose, mannose and mannobiose were analyzed using an Aminex HPX-87P column (Bio-Rad, Hercules, CA, USA) fitted with a Carbo-P micro-guard cartridge. The mobile phase used was doubly deionized water, and the flow rate was 1.0 ml/min at a column temperature of 80°C.
Saccharification analysis of the culture supernatant
Specific activities of cellulases and hemicellulases derived from A. cellulolyticus and T. reesei.
Specific activity (U mg-1 protein)
0.66 ± 0.13
0.26 ± 0.02
4.52 ± 1.32
1.20 ± 0.02
12.4 ± 0.15
0.011 ± 0.001
1.10 ± 0.15
0.00045 ± 0.0001
0.25 ± 0.05
0.11 ± 0.03
3.55 ± 1.01
0.072 ± 0.003
25.4 ± 2.32
0.049 ± 0.001
1.20 ± 0.06
0.0078 ± 0.0004
0.44 ± 0.01
0.25 ± 0.01
6.75 ± 0.34
2.85 ± 0.06
8.86 ± 0.85
0.023 ± 0.001
0.29 ± 0.03
0.00084 ± 0.0001
0.41 ± 0.02
0.19 ± 0.05
4.44 ± 0.87
2.09 ± 0.24
6.21 ± 0.21
0.0074 ± 0.0001
4.88 ± 0.87
0.051 ± 0.001
Eucalyptus and rice straw contain hemicellulose, mainly in the form of xylan, while Douglas fir mainly contains mannan [5, 6]. Thus, we examined xylose production from eucalyptus and rice straw, and mannose production from Douglas fir for each of the culture supernatants. The maximum xylose yield obtained from eucalyptus was 72% and from rice straw was 43% for SCDU-11 (Figure 1A and 1C). This data is consistent with that of the xylanase and β-xylosidase activities, which were higher for SCDU-11 than for SCF-2612 (Table 1). Mannose production from Douglas fir was similar between the two supernatants (Figure 1B). The HPLC column used in this study was not able to completely separate mannose and mannobiose, so the mannose production value presented also includes mannobiose. Mannanase specific activities of SCDU-11 and SCF-2612 were similar; however, β-mannosidase specific activity of SCDU-11 was 17-fold higher than that of SCF-2612 (Table 1). Thus, the SCF-2612 hydrolysate may contain more mannobiose than that of SCDU-11.
Saccharification analysis of commercial enzymes
We investigated the cellulase and hemicellulase specific activity of AC derived from A. cellulolyticus and of Accellerase 1000 derived from T. reesei. We revealed that the FPase, Avicelase, CMCase, β-glucosidase, xylanase and β-xylosidase specific activities of Accellerase 1000 were slightly higher than those achieved by AC (Table 1). In contrast, AC contained higher mannanase and β-mannosidase specific activities than Accellerase 1000 (Table 1).
The xylose yields from eucalyptus and rice straw were found to be similar for both Accellerase 1000 and AC (Figure 2A and 2C), although the xylanase and β-xylosidase activities of AC were lower than those of Accellerase 1000. In contrast, the maximum mannose yield obtained from Douglas fir by AC was 49% (Figure 2B). This result is consistent with the higher mannanase and β-mannosidase activity levels obtained by AC compared with those of Accellerase 1000.
T. reesei generally produce lower levels of β-glucosidase activity than A. cellulolyticus. This study demonstrated that the β-glucosidase specific activity of SCDU-11 was significantly lower than that of SCF-2612 (Table 1). This lower β-glucosidase activity is thought to trigger accumulation of cellobiose, which is a strong inhibitor of cellobiohydrolase and endoglucanase activities during cellulose hydrolysis . In fact, our data demonstrated that SCDU-11 hydrolyzed lignocellulosic materials more slowly than SCF-2612.
Analysis of saccharification of eucalyptus, Douglas fir and rice straw demonstrated that AC was able to produce more glucose than Accellerase 1000, even though it exhibited a lower cellulase specific activity. Similar results were also obtained during the analysis of xylose production. A potential explanation of this result may be the differences in substrate composition. The substrate used for the measurement of cellulase activity was highly purified and contained little material other than cellulose. However, lignocellulosic materials contain additional elements including lignin, which is known to inhibit the cellulose-hydrolyzing reaction . The hydrolysis curves of Avicel, that were pure for cellulose, were similar for both Accellerase 1000 and AC (Figure 3). This data suggest that Accellerase 1000 may be more sensitive to cellulase inhibitors, such as lignin, than AC. Enzyme sensitivity to inhibitors may aid in the explanation as to why xylose yield was equivalent for these enzymes. However, we do not have a clear explanation for this phenomenon.
