Accepted Manuscript Title: Effect of nitric acid on pretreatment and fermentation for enhancing ethanol production of rice straw Author: Ilgook Kim Bomi Lee Ji-Yeon Park Sun-A. Choi Jong-In Han PII: DOI: Reference:
S0144-8617(13)00876-X http://dx.doi.org/doi:10.1016/j.carbpol.2013.08.092 CARP 8082
To appear in: Received date: Revised date: Accepted date:
7-5-2013 26-8-2013 28-8-2013
Please cite this article as: Kim, I., Lee, B., Park, J.-Y., Choi, S.-A., & Han, J.-I., Effect of nitric acid on pretreatment and fermentation for enhancing ethanol production of rice straw, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.08.092 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of nitric acid on pretreatment and fermentation for enhancing ethanol
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production of rice straw
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Ilgook Kima, Bomi Leea, Ji-Yeon Parkb, Sun-A Choib, Jong-In Hana,*
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a
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Yuseong-gu, Daejeon 305-701, Republic of Korea
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b
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gu, Daejeon 305-343, Republic of Korea
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*
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Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong,
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Corresponding author: Jong-In Han (E-mail address: [email protected]; Tel: +82-42-350-
3629; Fax: +82-42-350-3610)
In this study, nitric acid (HNO3) was evaluated as an acid catalyst for rice straw
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Abstract
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Department of Clean Fuel, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-
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pretreatment, and, after neutralization, as a sole nitrogen source for subsequent fermentation.
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Response surface methodology was used to obtain optimal pretreatment condition with
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respect to HNO3 concentration (0.2−1.0%), temperature (120−160 °C) and reaction time
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(1−20 min). In a condition of 0.65% HNO3, 158.8 °C and 5.86 min, a maximum xylose
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yield of 86.5% and an enzymatic digestibility of 83.0% were achieved. The sugar solution
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that contained nitrate derived from the acid catalyst supported the enhancement of ethanol
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yield by Pichia stipitis from 10.92 g/L to 14.50 g/L. The results clearly reveal that nitric acid 1
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could be used not only as a pretreatment catalyst, but also as a nitrogen source in the
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fermentation process for bioethanol production. It is anticipated that the HNO3-based
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pretreatment can reduce financial burden on the cellulosic bioethanol industry by
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simplifying after-pretreatment-steps as well as providing a nitrogen source.
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Keywords
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Rice straw; RSM; HNO3 pretreatment; Enzymatic hydrolysis; Fermentation
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1.
Introduction
Due to the depletion of the world petroleum supply and increasing concerns about
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climate change, biomass-based renewable fuels such as lignocellulosic ethanol is generally
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counted as a potential solution to the energy crisis and global warming (Alvira et al., 2010).
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Lignocellulosic materials including agricultural residues, hardwood, and softwood are
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plentiful and generate low net greenhouse gas emissions (Glabe and Zacchi, 2007).
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However, its complex and heterogeneous cell walls make it hard downstream steps, such as
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enzymatic hydrolysis of sugar polymers. Therefore, pretreatment is absolutely required to
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increase the yield of fermentable sugars.
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Chemical pretreatment with diluted acid combined with enzymatic hydrolysis has
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been considered as a promising procedure for the conversion of lignocellulose to mono-
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sugars for ethanol-making process (Hendriks and Zeeman, 2009). This acid-catalyzed 2
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pretreatment causes the hydrolysis of hemicellulose and increases portion of amorphous
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cellulose, resulting in high recovery of hemicelluloses as monomers in the liquid fraction
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and high digestible cellulose in the solid fraction (Linde et al., 2008; Sassner et al., 2008;
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Yang and Wyman, 2008). Literally every strong acid has been investigated as a catalyst, e.g.,
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sulfuric acid (Hsu et al., 2010), hydrochloric acid (Marcotullio et al., 2011), phosphoric acid
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(Geddes et al., 2010), and also nitric acid (Zhang et al., 2011). Among these, sulfuric acid,
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the cheapest agent, is most commonly used. On the other hand, nitric acid can be massively
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produced via a NOx capture process from flue gases (Kim and Han, 2013), displaying a
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much shorter reaction time (Zhang et al., 2011) and higher efficiency for saccharification
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(Rodríguez-Chong et al, 2004). Furthermore, nitrate, formed upon being neutralized, could
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be used as a nitrogen source for fermentation process, as a variety of nitrogen sources
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including yeast extract and peptone (Laopaiboon et al., 2009), ammonium (Yue et al., 2012),
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amino acid (Albers et al., 1996) and urea (Jones and Ingledew, 1994) have been reported to
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be attempted for ethanol production.
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In this work, rice straw, the most abundant biomass feedstock in Korea, was chosen as a raw lignocellulosic material for ethanol production. Response surface methodology
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(RSM) was used to optimize pretreatment condition of rice straw with nitric acid (HNO3),
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particularly aiming at maximum xylose yield. Nitrate was also tested as the nitrogen source
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for the fermentation of Pichia stipitis.
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2.
Materials and methods 3
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2.1.
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Feedstock materials Rice straw was collected from Nonsan field near the city of Daejeon, South Korea.
The air-dried rice straw was milled to small particles by a grinding machine, screened to
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obtain a size of less than 2 mm, and stored in a drying oven prior to use. The rice straw was
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composed of 41.7% glucan, 18.3% xylan, and 16.6% lignin based on its dry weight.
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2.2.
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HNO3 Pretreatment
Pretreatment was carried out in a stainless steel cylindrical reactor with a total
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volume of 70 mL. Before pretreatment, 5g rice straw and 50 mL HNO3 solution were mixed
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together into the reactor. The reactor was then heated to set temperatures in an oil bath. The
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average heating rate was about 15 °C/min. Once the targeted temperature inside the reactor
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was reached, reaction time was counted. At the end of the pretreatment, solid residue was
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separated by filtration and then washed with deionized water for subsequent enzymatic
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hydrolysis, and liquid fraction was gathered for the analyses of sugar and inhibitor.
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2.3.
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Enzymatic hydrolysis
After pretreatment, an enzymatic hydrolysis experiment was conducted in a 150-mL
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Erlenmeyer flask containing 50 mM sodium citrate buffer (pH 4.8) at a solid loading of 10%
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(w/v). A novel commercial enzyme blend Cellic C-Tec (Novozymes, Denmark) derived
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from Trichoderma reesei with an activity loading of 30 FPU/g cellulose was added to the
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pretreated rice straw. The flask was incubated at 50 °C and 170 rpm for 72 h in a shaking
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incubator and the hydrolysate was sampled periodically for sugar analysis. 4
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2.4.
