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Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw
Accepted Manuscript Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw Youzhi Dai, Mengying Si, Yuehui Chen, Nianlei Zhang, Mo Zhou, Qi Liao, Deqiang Shi, Yine Liu PII: DOI: Reference:
S0960-8524(15)01370-X http://dx.doi.org/10.1016/j.biortech.2015.09.091 BITE 15600
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
2 August 2015 22 September 2015 23 September 2015
Please cite this article as: Dai, Y., Si, M., Chen, Y., Zhang, N., Zhou, M., Liao, Q., Shi, D., Liu, Y., Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.09.091
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Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw
Youzhi Dai a, Mengying Si a, Yuehui Chen a,∗, Nianlei Zhang a, Mo Zhou a, Qi Liao b, c Deqiang Shi d, Yine Liu a
a Department of Environmental Science and Engineering, Xiangtan University, Xiangtan City 411105, China. b Department of Environmental Engineering, School of Metallurgy and Environment, Central South University, Changsha, 410083, China. c Chinese National Engineering Research Centre for Control and Treatment of Heavy Metal Pollution, Changsha, 410083, China. d Guangxi Academy of Sciences, Nanning City, Guangxi Zhuang Autonomous Region 530007, China.
∗
Corresponding author
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1
Abstract A stepwise pretreatment of combination of bacterial treatment with NaOH/Urea (NU) treatment was conducted to enhance enzymatic hydrolysis of rice straw (RS). The results showed that the composition of RS changed significantly, the lignin and hemicellulose decreased while cellulose increased. The biological treatment with a bacterium Sphingobacterium sp. LD-1 achieved mild conditions for the sequential NU treatment, reducing the concentration of the NU solution from 7%/12% to 4%/6% and increasing the temperature from -20 °C to -10 °C. The saccharification of rice straw co-treated with bacterium Sphingobacterium sp. LD-1 and 4%/6% NU at -10 °C resulted in 1.396-fold and 1.372-fold increase of reducing sugar and glucose yield respectively than that of sole NU treatment. Keywords: Lignocellulose pretreatment, Sphingobacterium sp. LD-1, NaOH/Urea, Rice straw, Enzymatic hydrolysis
1. Introduction Recent years, to reduce the reliance on fossil fuels, governments have initiated the extensive research into the renewable energies (Dyk et al., 2012). The breakdown of plant lignocellulose (such as corn stover, bagasse, rice straw, and dedicated energy crops) to glucose monomers is the basis for second-generation cellulosic bio-ethanol production (Pickett et al., 2008). Among this rice straw is to be considered as the largest available biomass in the world which is about 731 million tons of dry rice straw annually and Asia is the largest region in the world which is responsible for 90% of the annual global production (Karimi et al., 2006). Since rice straw is high in cellulose (~45% w/w) content and hemicellulose (~25% w/w) content (Binod et al., 2010), it has the potential to be converted to more fermentable sugars by enzymatic saccharification. Conversion of lignocelluloses to ethanol employs three major steps: (1) pretreatment, to break down the recalcitrant structures of lignocellulose; (2) enzymatic hydrolysis, to hydrolyze polysaccharides (e.g., cellulose, hemicellulose) into fermentable sugars; and (3) fermentation, to convert sugars into ethanol (Huang et al., 2011). Nevertheless, pretreatment is essential to make biomass digestible by altering biomass structural features since structural modifications of lignocelluloses are highly dependent on the type of pretreatment employed and have a great effect on enzymatic hydrolysis (Zhu et al., 2010). Furthermore, the pretreatment constitutes more than one-third of the total production costs, which makes it to be a major barrier in the process of bio-ethanol conversion. Therefore, a number of different technologies have
been developed for pretreatment of lignocellulose. The typical pretreatment methods, such as acid, alkali, and steam explosion, have some disadvantages in the commercialization of lignocelluloses conversion, including inefficiency, special equipment requirements and a lot of energy consumption (Mosier et al., 2005). Microbial pretreatment, as an environmental friendly and low cost method, is attracting increasing attention in recent years (Jin et al., 2009; Salame et al., 2012). To date, the biological pretreatments mainly focused on white rot fungi which showed ability to break down and mineralize lignin (Wang et al., 2012). However, relative low efficiency, considerable loss of carbohydrates and long residence periods are the three major disadvantages for the fungal pretreatment (Yu et al., 2009). New strategies should be adopted to overcome these feeble sides. In recent years, a number of bacteria that were able to break down lignin were reported, and the lignin-degrading bacteria seemed to play an emerging role in decomposing lignin because of the rapider growth and easier genetic manipulation as compared with fungi (Bugg et al., 2011; Bugg et al., 2015). However, bacterial pretreatment in conversion of lignocellulose was rarely reported. In our previous study, a bacterial strain Sphingobacterium sp. LD-1 (CGMCC No.10920) was isolated from the effluent sludge of a pulp paper mill, which showed high activity in lignocellulose degradation. In addition, sodium hydroxide with urea was employed to dissolve cellulose completely to produce regenerated cellulose by Zhang’s group (Cai et al., 2005; Zhang et al., 2002; Zhou et al., 2004). These reports showed that
NaOH/Urea pretreatment had a great potential in conversion of lignocellulosic biomass to ethanol. To obtain a pretreatment method with short biological pretreatment period, low energy consumption and common equipment requirements, a stepwise pretreatment of combination of bacterial treatment with NaOH/Urea (NU) treatment was employed in this work. The RS was dealt with by Sphingobacterium sp. LD-1, and then treated with NU. To evaluate the effects of the combined pretreatment, changes in the components of the RS were investigated. Sugar yields of enzymatic hydrolysis of the pretreated RS were also tested. To gain insight into the mechanism of the combined pretreatment, Total organic carbon (TOC) and Ultraviolet-visible spectroscopy (UV-Vis) analysis of supernatant were measured. And scanning electron microscopy (SEM) and the Fourier transform infrared spectroscopy (FTIR) were used to observe the structural change of the RS after the pretreatments.