It is well established that cellulose-degrading fungi produce cellulase and hemicellulase complexes [7–9, 11]. In this study, the culture supernatant derived from A. cellulolyticus was found to exhibit a lower xylan-hydrolyzing activity than that derived from T. reesei. A. cellulolyticus CF-2612 has been screened previously as a high cellulase producer, rather than a high hemicellulase producer, in A. cellulolyticus Y-94 mutagenesis studies involving UV irradiation and chemical compounds [16, 17]. A. cellulolyticus Y-94 demonstrated a higher xylan-hydrolyzing activity than A. cellulolyticus CF-2612 (unpublished data), suggesting that the genes related to xylan-hydrolyzing performance of A. cellulolyticus CF-2612 may have been mutated, thus resulting in a reduction in xylan-hydrolyzing activity. The reduced xylan-hydrolyzing activity of A. cellulolyticus CF-2612 may also prove problematic for the saccharification of lignocellulosic materials and may require improvement. The maximum mannose yield produced from Douglas fir was less than 50% in all experiments in this study. This finding indicates that the mannan-hydrolyzing activity of A. cellulolyticus and T. reesei strains used was insufficient. We are currently focusing on producing an A. cellulolyticus strain that exhibits a higher mannan-hydrolyzing activity.
In this study, AC generated glucose from lignocellulosic materials more efficiently than SCF-2612, while the glucose production curves were similar for both Accellerase 1000 and SCF-2612 (Figures 1 and 2). We also found that the FPase and Avicelase specific activities of AC and Accellerase 1000 were lower than those of SCF-2612 (Table 1). However, the β-glucosidase specific activity of AC and Accellerase 1000 was 2.4 and 1.7-fold higher than that of SCF-2612, respectively. The ratio of β-glucosidase/FPase activity for SCF-2612 (1.8) was also lower than that for AC (5.1) and Accellerase 1000 (6.5). These results suggest that β-glucosidase is important for hydrolyzing lignocellulosic materials. The improvement of β-glucosidase activity in fungal strains involved in the hydrolysis of lignocellulosic materials requires further study. However, given that commercial enzymes often contain additional compounds including enzyme stabilizers, it may prove difficult to directly compare the activities of these enzymes with those present in culture supernatants.
In this study, we investigated the cellulase and hemicellulase specific activities of both culture supernatants and commercial enzymes derived from A. cellulolyticus and T. reesei, and examined their performance during the saccharification of three lignocellulosic materials. The culture supernatant derived from A. cellulolyticus demonstrated a higher cellulase specific activity and glucose yield from lignocellulosic materials than the T. reesei supernatant. In contrast, the enzymes derived from T. reesei demonstrated a superior xylan-hydrolyzing activity than those derived from A. cellulolyticus. AC produced a greater amount of glucose from lignocellulosic materials and a higher mannan-hydrolyzing activity than Accellerase 1000. Further studies will aid in the development of cellulases and hemicellulases that hydrolyze lignocellulosic materials more efficiently during the bioethanol production process, for advancing the generation of alternative fuel sources.
We thank Mr Osamu Takimura, Mr Shinichi Yano, Dr Kenichiro Tsukahara, Dr Akinori Matsushika and Dr Akihiro Hideno (AIST) for helpful discussions.
