Self-sufficient bioethanol production system using a lignin-derived

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Self-sufficient bioethanol production system using a lignin-derived adsorbent of fermentation inhibitors Koichi Yoshioka, Masakazu Daidai, Yoshihiro Matsumoto, Rie Mizuno, Yoko Katsura, Tatsuya Hakogi, Hideshi Yanase, and Takashi Watanabe ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02915 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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ACS Sustainable Chemistry & Engineering

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Self-sufficient bioethanol production system using a

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lignin-derived adsorbent of fermentation inhibitors

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Koichi Yoshiokaa, Masakazu Daidaib, Yoshihiro Matsumotoa, Rie Mizunoa, Yoko Katsurab,

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Tatsuya Hakogic, Hideshi Yanasec and Takashi Watanabea*

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a

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University, Gokasho, Uji, Kyoto 611-0011, Japan

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b

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Osaka 332-0012, Japan

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c

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Laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere, Kyoto

Japan Chemical Engineering & Machinery Co. Ltd.4-6-23 Kashima, Yodogawaku, Osaka,

Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori

University, 4-101 Koyamacho-Minami, Tottori, Tottori 680-8552, Japan

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*Corresponding author

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Research Institute for Sustainable Humanosphere, Kyoto University

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Gokasho, Uji, Kyoto 611-0011, Japan

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TEL: +81-774-38-3640, FAX: +81-774-38-3681

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E-mail: [email protected]

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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KEYWORDS: fermentation inhibitor; lignin; bioethanol; adsorbent; microwave-assisted

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pretreatment; simultaneous saccharification and co-fermentation; Zymomonas mobilis;

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Eucalyptus globulus

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ABSTRACT: We have developed a new self-sufficient bioethanol producing system that

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suppresses the inhibition of fermentation by thermally-processed residual lignin in a separate

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hydrolysis and fermentation (SHF) and one-pot simultaneous saccharification and co-

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fermentation (SSCF). The new fermentation process incorporates detoxification with the lignin-

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derived adsorbent; thus, needs no purchased adsorbent, produces no waste adsorbent and relieves

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waste water treatment load. Eucalyptus globulus wood was pretreated by microwave (MW)-

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assisted hydrothermolysis in aqueous maleic acid and separated into soluble and insoluble

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fractions. The insoluble fraction was hydrolyzed with cellulolytic enzymes, and the residual

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lignin was separated. We found that thermal processing of the lignin under a normoxic

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atmosphere efficiently adsorbed fermentation inhibitors without affecting monosaccharide

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concentration by enzymatic saccharification. The processing was achieved at 250–350 °C, which

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are much lower temperatures for wood charcoal production and resulted in higher yields of the

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adsorbent. The residual lignin formed after SSCF was also converted to the selective adsorbent.

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Using the lignin-derived adsorbent and genetically engineered Zymomonas mobilis, bioethanol

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was produced at 54 g/L from the pretreated biomass mash by one-pot SSCF processes coupled

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with prehydrolysis. The lignin-derived adsorbent is recyclable and potentially applicable to a

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wide range of fermentation processes of lignocellulosics.

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INTRODUCTION: Excessive use of fossil resources causes global warming and depletes

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accessible crude oil.1 Therefore, there is a growing demand to produce biofuels and chemicals

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from renewable lignocellulosic biomass. Bioethanol production from lignocellulosic materials

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relies on technologies that disintegrate cell wall structures to expose cellulose and

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hemicelluloses, hydrolyze the cell wall polysaccharides to monosaccharides and ferment sugars

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to bioethanol.2–9

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To increase the economic feasibility of bioethanol production from lignocellulosics, a

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decrease in the dosage of cellulolytic and hemicellulolytic enzymes, the use of monosaccharides

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derived from hemicelluloses and a high rate of ethanol fermentation of sugars should be

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accomplished, together with an optimized pretreatment for the overall process. The exposure of

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cell wall polysaccharides for efficient enzymatic hydrolysis requires harsh pretreatment

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conditions, but this process results in co-production of a higher amount of fermentation

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inhibitors. Therefore, a new technology for suppression of fermentation inhibition contributes to

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the overall processes of bioethanol production through intensive pretreatment, hydrolysis with a

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smaller amount of enzyme and rapid fermentation.

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In bioethanol production, pretreatments separating cell wall components result in the

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production of fermentation inhibitors such as furfural, 5-hydroxymethylfurfural (5-HMF),

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vanillin, vanillic acid, syringaldehyde, syringic acid, acetic acid and other organic compounds

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from the cell wall polysaccharides and lignin (Figure 1).7–20 Molecular breeding of inhibitor-

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resistant ethanologenic microorganisms and process engineering to remove fermentation

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inhibitors have been studied for the production of bioethanol. Higher substrate concentration is

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needed to produce bioethanol at a lower cost. However, this demand, like the harsh pretreatment

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conditions, requires a concomitantly high level of technology to suppress the fermentation 3



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inhibition. So far, a number of technologies have been employed for removing inhibitors. These

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include adsorption of inhibitors on ion exchange resin and activated carbon, treatments with

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calcium hydroxide, calcium carbonate, sodium hydroxide, ammonium hydroxide, dithionite,

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hydrogen sulfite, membrane filtration, enzymatic and microbial detoxification, extraction with

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organic solvents or supercritical fluid and evaporation before or during fermentation.7–20

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However, the cost of bioethanol production is increased by additional processes, and the use of

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purchased adsorbent for fermentation inhibition is impractical for the production of biofuels,

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with a low cost that is competitive in the market.

