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Construction of the Biological Saccharification Process from Lignocellulosic Biomass Using a Filamentous Fungus Trichoderma reesei
2025 Volume 72 Issue 2 Article ID: 7202203
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2025 Volume 72 Issue 2 Article ID: 7202203
Here, we aimed to construct a biological saccharification process that combines the steps of enzyme production and enzymatic saccharification using an aerobic fungus Trichoderma reesei, an excellent cellulase producer. Sugar production consists of the growth phase at 28 °C and the saccharification phase at 50 °C. Final sugar yields from alkali-treated rice straw and microcrystalline cellulose using the T. reesei M2-1 strain were greatly affected by mycelial inoculum size and growth phase periods. The optimization of these factors yielded 74.5 % and 60.6 % of sugar from the alkali-treated rice straw and microcrystalline cellulose, respectively, at 120 h of the biological saccharification process. In comparison with the process employing anaerobic microorganisms, a relatively higher yield of sugars was achieved within a shorter period and the use of non-GM fungal strain. However, large variability in sugar yields based on feedstocks suggests imperceptible differences in initial conditions.
non-GM, non-genetically modified; CBS, consolidated bio-saccharification; BM, basal medium; MCC, microcrystalline cellulose; RS, rice straw; FMOC-Cl, 9-fluorenylmethyl chloroformate; HPLC, high-performance liquid chromatography.
Lignocellulosic biomass is the most abundant sustainable carbon sources on earth and is considered an alternative resource to substitute fossil resources to obtain value-added products including biofuels, bioplastics, and biochemicals [1]. Although the conversion and utilization of this biomass may resolve some environmental issues, it is not easy to utilize it because of its recalcitrant structure. The enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars, such as glucose and xylose, is a key step for the production of value-added products. Enormous amount of studies on enzyme production and saccharification process have been reported [2, 3, 4, 5]. However, the high cost of the saccharification step remains a major hurdle in the implementation of economically friendly biorefineries.
Recently, a sugar production process that combines enzyme synthesis and enzymatic saccharification into one step, called consolidated bio-saccharification (CBS), has been reported. An anaerobic thermophilic bacteria Clostridium thermocellum is well known as a producer of cellulosome, a highly organized multiprotein complex consisting of enzymatic subunits and non-catalytic scaffoldins, which can effectively hydrolyze lignocellulose. There are several reports of using C. thermocellum-based CBS that effectively accumulates glucose by the cultivation of the bacterium on lignocellulosic material with the supplementation of β-glucosidase [6, 7, 8, 9]. The biological conversion of lignocellulosic material using an anaerobic fungal strain was also reported [10]. The fungal growth during the cultivation of an anaerobic strain on pretreated lignocellulosic material was inhibited by the addition of antibiotics leading to the accumulation of sugars in the culture medium. Although these CBS processes using anaerobic microorganisms are simple and effective for sugar production, they take relatively longer periods to obtain adequate sugar yields.
Thus, we aimed to construct a biological saccharification process using an aerobic fungus Trichoderma reesei, which is a well-known, hyper cellulase producer. In this study, T. reesei M2-1, a mutant of T. reesei ATCC 66589 that can produce cellulases in media with glucose as the sole carbon source [2], was used. In our preliminary experiment, this strain was inactivated at 50 °C, the optimum temperature for cellulolytic enzymes from this fungus (data not shown). Basal medium (BM) [11] supplemented with a carbon source was utilized. For seed cultivation, 107 conidia were inoculated into 100 mL of BM containing 20 mg/mL of glucose in a 500 mL flask, and then incubated while rotating at 180 rpm at 28 °C for 3 days. Sugar production consists of the growth phase at 28 °C and the saccharification phase at 50 °C (Fig. 1A). In the former, the fungus grows using the feedstocks and produces cellulolytic enzymes. After elevating