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Fermentation Pichia stipitis KCTC 7222 was obtained from Korean Collection for Type Culture
(Daejeon, Korea). The yeast was maintained on YM agar plates at 4 °C and transferred
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monthly. YM agar contained yeast extract 3 g/L, malt extract 3 g/L, peptone 5 g/L, glucose
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10 g/L, and agar 20 g/L. In order to increase cell density, the yeast was transferred into a
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250-mL Erlenmeyer flask with 100 mL of the inoculation medium containing yeast extract 3
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g/L, peptone 5 g/L, glucose 35g/L, and xylose 15g/L. After incubation for 16 h, cells were
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harvested and applied as an inoculum for fermentation test. The yeast cell concentration in
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the inoculum was determined to 5 g dry weight of cells/L.
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Fermentation experiment was carried out in sterile 250-mL Erlenmeyer flasks with
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cap and needle to remove CO2 in a shaking incubator at 30 °C and 170 rpm for 48h. Each
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flask was filled with 50 mL fermentation medium inoculated with 10% (v/v) growth culture.
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The fermentation medium was supplied with glucose 35 g/L, xylose 15g/L, KH2PO4 5g/L,
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MgSO4 0.4 g/L and trace element. The trace element solution contains (in 1L 0.08M HCl):
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FeCl2∙4H2O 1.5 g, ZnCl2 70 mg, MnCl2∙4H2O 100 mg, H3BO3 6mg, CoCl2∙6H2O 190 mg,
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CuCl2∙2H2O 2 mg, NiCl2∙6H2O 240 mg, Na2MoO4∙2H2O 36 mg. Diluted HNO3 solution
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was added as a nitrogen source and pH was adjusted to 5.4. The samples were withdrawn at
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designated times for analysis.
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2.5.
Analytical methods The composition of rice straw was determined following National Renewable
Energy Laboratory (NREL) protocols (Sluiter et al., 2008). At first, a sample was treated 5
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with 72% H2SO4 at 30 °C in a shaking water bath for 2 h. The reaction mixture was then
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diluted to 4% H2SO4 and autoclaved at 121 °C for 1 h. The hydrolysis solution was filtered
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and analyzed for sugar contents by HPLC. The remaining solid part was dried at 105 °C
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overnight and further placed in the muffle furnace at 575 °C for lignin analysis.
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The amount of sugars and inhibitors in hydrolysate and ethanol concentration were quantified using HPLC system (Agilent 1200 series, USA) with an Aminex HPX-87P
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column (Bio-Rad Inc., USA) and a reflective index detector. The mobile phase was 4mM
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H2SO4 at a flow rate of 0.6 mL/min.
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Experimental design
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Response surface methodology (RSM) was utilized to optimize the condition of pretreatment with HNO3 and to evaluate the effect of variables including HNO3
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concentration (X1), pretreatment temperature (X2), and reaction time (X3) on xylose yield
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(Y). The pretreatment conditions were HNO3 concentrations of 0.2−1.0% (w/w),
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temperatures of 140−180 °C, and reaction times of 1−20 min. The effect of three variables
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on the response was studied according to Box-Behnken Design (Box and Behnken, 1960)
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using Design-Expert software (version 8.0, Stat-Ease, USA).
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3.
Results and discussions
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3.1.
Compositional changes of pretreated rice straw
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Table 1 summarizes the compositions of liquid phase (hydrolysate) and solid residue 6
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produced by the HNO3 pretreatment under different conditions. Furfural and HMF were
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regarded as the main inhibitors in this process (Chen et al., 2012). The mass recovery of
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solid residue ranged from 49.5% to 76.6% depending on the pretreatment condition. Also,
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xylan content decreased at more severe conditions. This happened because at high
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temperature a strong acid such as HNO3 does hydrolyze and dissolve hemicellulose fraction
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and does so too much especially when the treatment is prolonged. Because of the removal of
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hemicellulose, on the other hand, relative amount of glucan and lignin increased in the solid
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residue.
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Since the mechanism of acid pretreatment is primarily to dissolve out hemicellulose and it certainly improves the enzymatic hydrolysis, the amount of xylose in the liquid
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fraction can be used as an indicator of the pretreatment efficiency. Yield from the conversion
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of hemicellulose to xylose varied from 24.8 to 89.9% depending on the pretreatment
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condition. The maximum xylose yield of 89.9% was obtained at a condition of 0.6% HNO3
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at180 °C for 1 min. The amount of released xylose was measured to be 16.44 g for 100 g of
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raw dry biomass. When either acid concentration or reaction time increased, the xylose
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recovery decreased. This occurred probably because of the partial decomposition of the
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released xylose to furfural. It is typical that such degradation products are accumulated in
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the hydrolysate under harsh reaction conditions. When the xylose yield was greater than
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80%, the maximum concentrations of acetic acid, furfural and HMF were 1.68 g/L, 1.18 g/L
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and 0.15 g/L, respectively. These amounts, lower than values reported to be inhibitory to P.
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stipitis fermentation (Bellido et al., 2011), were anticipated not to interfere with the
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subsequent fermentation process. Most of existing pretreatment technologies generate the
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byproducts higher than critical levels and thus suffer the degraded ethanol productivity; thus
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an additional detoxification step is required. Adjustment of pH is attempted to reduce the
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detrimental effect of acetic acid on xylose fermentation and various detoxification methods,
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such as over-liming, vacuum evaporation, and activated charcoal, are also proposed for
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furfural (Hsu et al., 2010). Therefore, the unnecessity of this extra step is an added
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advantage of the proposed nitric acid-based pretreatment.
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3.2.
Optimization of nitric acid pretreatment condition
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The optimization of the pretreatment condition was done using Box-Behnken
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analysis and the quadratic equation for xylose yield in terms of actual parameters was
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obtained:
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Y = − 1101.43344 + 493.74063X1 + 11.58386X2 + 16.55193X3 – 1.80719X1X2 –
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2.27500X1X3 – 0.080461X2X3 – 121.99479X12 − 0.029454X22 − 0.12099X32
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(1)
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where X1, X2, X3, and Y represent the HNO3 concentration, temperature, time, and xylose
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yield respectively. In order to study the significance and adequacy of this model, analysis of
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variance (ANOVA) was carried out and summarized in Table 2. The model F-value of 48.07
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and a low p-value (p < 0.001) implies that this model was statistically significant. In
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addition, the p-value for each model term that X1, X2, X1X2, X1X3, X2X3, X12, X22, and X32
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had significant effect on the xylose yield. Although X3 was insignificant (p > 0.05), this
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factor could not be eliminated to support hierarchy of the model, because the value of
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adjusted determination coefficient (Adj. R2= 0.9673) indicated a high significance of the
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model. The optimized condition for maximum xylose yield considering efficiency were X1 =
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0.65%, X2 = 158.8 °C and X3 = 5.86 min with a predicted xylose yield of 87.3%. To verify
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the validity of the predicted value, experiment was performed at the optimized condition,
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and the xylose yield was 86.5%.
