Pretreatment of Rice Straw by a Hot-Compressed Water Process for Enzymatic Hydrolysis
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- Yu, G., Yano, S., Inoue, H. et al. Appl Biochem Biotechnol (2010) 160: 539. doi:10.1007/s12010-008-8420-z
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Hot-compressed water (HCW) is among several cost-effective pretreatment processes of lignocellulosic biomass for enzymatic hydrolysis. The present work investigated the characteristics of HCW pretreatment of rice straw including sugar production and inhibitor formation in the liquid fraction and enzymatic hydrolysis of pretreated material. Pretreatment was carried out at a temperature ranging from 140 to 240 °C for 10 or 30 min. Soluble oligosaccharides were found to constitute almost all the components of total sugars in the liquid fraction. The maximal production of total glucose at 180 °C and below accounted for 4.4–4.9% of glucan in raw material. Total xylose production peaked at 180 °C, accounting for 43.3% of xylan in raw material for 10-min pretreatment and 29.8% for 30-min pretreatment. The production of acetic acid increased at higher temperatures and longer treatment time, indicating more significant disruption of lignocellulosic structure, and furfural production achieved the maximum (2.8 mg/ml) at 200 °C for both 10-min and 30-min processes. The glucose yield by enzymatic hydrolysis of pretreated rice straw was no less than 85% at 180 °C and above for 30-min pretreatment and at 200 °C and above for 10-min pretreatment. Considering sugar recovery, inhibitor formation, and process severity, it is recommended that a temperature of 180 °C for a time of 30 min can be the most efficient process for HCW pretreatment of rice straw.
KeywordsRice straw Pretreatment Hot-compressed water Sugar production Inhibitor formation Enzymatic hydrolysis Fermentation
Lignocellulosic biomass is one potential resource for the production of fuels such as ethanol, and the bioconversion of lignocellulosic biomass to ethanol is a multi-step process consisting of pretreatment, enzymatic hydrolysis, and ethanol fermentation. Among these steps, pretreatment is particularly crucial in view of the recalcitrance of lignocellulosic structure to enzymatic hydrolysis, i.e., the lignin seal and the hemicellulose sheath of cellulose and the crystallinity of cellulose itself, and often dominates the cost of the whole conversion process [1, 2].
Hot-compressed water (HCW) pretreatment, in which biomass is exposed to pressured hot water, is one of several most promising pretreatment methods [1, 2, 3, 4]. Water under pressure penetrates the cell structure of biomass, hydrates cellulose, and dissolves hemicellulose and lignin, and the acidity of water at high temperature (around 200 °C) and organic acids released from hemicellulose facilitate the disruption of lignocellulosic structure during pretreatment. HCW pretreatment does not require the addition of any chemical, can generate reactive cellulose fiber and recover most of the pentosans, and produce only an amount of degradation products with little inhibition to subsequent hydrolysis and fermentation [1, 2, 5]. So far, some research work has been conducted on HCW pretreatment of lignocellulosic biomass for sugar production, outlining fundamental characteristics of the process [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23]. However, specific features of the process are still to be sufficiently clarified for improvement of the promising technology such as details incorporating both sugar production and inhibitor formation during the hot-water extraction process. Furthermore, different types of biomass possess different structures and compositions, which would give rise to different characteristics of pretreatment. An advanced pretreatment process is required to be tailored to the unique compositional and structural features of lignocellulosic biomass .
Rice straw is one major agricultural residue and one potential lignocellulosic substrate for fuel ethanol production. Until now, the only work on HCW pretreatment of rice straw was mainly restricted to the analysis of solubilization by pretreatment , and no detailed study has been reported to our knowledge. The present work examined in detail the characteristics of HCW pretreatment of rice straw including sugar production and inhibitor formation in the liquid fraction as well as enzymatic hydrolysis and fermentation of the pretreated material.
Materials and Methods
Rice straw (Oryza sativa cv. Binan-mochi) used in this work was obtained from a local farmer in Hiroshima prefecture, Japan in November 2006. Rice straw was cut to 5 cm, air-dried, and then milled by a cutter mill (Fritsch, Germany) to 2 mm. After mesh sieving, particles of 250–420 μm were obtained and used for HCW pretreatment.
The composition of raw material and solid fractions was analyzed according to the analytical procedure of the National Renewable Energy Laboratory (NREL) . Acid-insoluble residue in the analysis of lignin content was taken as acid-insoluble lignin. The ash content was determined by placing 1.5-g sample in a muffle furnace at 600 °C for 24 h, cooling in a desiccator, and then weighing for ash.
