Short-time alkaline peroxide pretreatment for rapid pulping and efficient enzymatic hydrolysis of rice straw.

To improve utilization of agricultural residues in biorefineries, the effects of alkaline peroxide (AP) pretreatment on thermal degradation and enzymatic digestibility of rice straw were investigated. A high-cellulose (>70%) pulp with a width of a few micrometers was obtained by rapid (10min) treatment with AP, without prior heating or other treatment. Moreover, enzymatic hydrolysis of the pulp produced a high glucose yield (approximately 80%). Microfibril networks were exposed, and many nano-scale pores that are easily penetrated by cellulases were observed on the surface of the AP-treated sample. For enzymatic hydrolysis of the pulp, increasing the dosage of cellulases was more effective for improving the glucose yield than addition of a grinding treatment. This AP treatment has the potential for on-site application because it is simple, highly efficient, and can be performed in a short time.

Preparation using pectinase and characterization of nanofibers from orange peel waste in juice factories.

UNLABELLED: This study reports the preparation and characterization of nanofibers consisting mainly of cellulose microfibrils from orange peel (OP), which is a significant byproduct of orange juice production. Three treatments (boiling, alkaline, and pectinase) were investigated with and without subsequent grinding treatment. It was possible to prepare the cellulose nanofibers (CNFs) using these methods, except for the boiling treatment with grinding. Interestingly, only pectinase and a mild-physical blender treatment without grinding produced nanofibers. The width of the nanofibers from OP was approximately 10 to 50 nm. The microfibril bundles of OP were considered to be thinner than those of commercial CNFs. Our data indicated that the removal of pectic polysaccharides and hemicelluloses covering the cellulose microfibrils was important for the preparation of nanofibers from OP. These nanofibers from OP using pectinase are proposed to be applicable as food materials, pharmaceuticals, and filters for the tractive characteristics of the sheet. PRACTICAL APPLICATION: This study demonstrates: (1) it was possible to prepare the nanofibers from orange peel using pectinase and (2) the width of the nanofibers from orange peel was approximately 10 to 50 nm. (3) Removal of polysaccharides such as pectin and hemicelluloses covering cellulose microfibrils was very important for preparation of nanofibers from OP. Considering the tractive characteristics of the sheets from nanofibers and the origin of orange peel, they are suitable for application of food materials, pharmaceuticals, and filters.

Comparison of the enzymatic digestibility of physically and chemically pretreated selected line of diploid-Miscanthus sinensis Shiozuka and triploid-M.×giganteus.

The diploid Miscanthus sinensis "Shiozuka" which was selected as a high-biomass producing line, and the triploid M. × giganteus (M×G) were treated by ball milling (physical treatment) and alkaline hydrogen peroxide treatment (AHP; chemical treatment), and their structural sugar compositions and enzymatic digestibility were compared. The structural sugar content of Shiozuka was moderate and lower than that of M×G. The Klason lignin content of Shiozuka was also lower than that of M×G. However, Shiozuka was sensitive to ball milling and AHP treatment; ball milled and AHP-treated Shiozuka had higher enzymatic digestibility than ball milled and AHP-treated M×G. Shiozuka would be promising feedstock to obtain fermentable sugars with low energy consumption. Finally, enzymes for the hydrolysis of chemically treated Miscanthus were isolated from Trichoderma reesei ATCC 66589 and Penicillium pinophilum. The sugar yield could be increased by enzymatic hydrolysis of AHP-treated samples with NaOH and H2O2 and the isolated enzymes.

Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface.

The effects of adding trace acids in ethanol based organosolv treatment were investigated to increase the enzymatic digestibility of Japanese cypress. A high glucose yield (60%) in the enzymatic hydrolysis was obtained by treating the sample at 170 °C for 45 min in 50% ethanol liquor containing 0.4% hydrochloric acid. Moreover, the enzymatic digestibility of the treated sample was improved to ∼70% by changing the enzyme from acremonium cellulase to Accellerase1500. Field emission scanning electron microscopy revealed the presence of lignin droplets and partial cellulose nanofibers on the surface of the treated sample. Simultaneous saccharification and fermentation of the treated samples using thermotolerant yeast (Kluyveromyces marxianus NBRC1777) was tested. A high ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/HCl0.4-treated sample compared with samples from other treatments.

High-coverage gene expression profiling analysis of the cellulase-producing fungus Acremonium cellulolyticus cultured using different carbon sources.

The gene expression of a cellulase-producing fungus, Acremonium cellulolyticus, was investigated after culturing with three different carbon sources: glycerol, lactose, and Solka-Floc powdered cellulose (SF). High-coverage gene expression profiling (HiCEP) analysis, a method requiring no prior sequence knowledge, was used to screen genes upregulated at the early stage of cellulase production. SF was used as a strong inducer of cellulase production, lactose was used as an inducer of the expression of cellulase genes at the early stage of the culture, and glycerol was used as a negative control. Approximately 15,000 transcript-derived fragments (TDFs) were detected in each sample prepared from the culture grown for 16 h. Based on the expression profiles of the cultured cells, 36 fragments upregulated in both the SF and lactose cultures were selected and sequenced. The deduced gene products of 31 TDFs were likely related to biomass degradation, sugar metabolism, transcriptional regulation, protein modification and metabolism, cell wall recycling, fatty acid and polyketide biosynthesis, and other functions. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis verified that almost all of the transcripts obtained by HiCEP analysis were upregulated in the SF and lactose cultures grown for 18 h. Some of the TDFs in the SF culture were further upregulated over the course of 72 h. The gene products from these TDFs would provide insight into improving the cellulase productivity of A. cellulolyticus.

Combination of hot compressed water treatment and wet disk milling for high sugar recovery yield in enzymatic hydrolysis of rice straw.