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In order to determine the interactive effects of independent variables on the response, three-dimensional surface plots were described in Fig. 1. The response surface
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plot of xylose yield at a fixed time of 10.5 min with different HNO3 concentration and
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temperature is shown in Fig. 1a. Increases in acid concentration and temperature provided
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indeed better solubilization of hemicellulose, leading to an increase in the amount of
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released xylose. However, a further increase in HNO3 concentration and temperature
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resulted in a decrease in xylose yield due to secondary decomposition of the xylose to other
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derivatives such as furfural. Fig. 1b shows the interaction between HNO3 concentration and
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pretreatment time at the temperature of 160 °C. It was found that the effect of HNO3
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concentration was more significant than that of the pretreatment time, which was also
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evident from the ANOVA test. Fig. 1c suggested that the effect of the temperature and
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pretreatment time on the xylose yield was significant. It was observed that temperature had
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a strong positive interaction with reaction time as indicated by the high F-value and
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temperature was more influential compared to reaction time.
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3.3.
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Enzymatic hydrolysis
In order to evaluate the effectiveness of the pretreatment, enzymatic hydrolysis was
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also carried out using rice straw pretreated under the optimal condition, i.e., 0.65% HNO3 at
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158.8 °C for 5.86 min. Sulfuric acid (H2SO4) was used for comparison. Enzymatic
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hydrolysis yield was monitored by quantifying glucose released from the pretreated solid 9
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fraction over time (Fig. 2). The HNO3-treated rice straw exhibited much higher enzymatic
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digestibility than that of the untreated control, indicating that HNO3, as an effective catalytic
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agent, indeed improved the enzyme accessibility to cellulose. The highest glucose yield was
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achieved after 72 h hydrolysis (47.7 g/L), and it was higher than that with H2SO4 (43.8 g/L).
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In addition, most of xylan (> 99%) was converted into xylose monomer by HNO3, while
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some, albeit a small part of xylan (6.3%), still remained after H2SO4 pretreatment (data not
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shown). These results support that HNO3 is indeed a potent acid-catalyst, which is effective
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for xylose release from rice straw and enhances the recovery of hemicellulose and the
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enzymatic hydrolysis of cellulose.
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3.4.
Effect of nitrate on ethanol production
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One of distinct benefits to use nitric acid for the pretreatment lies in the downstream step; it is possible that the neutralized catalyst serves as a nitrogen source for yeast
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fermentation. Since a typical medium for ethanol fermentation contains 3 g/L yeast extract
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and 5 g/L of peptone, corresponding to a nitrogen amount of 1.13 g/L (Laopaiboon et al.,
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2009), the optimal amount of 0.65% HNO3, corresponding to a nitrogen content of 1.44
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g/L, might fall into a right range required for active ethanol production. To explore this
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possibility, fermentation experiments were conducted with P.stipitis with and without
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HNO3 (Fig. 3). The total nitrogen concentration in the fermentation medium with 0.65%
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HNO3 was found to be 1.71g/L, which was slightly higher than that of pretreatment
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hydrolysate (1.63 g/L). As expected, the control medium containing yeast extract and
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peptone supported the best fermentation; even after 24h, glucose was almost completely
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exhausted and after that xylose was gradually consumed, which is a typical pattern with
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P.stipitis. Ethanol concentration reached the highest value of 17.68 g/L after 120 h. With
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nitrate as a sole nitrogen source, the rates of sugar consumption and ethanol production
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yield were relatively lower than those obtained in the control. This result suggested that the
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type of nitrogen sources did affect the fermentation step: complex organic nitrogen sources
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are better than the inorganic source. Wang et al. (2012) also found the similar phenomenon;
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urea was better than ammonium in terms of ethanol production. Even so, the presence of
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nitrate in medium obviously enhanced the fermentation, e.g., final ethanol concentration
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from 10.92 g/L to 14.50 g/L as shown in Fig. 3b. This finding clearly supports a possibility
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that nitrate, derived from the nitric acid pretreatment, could be directly used as a nitrogen
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source in the subsequent fermentation process. This possibility expects not only to
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substantially simplify after-pretreatment-steps, since the acid catalyst needs not to be
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removed unlike the other acid catalysts, but also to reduce the overall process cost by
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providing an additional nitrogen source.
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Conclusions
The present study demonstrated that nitric acid is a potent acid catalyst able to
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effectively release xylose from rice straw and allow for efficient enzymatic hydrolysis of
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cellulose. The maximum xylose yield of 86.5% in the pretreatment and enzymatic
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hydrolysis yield of 83.0% were obtained with 0.65% HNO3 at 158.8 °C for the rather short
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reaction time (5.86 min). Furthermore, HNO3 at the optimal amount improved ethanol yield
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by Pichia stipitis (14.50 g/L compared with 10.92 g/L of the control). Nitrate, the 11
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neutralized form of nitric acid, was found to be used as an acceptable nitrogen source for the
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bioethanol production. This powerful acid-catalyst is expected to reduce overall cost of
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bioethanol production.
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Acknowledgements
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This study was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project (ABC-2012053875) and the National Research Foundation of Korea (NRF)
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(NRF-2012M1A2A2026587) funded by the Korea government Ministry of Education,
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Science and Technology.
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Table 1
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The composition of solid and liquid phase (hydrolysate) after nitric acid pretreatment of RS. HNO3
Temp.
