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Enzyme pretreatment enhancing biogas yield from corn stover: feasibility, optimization, and mechanism analysis Siqi Wang, Fan Li, Dan Wu, Panyue Zhang, Hongjie Wang, Xue Tao, Junpei Ye, and Mohammad Nabi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03086 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 11, 2018
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Enzyme pretreatment enhancing biogas yield from corn stover:
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feasibility, optimization, and mechanism analysis
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Siqi Wang†‡, Fan Li†, Dan Wu†, Panyue Zhang*,†, Hongjie Wang‡, Xue Tao†, Junpei Ye†,
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Mohammad Nabi†
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†
Beijing Key Lab for Source Control Technology of Water Pollution, Beijing Forestry
University, Beijing 100083, China ‡
Xiong’an Institute of Eco-Environment, Hebei University, Baoding, 071002, China
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ABSTRACT:
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In this study, feasibility, optimization and mechanisms of enzyme pretreatment to
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enhance anaerobic digestion of corn stover were investigated. Results showed that the
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enzyme pretreatment efficiently enhanced the biogas yield, and the optimal conditions
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of enzyme pretreatment were enzyme load of 30 FPU/g, pretreatment time of 24 h,
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and solid content of 60 g/L. Under the optimal conditions, the cumulative biogas yield
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increased by 36.9%, which was mainly attributed to disruption of surface structure
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and degradation of non-crystalline cellulose in the enzyme-pretreated corn stover. The
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kinetic analysis indicated that enzyme pretreatment significantly enhanced the
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hydrolysis rate and biogas production rate to 0.15/d and 23.89 ml/gVS, shortened the
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lag phase time to 1.2 d. Correlation analysis illustrated that the SCOD yield of 1
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250-350 mg/g from corn stover after enzyme pretreatment was suitable for the further
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anaerobic digestion of corn stover.
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KEYWORDS:
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Lignocellulosic structure, Kinetic analysis
Corn
stover,
Enzyme
pretreatment,
Biogas
production,
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INTRODUCTION
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The rapid development of global economy and the fast increase of population
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have led to the growing shortage of fossil energy. As a new and clean energy,
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bioenergy is becoming more and more important, and is increasingly used as
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replacement for fossil fuels.1 Biogas production from organic wastes (e.g.,
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food-processing wastes, fruit wastes and agriculture wastes) through anaerobic
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digestion is promising.2,3 Anaerobic digestion not only produces the bioenergy but
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also reduces the organic wastes.4,5 Meanwhile, biogas residues after anaerobic
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digestion can be used as an efficient organic fertilizer to improve soil fertility.
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Lignocellulosic biomass is the most abundant source of renewable energy on the
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planet. Corn is one of the largest grains in the world, therefore, corn stover of billion
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tons is produced every year as agriculture wastes. The corn stover is a typical
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lignocellulosic biomass and has great potential in biofuel production. Corn stover
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consists of high content of cellulose, hemicellulose, and relatively low content of
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lignin.6 Exactly, cellulose and hemicellulose are the main utilized components during
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anaerobic digestion of corn stover. Accelerating their degradation is critical to
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improve the anaerobic digestion of corn stover. 2
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However,
due
to
the
recalcitrant
lignocellulose
structure
(cellulose,
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hemicellulose and lignin are tightly held together) and subsequent low hydrolysis rate,
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the biogas production efficiency of lignocellulosic biomass is low.7 Therefore,
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efficient pretreatments for biomass hydrolysis are key technologies. Various
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pretreatments have been explored to improve the microbial degradation and biogas
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production from biomass, including physical pretreatment, chemical pretreatment,
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combined physical and chemical pretreatment, and biological pretreatment.8-16
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Physical pretreatment requires high energy consumption, which may hold back its
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industrial application. Chemical pretreatment (such as using acid or alkali) is effective,
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but the liquid after pretreatment is difficult to be recycled and may lead to significant
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environmental risks. Biological pretreatment exhibits great superiority compared to
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chemical or physical processes for its mild conditions and low energy consumption.10
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Biological pretreatment of lignocellulosic biomass involves using enzymes
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and/or microorganisms to enhance the conversion of recalcitrant feedstock to easily
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biodegradable materials.17. Hydrolysis of cellulose and hemicellulose under the action
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of enzymes yields monosaccharide, such as glucose, xylose, glucose, galactose,
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arabinose and mannose.18 When enzymes are applied in the hydrolysis of
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lignocellulose to produce fermentable sugar, the cost of enzymatic hydrolysis is much
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lower compared to the other alternative methods.19 Researchers have reported
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significant improvement in biogas production with the pretreatment of crude and
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commercial enzymes for complex organic matters, such as sludge, food-processing
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wastes and agriculture wastes etc. Recktenwald et al.20 reported that addition of 3
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enzyme mixture to an anaerobic sludge digester improved biogas production and
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dewatering properties. A fungal mash rich in hydrolytic enzymes was applied for
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pretreatment of mixed food wastes, and the biomethane yield and production rate
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were found to be respectively about 2.3 and 3.5-times higher than that without
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pretreatment.21 Romano et al.22 used multi-enzymes to pretreat wheat grass prior to
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anaerobic digestion or added the enzymes in hydrolysis stage, the biogas production
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rate was significantly accelerated, and the biogas yield increased from 0.44 to 0.53
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L/g VS. However, few studies involved the enzymes pretreatment of corn stover for
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enhancing biogas production. The efficiency and underlying mechanisms are still
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unknown.