Rice straw has attracted significant interest in Japan as a potential raw material for biorefineries. Combination of hot-compressed water treatment (HCWT) and wet disk milling (WDM) was investigated to improve the enzymatic digestibility of rice straw and enhance sugar recovery yield. Rice straw, cut to <3 mm, was autoclaved at 121, 135, and 150 °C for 60 min, and subsequently treated by wet disk milling. WDM with HCWT at 135 °C for 60 min produced maximum xylose and glucose yields of 79% and 90%, respectively, at 10 FPU/g-substrate cellulase loading. Autoclaving at 150 °C leaked a 35% arabinose effluence in the liquid phase. Hydrolysis via WDM with HCWT required a lower enzyme loading (5 FPU/g-substrate) than either pretreatment process in isolation for >70% xylose and 80% glucose yield. Economical analysis indicate that enzymes cost for ethanol production is reduced by 19-67% by WDM with HCWT.

Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source.

Cellulases and hemicellulases are key enzymes in the production of alternative fuels and chemicals from lignocellulosic biomass-an abundant renewable resource. Carbon source selection is an important factor in the production of cellulases and hemicellulases. Rice straw--a potential ethanol source--has recently gained considerable interest in Asian countries. Here, we investigated the production of cellulases by using rice straw subjected to various pretreatments as substrates in order to produce cellulases at low costs; we also identified the enzymes' characteristics. Rice straw cutter milled to <3mm was pretreated by wet disk milling, dry ball milling, or hot-compressed water treatment (HCWT). Pretreated rice straw and commercial cellulose, Solka Floc (SF), were used as carbon sources for cellulase production by the fungus Acremonium cellulolyticus. Filter paper cellulase, ß-xylanase, and ß-xylosidase production from ball- and disk-milled samples were higher than those from SF. Enzymatic activity was absent in cultures where HCWT rice straw was used as carbon source. Wet disk-milled rice straw cultures were more suitable for enzymatic hydrolysis of pretreated rice straw than SF cultures. Thus, wet disk milling may be a suitable pretreatment for producing substrates for enzymatic hydrolysis and generating inexpensive carbon sources for cellulase production.

A chimeric NST repressor has the potential to improve glucose productivity from plant cell walls.

Bioethanol might be produced more economically and with less ecological impact (with reduced exploitation of food crops) if we could increase the production of glucose from the cellulosic materials in plant cell walls. However, plant cell walls are relatively resistant to enzymatic and physicochemical hydrolysis and, therefore, it is necessary to develop methods for reducing such resistance. Changes in plant cell wall materials, by genetic engineering, that render them more easily hydrolyzable to glucose might be a valuable approach to this problem. We showed previously that, in Arabidopsis, NAC secondary wall thickening-promoting factor1 (NST1) and NST3 are key regulators of secondary wall formation. We report here that transgenic Arabidopsis plants that expressed a chimeric repressor derived from NST1 produced cell wall materials that were twice as susceptible to both enzymatic and physicochemical hydrolysis as those from wild-type plants. The yields of glucose from both fresh and dry biomass were increased in the chimeric repressor lines. Use of the NST1 chimeric repressor might enhance production of glucose from plant cell walls, by changing the nature of the cell walls themselves.

Wet disk milling pretreatment without sulfuric acid for enzymatic hydrolysis of rice straw.

Rice straw has recently attracted interest in Japan as a potential source of raw material for ethanol production. Wet disk milling, a continuous pretreatment to enhance the enzymatic digestibility of rice straw, was compared with conventional ball milling and hot-compressed water treatment. Pretreated rice straw was evaluated by enzymatic hydrolysis using Acremonium cellulase and characterized by X-ray diffraction and scanning electron microscopy. Glucose and xylose yields by wet disk milling, ball milling, and hot-compressed water treatment were 78.5% and 41.5%, 89.4% and 54.3%, and 70.3% and 88.6%, respectively. Wet disk milling and hot-compressed water treatment increased sugar yields without decreasing their crystallinity. The feature size of the wet disk milled rice straw was similar to that of hot-compressed water-treated rice straw. The energy consumption of wet disk milling was lower than that of other pretreatments. Thus, wet disk milling is an economical, practical pretreatment for the enzymatic hydrolysis of lignocellulosic biomass, especially herbaceous biomass such as rice straw.

Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulase.

The feasibility of using loofa sponge for immobilization of cellulase-producing microorganisms was investigated by acetylating loofa sponge. Acetylation was achieved by autoclaving process of loofa sponge immersed in acetic anhydride at various temperatures for various times. The degree of acetylation, as inferred by the weight percentage gain (WPG), was enhanced by increasing both temperature and the duration of acetylation. The acetylation of a piece of loofa sponge in an autoclave at 120 degrees C for 20 min resulted in a WPG of about 8%, which was sufficient to protect the loofa sponge against cellulose degradation. The acetylated loofa sponge prepared under this condition was not decomposed by commercial cellulase and its structure was maintained for more than 720 h during repeated-batch treatments with commercial cellulase. A flocculating yeast (Saccharomyces cerevisiae IR-2) and a fungus (Trichoderma reesei QM9414) were successfully immobilized in the acetylated loofa sponge. In each case, the percentage of immobilized cells was as high as that obtained using nonacetylated loofa sponge. Acetylation had no adverse effects on cell growth and immobilization of T. reesei QM9414, as well as on cell growth and ethanol production by S. cerevisiae IR-2. T. reesei QM9414 immobilized on an acetylated loofa sponge was successfully used for repeated-batch cellulase production from commercial cellulose powder. Although the acetylated loofa sponge showed a slight weight loss, it was not disintegrated by activated sludge. The results obtained in this study showed that acetylated loofa sponge is suitable as an immobilization carrier for bioprocesses involving cellulase.