Time
Solid
Solid composition (%)
(%)
(℃)
(min)
recovery
Glucan
Xylan
Lignin
Liquid composition (g/L) Glucose
Xylose
(%)
325
326 327
HMF
(%)
acid
140
10.5
76.57
43.49
21.05
18.03
3.26
4.53
2
0.2
160
1.0
70.91
45.11
17.44
18.57
2.76
6.12
3
0.2
160
20.0
61.62
56.43
9.97
21.33
3.92
9.47
4
0.2
180
10.5
55.96
56.03
6.45
22.27
5.99
11.43
5
0.6
140
1.0
65.45
50.41
12.56
19.90
4.08
6
0.6
140
20.0
55.15
55.77
5.51
24.57
7.99
7
0.6
160
10.5
53.94
58.38
4.33
24.10
8
0.6
160
10.5
53.33
61.19
2.44
25.13
9
0.6
160
10.5
54.34
60.32
2.88
23.40
10
0.6
180
1.0
52.37
61.88
2.10
11
0.6
180
20.0
48.28
53.63
0
12
1.0
140
10.5
57.17
56.38
4.19
13
1.0
160
1.0
52.93
56.70
14
1.0
160
20.0
50.10
56.17
15
1.0
180
10.5
49.49
0
0
24.78
1.37
0
0
33.48
1.54
0.24
0
51.81
1.58
0.56
0
62.53
1.60
0
0
49.84
12.93
1.62
0.61
0.06
70.73
us
9.11
15.78
1.65
1.03
0.12
86.32
9.04
16.33
1.66
1.06
0.13
89.33
8.01
16.01
1.65
1.13
0.15
87.58
24.00
9.07
16.44
1.68
1.18
0.15
89.93
31.17
11.09
9.08
1.72
1.74
0.25
49.67
22.20
7.78
15.49
1.68
0.88
0.10
79.27
4.60
23.57
9.56
14.64
1.84
0.83
0.08
80.09
2.24
25.27
10.95
12.67
2.18
1.31
0.18
63.84
27.93
10.41
10.82
2.32
1.73
0.27
59.19
M
an
7.85
ed 56.26
1.31
ip t
0.2
cr
1
yield
0
pt
324
Furfural
Ac ce
323
Acetic
Xylose
328 329 330
16
Page 16 of 22
331 332 333
Table 2
336
Analysis of variance (ANOVA) for the quadratic model. Source
Sum of squares
df
Mean square
F Value
cr
335
ip t
334
P Value
5863.11
9
651.46
46.98
0.0003
X1-HNO3
1506.73
1
1506.73
108.67
0.0001
X2-Temp.
168.36
1
168.36
X3-Time
37.37
1
37.37
X1 X 2
836.08
1
X1 X 3
298.94
1
X2 X 3
934.83
0.0176
2.70
0.1616
836.08
60.30
0.0006
298.94
21.56
0.0056
ed
M
12.14
934.83
67.42
0.0004
1
1406.76
101.46
0.0002
1
36.96
0.0017
1
440.23
31.75
0.0024
9.45
0.0972
1406.76
2
512.52
512.52
X3
2
440.23
pt
1
2
Residual
69.33
5
13.87
Lack of fit
64.76
3
21.59
Pure error
4.57
2
2.29
Ac ce
X2
an
Model
X1
337
us
Prob>F
significant
not significant
338 339
17
Page 17 of 22
339
Figure captions
340
Fig. 1. The surface response plots of the effects of HNO3 concentration, temperature and
342
time on the xylose yield: (a) fixed tiem at 10.5 min; (b) fixed temperature at 160 °C; (c)
343
fixed HNO3 concentration at 0.6%.
ip t
341
344
Fig. 2. Enzymatic hydrolysis yield of pretreated rice straw (HNO3 or H2SO4 0.65%,
346
158.8 °C, and 5.86 min). Enzymatic hydrolysis was conducted at 50 °C and 170 rpm in 50
347
mM citrate buffer (pH 4.8) using 30 FPU enzyme blend Cellic C-Tec per gram of cellulose.
us
cr
345
348
Fig. 3. (a) Sugar consumption and (b) ethanol production during fermentation of P. stipitis.
an
349 350
M
351
Ac ce
pt
ed
352
18
Page 18 of 22
352
Highlights
353 354
356
HNO3 is a potent acid catalyst to release xylose from rice straw and allow for efficient enzymatic hydrolysis.
ip t
355
RSM was applied to optimize HNO3 pretreatment condition for maximum xylose yield.
358
Under the optimal condition, a maximum xylose yield of 86.5% in the pretreatment and
361
Nitrate, the neutralized form of nitric acid, was found to be used as a nitrogen source in the fermentation process.
M
362
pt Ac ce
[22]
ed
363 364
us
360
an enzymatic digestibility of 83.0% were achieved.
an
359
cr
357
19
Page 19 of 22
Fig.1.
Fig. 1.
sign-Expert?Software
ose yield 89.93
(a)
24.78 91
= A: HNO3 conc. = B: Temperature 74
57
ip t
Xylose yield
Xylose yield
ual Factor Time = 10.50
40
1 80 .0 0
1.0 0 0.8 0 1 60 .0 0
0.6 0 1 50 .0 0
B: Temp. B: Temperature
0.4 0 1 40 .0 0
0.2 0
HNO3 conc. A:A:HNO 3
an
sign-Expert?Software
(b)
24.78 91
= A: HNO3 conc. = C: Time
62
4 7.5
ed
Xylose yield
Xylose yield
M
7 6.5
ual Factor Temperature = 160.00
33
1 5.25
ce pt
2 0.00
1 0.50
C:C:Time Time
Design-Expert?Software
(c)
24.78
0.4 0
79.25
Xylose yield
Xylose yield
Actual Factor A: HNO3 conc. = 0.60
A:A: HNO 3 HNO3 conc.
0.2 0
Ac
90
X1 = B: Temperature X2 = C: Time
1.0 0
0.8 0
0.6 0
5 .7 5
1 .0 0
Xylose yield 89.93
us
1 70 .0 0
ose yield 89.93
cr
23
68.5
57.75
47
20.00
180.00 15.25
170.00 10.50
C: C: Time
160.00 5.75
150.00 1.00
Temperature B: B: Temp.
140.00
Page 20 of 22
Fig.2.
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 2.
Page 21 of 22
Fig.3.
Fig. 3.
M
an
us
cr
ip t
(a)
Ac
ce pt
ed
(b)
Page 22 of 22
S0144-8617(13)00876-X http://dx.doi.org/doi:10.1016/j.carbpol.2013.08.092 CARP 8082
To appear in: Received date: Revised date: Accepted date:
7-5-2013 26-8-2013 28-8-2013
Please cite this article as: Kim, I., Lee, B., Park, J.-Y., Choi, S.-A., & Han, J.-I., Effect of nitric acid on pretreatment and fermentation for enhancing ethanol production of rice straw, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.08.092 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Effect of nitric acid on pretreatment and fermentation for enhancing ethanol
2
production of rice straw
3
Ilgook Kima, Bomi Leea, Ji-Yeon Parkb, Sun-A Choib, Jong-In Hana,*
5
a
6
Yuseong-gu, Daejeon 305-701, Republic of Korea
7
b
8
gu, Daejeon 305-343, Republic of Korea
9
*
cr
Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong,
an
Corresponding author: Jong-In Han (E-mail address: [email protected]; Tel: +82-42-350-
3629; Fax: +82-42-350-3610)
In this study, nitric acid (HNO3) was evaluated as an acid catalyst for rice straw
Ac ce
14
Abstract
pt
12
ed
11
13
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Department of Clean Fuel, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-
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10
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4
15
pretreatment, and, after neutralization, as a sole nitrogen source for subsequent fermentation.