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To overcome problems caused by fermentation inhibitors, development of new adsorbent,

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which is highly efficient, commercially available, environmentally friendly and easily

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obtainable, is strongly required. Additionally, construction of highly efficient bioethanol process

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is necessary for industrial use. In this paper, we found that thermally-processed residual lignin

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produced after ethanol fermentation efficiently adsorbed fermentation inhibitors without

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affecting monosaccharide concentration by enzymatic saccharification, and developed self-

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sufficient bioethanol production system using the novel lignin-derived adsorbent for inhibitors.

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EXPERIMENTAL SECTION:

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Materials and general methods. All the reagents and activated carbon were of analytical

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grade and purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and Nacalai

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Tesque (Osaka, Japan). Ion exchange resin was obtained from Mitsubishi Chemical Corporation

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(Tokyo, Japan). E. globulus wood chips were purchased from Oji paper Co., Ltd. (Tokyo, Japan).

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The wood chips were ground to 20 mesh using a Wiley mill, air-dried to approximately 10 %

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moisture content and used for experiments. The wood contained 46.6 % cellulose, 30.2 %

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hemicellulose and 22.2 % lignin.

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The holocellulose and α-cellulose contents were determined according to the Wise chlorite

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method 21 and TAPPI T203 om-93,22 respectively. Klason lignin was determined from the weight

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ratio of solid residue after a two-step acid hydrolysis using 72 % and 1.9 % sulphuric acid

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according to TAPPI T222 om-98 procedure.23

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Preparation of the adsorbents from the residual lignin by enzymatic hydrolysis of

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microwave-assisted pretreated pulp. The pretreatment of E. globulus for preparation of the

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adsorbents was conducted using a tower-shaped 2.45 GHz microwave (MW) reactor equipped

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with eight sets of 1.5 kW magnetrons and a 50 L reaction vessel (Japan Chemical Engineering &

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Machinery Co. Ltd., Shiga, Japan).24 Wood powder of E. globulus (7.5 kg) was added to 42 kg of

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tap water containing 0.5 kg of maleic acid. Microwave-assisted pretreatment of E. globulus wood

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was performed at 170 °C for 30 min. After the treatment, the pulp residue was separated by

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centrifugation and washed three times with tap water.

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The pulp residue separated by microwave pretreatments was hydrolyzed with a commercial

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cellulase preparation, Cellic CTec2 from Trichoderma reesei (Novozymes A/S, Bagsvaerd,

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Denmark). Cellulase enzyme loading was 10 FPU/g of dry pulp. Enzymatic hydrolysis was

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performed in 50 mM sodium succinate buffer (pH 4.5) at 50 °C for 72 h. After enzymatic

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hydrolysis, the residual lignin was separated by filtration and washed with water. The residue

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was added into hot water and stirred to remove buffer. After the solution was filtered and washed

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with water, the purified residue was dried at 105 °C overnight.

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For preparation of the adsorbent by thermal treatment under normoxic conditions, the dried

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residual lignin was placed in a stainless steel tray, covered with an aluminium sheet with ten

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small pinholes per 20 cm2, and heated in air at 250–350 °C for 1–4 h in an electric furnace

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(FO810, Yamato Scientific Co. Ltd., Tokyo, Japan). The yield of the adsorbent treated at 350 °C

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for 2 h was 39 % from the residual lignin. The yield of the adsorbent was calculated using the

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following equation (1).

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(1) Yield (wt%) = W1/W0 × 100, where W0 is the dry weight of the residual lignin and W1 is the dry weight of the adsorbent obtained by thermal treatment.

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Recycled adsorbent was prepared by heating the treated adsorbent at 350 °C for 1 h in air

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using the same apparatus. Yield of the recovered adsorbent was 69 %. The yields of the

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adsorbent were calculated using the following equation (2).

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(2) Yield (wt%) = W3/W2 × 100, where W2 is the dry weight of the treated adsorbent and W3 is the dry weight of the treated adsorbent obtained by thermal treatment.

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For preparation of the lignin-derived adsorbents under anaerobic conditions, the dried

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residue in a stainless steel dish was set in a gas-sealed electric furnace (Tokai Denki Co. Ltd.,

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Osaka, Japan) under a nitrogen atmosphere. The thermal treatment was carried out at 250–350 ºC

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for 2 h. The yield of each adsorbent was 52–82 % from the residual lignin. The yields of the

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adsorbent were calculated using the equation (1).

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Pretreatment of E. globulus wood using microwave irradiation for the fermentation

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test and one-pot SSCF. Wood powder of E. globulus (7.5 kg) was added to 42.49 kg of tap

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water containing 0.015 kg of maleic acid. Microwave-assisted pretreatment of E. globulus wood

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was performed at 190 °C for 30 min. After the treatment, the pulp residue and the soluble

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fraction were separated by centrifugation and washed three times with distilled water.

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Analysis of fermentation inhibitors. Fermentation inhibitors were quantified using high

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performance liquid chromatography (HPLC). Shimadzu Prominence system (Shimadzu Co., Ltd,

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Kyoto, Japan) equipped with an LC-20AD pump, a CTO-20A column oven, and an SPD-M20A

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photodiode array were used for HPLC analysis. Acetic acid, furfural and 5-HMF were analyzed

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using an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad Laboratories, Inc., Hercules,

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CA), with 8 mM aqueous sulfuric acid solutions as a mobile phase at a column temperature of

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35 °C and detected at UV 210 nm. The elution was performed using a Unison UK-Phenyl

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column (Imtakt, Inc., Kyoto, Japan), with 10 mM ammonium acetate buffer (pH 6.8) and

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acetonitrile at 40 °C to quantify lignin-derived inhibitors vanillin, syringaldeyde, vanillic acid

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and syringic acid with the detection at UV 280 nm.

