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This study aimed to characterize the interactions between cereal flour (rice, wheat, and barley) and "nata puree" (NP), a disintegrated bacterial cellulose (BC) in the presence of a water-soluble polysaccharide, with powder-dispersion activity. Pasting properties of cereal flour with additives were analyzed using a Rapid Visco Analyzer, and disintegrated BC in water (BCW), three water-soluble polysaccharides: (1,3)(1,4)-β-glucan, tamarind seed gum, and birchwood xylan, and the corresponding NPs were used as additives. For rice flour, additional BCW or NPs increased the initial and the peak viscosity. The addition of water-soluble polysaccharides produced the opposite trend: viscosity increased from the peak time to the end of measurements. For wheat flour, the addition of BCW or NP delayed the peak time and increased peak viscosity; the increase was maintained till the end of measurements. For barley flour, the additional BCW or NP caused a higher gelatinization rate and increased viscosity at the starch-retrogradation stage. Next, static gelatinization of a rice flour suspension in NP was successfully accomplished before placing it in a vessel; NP concentration in the gel significantly affected the firmness. Thus, the dynamic and unique interactions between various cereal flours and cell-wall polysaccharides in NPs can increase the flours' potential; static gelatinization of cereal flour with NP could expand flours' application range in both current and next-generation cooking.

Nata puree (NP)—obtained by disintegrating nata de coco (bacterial cellulose [BC]) using a household blender—can be combined with tamarind seed gum (TG) to generate NPTG. In this study, BC fibrils (BC-TG) were prepared by removing free TG from NPTG and characterized. BC-TG exhibited high water dispersibility and relatively long nanofibrils (> 20 µm). We examined the distribution of xyloglucan, the main component of TG, on BC nanofibrils using immunofluorescence staining with calcofluor white, which stains the hydrophilic cellulose surface, and found that xyloglucan was adsorbed at different sites along the fibers. This indicated that BC-TG was a composite nanofibril of xyloglucan and BC. Furthermore, BC-TG showed a higher degree of adsorption on hydrophobic plastic substrates than BC did, suggesting a change in the surface properties of BC. Because the BC-TG preparation process is simple, requires only water and raw materials, and does not involve chemical reactions, it is expected to be an environmentally friendly method for the preparation and modification of BC nanofibrils.

Pulverization is a potentially powerful solution for the resource management of surplus- and non-standard agricultural products, maintaining their nutritional values for long and ensuring their homogeneity, whereas their original textures could disappear to narrow the application ranges. Therefore, new technologies should be developed for reconstructing the powders to provide them with new physical characteristics. Herein, we developed a novel food material, nata puree (NP), by nata de coco (bacterial cellulose gel) disintegration with a water-soluble polysaccharide using a household blender. The process worked well with (1,3)(1,4)-β-glucan (BGL) as the polysaccharide, which could be substituted with barley extract. Lichenase treatment of the NP dramatically modified its physical properties, suggesting the importance of the BGL polymeric forms. NP exhibited distinct potato powder and starch binding activities, which would be attributed to its interactions with the cell wall components and a physical capture of powders by the NP network, respectively. NP supplementation into the potato paste improved its firmness and enabled its printable range shift for 3D food printing to a lower powder-concentration. NP also promoted the dispersion of powders in its suspension, and designed gelation could also be successfully performed by the laser irradiation of an NP suspension containing dispersed curdlan and turmeric powders. Therefore, NP could be applied as a powder modifier to a wide range of products in both conventional cooking, food manufacturing, and next generation processes such as 3D food printing.

