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A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover
Bioresource Technology xxx (2015) xxx–xxx
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A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover Heng Yu a, Jiwei Ren b, Lei Liu a, Zhaojuan Zheng a, Junjun Zhu a, Qiang Yong c, Jia Ouyang a,c,⇑ a
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China College of Forestry, Nanjing Forestry University, Nanjing 210037, People’s Republic of China c Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education, Nanjing Forestry University, Nanjing 210037, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 25 June 2015 Received in revised form 24 August 2015 Accepted 25 August 2015 Available online xxxx Keywords: Magnesium bisulfite pretreatment Enzymatic hydrolysis Bio-ethanol production Nonwoody lignocelluloses
a b s t r a c t This study established a new more neutral magnesium bisulfate pretreatment (MBSP) using magnesium bisulfate as sulfonating agent for improving the enzymatic hydrolysis efficiency of corn stover. Using the MBSP with 5.21% magnesium bisulfate, 170 °C and pH 5.2 for 60 min, about 90% of lignin and 80% of hemicellulose were removed from biomass and more than 90% cellulose conversion of substrate was achieved after 48 h hydrolysis. About 6.19 kg raw corn stover could produce 1 kg ethanol by Saccharomyces cerevisiae. Meanwhile, MBSP also could protect sugars from excessive degradation, prevent fermentation inhibition formation and directly convert the hemicelluloses into xylooligosaccharides as higher-value products. These results suggested that the MBSP method offers an alternative approach to the efficient conversion of nonwoody lignocellulosic biomass to ethanol and had broad space for development. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass is abundant, renewable and environmentally friendly resource, which can be converted into bioethanol (Hamelinck et al., 2005). But for the conversion of biomass into bioethanol, the degradation of lignocelluloses is still a major barrier to obtain a high overall ethanol yield. The pretreatment is necessary to breakdown the recalcitrance of lignocellulosic biomass for the second generation bio-ethanol production (Galbe and Zacchi, 2012). An effective pretreatment method can increase the enzymatic digestion performance of lignocelluloses and reduce consumption of enzyme in saccharification stage (Biswas et al., 2015). In the past two decades, variety of pretreatment methods had been discovered, such as lime, dilute acid, steam explosion and organic solvent pretreatment (Singh et al., 2015). However, several problems, including equipment corrosion, environmental pollution, high inhibitor production, and low sugar recovery still restricted the development of pretreatment technology. Dilute sulfuric acid (DA) pretreatment is the most common method due to the low price and the extensive applicability to raw material (Hu and Ragauskas, 2012). But the low pH would cause serious equip⇑ Corresponding author at: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China. Tel.: +86 025 85427129; fax: +86 025 85427587. E-mail address: [email protected] (J. Ouyang).
ment corrosion and produce a great amount of fermentation inhibitors (Bellido et al., 2011). Steam explosion is an effective pretreatment method for improving the enzymatic degradation of lignocellulosic materials. After years of improvement, various steam explosion methods, such as ammonia fiber explosion (AFEX), CO2 explosion and SO2 explosion (Bals et al., 2011), have been developed to adapt different feedstock or further improve the enzymatic degradation of pretreated lignocellulosic material (Singh et al., 2015). However, the main technical barriers for the steam explosion process includes its scalability for commercialization, the high energy consumption and harmful degradation products formation (Zhu and Zhuang, 2012). As for lime pretreatments, the recovery of hemicelluloses sugars and NaOH recovery due to the high cost of lime are still the problems. Therefore, it is necessary to develop new approaches to treat biomass. Recently, a kind of novel sulfite pretreatment (SPORL) derived from sulfite pulping has been extensively studied for wood biomass by Zhu’s groups (Wang et al., 2009; Zhu et al., 2009). Feedstock is firstly treated using a sulfite salt at 160–180 °C and pH 2–4 (H2SO4 + Na2SO3) and then size-reduced using a disk mill (Zhu and Pan, 2010). This pretreatment process has combined effects on dissolution of hemicelluloses, depolymerization of cellulose, and partial delignification (Zhu and Pan, 2010). As a result, pretreated substrate shows great degradability in enzyme hydrolysis and highly adapt to the existing equipment in pulp and paper
http://dx.doi.org/10.1016/j.biortech.2015.08.090 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx
industry (Leu et al., 2013; Li et al., 2014). Additionally, the lignin dissolved in SPORL hydrolysate is sulfated (lignosulfonate), which has more potential application than other lignin forms in established market (Shuai et al., 2010). SPORL is an efficient pretreatment for woody materials. But its effect on nonwoody biomass still needs to be executed. Additionally, the low pH during SPORL is another concern in application. Before the enzymatic hydrolysis, much alkali to neutralize the low pH value is needed because pH 4.8 is the common condition in the enzymatic hydrolysis of cellulose. In order to overcome these defects, in this study, a new more neutral bisulfate pretreatment method, which used magnesium bisulfate (Mg(HSO3)2, pH 5.2) as sulfonating agent, was proposed for bio-refinery of a typical non wood biomass corn stover. The main factors of pretreatment were optimized and the effects of pretreatment conditions on the composition changes and enzymatic digestibility were clearly argued to clarify their connections. Finally, mass balance of corn stover from the raw material to ethanol was established by magnesium bisulfite pretreatment (MBSP).
tank at a solid/liquid ratio of 1:6 (w/v) for pretreatment. In pretreatment process, magnesium bisulfite dose, pretreatment temperature and pretreatment time have been optimized respectively for investigating their effects. After pretreatment, the reactor was immediately cooled down in a water bath. The pretreated substrate was collected by filtration, washed with warm water for at least three times and stored at 4 °C. Solid loss was determined from the measure wet weight and moisture content of the solid. The spent liquor was stored in 20 °C for further analysis. All the experiments were carried out in two replicates. 2.5. Enzymatic hydrolysis Enzymatic hydrolysis was performed at 3% cellulose in 30 mL citrate buffer (pH 4.8, 50 mM), at 50 °C and 150 rpm on a shaker incubator for 48 h. A mixture of Celluclast 1.5 L at 15 FPU/g cellu100
A
2.1. Materials Corn stover was harvested from Lianyungang of Jiangsu province in China. After air-dried and crushed at Biochemistry Institute of Nanjing Forestry University, the corn stover was sieved to achieve the fraction between 20 and 80 mesh for avoiding the interference because of ash. The prepared biomass was stored at room temperature until use. Two commercial enzyme solutions, Celluclast 1.5 L (Cat C2730) and Novozyme 188 (Cat C6105) from Sigma–Aldrich were used for enzymatic hydrolysis. Sulfur dioxide solution (ACS reagent, P6%) was purchased from Sigma–Aldrich. Anhydrous sodium sulfite and magnesium chloride were purchased from Nanjing Chemical Reagent Co., LTD. 2.2. Magnesium bisulfite solution preparation Magnesium bisulfite solution was prepared by mixing solid magnesium sulfite with sulfur dioxide solution in a mole ratio of 1:1 (pH approximately 5.2). The mixture was stirred until insoluble magnesium sulfite particle disappeared. The above mentioned solid magnesium sulfite was prepared by mixing sodium sulfite with magnesium chloride in a mole ratio of 1:1. And then, the residual soluble reactants were eluted three times with water. Afterward, magnesium sulfite was dried by lyophilization.
