Bioethanol production from rice straw by popping pretreatment
© Wi et al.; licensee BioMed Central Ltd. 2013
Received: 29 May 2013
Accepted: 24 September 2013
Published: 29 November 2013
Rice straw has considerable potential as a raw material for bioethanol production. Popping pretreatment of rice straw prior to downstream enzymatic hydrolysis and fermentation was found to increase cellulose to glucose conversion efficiency. The aim of this study was to investigate the influence of popping pretreatment and determine the optimal enzyme loading using a surface response design.
The optimal doses of cellulase and xylanase enzymes were 23 FPU and 62 IU/g biomass, respectively. Using the optimized enzyme condition and popping pretreatment of rice straw (15% substrate loading, w/v), a sugar recovery of 0.567 g/g biomass (glucose; 0.394 g/g) was obtained in 48 h, which was significantly higher than that from untreated rice straw (total sugar recovery; 0.270 g/g biomass). Fermentation of the hydrolyzates by Saccharomyces cerevisiae resulted in 0.172 g ethanol/g biomass after 24 h, equivalent to 80.9% of the maximum theoretical yield (based on the amount of glucose in raw material). Changes in the chemical composition and surface area of rice straw were also investigated before and after popping pretreatment. The results showed little or no difference in chemical composition between the pretreated rice straw and the control. However, the surface area of pretreated rice straw increased twofold compared to the control.
Popping pretreatment of rice straw can effectively improve downstream saccharification and fermentation, important for bioethanol production.
KeywordsPopping pretreatment Rice straw Bioethanol Enzymatic hydrolysis Fermentation
Bioethanol is currently produced primarily from sugar and starch sourced from crops (first-generation biomass) such as sugar cane, wheat and corn, which have a high concentration of sugar [1, 2]. However, because these crops are also important food sources bioethanol produced from them can have a significant impact on food prices and food security . In contrast, lignocellulosic biomass, residues from wood or dedicated energy crops (second generation) is an attractive alternative because there is no competition with food and animal feed production, and these materials are also cheaper than first-generation biomass [3, 4]. Additionally, the use of lignocellulosic materials as liquid fuels can aid in reducing greenhouse gas emissions [5–7].
Lignocellulosic biomass is the largest source of hexose and pentose sugars, which can be used for the production of bioethanol . Unlike first-generation biomass, in second-generation lignocellulosic substrates cellulose in the cell wall is encased within hemicellulose and lignin matrix, and thus accessibility of cellulose is a major problem in bioethanol production from such sources. Thus, the cost of bio-fuel production is high due to intensive labor and increased processing steps. These economic and technical obstacles must be overcome for efficient and cost effective biological conversion of lignocellulosic biomass into biofuels.
Rice straw is an abundant lignocellulosic waste material in many parts of the world. Rice straw production amounts to approximately 731 million tons per year globally, with distribution in Africa (20.9 million tons), Asia (667.6 million tons), and Europe (3.9 million) . Rice straw is one of the largest biomass feedstocks, and potentially 730 billion liters of bioethanol can be produced per year from the above quantity of available biomass. It is the largest amount from a single biomass feedstock. Presently, high value utilization potential of this biomass remains largely uptapped. Its accumulation in the soil deteriorates the ecosystem via disposal as a waste, and burning in the field air pollution thus which can affect human health .
Rice straw consists of cellulose, hemicellulose and lignin. Because cellulose is embedded in a lignin matrix, pretreatment of the lignocellulosic material is needed to enhance the accessibility of this substrate for the conversion of cellulose to glucose. There are a number of biological, physical and chemical technologies available for the pretreatment of lignocellulosic biomass, including use of enzymes, ball milling, steam explosion, acid, alkali, lime and wet oxidation. The slow action of biologically-based pretreatment processes , and high cost of ammonia fiber explosion and hot water pretreatment make the processes economically infeasible [11, 12]. Therefore, the development of an efficient, cost-effective and environmentally friendly pretreatment method is important .