Erianthus arundinaceus (ER) is greatly appreciated among domestic energy crops in Japan for the production of fermentable sugars from lignocellulosic polysaccharides. In this study, we developed an efficient Ca(OH)2-based pretreatment of both stems and leaves of ER at ambient temperature with the addition of a washing step for enzymatic saccharification. The recoveries of glucans and xylans in the pretreated ER after four countercurrent washing cycles were 91 and 76 %, respectively, the former being considerably higher than that of rice straw (RS) (72 %). Their saccharification ratios in the washed sample under the pressure of 1 atm CO2 were 80 and 92.5 %, respectively. The application of this simple sugar production process from ER would further support the domestic bioprocess development. ER is also foreseen to provide the additional feedstock favorable for harvesting from winter to spring in Japan, preventing a risk for feedstock shortage generated by single harvesting such as RS.

In this study, the selection of target sugars in lignocellulosic hydrolysate and the implementation of a bioprocess strategy led to efficient production of a catalyst for chitinous bioresource conversion. Streptomyces sp. K5, a chitinase producer in the presence of glucose, was isolated at 50 °C on an agar plate with glucose and colloidal chitin. The K5 was found to produce chitinase with a maximum activity of 70 U/L in a medium containing glucose and xylose as well as colloidal chitin as an inducer. The assimilation of glucose and xylose, however, was extremely slow, with significant residual being present even after 168 h of incubation. Assimilation tests of glucose, xylose, and cellobiose confirmed that K5 produces chitinase without chitinous inducer and assimilates cellobiose much more rapidly than either glucose or xylose. At the same time, the complete exhaustion of each sugar initiated chitinase inactivation. A pulse- feeding strategy was adopted for the cellobiose, taking its rapid assimilation, β-glucosidase activity, and chitinase inactivation into account, and a maximum chitinase activity of 235 U/L was achieved under pulse- feeding conditions that included four pulses.

The aim of this study was to investigate the effect of pH control by CO 2 pressurization on the enzymatic hydrolysis of herbaceous feedstock in the calcium capturing by carbonation (CaCCO) process for fermentable sugar production. The pH of the slurry of 5 % (w/w) Ca(OH) 2 -pretreated/CO 2 -neutralized rice straw could be controlled between 5.70 and 6.38 at 50 °C by changing the CO 2 partial pressure ( p CO 2 ) from 0.1 to 1.0 MPa. A mixture of fungal enzyme preparations, namely, Trichoderma reesei cellulases/hemicellulases and Aspergillus niger β-glucosidase, indicated that pH 5.5-6.0 is optimal for solubilizing sugars from Ca(OH) 2 -pretreated rice straw. Enzymatic saccharification of pretreated rice straw under various p CO 2 conditions revealed that the highest soluble sugar yields were obtained at p CO 2 0.4 MPa and over, which is consistent with the expected pH at the p CO 2 without enzymes and demonstrates the effectiveness of pH control by CO 2 pressurization.

Rice straw (RS), an agricultural resource for lignocellulosic biorefineries, can deteriorate when sun-drying is ineffective. Poultry litter ash (PLA) has been considered as a renewable phosphorus source for crops but is highly alkaline. Here, a simple process was developed for their reciprocal upgrading. RS, PLA, and water were mixed for wet storage and alkali pretreatment of the RS at 25 °C for 14 d, and solid–solid separation was performed to obtain PLA-treated RS (PT-RS) and RS-treated PLA (RT-PLA). PT-RS was susceptible to enzymatic saccharification, and 65.5-68.6% of total sugar residues in PT-RS was converted to lactic acid by its nonsterile application for simultaneous saccharification and fermentation using Bacillus coagulans. RT-PLA exhibited 1.8-points lower pH and a more sensitive response of phosphorus solubilization to acid than those of PLA. This process could thus provide a breakthrough for the rural bioeconomy by manufacturing two strategic primary products for various commercial bioproducts., Graphical abstract Unlabelled Image, Highlights • Rice straw, poultry litter ash, and water were mixed for reaction during storage. • The treatment made the straw susceptible to enzymatic saccharification. • Calcium in the ash was moved to the straw and water to lower the ash alkalinity. • Strategic primary products were created to activate the rural bioeconomy.