60 40 20 0 0
1.76
3.51
5.27
7.02
Magnesium bisulfite concentration (%) 100
B 80
Removal (%)
2. Methods
Removal (%)
80
60 40 20 0 140
150
160
170
180
o
Temperature ( C)
100
C
2.3. Microorganism and cultivation Saccharomyces cerevisiae AQ was provided by Anqi Company, China, and grown at the medium for inoculation contained (g/L): glucose, 20; peptone, 20; yeast extract, 10 at natural pH (YPD). The seed culture was prepared as follows: a loop of cells from the fully grown slant was inoculated into 100 ml of the above medium in 250 ml Erlenmeyer flasks and incubated for 24 h at 30 °C with 150 rpm. Then, the seed cells were harvested, washed and inoculated into Erlenmeyer flasks or bioreactors for ethanol production. 2.4. Pretreatment This laboratory scale pretreatment was carried out in an electrically heated oil bath. A total of 3 g of biomass sample and 18 mL pretreated solution was placed in a 30 mL sealed stainless steel
Removal (%)
80 60 40 20 0 0
20
40
60
80
Pretreatment time (min) Solids Cellulose Hemicellulose Lignin Fig. 1. Influences of different MBSP process conditions on removal ratio of solid and three main compositions in corn stovers (all not listed conditions in figure were: magnesium bisulfite content, 3.51% (w/v); temperature, 170 °C; pretreatment time, 60 min).
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx Table 1 Content of five known fermentation inhibitors and sugars in spent liquor collected from MBSP process with different condition. Pretreatment conditions
Fermentation inhibitors concentrations (g/L)
Sugar concentrations (g/L)
Mg(HSO3)2 concentration (%, w/v)
Temperature (°C)
Pretreatment time (min)
Formic acid
Acetic acid
Furfural
HMF
Levulinic acid
Glucose
Xylose
XOS
0 1.76 3.51 5.27 7.02
170
60
0.433 0.081 n/a n/a n/a
2.750 3.693 4.337 4.410 3.845
0.734 1.404 0.201 0.080 0.067
0.039 0.062 n/a n/a n/a
0.016 n/a n/a n/a n/a
0.385 0.433 0.319 0.339 0.323
0.993 3.036 2.226 1.747 1.535
10.306 10.714 9.903 10.880 9.739
3.51
140 150 160 170 180
60
n/a n/a n/a n/a n/a
0.815 1.969 3.101 4.337 5.201
n/a n/a 0.024 0.201 2.076
n/a n/a n/a n/a 0.307
n/a n/a n/a n/a n/a
0.500 0.307 0.243 0.319 0.363
0.129 0.126 0.457 2.226 0.875
4.397 10.247 19.710 9.903 2.193
3.51
170
0 20 40 60 80
n/a n/a n/a n/a n/a
1.708 2.623 3.467 4.337 4.678
n/a n/a 0.022 0.201 0.705
n/a n/a n/a n/a 0.115
n/a n/a n/a n/a n/a
0.335 0.266 0.238 0.319 0.303
0.125 0.323 0.881 2.226 3.101
8.660 18.232 16.579 9.903 7.709
n/a: not available.
lose and Novozyme 188 at 30 CBU/g cellulose was used for enzymatic hydrolysis. After hydrolysis, the samples were withdrawn and centrifuged to remove the insoluble materials. The supernatants were subsequently filtered through a 0.22 lm syringe filter (Millipore, Co., LTD) and used for subsequent HPLC analysis. All experiments were performed in replicates and each data point was the average of two replicates.
hemicellulose or lignin) in pretreated material. The units of measurement were g. The cellulose hydrolysis yield was calculated according to the following equation:
2.6. Fermentation
where Cglu was the concentration of glucose in enzymatic hydrolysate; Cbiose was the concentration of cellobiose in enzymatic hydrolysate; CCel was the initial concentration of cellulose. The units of measurement were g/L. The ethanol yield was calculated according to the following equation:
Fermentation was carried out in 250 mL Erlenmeyer flask at 30 °C and 150 rpm for 24 h with 50 mL of enzymatic hydrolysate adding 6 g/L yeast extract, 10 g/L peptone, 5 g/L (NH4)2SO4, 10 g/L KH2PO4, 0.5 g/L MgSO47H2O and 0.15 g/L CaCl2H2O. Each flask was equipped with a needle-pierced silicone stopper to allow removal of the produced CO2 during fermentation. The initial yeast inoculum was OD 10 (600 nm) and initial pH was controlled in 4.8. At the end of fermentation, samples of the broth were centrifuged at 5000g for 10 min for sugar and ethanol analysis. Reported data is the average of duplicates.
Enzymatic hydrolysis yield ð%Þ ¼
ðC glu þ 1:053 C biose Þ 0:9 100 C cel
Ethanol yield ð%Þ ¼
C eth 100 DC glu 0:51
ð2Þ
ð3Þ
where Ceth was the concentration of ethanol; DCglu was the consumption of glucose in fermentation process; 0.51 was the transformation factor from glucose to ethanol. The units of measurement were g/L.