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HPLC analysis of monosaccharides. Neutral carbohydrates obtained by enzymatic

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saccharification and adsorption experiments were determined by HPLC using a Prominence

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HPLC post-labelling system (Shimadzu, Co., Ltd., Kyoto, Japan) equipped with a F-1080

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fluorescence detector (HITACHI, Tokyo, Japan) and two tandemly connected Aminex HPX-87P

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columns (300 mm × 7.8 mm: Bio-Rad Laboratories, Inc., Hercules, CA). For fluorescence

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detection, samples were labelled at 150 °C using an aqueous solution of L-arginine (1 %) and

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boric acid (3 %).25 The sample solution (5 µL) was injected and analyzed at 58 °C using water as

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an eluent at a flow rate of 0.3 mL min−1. D-Glucoheptose was used as an internal standard.

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Adsorptive removal of fermentation inhibitors and determination of monosaccharides in

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MW-filtrate. The adsorbent (2.5 g) was added to 25 g of the soluble fraction obtained from the

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microwave pretreatment of E. globulus at 190 °C. Each mixture was stirred at room temperature.

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After 24 h, 1 mL of the solution was filtered through a Chromato-disc syringe filter with 0.45 µm

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pore size (GL science Inc., USA) and subjected to the HPLC analysis of fermentation inhibitors

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and monosaccharides. All adsorption tests were performed in triplicate.

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Adsorption test and analysis of lignin-derived inhibitors. The adsorbent (2.5 g) was added

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to 25 g of model aqueous solutions containing 20 mM of vanillin, syringaldehyde and p-

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hydroxybenzaldehyde in each. Each mixture was stirred at room temperature. After 24 h, 1 mL

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of the solution was filtered through the Chromato-disc syringe filter and subjected to the HPLC

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analysis of lignin-derived inhibitors from the soluble fraction. All adsorption tests were

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performed in triplicate.

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Adsorption test and sugar analysis of xylooligosaccharides. The adsorbent (0.5 g) was

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added to 5 g of the model aqueous solution containing 1 % of xylooligosaccharide (Wako Pure

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Chemical Industries Ltd., Osaka, Japan). The solution was stirred at room temperature. After 24

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h, total carbohydrates in the supernatant of mixture were determined by phenol-sulfuric acid

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method.26 All adsorption tests were performed in triplicate.

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Fermentation test using Z. mobilis. After preculturing the recombinant Z. mobilis6 for 48 h

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in 10 ml of basal medium (RM) containing 10 g/L yeast extract, 2 g/L KH2PO4 and 20 g/L

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glucose, cells were harvested and washed with the same volume of RM medium, except lacking

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a carbon source. The washed cells were then inoculated into 100 ml of the following media (8:2

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and 5:5) in a 200 ml bottle with a screw cap. The 8:2 medium was prepared by mixing eight parts

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of the soluble fraction from MW-pretreated E. globulus at 190 °C for 30 min and a solution

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obtained by treating the soluble fraction with various adsorbents, one part of the 10-fold

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concentrate of RM medium without a carbon source and one part of 200 g/L of glucose. The 5:5

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medium was prepared by mixing five parts of the soluble fraction, one part of the 10-fold

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concentrate RM medium without a carbon source, one part of 200 g/L glucose and three parts of

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the distilled water. The test media were adjusted to pH 5.5 and statically cultivated at 30 °C.

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Structural analyses of adsorbents. The surface morphologies of adsorbents were analyzed

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using scanning electron microscopy (SEM; JSM-6320, JEOL, Tokyo, Japan) at an acceleration

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voltage of 5 kV. The surface area and pore size of adsorbents were analyzed using ASAP 2000

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(Shimadzu, Co., Ltd., Kyoto, Japan) using nitrogen.

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Enzymatic hydrolysis in the presence of adsorbents. Sodium succinate buffer (1 M),

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distilled water and Cellic CTec2 were added to the soluble fraction (10 g) separated by MW

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pretreatment from E. globulus at 190 °C for 30 min so that total amount of the solution was 20 g.

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Cellulase enzyme loading was 5 mg protein per 20 g of the solution. After an adsorbent (1 g) was

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added to the solution, enzymatic hydrolysis was performed in 50 mM sodium succinate buffer

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(pH 4.5) at 50 °C on a rotary shaker (NTS-4000C, Rikakikai, Japan) at 140 rpm for 72 h. The

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sugar concentration was based on the weight percentage of each sugar to the soluble fraction

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using HPLC. All enzymatic hydrolysis experiments were performed in triplicate.