Generally, Ca(OH)2 pretreatment of lignocellulosics for fermentable sugar recovery requires a subsequent washing step for calcium removal and pH control for optimized saccharification. However, washing Ca(OH)2-pretreated feedstock with water is considered problematic because of the low solubility of Ca(OH)2 and its adsorption to biomass. In this study, we estimated the availability of carbonated water for calcium removal from the slurry of Ca(OH)2-pretreated rice straw (RS). We tested two kinds of countercurrent washing sequences, four washings exclusively with water (W4) and two washings with water and subsequent two washings with carbonated water (W2C2). The ratios of calcium removal from pretreatment slurry after washing were 64.2 % for the W4 process and 92.1 % for the W2C2 process. In the W2C2 process, 49 % of the initially added calcium was recovered as CaO by calcination. In enzymatic saccharification tests under a CO2 atmosphere at 1.5 atm, in terms of recovery of both glucose and xylose, pretreated, feedstock washed through the W2C2 process surpassed that washed through the W4 process, which could be attributed to the pH difference during saccharification: 5.6 in the W2C2 process versus 6.3 in the W4 process. Additionally, under an unpressurized CO2 atmosphere at 1 atm, the feedstock washed through the W2C2 process released 78.5 % of total glucose residues and 90.0 % of total xylose residues. Thus, efficient removal of calcium from pretreatment slurry would lead to not only the recovery of added calcium but also the proposal of a new, simple saccharification system to be used under an unpressurized CO2 atmosphere condition.

To expand the range of soluble carbon sources for our enzyme production system, we investigated the properties of sucrose utilization and its effect on cellulase production by Trichoderma reesei M2-1. We performed batch cultivation of T. reesei M2-1 on sucrose and related sugars along with cellobiose, which was used as a cellulase inducer. The results clearly revealed that the hydrolysis products of sucrose, i.e. glucose and fructose, but not sucrose, can be used as a carbon source for enzyme production. In a 10-day continuous feeding experiment using invertase-treated sucrose/cellobiose, the fungal strain produced cellulases with a filter paper-degrading activity of 20.3 U/mL and production efficiency of 254 U/g-carbon sources. These values were comparable with those of glucose/cellobiose feeding (21.2 U/mL and 265 U/g-carbon sources, respectively). Furthermore, the comparison of the specific activities clearly indicated that the compositions of both produced enzymes were similar. Therefore, enzymatically hydrolyzed sucrose can be utilized as an alternative carbon source to glucose in our enzyme production system with T. reesei M2-1.

Novel bioreactor beads for simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw (RS) into ethanol were prepared. Genetically modified Saccharomyces cerevisiae cells expressing genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase were immobilized in calcium alginate beads containing inorganic lightweight filler particles to reduce specific gravity. For SSF experiments, the beads were floated in slurry composed of lime-pretreated RS and enzymes and incubated under CO2 atmosphere to reduce the pH for saccharification and fermentation. Following this reaction, beads were readily picked up from the upper part of the slurry and were directly transferred to the next vessel with slurry. After 240 h of incubation, ethanol production by the beads was equivalent to that by free cells, a trend that was repeated in nine additional runs, with slightly improved ethanol yields. Slurry with pre-saccharified lime-pretreated RS was subjected to SSF with floating beads for 168 h. Although higher cell concentrations in beads resulted in more rapid initial ethanol production rates, with negligible diauxic behavior for glucose and xylose utilization, no improvement in the ethanol yield was observed. A fermentor-scale SSF experiment with floating beads was successfully performed twice, with repeated use of the beads, resulting in the production of 40.0 and 39.7 g/L ethanol. There was no decomposition of the beads during agitation at 60 rpm. Thus, this bioreactor enables reuse of yeast cells for efficient ethanol production by SSF of lignocellulosic feedstock, without the need for instruments for centrifugation or filtration of whole slurry.