16
Response surface methodology was used to obtain optimal pretreatment condition with
17
respect to HNO3 concentration (0.2−1.0%), temperature (120−160 °C) and reaction time
18
(1−20 min). In a condition of 0.65% HNO3, 158.8 °C and 5.86 min, a maximum xylose
19
yield of 86.5% and an enzymatic digestibility of 83.0% were achieved. The sugar solution
20
that contained nitrate derived from the acid catalyst supported the enhancement of ethanol
21
yield by Pichia stipitis from 10.92 g/L to 14.50 g/L. The results clearly reveal that nitric acid 1
Page 1 of 22
could be used not only as a pretreatment catalyst, but also as a nitrogen source in the
23
fermentation process for bioethanol production. It is anticipated that the HNO3-based
24
pretreatment can reduce financial burden on the cellulosic bioethanol industry by
25
simplifying after-pretreatment-steps as well as providing a nitrogen source.
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22
26
Keywords
28
Rice straw; RSM; HNO3 pretreatment; Enzymatic hydrolysis; Fermentation
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32
1.
Introduction
Due to the depletion of the world petroleum supply and increasing concerns about
ed
31
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30
climate change, biomass-based renewable fuels such as lignocellulosic ethanol is generally
34
counted as a potential solution to the energy crisis and global warming (Alvira et al., 2010).
35
Lignocellulosic materials including agricultural residues, hardwood, and softwood are
36
plentiful and generate low net greenhouse gas emissions (Glabe and Zacchi, 2007).
37
However, its complex and heterogeneous cell walls make it hard downstream steps, such as
38
enzymatic hydrolysis of sugar polymers. Therefore, pretreatment is absolutely required to
39
increase the yield of fermentable sugars.
40
Ac ce
pt
33
Chemical pretreatment with diluted acid combined with enzymatic hydrolysis has
41
been considered as a promising procedure for the conversion of lignocellulose to mono-
42
sugars for ethanol-making process (Hendriks and Zeeman, 2009). This acid-catalyzed 2
Page 2 of 22
pretreatment causes the hydrolysis of hemicellulose and increases portion of amorphous
44
cellulose, resulting in high recovery of hemicelluloses as monomers in the liquid fraction
45
and high digestible cellulose in the solid fraction (Linde et al., 2008; Sassner et al., 2008;
46
Yang and Wyman, 2008). Literally every strong acid has been investigated as a catalyst, e.g.,
47
sulfuric acid (Hsu et al., 2010), hydrochloric acid (Marcotullio et al., 2011), phosphoric acid
48
(Geddes et al., 2010), and also nitric acid (Zhang et al., 2011). Among these, sulfuric acid,
49
the cheapest agent, is most commonly used. On the other hand, nitric acid can be massively
50
produced via a NOx capture process from flue gases (Kim and Han, 2013), displaying a
51
much shorter reaction time (Zhang et al., 2011) and higher efficiency for saccharification
52
(Rodríguez-Chong et al, 2004). Furthermore, nitrate, formed upon being neutralized, could
53
be used as a nitrogen source for fermentation process, as a variety of nitrogen sources
54
including yeast extract and peptone (Laopaiboon et al., 2009), ammonium (Yue et al., 2012),
55
amino acid (Albers et al., 1996) and urea (Jones and Ingledew, 1994) have been reported to
56
be attempted for ethanol production.
ed
M
an
us
cr
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43
57
pt
In this work, rice straw, the most abundant biomass feedstock in Korea, was chosen as a raw lignocellulosic material for ethanol production. Response surface methodology
59
(RSM) was used to optimize pretreatment condition of rice straw with nitric acid (HNO3),
60
particularly aiming at maximum xylose yield. Nitrate was also tested as the nitrogen source
61
for the fermentation of Pichia stipitis.
Ac ce
58
62 63 64
2.
Materials and methods 3
Page 3 of 22
65
2.1.
66
Feedstock materials Rice straw was collected from Nonsan field near the city of Daejeon, South Korea.
The air-dried rice straw was milled to small particles by a grinding machine, screened to
68
obtain a size of less than 2 mm, and stored in a drying oven prior to use. The rice straw was
69
composed of 41.7% glucan, 18.3% xylan, and 16.6% lignin based on its dry weight.
ip t
67
71
2.2.
cr
70
HNO3 Pretreatment
Pretreatment was carried out in a stainless steel cylindrical reactor with a total
73
volume of 70 mL. Before pretreatment, 5g rice straw and 50 mL HNO3 solution were mixed
74
together into the reactor. The reactor was then heated to set temperatures in an oil bath. The
75
average heating rate was about 15 °C/min. Once the targeted temperature inside the reactor
76
was reached, reaction time was counted. At the end of the pretreatment, solid residue was
77
separated by filtration and then washed with deionized water for subsequent enzymatic
78
hydrolysis, and liquid fraction was gathered for the analyses of sugar and inhibitor.
79 80 81
2.3.
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72
Enzymatic hydrolysis
After pretreatment, an enzymatic hydrolysis experiment was conducted in a 150-mL
82
Erlenmeyer flask containing 50 mM sodium citrate buffer (pH 4.8) at a solid loading of 10%
83
(w/v). A novel commercial enzyme blend Cellic C-Tec (Novozymes, Denmark) derived
84
from Trichoderma reesei with an activity loading of 30 FPU/g cellulose was added to the
85
pretreated rice straw. The flask was incubated at 50 °C and 170 rpm for 72 h in a shaking
86
incubator and the hydrolysate was sampled periodically for sugar analysis. 4
Page 4 of 22
87 88
2.4.
89
Fermentation Pichia stipitis KCTC 7222 was obtained from Korean Collection for Type Culture
(Daejeon, Korea). The yeast was maintained on YM agar plates at 4 °C and transferred
91
monthly. YM agar contained yeast extract 3 g/L, malt extract 3 g/L, peptone 5 g/L, glucose
92
10 g/L, and agar 20 g/L. In order to increase cell density, the yeast was transferred into a
93
250-mL Erlenmeyer flask with 100 mL of the inoculation medium containing yeast extract 3
94
g/L, peptone 5 g/L, glucose 35g/L, and xylose 15g/L. After incubation for 16 h, cells were
95
harvested and applied as an inoculum for fermentation test. The yeast cell concentration in
96
the inoculum was determined to 5 g dry weight of cells/L.
an
us
cr
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90
Fermentation experiment was carried out in sterile 250-mL Erlenmeyer flasks with
98
cap and needle to remove CO2 in a shaking incubator at 30 °C and 170 rpm for 48h. Each
99
flask was filled with 50 mL fermentation medium inoculated with 10% (v/v) growth culture.