2.7. Analytical methods 3. Results and discussion The chemical compositions of the original and pretreated corn stover were determined according to the National Renewable Energy Laboratory (NREL, Golden) analytical methods for biomass (Sluiter et al., 2011). The free fermentable sugars (glucose, cellobiose, and xylose), several known inhibitors (formic acid, acetic acid, levulinic acid, furfural, 5-hydroxymethylfurfural (HMF)) and ethanol were directly quantified using a HPLC system (1200 series, Agilent) equipped with a Bio-Rad Aminex HPX-87H column (300 7.8 mm). 5 mM H2SO4 as mobile phase was controlled at a flow rate of 0.6 mL/min at 55 °C, analysis signal was detected by a refractive index detector (Ouyang et al., 2013). As for the analysis of xylooligosaccharide (XOS), 1 mL sample was mixed with 1 mL of 5 N H2SO4 and hydrolyzed at 120 °C for 45 min to convert XOS to their constitutive monomers and analyzed by HPLC to quantify the total amount of XOS (Nabarlatz et al., 2007). The removal of each composition (%) was calculated according to the following equation:
Removal of composition ð%Þ ¼
ðmini mtre Þ 100 mini
ð1Þ
where mini was the mass of composition (cellulose, hemicellulose or lignin) in raw material; mtre was the mass of composition (cellulose,
3.1. Removal of the lignocellulosic components during pretreatment The original corn stover used in the present study had a dominant cellulose content of 37.5% and low lignin content of approximately 22.1%. The hemicellulose content was 18.5%. As described in the Section 2.4, corn stover was pretreated by MBSP at varied chemical loadings, pretreatment temperature and time. The loss of dry matter and three key components following MBSP employ different conditions was assessed and presented in Fig. 1. The effect of magnesium bisulfite dose on the loss of three components at the pretreatment temperature of 170 °C and the pretreatment time of 60 min is shown in Fig. 1A. With the increase of magnesium bisulfate loading, the removal of lignin is more pronounced than the removal of hemicellulose. Hot water pretreatment resulted that the removal of lignin and hemicellulose were 13.94% and 65.76%, respectively. The increase in magnesium bisulfite dose enhanced delignification significantly. Due to the sulfonation of lignin, about 60% of lignin was removed when the chemical dose was 1.76%. In terms of hemicelluloses, it was well known that the acid environment promotes the solubilization of hemicellulose (Garlock et al., 2011). Dilute acid pretreatment could dissolve all
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx
Enzymatic hydrolysis yield (%)
4
100
A 80 60 40 20 0 0
1.76
3.51
5.27
7.02
Enzymatic hydrolysis yield (%)
Magnesium bisulfite concentration (%) 100
B 80 60 40 20 0 140
150
160
170
180
o
Enzymatic hydrolysis yield (%)
Temperature ( C) 100
C 80
20 min and gradually approach the limitation. When pretreatment time was 60 min at 170 °C with 3.51% magnesium bisulfite, the maximum lignin and hemicellulose removal ratio were 82.45% and 80.73%, respectively. Several different pretreatment methods were applied to enhance the enzymatic hydrolysis of lignocellulosic material by changing the composition of the biomass. DA pretreatment could degrade almost hemicelluloses from the biomass, leaving only cellulose and lignin in the DA substrates (Li et al., 2014). In SPORL pretreatment, above 90% hemicellulose could be removed while 20–40% lignin loss (Wang et al., 2009). Comparing with these pretreatment methods, MBSP showed more effect on the removal of lignin and hemicellulose while cellulose was relatively stable during pretreatment process. A large portion of lignin and hemicellulose were degraded and dissolved during the pretreatment. By contrast, only minor cellulose loss (approximately 5.5%) was discovered. When the corn stover was treated by 5.27% magnesium bisulfite at 170 °C for 60 min, the loss of hemicellulose and lignin could reach 89.10% and 80.87%, respectively. Correspondingly, the chemical composition of pretreated corn stover was 82.22% cellulose, 8.25% hemicellulose and 5.62% lignin. Lignin was universally regarded as main obstacle in enzymatic saccharification process, which led to the inactivation of cellulase due to its nonproductive adsorption of enzyme (Wang et al., 2011). MartinSampedro deemed that this phenomenon would become worse in high solid concentration enzymatic hydrolysis due to the lignin accumulation (Martin-Sampedro et al., 2013). The BSG hemicelluloses also exerts a negative influence on the enzymes attack to cellulose (Mussatto et al., 2008). These reports revealed that the partial removal of hemicelluloses and lignin would be beneficial for improving the enzymatic hydrolysis of lignocelluloses after MBSP pretreatment. Additionally, it was worth mentioning that, the detached lignin existed as soluble lignosulfonate, which was valuable in many areas (Xavier et al., 2010; Yu et al., 2014).
60
3.2. Pretreatment spent liquor (hydrolysate) composition
40
As discussed above, a large amount of hemicelluloses and lignin were removed from the biomass to the spent liquor during the pretreatment process. An important point that must be considered is regarding the changes of their existing forms in hydrolysates. Especially some of compounds derived from the sugar degradation had been known as potential inhibitors for subsequent fermentation (Palmqvist and Hahn-Hägerdal, 2000). Considering that the formation of these compounds was closely correlated with pretreatment conditions, the amount of several known fermentation inhibitors and soluble sugars in spent liquor were listed in Table 1 as a standard to evaluate the degradation of three key components during MBSP process. The furan aldehydes furfural and HMF, which are formed by dehydration of pentose and hexose sugars, respectively, are commonly found in lignocellulose hydrolysates (Jönsson et al., 2013). In present study, HMF and furfural decreased with increased magnesium bisulfite dose. An explanation is that more magnesium bisulfate depressed the decomposition of the sugar to HMF and furfural in a relatively higher pH (5.2–2.7). A similar phenomenon was also discovered in SPORL pretreatment (Zhu et al., 2009). The data in Table 1 also indicated that increasing pretreatment temperature and pretreatment time led to the production of HMF and furfural as expected. At high temperature, HMF would be further degraded and form levulinic acid and formic acid, the latter was also formed from furfural under acidic conditions (Palmqvist and Hahn-Hägerdal, 2000). Only little formic acid and levulinic acid were detected in the pretreatment spent liquor while magnesium bisulfite dose was in a low level (1.76%). It suggested that the degradation of HMF and furfural was negligible under studied
20 0 0
20
40
60
80
Pretreatment time (min) Fig. 2. Influences of different MBSP process conditions on enzymatic hydrolysis yields of corn stovers (all not listed conditions in figure were: magnesium bisulfite content, 3.51% (w/v); temperature, 170 °C; pretreatment time, 60 min).
hemicellulose from the biomass (Shuai et al., 2010). In the present study, the removal of hemicellulose ranged from 65.76% to 80.87%, which indicated MBSP also could dissolve part of hemicelluloses under pH 5.2. About 90% of lignin and 80% of hemicellulose removal were obtained by adding 5.27% magnesium bisulfite. Accordingly, increasing pretreatment temperature not only created the condition for dissolving out of hemicellulose, but also promoted the sulfonation of lignin by magnesium bisulfate (Fig. 1B). Alvarez-Vasco and Zhang reported that the degradation of hemicellulose in high temperature was due to thermal instability of hemicelluloses (Alvarez-Vasco & Zhang, 2013). An evident loss of hemicelluloses was started at 150 °C in MBSP process. The lignin removal also began at 150 °C. But when temperature reached 180 °C, the sulfurization was weakened, which reflected the higher temperature (>180 °C) was adverse to sulfonation of lignin in MBSP process. As for pretreatment time (Fig. 1C), the removal of the lignin, hemicelluloses and solid were sharply increased in first
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx
5
Fig. 3. Mass balance of the optimum MBSP process for bio-ethanol product.