The aim of this study was to investigate the effect of pH control by CO 2 pressurization on the enzymatic hydrolysis of herbaceous feedstock in the calcium capturing by carbonation (CaCCO) process for fermentable sugar production. The pH of the slurry of 5 % (w/w) Ca(OH) 2 -pretreated/CO 2 -neutralized rice straw could be controlled between 5.70 and 6.38 at 50 °C by changing the CO 2 partial pressure ( p CO 2 ) from 0.1 to 1.0 MPa. A mixture of fungal enzyme preparations, namely, Trichoderma reesei cellulases/hemicellulases and Aspergillus niger β-glucosidase, indicated that pH 5.5–6.0 is optimal for solubilizing sugars from Ca(OH) 2 -pretreated rice straw. Enzymatic saccharification of pretreated rice straw under various p CO 2 conditions revealed that the highest soluble sugar yields were obtained at p CO 2 0.4 MPa and over, which is consistent with the expected pH at the p CO 2 without enzymes and demonstrates the effectiveness of pH control by CO 2 pressurization.

Sorghum bagasse samples from two sets (n6 and bmr6; n18 and bmr18) of wild-type and corresponding “brown midrib” (bmr) mutant strains of sweet sorghum were evaluated as the feedstock for fermentable sugar recovery via the calcium capturing by carbonation (CaCCO) process, which involves Ca(OH)2 pretreatment of bagasse with subsequent neutralization with CO2 for enzymatic saccharification. Saccharification tests under various pretreatment conditions of the CaCCO process at different Ca(OH)2 concentrations, temperatures or residence periods indicated that bmr strains are more sensitive to the pretreatment than their counterparts are. It is expected that variant bmr6 is more suitable for glucose recovery than its wild-type counterpart because of the higher glucan content and better glucose recovery with less severe pretreatment. Meanwhile, bmr18showed higher scores of glucose recovery than its counterpart did, only at low pretreatment severity, and did not yield higher sugar recovery under the more severe conditions. The trend was similar to that of xylose recovery data from the two bmr strains. The advantages of bmr strains were also proven by means of simultaneous saccharification and fermentation of CaCCO-pretreated bagasse samples by pentose-fermenting yeast strain Candida shehatae Cs 4R. The amounts needed for production of 1 L of ethanol from n6, bmr6, n18, and bmr18samples were estimated as 4.11, 3.46, 4.03, and 3.95 kg, respectively. The bmr strains seem to have excellent compatibility with the CaCCO process for ethanol production, and it is expected that integrated research from the feedstock to bioprocess may result in breakthroughs for commercialization.

This study evaluates the life cycle of ethanol produced from soft carbohydrate (SC)-rich rice straw (cv. Leafstar ) and common rice straw (cv. Koshihikari ) while considering land-use change to estimate CO 2 emissions, energy balance (expressed as Net Energy Ratio, NER), and production costs. Three different pretreatment methods were considered: the DiSC (direct saccharification of culms), RT-CaCCO (room temperature-CaCCO) and CaCCO (calcium capturing by carbonation) processes. Although the reduction in CO 2 emission was found to be 59%, 42% and −3.5% for the DiSC, RT-CaCCO and CaCCO processes, respectively, the CO 2 emission reduction decreased significantly when land-use change was considered. This result clearly shows that the biomass (rice straw) should be obtained from paddy fields without land-use change. The NER values for the bioethanol produced by the DiSC, RT-CaCCO and CaCCO processes were estimated to be 2.7, 2.1 and 1.0, respectively, and the total costs were estimated to be 102, 134 and 151 Yen/L ethanol, respectively (US $1 = 100 Yen). The use of the SC-rich rice straw contributes to a reduction of the environmental load and costs for the pretreatment, enzyme production, and enzymatic hydrolysis processes. Therefore, the use of SC-rich rice straw for bioethanol production reduces the total cost of production, reduces CO 2 emissions, and improves the NER. Our results suggest that the DiSC and RT-CaCCO pretreatment processes are promising pretreatment techniques and that SC-rich rice straw is a promising resource for bioethanol production.