Recently, some new pretreatment technologies have attracted much attention, one of which is popping pretreatment [14–16]. This method is similar to water impregnated steam explosion method, which combines mechanical forces of the sudden explosion with chemical effects from hydrolysis in high temperature water and acetic acid formed from acetyl groups in the biomass. Unlike this method, however, the machine used to undertake popping pretreatment is a very simple system consisting of direct burner and rotary reactor without steam generator. This method offers key advantages over other processes, including significantly lower environmental impact and greater saccharification efficiency over similar methods used conventionally , with greater efficiency likely resulting from modification of the substrate that greatly enhances accessibility of desired cell wall components to enzymes. We examined the use of rice straw for ethanol production using the popping pretreatment method developed in our laboratory. Furthermore, the effect of pretreatment on rice straw was tested using downstream processing technologies. Although cellulose enzyme was the main focus of enzymatic saccharification in our study, xylanase was also included with a view to achieving fermentation also xylose with xylose specific yeast in future studies. Additionally, xylanase seemed to have worked synergistically with cellulase.
Results and discussion
Sugar and lignin compositions of rice straw, expressed as percentages of dry matter
3.3 ± 0.2
20.7 ± 0.2
0.3 ± 0.0
0.5 ± 0.2
1.2 ± 0.2
41.7 ± 2.2
67.8 ± 3.2
13.0 ± 0.2
2.3 ± 0.1
11.0 ± 0.5
1.8 ± 0.0**
19.3 ± 0.2**
0.4 ± 0.0
0.5 ± 0.0
0.9 ± 0.2
41.5 ± 3.6
64.5 ± 4.5
12.2 ± 0.7
2.1 ± 0.1
11.4 ± 0.1
Characterization of surface area
The morphology of rice straw was studied using FE-SEM (Additional file 1: Figure S1). The surface morphology of pretreated rice straw (Additional file 1: Figure S1d-f) differed markedly from that of control rice straw (Additional file 1: Figure S1a-c). Pretreated rice straw had a rough and porous surface with identifiable micropores (Additional file 1: Figure S1f). The rougher surface and a higher surface area resulting from the removal of hemicelluloses by the popping method enhanced enzymatic hydrolysis, as has generally been considered . These results are consistent with those for rapeseed straw pretreated by the popping method .
Optimization of enzyme loading and saccharification
Experimental matrix for the factorial design and center points
Reducing sugar (mg/mL)
Response = 5.78 + 0.53∙cellulase + 0.047∙xylanase - 0.088∙cellulase∙xylanase - 0.39·cellulase2 - 0.14·xylanase2.
Separate hydrolysis and fermentation (SHF)
Popping pretreatment of rice straw prior to downstream enzymatic hydrolysis and fermentation increased the efficiency of conversion of cellulose to glucose. The optimal cellulase and xylanase doses for popping pretreated rice straw at 220°C and 1.96 MPa were 23 FPU and 62 IU/g, respectively. Using the optimized enzyme condition and popping pretreatment (15% substrate loading, w/v), sugar recovery of 0.567 g/g biomass (glucose; 0.394 g/g biomass) was achieved in 48 h, which was significantly higher than that obtained from rice straw that had not been pretreated (total sugar recovery; 0.270 g/g biomass). Fermentation of the hydrolyzates with S. cerevisiae yielded 0.172 g ethanol/g untreated biomass after 24 h, equivalent to 80.9% of the theoretical yield based on the glucose content of raw material. There was little or no difference between the chemical composition of control and pretreated rice straw. However, the surface area of pretreated rice straw increased twofold over the control. The results obtained suggest that popping pretreatments brought about favorable changes to the substrate, such as increased surface area and larger pore volume, resulting from hemicellulose degradation, which greatly enhanced enzymatic accessibility of the substrate, leading to more efficient hydrolysis of cellulose. Popping pretreatment of rice straw can effectively improve downstream saccharification and fermentation, important for bioethanol production.
Materials and Methods
Raw material and popping pretreatment
Rice straw harvested in 2011 was chopped into small pieces of ~2 cm in length with a cutter, ground with a wet-disc mill (particle size: 0.7 ± 0.2 cm) and then kept refrigerated until use. Popping pretreatment was performed in a laboratory-scale cast iron cylindrical reactor with a total volume of 3 L, as described in a previous work . The reactor was filled with 400 g of disc-milled feedstock (moisture content 75%) per batch. That was directly heated with a gas burner at a rate of between 15 and 20°C/min and rapidly open the hatch at 220°C and 1.96 MPa. After popping, the material was recovered in a storage tank and the wet material was cooled to ambient temperature.