pretreatment conditions. As for acetic acid, it is a main co-product, which is formed primarily by hydrolysis of acetyl groups of hemicelluloses (Palmqvist and Hahn-Hägerdal, 2000). The amount of acetic acid increased greatly with increasing the temperature and time while the chemical dose did not show an obvious effect. When magnesium bisulfate concentration achieved 3.51%, acetic acid concentration increased to 4.337 g/L. In addition, the analysis of soluble sugar showed that MBSP produced less xylose and glucose in pretreatment spent liquor than SPORL and DA pretreatment. It might be caused by a higher pH environment, compared with pH 1.2 in the DA pretreatment and 2.7 in the SPORL pretreatment (Shuai et al., 2010). More interestingly, products from the hemicelluloses in the liquid phase were present in form of XOS. XOS is a class of non-digestible food ingredient, which could be produced by chemical methods, auto hydrolysis and enzymatic hydrolysis (Aachary & Prapulla, 2009). For the MBSP, a maximum amount of XOS (19.71 g/L) in the hydrolysate was found at 160 °C, 60 min and 3.51% magnesium bisulfite dose. This phenomenon meant magnesium bisulfite not only could prevent excessive degradation of sugar, but also contributed to the production of XOS by the restricted degradation (Yu et al., 2014). 3.3. Enzymolysis of the pretreated biomass To evaluate the effect of MSBP on the enzymatic hydrolysis of corn stover, the pretreated biomass was hydrolyzed for 48 h at enzyme loading of 15 FPU cellulase and 30 CBU b-glucosidase per gram of cellulose (Fig. 2). The enzymatic digestibility of the biomass was greatly improved after MBSP process. Hydrolysis yield was positively correlated with magnesium bisulfite concentration. The MBSP sample presented the highest hydrolysis yield of 90.44% at 5.27% magnesium bisulfite concentration. Otherwise, hot water pretreated corn stover only showed a 31.02% yield at the same condition. It indicated that adding magnesium bisulfite was able to effectively improve the enzymatic degradation of corn stover, which might be mainly due to the lignin removal (Fig. 2A). The lignin is generally believed as one of the most limiting factors in enzymatic hydrolysis of cellulose (Donohoe et al., 2008). Furthermore, according to the results from Fig. 1A, increasing magnesium
bisulfate dose influenced the lignin removal more than the hemicelluloses loss. The temperature was also the main factor in improving enzymatic degradation of corn stover (Fig. 2B). Increase of pretreatment temperature is contributed to the removal of hemicelluloses, especially over 150 °C (Fig. 1B). When pretreatment temperature reached to 170 °C, the enzymatic hydrolysis yield was 80.12%. Furthermore, the maximum hydrolysis yield (93.78%) was achieved at 180 °C. Similar result was achieved by Wei et al. in the process of dilute acid pretreated eucalyptus chips (Wei et al., 2012). It was noteworthy that the high temperature (above 180 °C) made the harmful degradation of sugar. Some strong inhibitor of fermentation was detected such as furfural and HMF. Considering that the presence of theses by-products of pretreatment is disadvantage of sequent fermentation, 170 °C seems to be a suitable temperature for pretreatment. As for pretreatment time, both biomass loss and enzymatic hydrolysis yield exhibited a clear increasing trend with time. The enzymatic hydrolysis yield increased to the maximum at 80.12% when the removal of lignin and hemicellulose approach saturated at pretreatment time of 60 min (Fig. 2C). Continuing to increase pretreatment time did not further improve the degradation of pretreated corn stover, but more harmful degradation product would form (Table 1). In summary, the enzymatic digestibility of the pretreated biomass improved with increasing pretreatment severity. The above findings from components analysis and hydrolysis results suggested that the lower the hemicelluloses and lignin contents in the pretreated biomass, the better the performance of cellulose enzymatic hydrolysis. It was agreement with many previous studies (Öhgren et al., 2007; Qing et al., 2010). The effect of MBSP on can be attributed to the solubilization of the hemicelluloses and lignin fractions. In this study, the pretreatment condition (5.27% magnesium bisulfite, at 170 °C for 60 min) was selected for the follow-up fermentation evaluation and mass balance according to optimization experiments. 3.4. Mass balance of MBSP process for bio-ethanol production During bio-ethanol production process, an ideal pretreatment is not only able to produce an effective enzymatic hydrolysis and
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx
excellent fermentation, but also to make full use of available sugar in biomass. According to this objective, enzymatic hydrolysate of MBSP pretreated corn stover was directly used to ethanol fermentation by S. cerevisiae and the ethanol yield of 93.46% were achieved after 12 h fermentation, which did not show obviously inhibitory effect from the enzymatic hydrolysate of MBSP pretreated corn stover. Afterward, preliminary mass balance of the MBSP process with 5.21% magnesium bisulfite, at 170 °C for 60 min for ethanol production was summarized in Fig. 3. There were 375.2 g cellulose, 185.4 g hemicellulose, and 221.4 g lignin from 1000 g oven dry raw corn stover. After MBSP, 429.8 g pretreated corn stover (containing 353.5 g cellulose, 35.5 g hemicellulose and 24.1 g lignin) was achieved. Enzymatic hydrolysis of the pretreated biomass resulted 341.9 g glucose, 12.6 g cellobiose and 30.6 g xylose produced in hydrolysate with low dosage of enzyme (15 FPU and 30 CBU/g cellulose). When ethanol fermentation was performed by S. cerevisiae, about 162.2 g ethanol was obtained from 1000 g oven dry raw corn stover, even xylose did not be utilized during the fermentation. The results clearly indicated that MBSP is an effective pretreatment method and about 6.17 kg of raw corn stover could transform to 1 kg ethanol by MBSP. Total detected XOS in the pretreatment spent hydrolysate was 65.28 g. Besides this, the spent liquor of MBSP also contained abundant valuable lignosulfonate (data not shown), which made the spent liquor had applicable value in many respects, such as: concrete technology (Ouyang et al., 2009), dispersant (Yang et al., 2007) and enzyme catalysis (Wang et al., 2013). 4. Conclusions MBSP method proposed in this study offers an alternative approach to efficient convert nonwoody lignocellulosic biomass to ethanol. After MBSP, about 90% of lignin and 80% of hemicellulose was removed from biomass and more than 90% cellulose conversion of substrate was achieved for 48 h hydrolysis. In bioethanol production using MBSP, 6.19 kg raw corn stover could produce 1 kg ethanol. Besides these, the great advantage of MBSP is that it could protect sugars from excessive degradation, prevent fermentation inhibition formation and directly convert the hemicelluloses into xylooligosaccharides as higher-value products. Acknowledgements This study was supported by the National Natural Science Foundation of China (31200443) and Excellent Youth Foundation of Jiangsu Province of China (BK2012038). The authors are also grateful to the National Hi-Tech Research and Development Program of China (2012AA022301) and Priority Academic Program Development (PAPD) for partial funding of this study. References Aachary, A.A., Prapulla, S.G., 2009. Value addition to corncob: production and characterization of xylooligosaccharides from alkali pretreated ligninsaccharide complex using Aspergillus oryzae MTCC 5154. Bioresour. Technol. 100 (2), 991–995. Alvarez-Vasco, C., Zhang, X., 2013. Alkaline hydrogen peroxide pretreatment of softwood: hemicellulose degradation pathways. Bioresour. Technol. 150, 321– 327. Bals, B.D., Teymouri, F., Campbell, T., Jin, M., Dale, B.E., 2011. Low temperature and long residence time AFEX pretreatment of corn stover. BioEnergy Res. 5 (2), 372–379. Bellido, C., Bolado, S., Coca, M., Lucas, S., González-Benito, G., García-Cubero, M.T., 2011. Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis. Bioresour. Technol. 102 (23), 10868–10874. Biswas, R., Uellendahl, H., Ahring, B.K., 2015. Wet explosion: a universal and efficient pretreatment process for lignocellulosic biorefineries. BioEnergy Res., 1–16