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One-pot SSCF with prehydrolysis in a medium bottle. The pulp (3 g of dry weight),

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soluble filtrate (13 ml) prepared by microwave-assisted reactions at 190 °C for 30 min, Cellic

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CTec2 containing 15 mg protein and 0.15 g of 5 % polyethylene glycol 20,000 solution were

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mixed in a bottle with a screw cap. Total volume of the fermentation broth was 20 mL. The

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prehydrolysis was conducted at 50 °C on a shaker at 100 rpm for 24 h. After prehydrolysis, 2 g

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of the lignin-derived adsorbent prepared by heating in air at 350 °C for 2 h (Entry 2 in Table 1)

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was added to the bottle. After 24 h, the solution was cooled to 35 °C, and 2 ml of the 10-fold

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concentrated RM medium without a carbon source and 2 ml of the seed culture were mixed to

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start SSCF. The seed culture was prepared by cultivating the recombinant Z. mobilis6 with the

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hydrolysates of MW-pretreated pulp. The concentration of glucose, xylose and ethanol was

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monitored using HPLC as previously reported.6

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One-pot SSCF with prehydrolysis in a jar fermenter. The pulp (90 g of dry weight) and

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soluble filtrate (390 ml) prepared by microwave-assisted reactions at 190 °C for 30 min, Cellic

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CTec2 containing 450 mg protein and 30g of the lignin-derived adsorbent prepared by heating in

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air at 350 °C for 2 h (Entry 2 in Table 1) were mixed and adjusted to pH 5.0 with 5 N NaOH

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solution in a 1 L jar fermenter. Total volume of the fermentation broth was 600 mL. The

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prehydrolysis was conducted at 50 °C with gentle stirring at 100 rpm. After 48 h prehydrolysis,

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the solution was cooled to 35 °C, and 60 ml of the 10-fold concentrated RM medium without a

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carbon source and 60 ml of the seed culture were mixed to start SSCF. The seed culture was

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prepared by cultivating the recombinant Z. mobilis

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pulp. The concentration of glucose, xylose, cellobiose and ethanol was monitored using HPLC as

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previously reported.6

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with the hydrolysates of MW-pretreated

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RESULTS and DISCUSSION:

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Preparation and properties of lignin-derived adsorbents. E. globulus wood was

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pretreated by microwaves in an aqueous solution containing 1 % maleic acid at 170 °C for 30

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min. The pretreated biomass was separated into soluble and insoluble fractions. The insoluble

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pulp fraction was hydrolyzed with cellulolytic enzymes, and the residual lignin was separated

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from the enzymatic hydrolysates by centrifugation. The residual lignin was washed with distilled

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water, dried and heated at 250 °C or 350 °C for 1–4 h under normoxic or nitrogen atmosphere in

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an electric furnace (Table 1). The lignin-derived adsorbent was subjected to adsorption

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experiments for inhibitors. In adsorption experiments for pretreated solutions, we used the filtrate

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separated by microwave pretreatment in an aqueous solution containing 0.02 % maleic acid at

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190 °C for 30 min. After mixing the soluble fraction with the lignin-derived adsorbent, the

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solution was filtrated, and the concentration of inhibitors in the filtrate was determined by HPLC.

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The concentration of furfural and 5-HMF in the original soluble fraction was 22.62 mM and 4.79

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mM, respectively (Table 1). When the solution was treated with the lignin-derived adsorbents

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prepared by thermal processing at 350 °C or 250 °C under normoxic oxygen atmosphere,

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concentration of furfural and 5-HMF decreased to 0.83–1.40 mM and 0.58–0.90 mM,

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respectively (Entry 1–4 in Table 1). The drastic decrease in the concentration of fermentation

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inhibitors was also found for lignin-derived degradation compounds, such as vanillin and

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syringaldehyde (Table 2). The concentration of vanillin decreased from 0.17 mM to a trace

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amount and that of syringaldehyde decreased from 0.70 mM to 0.01 mM. Removal of acetic acid

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was less effective, but the adsorbent decreased the concentration of the organic acid from 1.20 %

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to 0.59–0.73 %. Thus, we found that thermal processing of residual lignin at 350 and 250 °C

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under normoxic atmosphere gave the adsorbent a high adsorptivity (Entry 1–4 in Table 1). The

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high adsorptivity of the lignin-derived adsorbent against the five fermentation inhibitors (Table

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1) was comparable with that of activated carbon and anion exchange resin. The adsorbent

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processed at 250 °C removed the inhibitors as effective as that of 350 °C but much less effective

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to improve of fermentability. When the treatment was conducted with the lignin-derived

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adsorbent prepared at the same temperature under nitrogen atmosphere, removal of these

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inhibitors was much less effective as found in the very low levels of decrease in the

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concentrations of 5-HMF, acetic acid and syringaldehyde (Entry 5 in Table 1).

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Adsorptive removal of lignin-derived inhibitors was evaluated using a model solution

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containing 20 mM vanillin, syringaldehyde and 4-hydroxybenzaldehyde (Table 2). As found in

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adsorption treatments of the soluble fraction from E. globulus wood, the adsorbent made in air

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was effective at removing inhibitors as well as activated carbon and ion exchange resin. The

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lignin-derived adsorbent prepared under nitrogen atmosphere was ineffective at removing the

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lignin monomers both in the pretreated biomass (Table 1) and in the model solution containing

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inhibitors (Table 2).

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The effect of inhibitor removal on bacterial growth, carbohydrate consumption and ethanol

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production by the lignin-derived adsorbent was examined by fermentation experiments with Z.

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mobilis (Figure 2 and Table 1). When the untreated soluble fraction was subjected to

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fermentation experiments, at either ratio of the filtrate and RM medium (8:2 or 5:5), the bacterial

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growth was very slow, and almost no ethanol production was observed. Dilution of the solution

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to 2:8 ratio was necessary to produce ethanol without removal of inhibitory compounds.

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However, treatment of the soluble fraction with the lignin-derived adsorbent drastically

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improved the fermentability.