- Lynd LR: Overview and evaluation of fuel ethanol from cellulosic biomass: technology, economics, the environment, and policy. Annu Rev Energy Environ 1996, 21: 403-465. 10.1146/annurev.energy.21.1.403View ArticleGoogle Scholar
- Sun Y, Cheng J: Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002, 83: 1-11. 10.1016/S0960-8524(01)00212-7View ArticleGoogle Scholar
- Hendriks ATWM, Zeeman G: Pretreatment to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 2008, 100: 10-18. 10.1016/j.biortech.2008.05.027View ArticleGoogle Scholar
- Rubin EM: Genomics of cellulosic biofuels. Nature 2008, 454: 841-845. 10.1038/nature07190View ArticleGoogle Scholar
- Inoue H, Yano S, Endo T, Sakaki T, Sawayama S: Combining hot-compressed water and ball milling pretreatments to improve the efficiency of the enzymatic hydrolysis of eucalyptus. Biotechnol Biofuels 2008, 1: 2. 10.1186/1754-6834-1-2View ArticleGoogle Scholar
- Hideno A, Inoue H, Tsukahara K, Inoue S, Endo T, Sawayama S: Wet disk milling pretreatment without sulfuric acid for enzymatic hydrolysis of rice straw. Bioresour Technol 2009, 100: 2706-2711. 10.1016/j.biortech.2008.12.057View ArticleGoogle Scholar
- Krogh KB, Mørkeberg A, Jørgensen H, Frisvad JC, Olsson L: Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes. Appl Biochem Biotechnol 2004, 114: 389-401. 10.1385/ABAB:114:1-3:389View ArticleGoogle Scholar
- Wen Z, Liao W, Chen S: Production of cellulase/β-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure. Process Biochem 2005, 40: 3087-3094. 10.1016/j.procbio.2005.03.044View ArticleGoogle Scholar
- Sehnem NT, Bittencourt LR, Camassola M, Dillon AJP: Cellulase production by Penicillium echinulatum on lactose. Appl Microbiol Biotechnol 2006, 72: 163-167. 10.1007/s00253-005-0251-zView ArticleGoogle Scholar
- Goyal A, Ghosh B, Eveleigh D: Characteristics of fungal cellulases. Bioresour Technol 1991, 36: 37-50. 10.1016/0960-8524(91)90098-5View ArticleGoogle Scholar
- Shallom D, Shoham Y: Microbial hemicellulases. Curr Opin Microbiol 2003, 6: 219-228. 10.1016/S1369-5274(03)00056-0View ArticleGoogle Scholar
- Esterbauer H, Steiner W, Labudova I, Hermann A, Hayn M: Production of Trichoderma cellulase in laboratory and pilot scale. Bioresour Technol 1991, 36: 51-65. 10.1016/0960-8524(91)90099-6View ArticleGoogle Scholar
- Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, Danchin EG, Grigoriev IV, Harris P, Jackson M, Kubicek CP, Han CS, Ho I, Larrondo LF, de Leon AL, Magnuson JK, Merino S, Misra M, Nelson B, Putnam N, Robbertse B, Salamov AA, Schmoll M, Terry A, Thayer N, Westerholm-Parvinen A, et al.: Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina ). Nat Biotechnol 2008, 26: 553-560. 10.1038/nbt1403View ArticleGoogle Scholar
- Sternberg D, Vijayakumar P, Reese ET: β-glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can J Microbiol 1977, 23: 139-147. 10.1139/m77-020View ArticleGoogle Scholar
- Yamanobe T, Mitsuishi Y, Takasaki Y: Isolation of a cellulolytic enzyme producing microorganism, culture conditions and some properties of the enzymes. Agric Biol Chem 1987, 51: 65-74.View ArticleGoogle Scholar
- Fang X, Yano S, Inoue H, Sawayama S: Strain improvement of Acremonium cellulolyticus for cellulase production by mutation. J Biosci Bioeng 2009, 107: 256-261. 10.1016/j.jbiosc.2008.11.022View ArticleGoogle Scholar
- Fang X, Yano S, Inoue H, Sawayama S: Lactose enhances cellulase production by the filamentous fungus Acremonium cellulolyticus . J Biosci Bioeng 2008, 106: 115-120. 10.1263/jbb.106.115View ArticleGoogle Scholar
- Ado Y: Cellulase production by Trichoderma reesei . Biosci Ind 1989, 47: 840-843.Google Scholar
- Kawamori M, Ado Y, Takasawa S: Preparation and application of Trichoderma reesei mutants with enhanced β-glucosidase. Agric Biol Chem 1986, 50: 2477-2482.View ArticleGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin-phenol reagent. J Biol Chem 1951, 193: 265-275.Google Scholar
- Ghose TK: Measurement of cellulase activities. Pure Appl Chem 1987, 59: 257-268. 10.1351/pac198759020257Google Scholar
- Mandels M, Hontz L, Nystrom J: Enzymatic hydrolysis of waste cellulose. Biotechnol Bioeng 1974, 16: 1471-1493. 10.1002/bit.260161105View ArticleGoogle Scholar
- Mosier N, Wyman CE, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M: Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005, 96: 673-686. 10.1016/j.biortech.2004.06.025View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.