An advanced sugar-platform bioprocess for lignocellulosic feedstocks by adding a phenolic-acid (PA: p -coumaric acid and ferulic acid) recovery step to the CaCCO process was designed. For efficient PA extraction, pretreatment was 95 °C for 2 h, producing a yield of 7.30 g/kg-dry rice straw (65.2% of total ester-linked PAs) with insignificant effects on saccharification. PAs were readily recovered in solution during the repeated washings of solids, and the glucose yield, after 72-h saccharification of the washed solids, was significantly improved from 65.9% to 70.3–72.7%, suggesting the removal of potential enzyme inhibitors. The promotion of xylose yield was insignificant, probably due to 13.1–17.8% loss of xylose residues after washing(s). This new bioprocess, termed the SRB (simultaneous recovery of by-products)-CaCCO process, would effectively produce fermentable sugars and other valuables from feedstocks, strengthening the platform in both economic and environmental terms.

We successfully expressed the neutral β-glucosidase (BGL4) from Scytalidium thermophilum in the thermotolerant yeast Candida glabrata. Compared to the strain expressing Aspergillus acidic β-glucosidase (BGL1), the BGL4-expressing strain showed a higher cellobiose fermentation ability at pH 6.0 and 40°C, leading to a higher ethanol production from alkaline-pretreated rice straw.

A mixture of 5% (w/v) glucose, 4% (w/v) xylose and 5% (w/v) cellobiose was fermented into ethanol using non-recombinant yeasts. Two series of experiments were carried out: (1) sequential fermentation with Candida shehatae D45-6 and Saccharomyces cerevisiae (Cs-Sc), and (2) sequential fermentation with C. shehatae D45-6 and Brettanomyces bruxellensis (Cs-Bb). C. shehatae D45-6 was initially used for glucose and xylose fermentation before adding highly ethanol-tolerant yeasts, either S. cerevisiae or B. bruxellensis, for cellobiose fermentation. For the sequential fermentation using S. cerevisiae, β-glucosidase was also included in the second step. In these two experiments, ethanol concentration reached 5.6–5.8% (w/v) and 99% sugar was consumed. Our results suggest that restricted glucose production from cellulose by saccharification could allow D45-6 to complete monosaccharide fermentation before the ethanol concentration exceeded its tolerance level.

The objective of this study was to develop an efficient production system for cellulase preparation with a high level of xylanolytic enzymes using soluble carbon sources. When xylose and arabinose were simultaneously fed with glucose and cellobiose, a mutant of Trichoderma reesei, M3-1, showed sufficient levels of cellulolytic and xylanolytic activities, indicating that xylose and arabinose are good inducers for the production of xylanolytic enzymes. In a continuous feeding experiment using glucose/cellobiose and glucose/xylose/cellobiose, cellulase preparations with various levels of xylanolytic enzymes were obtained by altering the feeding solutions and the timing of their addition. The volumetric production rates for xylanolytic activities at the glucose/xylose/cellobiose-feeding phase were significantly higher than at the glucose/cellobiose-feeding phase, while those for cellulolytic activities were comparable under the two conditions. Thus the composition of the enzyme preparation produced by the mutant was readily controlled by varying the inducers and the pattern of their addition, facilitating the tailored production of enzymes in a diversity of bioconversion processes.

The present study was aimed to improve the efficiency of bioconversion process, namely the calcium capturing by carbonation at room temperature (RT-CaCCO) process. Rice endosperm starch was used as a model substance to observe the effects of lime treatments on the gelatinization of starch granules. Effect of subsequent carbonation and saccharification behaviors were also evaluated. With increasing lime concentration, significant rise in the gelatinization temperatures, and decrease in the gelatinization enthalpy were observed, suggesting enhanced granule stability and disruption of hydrogen bonds. These changes appeared to be leveling off near 9% lime concentration, at which the binding effect of calcium ions may be saturated. The mild heat treatment further promoted changes in gelatinization. It was postulated that it was not an annealing effect, but that mild heat treatment disrupted hydrogen bonds and provided more available sites for calcium binding. Carbondioxide (CO2) neutralization led to decrease in gelatinization temperatures. The optimum pretreatment conditions for efficient hydrolysis of mild heat treatment at 50°C for 24 h subsequent to lime pretreatment is effective in accelerating saccharification by 1.9-fold and CO2 neutralization should be carried out after heat treatment. This approach can be useful in improving the recovery of liberated glucose when applying the RT-CaCCO process to feedstocks which contain both starch and lignocellulosic material.