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We found that the efficiency of inhibitor removal increased with increasing the weight

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percentage of the adsorbent, and 10 % was the minimum concentration of the adsorbent under

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the fermentation conditions employed. The concentration of inhibitors in the broth is another

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factor affecting the fermentation rate. In this study, we used two different concentrations of the

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filtrate by changing the ratio between the filtrate and RM medium. When the soluble fraction

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was processed with 10 % lignin-derived adsorbent and fermented at a ratio of 8:2 of the filtrate

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and RM medium, glucose was consumed, and ethanol production increased during 72 h (Figure

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2C). When fermented at a ratio of 5:5, glucose was consumed within 24 h with concomitant

263

production of ethanol at the theoretical yield (Figure 2D). Thus, the new adsorbent derived from

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the residual lignin was effective for detoxification of the pretreated biomass.

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Recycling of the lignin-derived adsorbent increases the feasibility of practical applications

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to the bioethanol production process. Therefore, after ethanol fermentation, the used adsorbent,

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which was originally prepared at 350 °C for 2 h (Entry 2 in Table 1), was separated, processed at

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350 °C for 1 h in an electric furnace and repeatedly used for detoxification experiments. As

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shown in Figure 3, the recycled adsorbent slightly decreased the final ethanol concentration but

270

effective enough to produce ethanol at the ratio of the filtrate and RM medium (5:5). After

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repeated use, the adsorbent containing inhibitors from the filtrate could be burned to recover heat

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energy. This process is beneficial not only for energy recovery but also to decrease the

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biochemical oxygen demand and chemical oxygen demand in the waste water, which is one of

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the major cost factors in bioethanol production.

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To apply adsorbents to bioethanol production, a high level of sugar recovery from

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adsorbents is necessary. Therefore, loss of mono- and oligosaccharides by the adsorption process

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was evaluated using HPLC. The lignin-derived adsorbent made at 350 °C for 2 h did not adsorb

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monosaccharides. The low adsoptibity to carbohydrates is different from activated carbon and

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ion exchange resin, which adsorbed around 20 % of monosaccharides in MW-filtrate and more

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than 40 % of xylooligosaccharide in the model sugar solution (Entry 7 and 8 in Table 3). Thus,

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ion exchange resin and activated carbon are not selective to the inhibitors and economically

282

feasible for biofuel production. When 1 % of xylooligosaccharides were treated with these 13



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adsorbents, it was found that activated carbon and ion exchange resin strongly adsorbed

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xylooligosaccharides. Adsorption of oligosaccharides on the lignin-derived adsorbent prepared at

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350 °C in air (Entry 1–3 in Table 1) was much smaller than that on the activated carbon and ion

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exchange resin, but around 20 % loss of the oligosaccharides was found. The problem of

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adsorption of oligosaccharides can be avoided by prehydrolysis or simultaneous saccharification

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in the presence of adsorbent if enzymatic hydrolysis was not inhibited by the presence of the

289

adsorbent. The latter process can be extended to a one-pot SSCF process if co-presence of the

290

adsorbent did not inhibit ethanol fermentation. Therefore, we evaluated the effect of co-presence

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of the adsorbent on enzymatic saccharification and the SSCF process.

292

Enzymatic saccharification of pretreated biomass in the presence of lignin-derived

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adsorbents. To evaluate the applicability of the lignin-derived adsorbents to the SSCF process,

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we examined effects of co-presence of adsorbents on enzymatic saccharification of pretreated

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biomass. Yields of monosaccharides after hydrolysis with Cellic Ctec2 at 50 °C for 72 h are

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shown in Table 4. The sugar yields obtained in the presence of the lignin-derived adsorbents

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prepared in air were close to that obtained in the absence of adsorbents. When the residual lignin

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from the SSCF process was used for the adsorbent, higher sugar yields were obtained. These

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results are highly contrasted to activated carbon, which decreased the sugar yield by around 20 %

300

(Table 4). Thus, the thermally-processed lignin adsorbents are applicable to in situ enzymatic

301

hydrolysis of pretreated biomass.

302

One-pot SSCF after prehydrolysis using lignin-derived adsorbent. The high

303

adsorptivity of fermentation inhibitors without interference from enzymatic saccharification

304

attracts our interest to apply the lignin-derived adsorbents to the one-pot SSCF process using Z.

305

mobilis (Figure 4). We designed the process starting from prehydrolysis and subsequent SSCF in 14


Page 14 of 33

Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


306

the presence of the lignin-derived adsorbents prepared by heating in air at 350 °C for 2 h (Entry 2

307

in Table 1). The prehydrolysis increased the initial concentration of sugars and resulted in an

308

acceleration of the ethanol production rate by SSCF. We pretreated E. globulus wood by

309

microwave-assisted hydrothermolysis and separated wood into soluble and insoluble fractions.

310

Preliminary experiments of one-pot SSCF of the pulp and the MW-filtrate obtained from the

311

MW-pretreated E. globulus with and without the lignin-derived adsorbent in a bottle were

312

investigated using Z. mobilis (Figure 5). Figure 5A indicates that one-pot SSCF of the pulp and

313

MW-filtrate without the lignin-derived adsorbent cannot produce ethanol. Additionally, amount

314

of glucose and xylose was hardly consumed. In contrast, ethanol was gradually produced in the

315

mixture with the lignin-derived adsorbent until 48 h, and the concentration of ethanol reached at

316

around 20g/L (Figure 5B). Glucose was used up for fermentation in Figure 5B. From these

317

results, it was found that one-pot SSCF of the pulp and MW-filtrate with the lignin-derived

318

adsorbent is available for scale-up process.