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Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition. Bioresour. Technol. 74 (1), 25–33. Qing, Q., Yang, B., Wyman, C.E., 2010. Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresour. Technol. 101 (24), 9624–9630. Shuai, L., Yang, Q., Zhu, J.Y., Lu, F.C., Weimer, P.J., Ralph, J., Pan, X.J., 2010. Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production. Bioresour. Technol. 101 (9), 3106–3114. Singh, J., Suhag, M., Dhaka, A., 2015. Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods: a review. Carbohydr. Polym. 117, 624–631. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2011. Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory Analytical Procedure (LAP), NREL/TP-510-42618. National Renewable Energy Laboratory, Golden, Colorado. 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A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover Heng Yu a, Jiwei Ren b, Lei Liu a, Zhaojuan Zheng a, Junjun Zhu a, Qiang Yong c, Jia Ouyang a,c,⇑ a
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China College of Forestry, Nanjing Forestry University, Nanjing 210037, People’s Republic of China c Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education, Nanjing Forestry University, Nanjing 210037, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 25 June 2015 Received in revised form 24 August 2015 Accepted 25 August 2015 Available online xxxx Keywords: Magnesium bisulfite pretreatment Enzymatic hydrolysis Bio-ethanol production Nonwoody lignocelluloses
a b s t r a c t This study established a new more neutral magnesium bisulfate pretreatment (MBSP) using magnesium bisulfate as sulfonating agent for improving the enzymatic hydrolysis efficiency of corn stover. Using the MBSP with 5.21% magnesium bisulfate, 170 °C and pH 5.2 for 60 min, about 90% of lignin and 80% of hemicellulose were removed from biomass and more than 90% cellulose conversion of substrate was achieved after 48 h hydrolysis. About 6.19 kg raw corn stover could produce 1 kg ethanol by Saccharomyces cerevisiae. Meanwhile, MBSP also could protect sugars from excessive degradation, prevent fermentation inhibition formation and directly convert the hemicelluloses into xylooligosaccharides as higher-value products. These results suggested that the MBSP method offers an alternative approach to the efficient conversion of nonwoody lignocellulosic biomass to ethanol and had broad space for development. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass is abundant, renewable and environmentally friendly resource, which can be converted into bioethanol (Hamelinck et al., 2005). But for the conversion of biomass into bioethanol, the degradation of lignocelluloses is still a major barrier to obtain a high overall ethanol yield. The pretreatment is necessary to breakdown the recalcitrance of lignocellulosic biomass for the second generation bio-ethanol production (Galbe and Zacchi, 2012). An effective pretreatment method can increase the enzymatic digestion performance of lignocelluloses and reduce consumption of enzyme in saccharification stage (Biswas et al., 2015). In the past two decades, variety of pretreatment methods had been discovered, such as lime, dilute acid, steam explosion and organic solvent pretreatment (Singh et al., 2015). However, several problems, including equipment corrosion, environmental pollution, high inhibitor production, and low sugar recovery still restricted the development of pretreatment technology. Dilute sulfuric acid (DA) pretreatment is the most common method due to the low price and the extensive applicability to raw material (Hu and Ragauskas, 2012). But the low pH would cause serious equip⇑ Corresponding author at: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China. Tel.: +86 025 85427129; fax: +86 025 85427587. E-mail address: [email protected] (J. Ouyang).
ment corrosion and produce a great amount of fermentation inhibitors (Bellido et al., 2011). Steam explosion is an effective pretreatment method for improving the enzymatic degradation of lignocellulosic materials. After years of improvement, various steam explosion methods, such as ammonia fiber explosion (AFEX), CO2 explosion and SO2 explosion (Bals et al., 2011), have been developed to adapt different feedstock or further improve the enzymatic degradation of pretreated lignocellulosic material (Singh et al., 2015). However, the main technical barriers for the steam explosion process includes its scalability for commercialization, the high energy consumption and harmful degradation products formation (Zhu and Zhuang, 2012). As for lime pretreatments, the recovery of hemicelluloses sugars and NaOH recovery due to the high cost of lime are still the problems. Therefore, it is necessary to develop new approaches to treat biomass. Recently, a kind of novel sulfite pretreatment (SPORL) derived from sulfite pulping has been extensively studied for wood biomass by Zhu’s groups (Wang et al., 2009; Zhu et al., 2009). Feedstock is firstly treated using a sulfite salt at 160–180 °C and pH 2–4 (H2SO4 + Na2SO3) and then size-reduced using a disk mill (Zhu and Pan, 2010). This pretreatment process has combined effects on dissolution of hemicelluloses, depolymerization of cellulose, and partial delignification (Zhu and Pan, 2010). As a result, pretreated substrate shows great degradability in enzyme hydrolysis and highly adapt to the existing equipment in pulp and paper
http://dx.doi.org/10.1016/j.biortech.2015.08.090 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx
industry (Leu et al., 2013; Li et al., 2014). Additionally, the lignin dissolved in SPORL hydrolysate is sulfated (lignosulfonate), which has more potential application than other lignin forms in established market (Shuai et al., 2010). SPORL is an efficient pretreatment for woody materials. But its effect on nonwoody biomass still needs to be executed. Additionally, the low pH during SPORL is another concern in application. Before the enzymatic hydrolysis, much alkali to neutralize the low pH value is needed because pH 4.8 is the common condition in the enzymatic hydrolysis of cellulose. In order to overcome these defects, in this study, a new more neutral bisulfate pretreatment method, which used magnesium bisulfate (Mg(HSO3)2, pH 5.2) as sulfonating agent, was proposed for bio-refinery of a typical non wood biomass corn stover. The main factors of pretreatment were optimized and the effects of pretreatment conditions on the composition changes and enzymatic digestibility were clearly argued to clarify their connections. Finally, mass balance of corn stover from the raw material to ethanol was established by magnesium bisulfite pretreatment (MBSP).
tank at a solid/liquid ratio of 1:6 (w/v) for pretreatment. In pretreatment process, magnesium bisulfite dose, pretreatment temperature and pretreatment time have been optimized respectively for investigating their effects. After pretreatment, the reactor was immediately cooled down in a water bath. The pretreated substrate was collected by filtration, washed with warm water for at least three times and stored at 4 °C. Solid loss was determined from the measure wet weight and moisture content of the solid. The spent liquor was stored in 20 °C for further analysis. All the experiments were carried out in two replicates. 2.5. Enzymatic hydrolysis Enzymatic hydrolysis was performed at 3% cellulose in 30 mL citrate buffer (pH 4.8, 50 mM), at 50 °C and 150 rpm on a shaker incubator for 48 h. A mixture of Celluclast 1.5 L at 15 FPU/g cellu100
A
2.1. Materials Corn stover was harvested from Lianyungang of Jiangsu province in China. After air-dried and crushed at Biochemistry Institute of Nanjing Forestry University, the corn stover was sieved to achieve the fraction between 20 and 80 mesh for avoiding the interference because of ash. The prepared biomass was stored at room temperature until use. Two commercial enzyme solutions, Celluclast 1.5 L (Cat C2730) and Novozyme 188 (Cat C6105) from Sigma–Aldrich were used for enzymatic hydrolysis. Sulfur dioxide solution (ACS reagent, P6%) was purchased from Sigma–Aldrich. Anhydrous sodium sulfite and magnesium chloride were purchased from Nanjing Chemical Reagent Co., LTD. 2.2. Magnesium bisulfite solution preparation Magnesium bisulfite solution was prepared by mixing solid magnesium sulfite with sulfur dioxide solution in a mole ratio of 1:1 (pH approximately 5.2). The mixture was stirred until insoluble magnesium sulfite particle disappeared. The above mentioned solid magnesium sulfite was prepared by mixing sodium sulfite with magnesium chloride in a mole ratio of 1:1. And then, the residual soluble reactants were eluted three times with water. Afterward, magnesium sulfite was dried by lyophilization.