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and β-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12h, achieving 88% of the theoretical yield from the sugars and cellulose.

A simple process (the direct-saccharification-of-culms (DiSC) process) to produce ethanol from rice straw culms, accumulating significant amounts of soft carbohydrates (SCs: glucose, fructose, sucrose, starch and β-1,3-1,4-glucan) was developed. This study focused on fully mature culms of cv. Leafstar, containing 69.2% (w/w of dried culms) hexoses from SCs and cellulose. Commercially-available wind-separation equipment successfully prepared a culm-rich fraction with a SC recovery of 83.1% (w/w) from rice straw flakes (54.1% of total weight of rice straw). The fraction was suspended in water (20%, w/w) for starch liquefaction, and the suspension was subjected to a simultaneous saccharification and fermentation with yeast, yielding 5.6% (w/v) ethanol (86% of the theoretical yield from whole hexoses in the fraction) after 24 h fermentation. Thus, the DiSC process produced highly-concentrated ethanol from rice straw in a one vat process without any harsh thermo-chemical pretreatments.

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.

Cellulose II hydrate was prepared from microcrystalline cellulose (cellulose I) via its mercerization with 5 N NaOH solution over 1 h at room temperature followed by washing with water. The structure of cellulose II hydrate changed to that of cellulose II after drying. Compared with cellulose II, cellulose II hydrate exhibited a slightly (8.5%) expanded structure only along the [ 1 1 ¯ 0 ] direction. The hydrophobic stacking sheets of the cellulose II were conserved in the cellulose II hydrate, and water molecules could be incorporated in the inflated two-chain unit cell of cellulose II hydrate. Enzymatic hydrolysis of cellulose I, cellulose II hydrate, and cellulose II was carried out at 37 °C using solutions comprising a mixture of cellulase and β-glucosidase. The hydrolysis of cellulose II hydrate proceeded much faster than the hydrolysis of the other two substrates, while the saccharification ratio of cellulose II was only slightly higher than that of cellulose I. The alkaline mercerization treatment was also applied to sugarcane bagasse. After its direct mercerization, the cellulose in bagasse was converted from cellulose I to cellulose II hydrate, and then to cellulose II after drying. Similar to in the case of microcrystalline cellulose, the rate of the enzymatic hydrolysis of the mercerized bagasse without drying (cellulose II hydrate) was much faster than the enzymatic hydrolysis of the other two substrates. Thus, the wet forms of cellulose and cellulosic biomass after mercerization, and after hydrolysis with cellulolytic enzymes, afforded superior products with extremely high degradability.

Rice straw was manually dissected and two main fractions were recovered: a culm and a leaf sheath/blade fraction, in order to evaluate their potential as feedstocks for the recovery of fermentable sugars. In the case of cv. Koshihikari and Milkyqueen, most soft carbohydrates (SCs: glucose, fructose, sucrose, starch, and beta-1,3-1,4-glucan) were present in the culms, reaching 47.9% and 89.2% of total SCs in the two main fractions. The results also indicated that beta-glucans (cellulose and beta-1,3-1,4-glucan) and xylan in the culms were more susceptible to direct enzymatic attack than those in the leaf sheath/blades. Thus the culm has high potential as a new feedstock for the extraction of fermentable sugars in a concentrated form, as compared to whole rice straw and the leaf sheath/blade. In this study, a novel method of separating a culm from the whole rice straw by means of wind power was also evaluated.