319

To a 1 L jar fermenter, the insoluble pulp fraction, soluble fraction and lignin-derived

320

adsorbent were added. The prehydrolysis was started by addition of Cellic Ctec2. The

321

concentration of pretreated substrate and adsorbent was 15 % and 5 %, respectively. After

322

incubation at 50 °C for 48 h, the enzymatic hydrolysates were cooled down to 35 °C. To the

323

same reactor, the nutrient broth and seed culture of Z. mobilis were added and fermented at 35 °C

324

for 72 h. The profile of fermentation by the one-pot SSCF process is shown in Figure 6. Ethanol

325

was produced at an ethanol concentration over 5 % for 70 h after SSCF was started.

326

Thus, ethanol was successfully produced from pretreated biomass mash by the one-pot

327

SSCF using lignin-derived adsorbents and Z. mobilis. The one-pot SSCF reduces the production

15



328

cost of bioethanol by eliminating the solid-liquid separation process and adsorption facilities

329

using a large scale column.

330

Structural analyses of lignocellulose-based adsorbents. Structures of the lignin-derived

331

adsorbents were analyzed to understand the differential adsorptivity caused by the thermal

332

treatment. Table 5 shows the surface area and pore volumes of the series of adsorbents analyzed

333

by the N2-BET method. Surface area of the original residual lignin was 0.39 m2/g. The surface

334

area of adsorbent 2, which was prepared at 350 ºC for 2 h in air was 525 m2/g, indicating that

335

surface area of the lignin increased over 1,000-fold. The surface area of adsorbent 4 prepared at

336

250 °C for 2 h in air was 26.9 m2/g, whereas the surface area of adsorbent 6 prepared under

337

nitrogen atmosphere at the same temperature and heating time was 0.15 m2/g. Thus, thermal

338

treatment in air significantly increased the surface area. Pore volumes of the lignin-derived

339

adsorbent between 10 and 1000 Å increased from 0.001 to 0.239 ml/g, respectively, over 200

340

times by the thermal treatment at 350 °C in air, and the values were comparable with those of

341

activated carbon.27–30 SEM images of adsorbents prepared in air and in nitrogen atmosphere

342

supported the increase in pore structure by the thermal treatment under a normoxic atmosphere

343

(Figure 7). Lignin adsorbent recycled by thermal treatment in air after ethanol fermentation kept

344

the similar indices in the surface morphology as those of the adsorbent initially prepared

345

(Supporting information S1). We assume that the oxygenated aromatic core structures with a

346

propane side chain remained in part after the dehydration process at low temperature in air,

347

giving higher affinity to inhibitors by the balance of hydrophilic and hydrophobic interactions,

348

including π-π stacking.31–33 So far, activated carbon has been prepared by thermal treatments of

349

lignin for the adsorption of metal ions and environmental pollutants.34 Chemical or physical

350

activation was applied to produce the activated carbon with high surface area and pore volume.

16


Page 16 of 33

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


351

For instance Cotoruelo et al. converted kraft lignin into activated carbon by carbonization at

352

600–1100 °C followed by physical activation with CO2 to remove environmental pollutants. The

353

adsorbent effectively adsorbed a synthetic dye, crystal violet.35 Compared with the previous

354

studies, the residual lignin can be converted to the adsorbent at much lower temperatures without

355

physical and chemical activation, giving big advantage in terms of cost, energy and material

356

balance. Yields of the adsorbent from the original lignin prepared at 350 °C for 1h, 2 h and 4 h

357

were 69 %, 41 % and 13 %, respectively. Under the higher temperatures over 800 °C for

358

charcoal production, it would be impossible to complete the material flow using the biomass

359

components due to very low recovery of the thermally processed adsorbent.

360 361

CONCLUSIONS: In this study, we have developed novel lignin-derived adsorbent, which can

362

strongly adsorb fermentation inhibitors with less adsorption of saccharides and interference of

363

enzymatic saccharification, by thermally treating of residual lignin from E. globulus. The high

364

selectivity of lignin-derived adsorbent to inhibitors shown by the lower level of monosaccharide

365

adsorption, minimum interference to enzymatic saccharification and high adsorptivity to furfural,

366

5-HMF and lignin monomers can be emphasized. In addition, one-pot SSCF process coupled

367

with the novel lignin-derived adsorbent using Z. mobilis was successfully constructed, and

368

ethanol was produced at 54 g/L. This new self-sufficient bioethanol system has a lot of

369

advantages, such as no waste material, no purchase adsorbents, simplification and cost reduction

370

of facilities, recyclable, energy recovery from soluble inhibitors, decrease load of waste water.

371

The lignin-derived adsorbent and one-pot SSCF can be potentially applied to various

372

fermentation processes of lignocellulosics, depending on the tolerance of microorganisms to

373

remaining fermentation inhibitors. 17



374 375

FIGURES:

376 377 378 379 380 381

Figure 1. Chemical structures of fermentation inhibitors.

382

0.6

10

0.4

5

0.2

0

389 390 391 392 393 394 395 396 397 398

0.0 0

24 48 Time (h)

25

1.0

20

0.8

15

0.6

10

0.4

5

0.2

0

72

24 48 Time (h)

72

(D)

25

2.0

20

1.6

15

1.2

10

0.8

5

0.4

0

0.0 0

0.0 0

(C)

Growth (OD660nm)

15

Sugars and ethanol (g/l)

388

0.8


387

20

Growth (OD660nm)

386

1.0

Growth (OD660nm)

385


384

(B)

(A) 25

24 48 Time (h)

:Glucose

25

1.0

20

0.8

15

0.6

10

0.4

5

0.2 0.0

0 0

72

:Xylose

:EtOH

399

18


24 48 Time (h)

72

:Growth(OD)

Growth (OD660nm)

383


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

Page 19 of 33

400

Figure 2. Ethanol fermentation of the filtrate from the microwave-pretreated E. globulus using Z.