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The fermentation medium was supplied with glucose 35 g/L, xylose 15g/L, KH2PO4 5g/L,
101
MgSO4 0.4 g/L and trace element. The trace element solution contains (in 1L 0.08M HCl):
102
FeCl2∙4H2O 1.5 g, ZnCl2 70 mg, MnCl2∙4H2O 100 mg, H3BO3 6mg, CoCl2∙6H2O 190 mg,
103
CuCl2∙2H2O 2 mg, NiCl2∙6H2O 240 mg, Na2MoO4∙2H2O 36 mg. Diluted HNO3 solution
104
was added as a nitrogen source and pH was adjusted to 5.4. The samples were withdrawn at
105
designated times for analysis.
Ac ce
pt
100
106 107 108 109
2.5.
Analytical methods The composition of rice straw was determined following National Renewable
Energy Laboratory (NREL) protocols (Sluiter et al., 2008). At first, a sample was treated 5
Page 5 of 22
with 72% H2SO4 at 30 °C in a shaking water bath for 2 h. The reaction mixture was then
111
diluted to 4% H2SO4 and autoclaved at 121 °C for 1 h. The hydrolysis solution was filtered
112
and analyzed for sugar contents by HPLC. The remaining solid part was dried at 105 °C
113
overnight and further placed in the muffle furnace at 575 °C for lignin analysis.
ip t
110
114
The amount of sugars and inhibitors in hydrolysate and ethanol concentration were quantified using HPLC system (Agilent 1200 series, USA) with an Aminex HPX-87P
116
column (Bio-Rad Inc., USA) and a reflective index detector. The mobile phase was 4mM
117
H2SO4 at a flow rate of 0.6 mL/min.
us
cr
115
119
2.6.
120
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118
Experimental design
M
Response surface methodology (RSM) was utilized to optimize the condition of pretreatment with HNO3 and to evaluate the effect of variables including HNO3
122
concentration (X1), pretreatment temperature (X2), and reaction time (X3) on xylose yield
123
(Y). The pretreatment conditions were HNO3 concentrations of 0.2−1.0% (w/w),
124
temperatures of 140−180 °C, and reaction times of 1−20 min. The effect of three variables
125
on the response was studied according to Box-Behnken Design (Box and Behnken, 1960)
126
using Design-Expert software (version 8.0, Stat-Ease, USA).
Ac ce
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127 128 129
3.
Results and discussions
130
3.1.
Compositional changes of pretreated rice straw
131
Table 1 summarizes the compositions of liquid phase (hydrolysate) and solid residue 6
Page 6 of 22
produced by the HNO3 pretreatment under different conditions. Furfural and HMF were
133
regarded as the main inhibitors in this process (Chen et al., 2012). The mass recovery of
134
solid residue ranged from 49.5% to 76.6% depending on the pretreatment condition. Also,
135
xylan content decreased at more severe conditions. This happened because at high
136
temperature a strong acid such as HNO3 does hydrolyze and dissolve hemicellulose fraction
137
and does so too much especially when the treatment is prolonged. Because of the removal of
138
hemicellulose, on the other hand, relative amount of glucan and lignin increased in the solid
139
residue.
cr
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140
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132
Since the mechanism of acid pretreatment is primarily to dissolve out hemicellulose and it certainly improves the enzymatic hydrolysis, the amount of xylose in the liquid
142
fraction can be used as an indicator of the pretreatment efficiency. Yield from the conversion
143
of hemicellulose to xylose varied from 24.8 to 89.9% depending on the pretreatment
144
condition. The maximum xylose yield of 89.9% was obtained at a condition of 0.6% HNO3
145
at180 °C for 1 min. The amount of released xylose was measured to be 16.44 g for 100 g of
146
raw dry biomass. When either acid concentration or reaction time increased, the xylose
147
recovery decreased. This occurred probably because of the partial decomposition of the
148
released xylose to furfural. It is typical that such degradation products are accumulated in
149
the hydrolysate under harsh reaction conditions. When the xylose yield was greater than
150
80%, the maximum concentrations of acetic acid, furfural and HMF were 1.68 g/L, 1.18 g/L
151
and 0.15 g/L, respectively. These amounts, lower than values reported to be inhibitory to P.
152
stipitis fermentation (Bellido et al., 2011), were anticipated not to interfere with the
153
subsequent fermentation process. Most of existing pretreatment technologies generate the
154
byproducts higher than critical levels and thus suffer the degraded ethanol productivity; thus
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7
Page 7 of 22
an additional detoxification step is required. Adjustment of pH is attempted to reduce the
156
detrimental effect of acetic acid on xylose fermentation and various detoxification methods,
157
such as over-liming, vacuum evaporation, and activated charcoal, are also proposed for
158
furfural (Hsu et al., 2010). Therefore, the unnecessity of this extra step is an added
159
advantage of the proposed nitric acid-based pretreatment.
161
3.2.
Optimization of nitric acid pretreatment condition
cr
160
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155
The optimization of the pretreatment condition was done using Box-Behnken
163
analysis and the quadratic equation for xylose yield in terms of actual parameters was
164
obtained:
165
Y = − 1101.43344 + 493.74063X1 + 11.58386X2 + 16.55193X3 – 1.80719X1X2 –
166
2.27500X1X3 – 0.080461X2X3 – 121.99479X12 − 0.029454X22 − 0.12099X32
167
(1)
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162
where X1, X2, X3, and Y represent the HNO3 concentration, temperature, time, and xylose
169
yield respectively. In order to study the significance and adequacy of this model, analysis of
170
variance (ANOVA) was carried out and summarized in Table 2. The model F-value of 48.07
171
and a low p-value (p < 0.001) implies that this model was statistically significant. In
172
addition, the p-value for each model term that X1, X2, X1X2, X1X3, X2X3, X12, X22, and X32
173
had significant effect on the xylose yield. Although X3 was insignificant (p > 0.05), this
174
factor could not be eliminated to support hierarchy of the model, because the value of
175
adjusted determination coefficient (Adj. R2= 0.9673) indicated a high significance of the
176
model. The optimized condition for maximum xylose yield considering efficiency were X1 =
177
0.65%, X2 = 158.8 °C and X3 = 5.86 min with a predicted xylose yield of 87.3%. To verify
Ac ce
pt
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8
Page 8 of 22
178
the validity of the predicted value, experiment was performed at the optimized condition,
179
and the xylose yield was 86.5%.