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In this paper, enzyme pretreatment was used to enhance the biogas yield of corn
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stover, aiming to assesse the feasibility of enzyme pretreatment. Several influencing
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factors of enzyme pretreatment including enzyme load, pretreatment time and
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pretreatment solid content were examined since the enzyme pretreatment efficiency
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was greatly affected by these factors.21,23 In addition, the kinetic of biogas production
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processes were analyzed by modified Gompertz and Cone models. Structure changes
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in corn stover due to enzyme pretreatment were analyzed to study the fermentation
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mechanisms. Finally, the relationship between the soluble COD (SCOD) yield by
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enzyme pretreatment and biogas yield in the following anaerobic digestion was
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examined to guide the operation.
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MATERIALS AND METHODS 4
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Materials. Corn stover was harvested from a farm in Hebei, China. The raw corn
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stover was dried at room temperature for 72 h, and the dried corn stover was crushed
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to a particle size smaller than 0.425 mm by a laboratory hammer mill (DF-25S, Dade,
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China). The corn stover powders were stored in sealed plastic bags for further
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experiments. The seed sludge was obtained from a local biogas station in Beijing,
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China and stored at 4 °C before experiments. The characteristics of seed sludge and
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corn stover are shown in Table 1.
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Enzyme Pretreatment. Enzyme used in this study was cellulase generated by
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Aspergillus niger (powder, ≥ 0.3 units/mg solid) purchased from Sigma-Aldrich Co.
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LLC. Previous study showed that this enzyme could efficiently degrade
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lignocellulosic biomass,24,25 and thus was used for enzyme pretreatment in this study.
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The purchased enzyme was dissolved in sodium citrate buffer solution (0.05 mol/L,
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pH=4.8) as enzyme solution with a concentration of 10 g/L. For measuring filter paper
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activity (FPA) of this enzyme, 50 mg filter paper and 0.5 ml enzyme solution were
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incubated at 50 °C for 60 min in an air bath thermostat (THZ-82B, Jintan shenwei
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equipment Co., Ltd, China). The reaction was stopped by adding 3,5-dinitrosalicylic
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acid solution (DNS) of 0.75 ml and then boiled in 100 °C water bath for 5 min. After
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cooling to room temperature, the reaction mixtures were diluted to 10 ml. The
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reducing sugar released due to the enzymatic activities was estimated by DNS
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method.26 The enzyme activity is described as the reducing sugar (µmol) produced
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every ml enzyme solution every minute under the measurement conditions with a unit
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of FPU/ml. The FPA of this enzyme was determined to be about 18.6 FPU/ml. 5
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Serum bottles of 250 ml as the reactors were applied to carry out the enzyme
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pretreatments. Dry corn stover of 1g and enzyme solution of 0.54, 1.08 or 1.62 ml
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were added into reactors and the enzyme load of 10, 20 and 30 FPU/g was formulated,
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respectively. Total mixed volume was supplemented to 100 ml with citrate buffer (0.1
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mol/L, pH=4.8). The reactors were placed into an air bath thermostat (THZ-82B,
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Jintan shenwei equipment Co., Ltd, China) with a rotation speed of 100 r/min at 50 °C.
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After the enzyme pretreatment, the total materials including liquid and solid were
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used for subsequent anaerobic digestion. Moreover, for the determination of liquid
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SCOD and solid structural change in mixture due to enzyme pretreatment, 5 ml
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mixture sample was taken after enzyme pretreatment to be centrifuged at 8000 r/min
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for 10 min by a refrigerated high-speed centrifuge (3H16RI, Hunan Hersxi instrument
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& equipment Co., Ltd, China). The supernatant was filtered with a filter membrane of
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0.45 µm. Then the filtrate was used to analyze the SCOD concentration. The solid
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residues were dried at 105 ± 3 °C for at least 4 h. The dried solid sample was stored in
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sealed plastic bags for the structural analysis.
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Anaerobic Digestion. Seed sludge of 90 ml was added in the pretreatment
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reactors to carry out anaerobic digestion. All the reactors were filled with nitrogen gas
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in order to maintain anaerobic circumstance and sealed with rubber stoppers. The
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system pH was adjusted to 7.5 with HCl and NaOH solution (1 mol/L), and total
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volume was supplemented to 200 ml with deionized water. The reactors were placed
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in an air bath thermostat (THZ-82B, Jintan shenwei equipment Co., Ltd, China) with
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a rotation speed of 100 r/min at 37 °C for anaerobic digestion. 6
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Analytical Methods. VS and TS of corn stover and sludge were determined
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based on the standard methods. The SCOD concentration of supernatant after enzyme
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pretreatment was measured by water quality analyzer (5B-1(B), Lian-hua Tech Co.
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Ltd., China), based on potassium dichromate method. The volume of biogas was
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determined by the method of liquid displacement with diluted hydrochloric acid (pH