temperature in the later phase, the fungus is inactivated but the enzymes produced are still active, due to which sugars accumulate. Microcrystalline cellulose (Avicel® PH-101; Sigma-Aldrich Co., St. Louis, MO, USA) and Ca(OH)2-treated rice straw were used as feedstocks. The latter was prepared by employing the method described previously [12]. Fine powders (0.5-mm-mesh-pass) of rice straw (cv. ‘Koshihikari’) were mixed well with 15 % (g/g of rice straw) and Ca(OH)2 in a bottle, and added with distilled water (10 mL/g of rice straw). After heat treatment at 120 °C for 1 h, HCl-neutralization and washing were performed. The glucan and xylan contents of the pretreated materials were 38.0 % and 13.4 %, on a dry basis, respectively. All sugar production experiments were conducted in 30 g of fresh BM containing 1.5 g (dry-basis) of microcrystalline cellulose or Ca(OH)2-treated rice straw in 100 mL flasks. A portion of the seed culture (low inoculum size: 0.6 g, medium inoculum size: 1.5 g, high inoculum size: 3 g) was inoculated into fresh media and incubated at 28 °C for appropriate periods (growth phase). After that, the culture was incubated at an elevated temperature (50 °C) until the total process reached a duration of 120 h (saccharification phase). The total process of 120 h was set based on the results of a preliminary test, where the sugar yield reached almost plateau at that time (data not shown). The conditions used during the sugar production process in this study are summarized in Fig. 1B. Next, the supernatant was recovered by centrifugation (12,000 × G for 15 min at 4 °C). The sugar concentrations in the supernatant were analyzed using a high-performance liquid chromatography system (Prominence UFLC, Shimadzu Corporation, Kyoto, Japan) equipped with a refractive index detector (RID-10A, Shimadzu Corporation). Samples were desalted and separated at 60 °C with distilled water as a mobile phase (0.6 mL/min) employing a Deashing Micro-Guard pre-column and an Aminex HPX-87P column (Bio-Rad Laboratories, Inc., Hercules, CA, USA), respectively. The sugar yields were calculated applying the following equations: Glucose yield (%) = (glucose amount produced [g] × 0.9)/(glucan content in the feedstock; g-feedstock) × 100. Xylose yield (%) = (xylose amount produced [g] × 0.88)/(glucan content in the feedstock; g-feedstock) × 100. Solubilized C6 sugar yield (%) = (glucose amount produced [g] × 0.9 + cellobiose amount produced [g] × 0.95)/(glucan content in the feedstock; g-feedstock) × 100.
To investigate the effect of mycelial inoculum size and growth phase period on the final sugar yields, the 120 h-biological saccharification process was carried out per the conditions described in Fig. 1B using Ca(OH)2-treated rice straw or microcrystalline cellulose as feedstocks. Figure 2 indicates the amount of C6 sugar liberated (glucose + cellobiose) after the biological saccharification processes. Although differences in sugar yield occurred, similar trends were observed between both feedstocks. In the cases of 0 h (a, b) and 9 h (c) of the growth phase, more sugars were generated as the inoculum size was increased. Relatively high C6 sugar yields (68.5 % and 60.9 % from Ca[OH]2-treated rice straw and microcrystalline cellulose, respectively) were obtained in the 9 h-growth phase and large inoculum size process. Notably, sugar accumulation was observed in the 0 h-growth phase process because M2-1 can produce cellulases in media with glucose as the sole carbon source. With low and medium inoculum sizes, the maximum amount of sugar was produced in the 24 h-growth phase process; 74.5 % and 60.6 % for low inoculum size, 67.8 % and 60.0 % for medium inoculum size from Ca(OH)2-treated rice straw and microcrystalline cellulose, respectively (Figs. 2A, B). Conversely, with high inoculum size, similar levels of sugar yields were shown with the 9 h- and 24 h-growth phase processes (Fig. 2C). With all inoculum sizes, lower sugar yields were obtained in the 48 h-growth phase processes. These results indicate that final sugar yields are greatly affected by the mycelial inoculum size and growth phase periods; the optimization of these factors can enhance sugar yields.
Ca(OH)2-treated rice straw (RS) and microcrystalline cellulose (MCC) were used as feedstocks. (A) low inoculum size, (B) medium inoculum size, (C) high inoculum size. Process conditions (a-e) are shown in Fig. 1B.