60 40 20 0 0
1.76
3.51
5.27
7.02
Magnesium bisulfite concentration (%) 100
B 80
Removal (%)
2. Methods
Removal (%)
80
60 40 20 0 140
150
160
170
180
o
Temperature ( C)
100
C
2.3. Microorganism and cultivation Saccharomyces cerevisiae AQ was provided by Anqi Company, China, and grown at the medium for inoculation contained (g/L): glucose, 20; peptone, 20; yeast extract, 10 at natural pH (YPD). The seed culture was prepared as follows: a loop of cells from the fully grown slant was inoculated into 100 ml of the above medium in 250 ml Erlenmeyer flasks and incubated for 24 h at 30 °C with 150 rpm. Then, the seed cells were harvested, washed and inoculated into Erlenmeyer flasks or bioreactors for ethanol production. 2.4. Pretreatment This laboratory scale pretreatment was carried out in an electrically heated oil bath. A total of 3 g of biomass sample and 18 mL pretreated solution was placed in a 30 mL sealed stainless steel
Removal (%)
80 60 40 20 0 0
20
40
60
80
Pretreatment time (min) Solids Cellulose Hemicellulose Lignin Fig. 1. Influences of different MBSP process conditions on removal ratio of solid and three main compositions in corn stovers (all not listed conditions in figure were: magnesium bisulfite content, 3.51% (w/v); temperature, 170 °C; pretreatment time, 60 min).
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx Table 1 Content of five known fermentation inhibitors and sugars in spent liquor collected from MBSP process with different condition. Pretreatment conditions
Fermentation inhibitors concentrations (g/L)
Sugar concentrations (g/L)
Mg(HSO3)2 concentration (%, w/v)
Temperature (°C)
Pretreatment time (min)
Formic acid
Acetic acid
Furfural
HMF
Levulinic acid
Glucose
Xylose
XOS
0 1.76 3.51 5.27 7.02
170
60
0.433 0.081 n/a n/a n/a
2.750 3.693 4.337 4.410 3.845
0.734 1.404 0.201 0.080 0.067
0.039 0.062 n/a n/a n/a
0.016 n/a n/a n/a n/a
0.385 0.433 0.319 0.339 0.323
0.993 3.036 2.226 1.747 1.535
10.306 10.714 9.903 10.880 9.739
3.51
140 150 160 170 180
60
n/a n/a n/a n/a n/a
0.815 1.969 3.101 4.337 5.201
n/a n/a 0.024 0.201 2.076
n/a n/a n/a n/a 0.307
n/a n/a n/a n/a n/a
0.500 0.307 0.243 0.319 0.363
0.129 0.126 0.457 2.226 0.875
4.397 10.247 19.710 9.903 2.193
3.51
170
0 20 40 60 80
n/a n/a n/a n/a n/a
1.708 2.623 3.467 4.337 4.678
n/a n/a 0.022 0.201 0.705
n/a n/a n/a n/a 0.115
n/a n/a n/a n/a n/a
0.335 0.266 0.238 0.319 0.303
0.125 0.323 0.881 2.226 3.101
8.660 18.232 16.579 9.903 7.709
n/a: not available.
lose and Novozyme 188 at 30 CBU/g cellulose was used for enzymatic hydrolysis. After hydrolysis, the samples were withdrawn and centrifuged to remove the insoluble materials. The supernatants were subsequently filtered through a 0.22 lm syringe filter (Millipore, Co., LTD) and used for subsequent HPLC analysis. All experiments were performed in replicates and each data point was the average of two replicates.
hemicellulose or lignin) in pretreated material. The units of measurement were g. The cellulose hydrolysis yield was calculated according to the following equation:
2.6. Fermentation
where Cglu was the concentration of glucose in enzymatic hydrolysate; Cbiose was the concentration of cellobiose in enzymatic hydrolysate; CCel was the initial concentration of cellulose. The units of measurement were g/L. The ethanol yield was calculated according to the following equation:
Fermentation was carried out in 250 mL Erlenmeyer flask at 30 °C and 150 rpm for 24 h with 50 mL of enzymatic hydrolysate adding 6 g/L yeast extract, 10 g/L peptone, 5 g/L (NH4)2SO4, 10 g/L KH2PO4, 0.5 g/L MgSO47H2O and 0.15 g/L CaCl2H2O. Each flask was equipped with a needle-pierced silicone stopper to allow removal of the produced CO2 during fermentation. The initial yeast inoculum was OD 10 (600 nm) and initial pH was controlled in 4.8. At the end of fermentation, samples of the broth were centrifuged at 5000g for 10 min for sugar and ethanol analysis. Reported data is the average of duplicates.
Enzymatic hydrolysis yield ð%Þ ¼
ðC glu þ 1:053 C biose Þ 0:9 100 C cel
Ethanol yield ð%Þ ¼
C eth 100 DC glu 0:51
ð2Þ
ð3Þ
where Ceth was the concentration of ethanol; DCglu was the consumption of glucose in fermentation process; 0.51 was the transformation factor from glucose to ethanol. The units of measurement were g/L.