180
In order to determine the interactive effects of independent variables on the response, three-dimensional surface plots were described in Fig. 1. The response surface
182
plot of xylose yield at a fixed time of 10.5 min with different HNO3 concentration and
183
temperature is shown in Fig. 1a. Increases in acid concentration and temperature provided
184
indeed better solubilization of hemicellulose, leading to an increase in the amount of
185
released xylose. However, a further increase in HNO3 concentration and temperature
186
resulted in a decrease in xylose yield due to secondary decomposition of the xylose to other
187
derivatives such as furfural. Fig. 1b shows the interaction between HNO3 concentration and
188
pretreatment time at the temperature of 160 °C. It was found that the effect of HNO3
189
concentration was more significant than that of the pretreatment time, which was also
190
evident from the ANOVA test. Fig. 1c suggested that the effect of the temperature and
191
pretreatment time on the xylose yield was significant. It was observed that temperature had
192
a strong positive interaction with reaction time as indicated by the high F-value and
193
temperature was more influential compared to reaction time.
194 195 196
3.3.
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181
Enzymatic hydrolysis
In order to evaluate the effectiveness of the pretreatment, enzymatic hydrolysis was
197
also carried out using rice straw pretreated under the optimal condition, i.e., 0.65% HNO3 at
198
158.8 °C for 5.86 min. Sulfuric acid (H2SO4) was used for comparison. Enzymatic
199
hydrolysis yield was monitored by quantifying glucose released from the pretreated solid 9
Page 9 of 22
fraction over time (Fig. 2). The HNO3-treated rice straw exhibited much higher enzymatic
201
digestibility than that of the untreated control, indicating that HNO3, as an effective catalytic
202
agent, indeed improved the enzyme accessibility to cellulose. The highest glucose yield was
203
achieved after 72 h hydrolysis (47.7 g/L), and it was higher than that with H2SO4 (43.8 g/L).
204
In addition, most of xylan (> 99%) was converted into xylose monomer by HNO3, while
205
some, albeit a small part of xylan (6.3%), still remained after H2SO4 pretreatment (data not
206
shown). These results support that HNO3 is indeed a potent acid-catalyst, which is effective
207
for xylose release from rice straw and enhances the recovery of hemicellulose and the
208
enzymatic hydrolysis of cellulose.
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200
209
211
3.4.
Effect of nitrate on ethanol production
M
210
One of distinct benefits to use nitric acid for the pretreatment lies in the downstream step; it is possible that the neutralized catalyst serves as a nitrogen source for yeast
213
fermentation. Since a typical medium for ethanol fermentation contains 3 g/L yeast extract
214
and 5 g/L of peptone, corresponding to a nitrogen amount of 1.13 g/L (Laopaiboon et al.,
215
2009), the optimal amount of 0.65% HNO3, corresponding to a nitrogen content of 1.44
216
g/L, might fall into a right range required for active ethanol production. To explore this
217
possibility, fermentation experiments were conducted with P.stipitis with and without
218
HNO3 (Fig. 3). The total nitrogen concentration in the fermentation medium with 0.65%
219
HNO3 was found to be 1.71g/L, which was slightly higher than that of pretreatment
220
hydrolysate (1.63 g/L). As expected, the control medium containing yeast extract and
221
peptone supported the best fermentation; even after 24h, glucose was almost completely
222
exhausted and after that xylose was gradually consumed, which is a typical pattern with
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10
Page 10 of 22
P.stipitis. Ethanol concentration reached the highest value of 17.68 g/L after 120 h. With
224
nitrate as a sole nitrogen source, the rates of sugar consumption and ethanol production
225
yield were relatively lower than those obtained in the control. This result suggested that the
226
type of nitrogen sources did affect the fermentation step: complex organic nitrogen sources
227
are better than the inorganic source. Wang et al. (2012) also found the similar phenomenon;
228
urea was better than ammonium in terms of ethanol production. Even so, the presence of
229
nitrate in medium obviously enhanced the fermentation, e.g., final ethanol concentration
230
from 10.92 g/L to 14.50 g/L as shown in Fig. 3b. This finding clearly supports a possibility
231
that nitrate, derived from the nitric acid pretreatment, could be directly used as a nitrogen
232
source in the subsequent fermentation process. This possibility expects not only to
233
substantially simplify after-pretreatment-steps, since the acid catalyst needs not to be
234
removed unlike the other acid catalysts, but also to reduce the overall process cost by
235
providing an additional nitrogen source.
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239
4.
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237 238
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us
cr
ip t
223
Conclusions
The present study demonstrated that nitric acid is a potent acid catalyst able to
240
effectively release xylose from rice straw and allow for efficient enzymatic hydrolysis of
241
cellulose. The maximum xylose yield of 86.5% in the pretreatment and enzymatic
242
hydrolysis yield of 83.0% were obtained with 0.65% HNO3 at 158.8 °C for the rather short
243
reaction time (5.86 min). Furthermore, HNO3 at the optimal amount improved ethanol yield
244
by Pichia stipitis (14.50 g/L compared with 10.92 g/L of the control). Nitrate, the 11
Page 11 of 22
245
neutralized form of nitric acid, was found to be used as an acceptable nitrogen source for the
246
bioethanol production. This powerful acid-catalyst is expected to reduce overall cost of
247
bioethanol production.
ip t
248
Acknowledgements
us
250
cr
249
251
This study was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project (ABC-2012053875) and the National Research Foundation of Korea (NRF)
253
(NRF-2012M1A2A2026587) funded by the Korea government Ministry of Education,
254
Science and Technology.
M
an
252
256
[1]
259
formation. Applied and Environmental Microbiology, 62, 3187-3195.
[2]
262
Alvira, P., Tomás-Pejó, E., Ballesteros, M., & Nergo, M. J. (2010). Pretreatment technologies for an efficient bioethanol production process based on enzymatic
263 264
Albers, E., Larsson, C., Liden, G., Niklasson, C., & Gustafsson, L. (1996). Influence of the nitrogen source on Saccharomyces serevisiae anaerobic growth and product
260 261
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Zhang, R., Lu, X., Sun, Y., Wang, X., & Zhang, S. (2011). Modeling and
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ip t
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320
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Ac ce
pt
ed
M
an
321
us
319
15
Page 15 of 22
321
Table 1
322
The composition of solid and liquid phase (hydrolysate) after nitric acid pretreatment of RS. HNO3
Temp.