401

mobilis (A) and (B): Filtrate without detoxification was used; (C) and (D): Filtrate after

402

processing with lignin-derived adsorbent was used; (A) and (C): The filtrate was mixed with RM

403

medium at a ratio of 8:2; (B) and (D): The filtrate was mixed with RM medium at a ratio of 5:5.

404

Diamond and closed circle stand for glucose and xylose concentration. Closed triangle and

405

square show ethanol concentration and bacterial growth expressed by OD 660 nm. The lignin

406

adsorbent for (C) and (D) was prepared at 350 °C for 2 h under O2 atmosphere.

407

409 410 411 412 413 414 415 416

25

2.0

20

1.6

15

1.2

10

0.8

5

0.4

0

0.0 0

417

24

48

Growth (OD660nm)

408


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


72

Time (h)

418 419

:Glucose

:Xylose

:EtOH

:Growth(OD)

420 421

Figure 3. Ethanol fermentation of the filtrate from the microwave-pretreated E. globulus using Z.

422

mobilis after the treatment with the recycled lignin adsorbent (Entry 9 in Table 1). Diamond and

423

closed circle stand for glucose and xylose concentration. Closed triangle and square show

424

ethanol concentration and bacterial growth expressed by OD 660 nm.

19



425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

Figure 4. Bioethanol production system from lignocellulosic biomass by SSCF (A)

445

Conventional process. (B) One-pot SSCF using lignin-derived adsorbent.

446 447 448 449 450

20


Page 20 of 33

Page 21 of 33

451 452 453

457 458 459 460

80

6

60

5.5

40

5

20

4.5

0

461

24

48 72 Time (h)

96

100

6.5

80

6

60

5.5

40

5

20

4.5

0

4 0


456

6.5

pH

455

(B)

(A) 100

4 0

120

pH

454 Sugars and ethanol (g/l)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24

48 72 Time (h)

96

120

462

:Glucose

463

:Xylose

:EtOH

:pH

464 465

Figure 5. One-pot SSCF of the pulp and MW-filtrate obtained by microwave-pretreatment of E.

466

globulus (A) without detoxification and (B) with detoxification using the lignin-derived

467

adsorbent prepared at 350 °C for 2 h under O2 atmosphere using Z. mobilis. Closed diamond,

468

circle and triangle stand for glucose, xylose ethanol concentration. Opened square shows pH in

469

the solution.

470 471 472 473 474 475 476

21



477 478 479 480

120

Prehydrolysis

481

100

483

60

SSCF

Inoculate rec Zm. mobilis

482

EtOH

50

485 486 487 488 489 490

40

80

Glc

60

30

40

20

20

10

Ethanol and xylose (g/liter)

484 Released glucose (g/liter)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

491 492 493 Xyl

494 495

0

0 0

24

48

72

96

120

144

168

192

Time (hr)

496 497 498

Figure 6. One-pot SSCF coupled with prehydrolysis of microwave-pretreated E. globulus using

499

Z. mobilis and lignin-derived adsorbent.

500 501 502

22


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


503 504 505 506 507 508

(A)

(B)

(C)

(D)

509 510 511 512 513 514 515 516 517 518 519 520

Figure 7. SEM of adsorbents prepared by thermal treatment at 350 °C for 2 h under oxygen

521

atmosphere (A, B) and nitrogen atmosphere (C, D) with 1,000 (B, D) and 10,000 (A, C)

522

magnifications.

523 524 525 526 527 528

23



Page 24 of 33

529 530

TABLES:

531

Table 1. Concentrations of fermentation inhibitors in MW-filtrate after treatment with lignin-

532

derived adsorbents and fermentability by Z. mobilis.

Entry

Adsorbent

Furfural (mM)

5-HMF (mM)

Acetic acid (%)

Vanillin (mM)

Syringaldehyde (mM)

Fermentabilityb

—

Original soluble Fr.

22.62

4.79

1.20

0.17

0.70

2:8 24 h

1

350°C-4h-O2

1.40

0.65

0.59

n.d.

0.01

5:5 48 h

2

350°C-2h-O2

0.83

0.58

0.71

n.d.

0.01

5:5 24 h

3

350°C-1h-O2

1.29

0.65

0.60

n.d.

0.01

5:5 24 h

4

250°C-2h-O2

1.33

0.90

0.73

0.01

0.49

n.p.

5

350°C-2h-N2

18.52

4.96

1.19

0.05

0.64

n.p.

6

250°C-2h-N2

14.11

4.22

1.16

0.02

0.37

5:5 72 h

7

Activated carbon

1.12

0.50

0.46

n.d.

n.d.

5:5 72 h

8

Ion exchange resin

0.78

0.48

0.10

n.d.

0.01

8:2 24 h

9

350°C-2h-O2 recyclea

1.07

0.53

0.54

n.d.

0.01

5:5 24 h

533 534

a

535

b

536

n.d.: Not detected.

537

n.p.: No production of bioethanol at 8:2 and 5:5 ratios.

Residual lignin was recovered after adsorption treatment, thermally processed and used. Cultivation time and ratio between the filtrate and RM medium giving ethanol yield over 90 %.