Sugars and degradation products in the liquid fraction samples were analyzed by following the NREL procedure . Monomeric sugars in the liquid fraction were analyzed by high-performance liquid chromatography (HPLC; Jasco, Japan) using a Biorad Aminex HPX-87P column (300 × 7.8 mm) equipped with a refractive index detector . The mobile phase was degassed deionized water with a flow rate of 1.0 ml/min. The column temperature was 80°C. Total sugars consisting of monomeric and oligomeric sugars in the liquid fraction were analyzed by sulfuric acid hydrolysis method followed by HPLC determination .
Degradation products in the liquid fraction were analyzed by HPLC using a Biorad Aminex HPX-87H column (300 × 7.8 mm) equipped with a refractive index detector . The mobile phase was 0.01 N sulfuric acid with a flow rate of 0.6 ml/min. The column temperature was 60 °C.
Analysis was carried out in duplicate, and results are expressed as the mean values. Relative standard deviations in all cases were within 5%.
Solubilization of rice straw by pretreatment was calculated as the percentage of raw material dissolved by pretreatment by measuring dry weight of the solid fraction. pH in the liquid fraction was measured by a pH meter at room temperature.
Enzymatic hydrolysis of raw material and HCW solid fractions was carried out in 50 mM acetate buffer (pH 5.0) at 45 °C for 72 h. A substrate concentration of 2% was used to avoid possible substrate inhibition. Two enzyme loadings of Acremonium cellulase (Meiji Seika, Japan, specific activity determined as 307.7 FPU/g protein), i.e., 40 and 10 FPU/g substrate, were adopted. In addition, 5 IU/g substrate of Novozyme 188 (β-glucosidase, 617 IU/ml) and 2%(v/v)/g substrate of Optimash BG (β-glucanase/xylanase, Genencor, 11255 CMCU/g protein) were supplemented to improve the efficiency of enzymatic hydrolysis according to our previous work. Controls containing no substrate were performed simultaneously to eliminate the deviation in hydrolysis results caused by any sugars existing in enzyme preparations. Production of monomeric sugars was analyzed by HPLC as described above. The sugar yields were defined as the percentage of total sugars available in raw material or solid fraction enzymatically converted to monomeric sugars. Enzymatic hydrolysis was performed in duplicate, and results are presented as the average. Standard deviations were less than 5%.
Separate Hydrolysis and Fermentation
Separate hydrolysis and fermentation (SHF) was carried out to check the fermentability of pretreated rice straw, with the solid fraction from pretreatment at 180 °C for 30 min as the substrate. Hydrolysis was performed in a 13-ml vial magnetically stirred with a reaction volume of 8 ml. The substrate concentration was 5%, and the enzyme loadings were 40 FPU/g substrate of Acremonium cellulase, 5 IU/g substrate of Novozyme 188 and 2%(v/v)/g substrate of Optimash BG. Hydrolysis was carried out in 50 mM acetate buffer (pH 5.0) at 45 °C for 72 h. Upon completion of hydrolysis, 0.8 ml of YPD preculture of Saccharomyces cerevisiae type II (Sigma) was inoculated, and fermentation was carried out at 30 °C for 72 h. For comparison, the solid fraction together with about one-fourth strength of the hydrolysate from the same pretreatment was also tested for SHF. pH was checked and adjusted to the same value for the two cases before and after saccharification. Samples from hydrolysis and fermentation were analyzed by HPLC using a Biorad Aminex HPX-87P column under the same conditions as above.
Scanning Electron Microscopy
The morphology of rice straw untreated, pretreated under different HCW conditions, and enzymatically hydrolyzed was observed by a scanning electron microscope (HITACHI S-3400N).
Results and Discussion
The composition of rice straw used in this work was determined to be (on a dry weight basis): 36.40% glucan, 19.15% xylan, 3.04% galactan, 1.59% arabinan, 16.51% acid insoluble lignin, 1.71% acid soluble lignin, 1.45% acetyl, and 15.65% ash. The contents of most carbohydrates, such as glucan and xylan, and lignin were comparable to those of corn stover, whereas the contents of arabinan and acetyl group were less than half of those in corn stover . In addition, mannan was not detected in the raw material. It is noticed that the ash content was much higher compared with many kinds of lignocellulosic biomass especially woody biomass . The compositional differences may suggest a noteworthy structural distinction and would give rise to unique characteristics of HCW pretreatment of rice straw.