2.7. Analytical methods 3. Results and discussion The chemical compositions of the original and pretreated corn stover were determined according to the National Renewable Energy Laboratory (NREL, Golden) analytical methods for biomass (Sluiter et al., 2011). The free fermentable sugars (glucose, cellobiose, and xylose), several known inhibitors (formic acid, acetic acid, levulinic acid, furfural, 5-hydroxymethylfurfural (HMF)) and ethanol were directly quantified using a HPLC system (1200 series, Agilent) equipped with a Bio-Rad Aminex HPX-87H column (300 7.8 mm). 5 mM H2SO4 as mobile phase was controlled at a flow rate of 0.6 mL/min at 55 °C, analysis signal was detected by a refractive index detector (Ouyang et al., 2013). As for the analysis of xylooligosaccharide (XOS), 1 mL sample was mixed with 1 mL of 5 N H2SO4 and hydrolyzed at 120 °C for 45 min to convert XOS to their constitutive monomers and analyzed by HPLC to quantify the total amount of XOS (Nabarlatz et al., 2007). The removal of each composition (%) was calculated according to the following equation:
Removal of composition ð%Þ ¼
ðmini mtre Þ 100 mini
ð1Þ
where mini was the mass of composition (cellulose, hemicellulose or lignin) in raw material; mtre was the mass of composition (cellulose,
3.1. Removal of the lignocellulosic components during pretreatment The original corn stover used in the present study had a dominant cellulose content of 37.5% and low lignin content of approximately 22.1%. The hemicellulose content was 18.5%. As described in the Section 2.4, corn stover was pretreated by MBSP at varied chemical loadings, pretreatment temperature and time. The loss of dry matter and three key components following MBSP employ different conditions was assessed and presented in Fig. 1. The effect of magnesium bisulfite dose on the loss of three components at the pretreatment temperature of 170 °C and the pretreatment time of 60 min is shown in Fig. 1A. With the increase of magnesium bisulfate loading, the removal of lignin is more pronounced than the removal of hemicellulose. Hot water pretreatment resulted that the removal of lignin and hemicellulose were 13.94% and 65.76%, respectively. The increase in magnesium bisulfite dose enhanced delignification significantly. Due to the sulfonation of lignin, about 60% of lignin was removed when the chemical dose was 1.76%. In terms of hemicelluloses, it was well known that the acid environment promotes the solubilization of hemicellulose (Garlock et al., 2011). Dilute acid pretreatment could dissolve all
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
H. Yu et al. / Bioresource Technology xxx (2015) xxx–xxx
Enzymatic hydrolysis yield (%)
4
100
A 80 60 40 20 0 0
1.76
3.51
5.27
7.02
Enzymatic hydrolysis yield (%)
Magnesium bisulfite concentration (%) 100
B 80 60 40 20 0 140
150
160
170
180
o
Enzymatic hydrolysis yield (%)
Temperature ( C) 100
C 80
20 min and gradually approach the limitation. When pretreatment time was 60 min at 170 °C with 3.51% magnesium bisulfite, the maximum lignin and hemicellulose removal ratio were 82.45% and 80.73%, respectively. Several different pretreatment methods were applied to enhance the enzymatic hydrolysis of lignocellulosic material by changing the composition of the biomass. DA pretreatment could degrade almost hemicelluloses from the biomass, leaving only cellulose and lignin in the DA substrates (Li et al., 2014). In SPORL pretreatment, above 90% hemicellulose could be removed while 20–40% lignin loss (Wang et al., 2009). Comparing with these pretreatment methods, MBSP showed more effect on the removal of lignin and hemicellulose while cellulose was relatively stable during pretreatment process. A large portion of lignin and hemicellulose were degraded and dissolved during the pretreatment. By contrast, only minor cellulose loss (approximately 5.5%) was discovered. When the corn stover was treated by 5.27% magnesium bisulfite at 170 °C for 60 min, the loss of hemicellulose and lignin could reach 89.10% and 80.87%, respectively. Correspondingly, the chemical composition of pretreated corn stover was 82.22% cellulose, 8.25% hemicellulose and 5.62% lignin. Lignin was universally regarded as main obstacle in enzymatic saccharification process, which led to the inactivation of cellulase due to its nonproductive adsorption of enzyme (Wang et al., 2011). MartinSampedro deemed that this phenomenon would become worse in high solid concentration enzymatic hydrolysis due to the lignin accumulation (Martin-Sampedro et al., 2013). The BSG hemicelluloses also exerts a negative influence on the enzymes attack to cellulose (Mussatto et al., 2008). These reports revealed that the partial removal of hemicelluloses and lignin would be beneficial for improving the enzymatic hydrolysis of lignocelluloses after MBSP pretreatment. Additionally, it was worth mentioning that, the detached lignin existed as soluble lignosulfonate, which was valuable in many areas (Xavier et al., 2010; Yu et al., 2014).
60
3.2. Pretreatment spent liquor (hydrolysate) composition
40
As discussed above, a large amount of hemicelluloses and lignin were removed from the biomass to the spent liquor during the pretreatment process. An important point that must be considered is regarding the changes of their existing forms in hydrolysates. Especially some of compounds derived from the sugar degradation had been known as potential inhibitors for subsequent fermentation (Palmqvist and Hahn-Hägerdal, 2000). Considering that the formation of these compounds was closely correlated with pretreatment conditions, the amount of several known fermentation inhibitors and soluble sugars in spent liquor were listed in Table 1 as a standard to evaluate the degradation of three key components during MBSP process. The furan aldehydes furfural and HMF, which are formed by dehydration of pentose and hexose sugars, respectively, are commonly found in lignocellulose hydrolysates (Jönsson et al., 2013). In present study, HMF and furfural decreased with increased magnesium bisulfite dose. An explanation is that more magnesium bisulfate depressed the decomposition of the sugar to HMF and furfural in a relatively higher pH (5.2–2.7). A similar phenomenon was also discovered in SPORL pretreatment (Zhu et al., 2009). The data in Table 1 also indicated that increasing pretreatment temperature and pretreatment time led to the production of HMF and furfural as expected. At high temperature, HMF would be further degraded and form levulinic acid and formic acid, the latter was also formed from furfural under acidic conditions (Palmqvist and Hahn-Hägerdal, 2000). Only little formic acid and levulinic acid were detected in the pretreatment spent liquor while magnesium bisulfite dose was in a low level (1.76%). It suggested that the degradation of HMF and furfural was negligible under studied
20 0 0
20
40
60
80
Pretreatment time (min) Fig. 2. Influences of different MBSP process conditions on enzymatic hydrolysis yields of corn stovers (all not listed conditions in figure were: magnesium bisulfite content, 3.51% (w/v); temperature, 170 °C; pretreatment time, 60 min).
hemicellulose from the biomass (Shuai et al., 2010). In the present study, the removal of hemicellulose ranged from 65.76% to 80.87%, which indicated MBSP also could dissolve part of hemicelluloses under pH 5.2. About 90% of lignin and 80% of hemicellulose removal were obtained by adding 5.27% magnesium bisulfite. Accordingly, increasing pretreatment temperature not only created the condition for dissolving out of hemicellulose, but also promoted the sulfonation of lignin by magnesium bisulfate (Fig. 1B). Alvarez-Vasco and Zhang reported that the degradation of hemicellulose in high temperature was due to thermal instability of hemicelluloses (Alvarez-Vasco & Zhang, 2013). An evident loss of hemicelluloses was started at 150 °C in MBSP process. The lignin removal also began at 150 °C. But when temperature reached 180 °C, the sulfurization was weakened, which reflected the higher temperature (>180 °C) was adverse to sulfonation of lignin in MBSP process. As for pretreatment time (Fig. 1C), the removal of the lignin, hemicelluloses and solid were sharply increased in first
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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Fig. 3. Mass balance of the optimum MBSP process for bio-ethanol product.