Time
Solid
Solid composition (%)
(%)
(℃)
(min)
recovery
Glucan
Xylan
Lignin
Liquid composition (g/L) Glucose
Xylose
(%)
325
326 327
HMF
(%)
acid
140
10.5
76.57
43.49
21.05
18.03
3.26
4.53
2
0.2
160
1.0
70.91
45.11
17.44
18.57
2.76
6.12
3
0.2
160
20.0
61.62
56.43
9.97
21.33
3.92
9.47
4
0.2
180
10.5
55.96
56.03
6.45
22.27
5.99
11.43
5
0.6
140
1.0
65.45
50.41
12.56
19.90
4.08
6
0.6
140
20.0
55.15
55.77
5.51
24.57
7.99
7
0.6
160
10.5
53.94
58.38
4.33
24.10
8
0.6
160
10.5
53.33
61.19
2.44
25.13
9
0.6
160
10.5
54.34
60.32
2.88
23.40
10
0.6
180
1.0
52.37
61.88
2.10
11
0.6
180
20.0
48.28
53.63
0
12
1.0
140
10.5
57.17
56.38
4.19
13
1.0
160
1.0
52.93
56.70
14
1.0
160
20.0
50.10
56.17
15
1.0
180
10.5
49.49
0
0
24.78
1.37
0
0
33.48
1.54
0.24
0
51.81
1.58
0.56
0
62.53
1.60
0
0
49.84
12.93
1.62
0.61
0.06
70.73
us
9.11
15.78
1.65
1.03
0.12
86.32
9.04
16.33
1.66
1.06
0.13
89.33
8.01
16.01
1.65
1.13
0.15
87.58
24.00
9.07
16.44
1.68
1.18
0.15
89.93
31.17
11.09
9.08
1.72
1.74
0.25
49.67
22.20
7.78
15.49
1.68
0.88
0.10
79.27
4.60
23.57
9.56
14.64
1.84
0.83
0.08
80.09
2.24
25.27
10.95
12.67
2.18
1.31
0.18
63.84
27.93
10.41
10.82
2.32
1.73
0.27
59.19
M
an
7.85
ed 56.26
1.31
ip t
0.2
cr
1
yield
0
pt
324
Furfural
Ac ce
323
Acetic
Xylose
328 329 330
16
Page 16 of 22
331 332 333
Table 2
336
Analysis of variance (ANOVA) for the quadratic model. Source
Sum of squares
df
Mean square
F Value
cr
335
ip t
334
P Value
5863.11
9
651.46
46.98
0.0003
X1-HNO3
1506.73
1
1506.73
108.67
0.0001
X2-Temp.
168.36
1
168.36
X3-Time
37.37
1
37.37
X1 X 2
836.08
1
X1 X 3
298.94
1
X2 X 3
934.83
0.0176
2.70
0.1616
836.08
60.30
0.0006
298.94
21.56
0.0056
ed
M
12.14
934.83
67.42
0.0004
1
1406.76
101.46
0.0002
1
36.96
0.0017
1
440.23
31.75
0.0024
9.45
0.0972
1406.76
2
512.52
512.52
X3
2
440.23
pt
1
2
Residual
69.33
5
13.87
Lack of fit
64.76
3
21.59
Pure error
4.57
2
2.29
Ac ce
X2
an
Model
X1
337
us
Prob>F
significant
not significant
338 339
17
Page 17 of 22
339
Figure captions
340
Fig. 1. The surface response plots of the effects of HNO3 concentration, temperature and
342
time on the xylose yield: (a) fixed tiem at 10.5 min; (b) fixed temperature at 160 °C; (c)
343
fixed HNO3 concentration at 0.6%.
ip t
341
344
Fig. 2. Enzymatic hydrolysis yield of pretreated rice straw (HNO3 or H2SO4 0.65%,
346
158.8 °C, and 5.86 min). Enzymatic hydrolysis was conducted at 50 °C and 170 rpm in 50
347
mM citrate buffer (pH 4.8) using 30 FPU enzyme blend Cellic C-Tec per gram of cellulose.
us
cr
345
348
Fig. 3. (a) Sugar consumption and (b) ethanol production during fermentation of P. stipitis.
an
349 350
M
351
Ac ce
pt
ed
352
18
Page 18 of 22
352
Highlights
353 354
356
HNO3 is a potent acid catalyst to release xylose from rice straw and allow for efficient enzymatic hydrolysis.
ip t
355
RSM was applied to optimize HNO3 pretreatment condition for maximum xylose yield.
358
Under the optimal condition, a maximum xylose yield of 86.5% in the pretreatment and
361
Nitrate, the neutralized form of nitric acid, was found to be used as a nitrogen source in the fermentation process.
M
362
pt Ac ce
[22]
ed
363 364
us
360
an enzymatic digestibility of 83.0% were achieved.
an
359
cr
357
19
Page 19 of 22
Fig.1.
Fig. 1.
sign-Expert?Software
ose yield 89.93
(a)
24.78 91
= A: HNO3 conc. = B: Temperature 74
57
ip t
Xylose yield
Xylose yield
ual Factor Time = 10.50
40
1 80 .0 0
1.0 0 0.8 0 1 60 .0 0
0.6 0 1 50 .0 0
B: Temp. B: Temperature
0.4 0 1 40 .0 0
0.2 0
HNO3 conc. A:A:HNO 3
an
sign-Expert?Software
(b)
24.78 91
= A: HNO3 conc. = C: Time
62
4 7.5
ed
Xylose yield
Xylose yield
M
7 6.5
ual Factor Temperature = 160.00
33
1 5.25
ce pt
2 0.00
1 0.50
C:C:Time Time
Design-Expert?Software
(c)
24.78
0.4 0
79.25
Xylose yield
Xylose yield
Actual Factor A: HNO3 conc. = 0.60
A:A: HNO 3 HNO3 conc.
0.2 0
Ac
90
X1 = B: Temperature X2 = C: Time
1.0 0
0.8 0
0.6 0
5 .7 5
1 .0 0
Xylose yield 89.93
us
1 70 .0 0
ose yield 89.93
cr
23
68.5
57.75
47
20.00
180.00 15.25
170.00 10.50
C: C: Time
160.00 5.75
150.00 1.00
Temperature B: B: Temp.
140.00
Page 20 of 22
Fig.2.
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 2.
Page 21 of 22
Fig.3.
Fig. 3.
M
an
us
cr
ip t
(a)
Ac
ce pt
ed
(b)
Page 22 of 22