Production of Sugars in Liquid Fraction
It is found that pretreatment temperature basically dominated the pattern of sugar production for an HCW process, whereas the length of pretreatment time exerted an influence on the amount of the produced sugars. Pretreatment for 10 min tended to generate a higher amount of sugars than pretreatment for 30 min. A 30-min process gave threefold of R0 of a 10-min process for a given temperature, and solubilized oligosaccharides can be degraded to monomeric sugars and then further to smaller byproducts during a longer process, thus affecting the amount of sugars remained in the liquid fraction.
Formation of Inhibitors in Liquid Fraction
The production of acetic acid increased at higher temperatures, with the greater amounts for 30-min pretreatment than for 10-min pretreatment at each temperature (Fig. 3a), indicating that more significant breakdown of lignocellulosic structure occurred at higher temperatures and longer treatment time. The similar effect of temperature on acetic acid formation was also observed in other work . Furfural production achieved the maximum (2.8 mg/ml) at 200 °C for both 10-min and 30-min processes (Fig. 3b), corresponding to complete or nearly complete degradation of xylose and arabinose. Furthermore, 30-min pretreatment caused greater production of furfural at temperatures lower than 200 °C and more rapid degradation of furfural at temperatures higher than 200 °C compared with 10-min pretreatment. The formation of 5-hydroxymethyl-2-furaldehyde (HMF) was also enhanced at higher process severity (HMF can be further degraded under harsh conditions such as 240 °C for 30 min) and was largely consistent with the decomposition of glucose and galactose (Fig. 3c). Formic acid, which can be produced from the degradation of furfural and HMF, was also detected in the liquid fraction (Fig. 3d). However, the maximal production of HMF and formic acid was only about one third of that of acetic acid and furfural, suggesting that acetic acid and furfural may have a greater impact on the following biochemical transformation processes. Additionally, no production of levulinic acid, a degradation product of HMF, was found during HCW pretreatment under all conditions tested, implying that furfural may be the major source of formic acid.
Solubilization of Rice Straw
The solubilization by HCW gave rise to a great change in the composition of rice straw. Xylan and acetyl group were remarkably removed from raw material by pretreatment at 180 °C, with xylan contents of only 2.78% (30 min) and 7.96% (10 min) and acetyl contents of only 0.30% (30 min) and 0.66% (10 min) in solid fractions. The xylan content was decreased to nearly one seventh of that in raw material by pretreatment for 30 min. Galactan and arabinan were completely dissolved after pretreatment at 180 °C. In contrast, the glucan content was increased to more than 50% by pretreatment at 180 °C (52.97% for 30 min and 52.47% for 10 min) thanks to the removal of hemicellulose and other components. In fact, the highest content of glucan in solid fraction was 53.0% at 180 °C for 30-min pretreatment and 54.3% at 200 °C for 10-min pretreatment, respectively.
Considering sugar yields by enzymatic hydrolysis and process severity, it is recommended that pretreatment at 180 °C for 30 min can be the most cost-effective HCW process of rice straw. The solid fraction derived from the pretreatment (180 °C, 30 min) was therefore adopted for examination of the efficiency of ethanol fermentation.
Separate Hydrolysis and Fermentation
Morphology of HCW-Pretreated Rice Straw
This work has studied the characteristics of pretreatment of rice straw by hot-compressed water including the compositional features of the liquid fraction and enzymatic hydrolysis of pretreated material.
It was found that oligosaccharides constituted almost all the components of total sugars in the liquid fraction. The maximum production of total glucose accounted for 4.4−4.9% of glucan in raw material, and the highest production of total xylose at 180 °C accounted for 43.3% of xylan in raw material for 10-min pretreatment and 29.8% for 30-min pretreatment. The production of acetic acid indicated more significant breakdown of lignocellulosic structure occurring at higher temperature and longer pretreatment time. Furfural production peaked at 2.8 mg/ml at 200 °C for both 10- and 30-min processes, corresponding to complete or nearly complete degradation of pentoses. The glucose yield by enzymatic hydrolysis of pretreated rice straw increased with elevated temperature and was no less than 85% at 180 °C and above for 30-min pretreatment and at 200 °C and above for 10-min pretreatment. Approximately 100% of theoretical ethanol yield was obtained for glucose in SHF. SEM micrographs confirmed the significant disruption of lignocellulosic structure and the enhancement of enzymatic hydrolysis by pretreatment.
In terms of sugar recovery, inhibitor formation and process severity, a temperature of 180 °C for a time of 30 min can be taken as the most cost-effective process for pretreatment of rice straw by hot-compressed water.
We would like very much to thank Dr. Seung-Hwan Lee for his help in taking SEM photos. We appreciate Dr. Tsuyoshi Sakaki for his valuable comments on the experimental results. This work was partly supported by Japan International Cooperation Agency in the “Research on Biomass Technology” program (2006–2007).