In order to investigate the residues important for the catalysis of an exo-β- d -glucosaminidase from Trichoderma reesei (Gls93), we performed site-directed mutagenesis study on this enzyme. Seven acidic amino acid residues in putative catalytic TIM domain, Glu409, Glu415, Asp464, Glu470, Asp513, Glu539 and Asp655, were selected for the mutational studies, each of which is located close to a C-terminus of the predicted β-strands and conserved within exo-β- d -glucosaminidase (GlcNase) subgroup of glycoside hydrolase family (GHF) 2 except for Glu470. The mutants of Gls93 at the residue Asp464 and/or Glu539, which showed a similar CD spectrum pattern to that of the wild-type enzyme, hardly have activity, indicating that Asp464 and Glu539 function as the catalytic residues of Gls93. Furthermore, the sterically conserved mutation of Glu409, Glu415 and Asp513 (the mutants E409Q, E415Q and D513N, respectively) gave rise to the change in its kinetic parameters, suggesting that these carboxylic residues could contribute to substrate recognition/binding or arrangement of ionic environment of the catalytic residues.

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.

A mixed mash of sugar beet roots and potato tubers with a sugar concentration of 23.7% w/v was used as a feedstock for bioethanol production. Enzymatic digestion successfully reduced the viscosity of the mixture, enabling subsequent heat pretreatment for liquefaction/sterilization. An energy-consuming thick juice preparation from sugar beet for concentration and sterilization was omitted in this new process.

Rice-straw hydrolysate (RSH) prepared via the CaCCO (Calcium Capturing by Carbonation) process contains not only monosaccharides but also significant amounts of oligosaccharides. In this study, a glutathione-producing yeast, Candida utilis NBRC 0626, was found to assimilate those oligosaccharides. The yields of reduced glutathione (GSH) and dry cell weight (DCW) per consumed sugars in a medium with RSH after 72h incubation were 10.1mg/g-sugars and 0.49g/g-sugars, respectively. The yields were comparative to those in a medium containing a model monosaccharide mix, suggesting that the assimilated oligosaccharides contribute to additional GSH and DCW production. Glycosyl linkage analysis indicated that the yeast could cleave xylose-, galactose-, and arabinose residues as well as glucose residues at the non-reducing ends. After 72h incubation, 99.1% of the total glucose residues and 84.2% of the total xylose residues in RSH were depleted. Thus the yeast could be applied for efficient utilization of lignocellulosic hydrolysates.

The RT-CaCCO process for enzymatic saccharification was applied to readily-milled fractions of wet sugarcane bagasse. Wet bagasse immediately after juice extraction was crushed with shark-mill blades to prepare two fractions referred to as readily-milled (RF) and hardly-milled fraction (HF). Monosaccharide recoveries from RFs via the RT-CaCCO process were 1.03–1.21 times higher than those from HFs. Moreover, when the wet weight ratio of RF/HF was adjusted to 2/8, the hexose recovery from RF was 90.9%, which was 1.3 times higher than that of the wet bagasse before fractionation. The results show that this process can be used for efficient monosaccharide recovery from RF of wet bagasse. In addition, the process can be adapted to more fibrous HF for multiple uses such as fuel for boilers and fibers for particleboards.

A mutant Cs3512, which showed better fermentation of xylose and the mixtures of xylose and glucose, was obtained through mutation of Candida shehatae ATCC 22984 and screening with a medium containing antimycin A and TTC (2,3,5-triphenyltetrazolium chloride). Cs3512 produced 44.4 g/l of ethanol from 121.3 g/l of xylose, which was 13% higher than that by ATCC 22984. At the same time, xylitol production was reduced by 38% to 10.2 g/l from 16.3 g/l by ATCC 22984. Cs3512 also showed 8% increase in ethanol yield from 0.39 to 0.42 g/g comparing to ATCC 22984 when fermenting the sugar mixture composed of 52.9 g/l glucose and 21.2 g/l xylose. When Cs3512 was used in the simultaneous saccharification and fermentation of lime pretreated rice straw via CaCCO (calcium capturing by carbonation) process, it produced ethanol at 77% of the theoretical yield. The results imply that Cs3512 is a potential non-recombinant yeast strain for ethanol production from lignocellulosic biomass.