Next, we analyzed sugar yield (glucose, cellobiose, and xylose) and mycelial growth for a detailed investigation of the process. The process was carried out using Ca(OH)2-treated rice straw with low inoculum size, and growth phases of 24 or 48 h. During the process, an aliquot of the slurry (0.5 g) was collected at appropriate intervals, and the sugar content of the supernatants was ascertained. Mycelial growth was evaluated by measuring the amount of mycelial chitin in the slurry. Solids in the slurry were collected by centrifugation (12,000 × G for 5 min at 4 °C), followed by washing with water. The washed solids were dried at 105 °C, overnight and powdered with ball milling equipment (4 min at 15 Hz; MM400, Verder Scientific Co., Ltd., Tokyo, Japan). Fine powder of the solids (50 mg) was suspended in 10 mL of water and heated at 100 °C for 2 h. Then, the solids were collected by centrifugation (12,000 G for 5 min) and washed twice with water. Then, the sample was added with NaOH to a final concentration of 40 % (final volume: 5 mL) and heated at 120 °C for 1 h. After collecting the solids and several times of washing with water, the solids were resuspended by adding HCl to a final concentration of 5 M and heated at 95 °C for 6 h. An aliquot of the heated suspension (0.01 mL) was dried and applied for derivatization of glucosamine, which was produced from the mycelial cell wall in the slurry, with 9-fluorenylmethyl chloroformate (FMOC-Cl; Sigma-Aldrich Co.) per the method described previously [13]. The derivatized sugars were analyzed using a HPLC system equipped with an RF-20A fluorescence detector (excitation 254 nm, emission 322 nm; Shimadzu Corporation). Samples were separated at 40 °C with 45 % acetonitrile/0.1 % formic acid as a mobile phase (0.6 mL/min) on a CAPCELL PAK C18 MGⅡcolumn (Osaka Soda Co., Ltd., Osaka, Japan). Glucosamine solutions that were derivatized by FMOC-Cl as described above were used as standards.
In both cases, the amount of C6 sugars generated increased throughout the process; 75.0 % and 50.6 % were recovered at 120 h at 24 and 48 h growth phases, respectively (Fig. 3). On the contrary, glucose was detected in trace amounts during the growth phase and elevated after transfer to the saccharification phase, indicating that the fungus rapidly consumes the glucose liberated by the action of the enzymes produced. Although xylose accumulation was also observed during the saccharification phase (Fig. 3B), the monomeric sugar increased in the initial stage of growth and then decreased during prolonged growth. These results indicate that the amount of glucose generated was insufficient as the mycelia proliferated, and then the fungus consumed xylose. The accumulation of cellobiose (difference between C6 sugar and glucose) and xylose were observed during growth phase, indicating that these sugars induced the production of cellulolytic and hemicellulolytic enzymes. However, the enzymatic activities of cellulases and hemicellulases (cellobiohydrolases, β-glucosidases, β-xylosidases and α-L-arabinofuranosidases) in the supernatants were undetectable or very low. It was thought that most of produced enzymes were absorbed onto remaining feedstocks. As for fungal growth, the amounts of mycelia at the shifting point to the saccharification phase were evaluated at 3.4 and 5.1 mg/g-slurry in the 24 h- and 48 h-growth phase processes, respectively. As higher sugar yield was obtained with the 24 h-growth phase process, it will be ideal to transfer the saccharification phase at ~3.5 mg/g-slurry of mycelia. In this study, high sugar yields (~75 % and 60 % from Ca[OH]2-treated rice straw and microcrystalline cellulose, respectively) were achieved by the optimization of mycelial inoculum size and growth phase period during the 120 h-biological saccharification process of lignocellulosic feedstock. In the CBS process employing anaerobic microorganisms, for example, C. thermocellum ~70 % of sugar yield was achieved at 10 days-cultivation [6]. Several studies have reported improvements in the biological saccharification process, with > 80 % of sugar yields within a shortened cultivation period by using a genetically modified strain [7]. In comparison with the process using anaerobic microorganisms, it is believed that our biological saccharification process possesses several advantages, such as a shorter period and the use of non-GM fungal strains. Conversely, the problem of using Ca(OH)2-treated rice straw as a feedstock has to be solved: our results on large variability of sugar yields from different feedstocks suggest that imperceptible differences in the initial condition, such as the feedstock composition and the activity of the microorganism, might robustly affect fungal growth and final sugar yields (Fig 2). To solve this issue, we are conducting a more detailed analysis and technology development for process management.
Monitoring the accumulation of the sugars (C6 sugars [circles], glucose [squares], and xylose [triangles]) and quantity of mycelia (diamonds) produced during the hydrolysis of Ca(OH)2-treated rice straw by T. reesei M2-1. (A) Initial growth phase 24 h/saccharification phase 96 h, (B) Initial growth phase 48 h/saccharification phase 72 h.
We are grateful to H. Yamada and K. Hiramoto for their excellent technical assistance. The authors would also like to thank Enago (www.enago.jp) for the English language review.