pretreatment conditions. As for acetic acid, it is a main co-product, which is formed primarily by hydrolysis of acetyl groups of hemicelluloses (Palmqvist and Hahn-Hägerdal, 2000). The amount of acetic acid increased greatly with increasing the temperature and time while the chemical dose did not show an obvious effect. When magnesium bisulfate concentration achieved 3.51%, acetic acid concentration increased to 4.337 g/L. In addition, the analysis of soluble sugar showed that MBSP produced less xylose and glucose in pretreatment spent liquor than SPORL and DA pretreatment. It might be caused by a higher pH environment, compared with pH 1.2 in the DA pretreatment and 2.7 in the SPORL pretreatment (Shuai et al., 2010). More interestingly, products from the hemicelluloses in the liquid phase were present in form of XOS. XOS is a class of non-digestible food ingredient, which could be produced by chemical methods, auto hydrolysis and enzymatic hydrolysis (Aachary & Prapulla, 2009). For the MBSP, a maximum amount of XOS (19.71 g/L) in the hydrolysate was found at 160 °C, 60 min and 3.51% magnesium bisulfite dose. This phenomenon meant magnesium bisulfite not only could prevent excessive degradation of sugar, but also contributed to the production of XOS by the restricted degradation (Yu et al., 2014). 3.3. Enzymolysis of the pretreated biomass To evaluate the effect of MSBP on the enzymatic hydrolysis of corn stover, the pretreated biomass was hydrolyzed for 48 h at enzyme loading of 15 FPU cellulase and 30 CBU b-glucosidase per gram of cellulose (Fig. 2). The enzymatic digestibility of the biomass was greatly improved after MBSP process. Hydrolysis yield was positively correlated with magnesium bisulfite concentration. The MBSP sample presented the highest hydrolysis yield of 90.44% at 5.27% magnesium bisulfite concentration. Otherwise, hot water pretreated corn stover only showed a 31.02% yield at the same condition. It indicated that adding magnesium bisulfite was able to effectively improve the enzymatic degradation of corn stover, which might be mainly due to the lignin removal (Fig. 2A). The lignin is generally believed as one of the most limiting factors in enzymatic hydrolysis of cellulose (Donohoe et al., 2008). Furthermore, according to the results from Fig. 1A, increasing magnesium
bisulfate dose influenced the lignin removal more than the hemicelluloses loss. The temperature was also the main factor in improving enzymatic degradation of corn stover (Fig. 2B). Increase of pretreatment temperature is contributed to the removal of hemicelluloses, especially over 150 °C (Fig. 1B). When pretreatment temperature reached to 170 °C, the enzymatic hydrolysis yield was 80.12%. Furthermore, the maximum hydrolysis yield (93.78%) was achieved at 180 °C. Similar result was achieved by Wei et al. in the process of dilute acid pretreated eucalyptus chips (Wei et al., 2012). It was noteworthy that the high temperature (above 180 °C) made the harmful degradation of sugar. Some strong inhibitor of fermentation was detected such as furfural and HMF. Considering that the presence of theses by-products of pretreatment is disadvantage of sequent fermentation, 170 °C seems to be a suitable temperature for pretreatment. As for pretreatment time, both biomass loss and enzymatic hydrolysis yield exhibited a clear increasing trend with time. The enzymatic hydrolysis yield increased to the maximum at 80.12% when the removal of lignin and hemicellulose approach saturated at pretreatment time of 60 min (Fig. 2C). Continuing to increase pretreatment time did not further improve the degradation of pretreated corn stover, but more harmful degradation product would form (Table 1). In summary, the enzymatic digestibility of the pretreated biomass improved with increasing pretreatment severity. The above findings from components analysis and hydrolysis results suggested that the lower the hemicelluloses and lignin contents in the pretreated biomass, the better the performance of cellulose enzymatic hydrolysis. It was agreement with many previous studies (Öhgren et al., 2007; Qing et al., 2010). The effect of MBSP on can be attributed to the solubilization of the hemicelluloses and lignin fractions. In this study, the pretreatment condition (5.27% magnesium bisulfite, at 170 °C for 60 min) was selected for the follow-up fermentation evaluation and mass balance according to optimization experiments. 3.4. Mass balance of MBSP process for bio-ethanol production During bio-ethanol production process, an ideal pretreatment is not only able to produce an effective enzymatic hydrolysis and
Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090
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excellent fermentation, but also to make full use of available sugar in biomass. According to this objective, enzymatic hydrolysate of MBSP pretreated corn stover was directly used to ethanol fermentation by S. cerevisiae and the ethanol yield of 93.46% were achieved after 12 h fermentation, which did not show obviously inhibitory effect from the enzymatic hydrolysate of MBSP pretreated corn stover. Afterward, preliminary mass balance of the MBSP process with 5.21% magnesium bisulfite, at 170 °C for 60 min for ethanol production was summarized in Fig. 3. There were 375.2 g cellulose, 185.4 g hemicellulose, and 221.4 g lignin from 1000 g oven dry raw corn stover. After MBSP, 429.8 g pretreated corn stover (containing 353.5 g cellulose, 35.5 g hemicellulose and 24.1 g lignin) was achieved. Enzymatic hydrolysis of the pretreated biomass resulted 341.9 g glucose, 12.6 g cellobiose and 30.6 g xylose produced in hydrolysate with low dosage of enzyme (15 FPU and 30 CBU/g cellulose). When ethanol fermentation was performed by S. cerevisiae, about 162.2 g ethanol was obtained from 1000 g oven dry raw corn stover, even xylose did not be utilized during the fermentation. The results clearly indicated that MBSP is an effective pretreatment method and about 6.17 kg of raw corn stover could transform to 1 kg ethanol by MBSP. Total detected XOS in the pretreatment spent hydrolysate was 65.28 g. Besides this, the spent liquor of MBSP also contained abundant valuable lignosulfonate (data not shown), which made the spent liquor had applicable value in many respects, such as: concrete technology (Ouyang et al., 2009), dispersant (Yang et al., 2007) and enzyme catalysis (Wang et al., 2013). 4. Conclusions MBSP method proposed in this study offers an alternative approach to efficient convert nonwoody lignocellulosic biomass to ethanol. After MBSP, about 90% of lignin and 80% of hemicellulose was removed from biomass and more than 90% cellulose conversion of substrate was achieved for 48 h hydrolysis. In bioethanol production using MBSP, 6.19 kg raw corn stover could produce 1 kg ethanol. Besides these, the great advantage of MBSP is that it could protect sugars from excessive degradation, prevent fermentation inhibition formation and directly convert the hemicelluloses into xylooligosaccharides as higher-value products. Acknowledgements This study was supported by the National Natural Science Foundation of China (31200443) and Excellent Youth Foundation of Jiangsu Province of China (BK2012038). The authors are also grateful to the National Hi-Tech Research and Development Program of China (2012AA022301) and Priority Academic Program Development (PAPD) for partial funding of this study. References Aachary, A.A., Prapulla, S.G., 2009. Value addition to corncob: production and characterization of xylooligosaccharides from alkali pretreated ligninsaccharide complex using Aspergillus oryzae MTCC 5154. Bioresour. Technol. 100 (2), 991–995. Alvarez-Vasco, C., Zhang, X., 2013. Alkaline hydrogen peroxide pretreatment of softwood: hemicellulose degradation pathways. Bioresour. Technol. 150, 321– 327. Bals, B.D., Teymouri, F., Campbell, T., Jin, M., Dale, B.E., 2011. Low temperature and long residence time AFEX pretreatment of corn stover. BioEnergy Res. 5 (2), 372–379. Bellido, C., Bolado, S., Coca, M., Lucas, S., González-Benito, G., García-Cubero, M.T., 2011. Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis. Bioresour. Technol. 102 (23), 10868–10874. Biswas, R., Uellendahl, H., Ahring, B.K., 2015. Wet explosion: a universal and efficient pretreatment process for lignocellulosic biorefineries. BioEnergy Res., 1–16
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Please cite this article in press as: Yu, H., et al. A new magnesium bisulfite pretreatment (MBSP) development for bio-ethanol production from corn stover. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.08.090