538 539 540 541 542 543 544 24


Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


545 546

Table 2. Adsorptive removal of three lignin degradation products with adsorbents.

547

1

350°C-4h-O2a

Vanillin (mM) i 0.12

2

350°C-2h-O2b

0.28

0.37

0.36

3

350°C-1h-O2

c

0.65

0.98

0.67

250°C-2h-O2

d

13.90

18.27

3.60

e

19.31 14.46 n.d. 0.07

19.11 15.63 n.d. 0.02

20.22 14.81 0.02 0.12

Entry

4 5 6 7 8

Adsorbent

350°C-2h-N2 250°C-2h-N2f Activated carbong Ion exchange resinh

Syringaldehyde (mM) i 0.09

4-hydroxybenzaldehyde (mM) i 0.21

548 549

a

Entry 1, bEntry 2, cEntry 3, dEntry 4, eEntry 5, fEntry 6, gEntry 7 and hEntry 8 in Table 1.

550

i

Concentration of each compound in the original solution was 20 mM.

551

n.d.: Not detected.

552 553 554 555 556 557 558 559 560 561 562 563 564

25



Page 26 of 33

565 566

Table 3. Analysis of monosaccharides in MW-filtrate and xylooligosaccharide in water after

567

treatment with adsorbents.

Entry

Adsorbent

Monosaccharides (%)

—

MW-filtrate (No treatment)

1.60

100

1

350°C-4h-O2

a

1.47

77

2

350°C-2h-O2b

1.51

83

3

350°C-1h-O2c

1.53

82

4

250°C-2h-O2d

1.54

101

5

350°C-2h-N2e

1.54

100

6

250°C-2h-N2

f

1.60

102

7

Activated carbong

1.33

5

1.28

59

h

8

Ion exchange resin

9

350ºC-2h-O2 recyclei

568 569

a

b

c

570

n. d.: Not determined

d

Xylooligosaccharide (%)

1.56 e

f

n.d. g

Entry 1, Entry 2, Entry 3, Entry 4, Entry 5, Entry 6, Entry 7, Entry 8 and iEntry 9 given in Table 1.

571 572 573 574 575 576 577 578 579 580 581

26


h

Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


582

Table 4. Concentration of total monosaccharides after saccharification of MW-filtrate and MW-

583

filtrate under the adsorbent.

584

Entry

Adsorbent

Concentration of total monosaccharides (%)

—

MW-filtrate (No treatment)

1.95

2

350°C-2h-O2a

1.89

5

350°C-2h-N2b

1.85

7

Activated carbonc

1.65

10

SSCF-350°C-2h-O2

2.04

a

b

c

Entry 2, Entry 5 and Entry 7 in Table 1.

585 586 587 588

Table 5. Surface area and pore size of adsorbents. Temperature (°C)

Time (h)

Atmos -phere

Surface area (m2/g)

Pore volume 10-1000 Å (mL/g)

Pore volume < 10 Å (mL/g)

Total pore volume (mL/g)

Average pore diameter (Å)

Residual lignin

―

―

―

0.39

0.001/0.001

―

0.001

128

2

350°C-2h-O2a

350

2

Air

525

0.117/0.074

0.205

0.239

18

4

250°C-2h-O2

b

250

2

Air

26.9

0.015/0.002

0.006

0.017

25

6

250°C-2h-N2c

250

2

N2

0.15

n.d.f

n.d.f

n.d.f

126

7

Activated carbond

―

―

―

387g

―

―

―

―

350°C-2h-O2 recycle e

350

1

Air

565

0.131/0.092

0.219

0.261

18

Entry

Adsorbent

—

9

589

a

b

c

d

e

Entry 2, Entry 4, Entry 5, Entry 7 and Entry 9 given in Table 1.

590

f

Unmeasurable due to production of high amount of tar.

591

g

Value from the reference 31.

592 593 594 595

27



596

ASSOCIATED CONTENT:

597

Supporting Information. SEM images of the recycled adsorbents prepared by thermal treatment

598

of at 350 °C for 1 h under oxygen atmosphere (Figure S1).

599 600

AUTHOR INFORMATION:

601

Corresponding Author

602

E-mail: [email protected]; Tel: +81-774-38-3644; Fax: +81-774-38-3681

603 604

Present Addresses

605

†K. Y: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University,

606

Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.

607 608

Notes

609

The authors declare no competing financial interest.

610 611

ACKNOWLEDGMENT:

612

A part of this work was supported by New Energy and Industrial Technology Development

613

Organisation (NEDO) and Collaborative Research Program of RISH, Kyoto University. The

614

authors acknowledge Dr. Sensho Honma for preparing the adsorbent under N2 atmosphere and

615

Toyota motors for collaboration in the NEDO bioethanol project. The authors extend their

616

gratitude to Mr. Masakazu Kaneko, Mr. Masashi Tomita, Mr. Yosuke Kurosaki, Mrs. Yukari 28


Page 28 of 33

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617

Takahashi and Ms. Ai Yamada from RISH, Kyoto University for technical and scientific

618

discussion and support.

619 620

ABBREVIATIONS:

621

SHF, separate hydrolysis and fermentation; SSCF, simultaneous saccharification and co-

622

fermentation; 5-HMF, 5-hydroxymethylfurfural; HPLC, high performance liquid

623

chromatography;

624 625

REFERENCES:

626

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For Table of Contents Use Only:

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Synopsis: A new process suppressing the fermentation inhibition using the residual lignin from

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the bioethanol production system has been developed.

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Content graphic:

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Self-sufficient bioethanol production system using a lignin-derived

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