Chemical composition analysis
The ethanol-benzene soluble fraction was determined gravimetrically. Klason lignin, acid-soluble lignin and the ash of raw and pretreated rice straw were analyzed in accordance with TAPPI Standard Methods . Analyses of structural sugars (glucose, xylose, arabinose, mannose, galactose and rhamnose) were conducted using a gas chromatograph .
The commercial enzymes used in this study were cellulase (Celluclast 1.5 L, Novozyme) and xylanase (X2753, Sigma). Filter paper unit activity of cellulase was measured in terms of FPU/mL . One filter-paper unit (FPU) was defined as the amount of enzyme required to release 1 μmole of glucose from filter paper per min. Xylanase activity was measured on the basis of xylose released from birch wood xylan as a substrate and was expressed in terms of international units (IU)/mL. One IU was defined as the amount of enzyme required to release 1 μmole of xylose from birch wood xylan per min . The activities of cellulase and xylanase were 79 FPU/mL and 592 IU/mL, respectively.
Optimization of enzyme mixture
Experimental domain and level distribution used for enzyme ratio optimization
Cellulase (FPU/g biomass)
Xylanase (IU/g biomass)
Separate hydrolysis and fermentation
Enzymatic saccharification was conducted in a 500 mL Erlenmeyer flask with a total working volume of 100 mL at a substrate concentration of 15% DM (w/v) with 0.1% (w/v) yeast extract, 0.2% (w/v) peptone, and 0.05 M citrate buffer (pH 4.8). Reaction flasks were run in triplicate with an enzyme loading of 23 FPU cellulase and 62 IU xylanase/g biomass at 150 rpm for 48 h. The flasks were then stored at 4°C until required fermentation.
For the fermentation with S. cerevisiae KCTC 7906, 0.5 g of dry yeast was added as inoculum to 100 mL of hydrolyzates. Fermentation was carried out at 32°C for 48 h with agitation at 150 rpm. All experiments were performed in triplicate, and ethanol yield was calculated on the basis of total glucose content in the pretreated materials by dividing the quantity of ethanol produced by the total amount of glucose.
High-performance liquid chromatography (HPLC) analysis for liquid phase
During enzymatic hydrolysis and fermentation sugars (glucose and xylose) and ethanol were monitored using HPLC equipped with a refractive index detector (YoungLin Instruments, Anyang, Korea). A Rezex ROA organic acid column (Phenomenex, Torrance, CA) was used for compound identification (300 × 7.8 mm). The temperatures of the column and detector were maintained at 65 and 40°C, respectively, and 5 mM sulfuric acid was added to the mobile phase at a flow rate of 0.6 mL per min.
The surface morphologies of the samples were examined using field-emission scanning electron microscopy (FE-SEM) with a JSM-7500 F (Jeol, Japan) instrument operating at a beam voltage of 3 kV. Prior to observation, each sample was dehydrated with a graded ethanol series and freeze-dried. The external surface of the sample was then sputter-coated with osmium suing a sputter-coater.
Surface area measurement using a BET
The pore structures of rice straw and its popping pretreated materials were measured using BET nitrogen adsorption-desorption isotherms at -196°C in a surface-area analyzer (ASAP 2020, Micromeritics Co., USA). Prior to determination, the sample (~0.7 g) was degassed for 1.5 h at 110°C under vacuum (5 mmHg) to remove moisture and any other contaminants. The total pore volume was assessed by converting the amount of nitrogen gas adsorbed to the volume (cm3/g at STP) of liquid adsorbate, using a single point adsorption (at a relative pressure circa 0.99).
Brunauer Emmett and Teller
Field emission scanning electron microscopy
high performance liquid chromatography
- S. cerevisiae:
Separate hydrolysis and fermentation.
This work was supported by Priority Research Centers Program (2010-0020141) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, and by a grant (S2113131L010120) from Forest Science & Technology Projects, Forest Service, Republic of Korea.
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