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120 °C for 1 h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120 °C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl beta-D: -lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1-1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.

In order to establish an efficient bioethanol production system for rice straw, we developed a novel lime-pretreatment process (CaCCO process) that did not require a solid–liquid-separation step. This process adopted a step in which after pretreatment lime was neutralized by carbonation, resulting in a final pH of about 6. CaCO3 produced by the process was kept in the reaction vessel, and no significant inhibitory effects on enzymatic saccharification and fermentation were observed. In the CaCCO process, solubilized carbohydrates, such as xylan, starch, and sucrose were also kept in the vessel, enabling high recoveries of monomeric sugars. Simultaneous saccharification and fermentation (SSF) of pretreated rice straw, 10% (g-rice straw/g-water), using Saccharomyces cerevisiae and Pichia stipitis yielded 19.1 g L−1 ethanol that was 74% of the theoretical yield from glucose and xylose. Thus, this process represents a novel pretreatment method to utilize not only cellulose but also xylan, starch, and sucrose from biomass.

Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-beta-D-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of approximately 105 kDa (Gls93-F1) and approximately 100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 A, and diffracted to 1.8 A resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 A, and diffracted to 2.4 A resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.

We have previously reported on purification and characterization of an exo-beta-D-glucosaminidase (Gls93) from culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetyl-D-glucosamine (GlcNAc). The corresponding gene of Gls93 was cloned and characterized in this work. To our knowledge, this is the first report on cloning of the gene encoding fungal exo-beta-D-glucosaminidase. This gene has no introns and encodes a polypeptide of 892 amino acids (aa) containing a secretion signal of 28 amino acids. Comparison of the amino acid sequence to known proteins and phylogenetic analysis indicated that gls93 belongs to the glycoside hydrolase family (GHF) 2 and should be further classified into a new subgroup, exo-beta-D-glucosaminidase subgroup. The gls93 transcription was biphasic when T. reesei was grown on GlcNAc, suggesting that the expression of this gene may be regulated by a complex mechanism, in which multiple regulatory proteins are involved. Furthermore, gls93 could be expressed in Pichia pastoris (ca. 0.49-mg/ml culture). The recombinant Gls93 had the two molecular forms, ca. 105 and 100 kDa, whose difference is caused by N-glycosylation. Both of them had the same properties such as specific activity and substrate specificity and showed only the activity of exo-beta-D-glucosaminidase but not those of beta-galactosidase, beta-glucuronidase, and beta-mannosidase belonging to GHF2.

We purified a chitinase (named Chi46), with a molecular mass of 46 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from the culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetylglucosamine (GlcNAc). The relative activity of this enzyme reduced when the degrees of acetylation (DA) of chitosan decreased. Furthermore, the enzyme was able to hydrolyze colloidal chitin and ethylene glycol chitin. The gene chi46 was cloned and sequenced. chi46 encodes a protein of 424 amino acid residues containing a 35-amino acid prepro-type secretion signal peptide. The molecular mass of mature Chi46 calculated from deduced amino acid sequence was 42,265 Da. The chi46 transcript was biphasic when the mycelia were grown on GlcNAc, suggesting that the multiple regulatory proteins are involved in the chi46 expression. The chi46 cDNA was expressed in Escherichia coli (ca. 0.23 mg/ml culture). To determine substrate cleavage fashion of Chi46 in more detail, we carried out high-performance liquid chromatography analysis and viscosimetric assay using recombinant Chi46 (rChi46). Chi46 was shown to release mainly (GlcNAc)(2) from colloidal chitin (insoluble chitin) as an exo-type manner and to act on chitosan 7B (DA ca. 30%) and N-acetylchitooligosaccharides (soluble chitins) in an endo-type one.