2. Methods 2.1. Microorganism and Rice straw The degradation of alkali lignin, sodium carboxymethylcellulose and xylan by Sphingobacterium sp. LD-1 was all more than 40% in 5 days (data unpublished), which showed high potentiality for lignocellulose pretreatment. The bacterium LD-1 was cultured on a rotary shaker at 30 °C with a speed of 125 rpm in Luria-Bertani broth medium for 18 h. The Luria-Bertani broth grown bacterial culture was used as seed culture for biological pretreatment.
RS from countryside of Harbin was ground into powder and air-dried. The dried RS powder was then sifted by a 40-mesh griddle and used as the feedstock for the pretreatment. 2.2 Pretreatment All experiments were performed in duplicate for result confirmatory purposes. All analyses were carried out in triplicates and the average values were presented in this text. 2.2.1. Biological pretreatment 100 ml of the seed culture was centrifuged to harvest bacterial cells. The collected cells were then inoculated into 1000 ml sterile mineral salt medium containing 3 g RS. The composition of mineral salt medium was as follows (g l-1 de-ionized water): (NH4)2SO4 2, K2HPO4 1, MgSO4 0.2, CaCl2 0.1, FeSO4 0.05, MnSO4 0.02, KH2PO4 1, pH 7.0. The biological pretreatment was carried on a rotary shaker at 30 °C with a speed of 125 rpm. After 4 d incubation, the bio-treated RS sample was collected and washed with distilled water thoroughly, and then dried at 55 °C for further experiments. 2.2.2. NU pretreatment Series of condition experiments of NU concentration and temperature for NU pretreatment were done. 1 g untreated RS was soaked in a 20 ml solution of different concentration of NU (2%/3%, 4%/6%, 7%/12%) and then treated statically at different temperatures (-20 °C, -10 °C, 4 °C, 20 °C) for 4 h. Likewise, the bio-treated RS was performed aforementioned experiments.
Subsequently, the sole NU treated and co-treated RS samples were washed with distilled water thoroughly and then dried at 55 °C for further analysis. 2.3. Chemical composition measurement of RS The chemical composition of the RS were measured according to the laboratory analytical procedures of the National Renewable Energy Laboratory (Sluiter, 2008). 2.4. Enzymatic hydrolysis and measurement of sugar yield Commercial cellulase produced by Trichoderma viride was purchased from Sinopharm Chemical Reagent. A typical hydrolysis mixture consisted of 0.5 g RS sample, 20 ml of 50 Mm citric acid buffer (pH 4.8) supplemented with antibiotic (40 µg ml-1) and cycloheximide (30 µg ml-1) to prevent microbial contamination, and 300 U g-1 substrate of cellulase. The mixture was incubated at 47.5 °C in a rotary shaker at 100 rpm for 72 h. After incubation, samples were collected and centrifuged for sugar analysis. The reducing sugar and glucose were measured by the DNS assay (Miller, 1959) and enzyme-colorimetric method (the Standard of the People’s Republic of China, GB/T 16285-2008), respectively. 2.5. Analytical methods The washed and air-dried RS samples were used for SEM and FTIR analysis. The lixivium were used for TOC and UV-Vis analysis. 2.5.1. TOC and UV-Vis analysis of supernatant After LD-1 treatment, NU treatment and combination treatment, respectively, the samples were centrifuged and supernatant was collected for TOC and UV-Vis analysis.
The supernatant from untreated sample (0.5 g RS was soaked in 20 ml distilled water at room temperature for 4 h) was used as control. 1 ml sample was diluted with water to 10 ml then filtered with 0.45 µm membrane filters and the concentration of TOC and the UV-Vis were measured by TOC (Shimadzu TOC-VCPH) analyzer and Ultraviolet-visible spectroscopy (Shimadzu UV-2550), respectively. 2.5.2. SEM observation Some samples obtained after various pretreatments coated with gold using a sputter coater were observed under a SEM (JEOL JSM-6360LV) observation. 2.5.3. FTIR analysis. 2 mg RS sample was prepared by mixing with 120 mg of spectroscopic grade KBr then pressed in a standard device to produce 13 mm diameter pellets. The background spectrum of pure potassium bromide was subtracted from that of the sample spectrum. FTIR spectra of samples were obtained with analysis performed on a Thermo Scientific Nicolet-380 FTIR spectrometer in the wave number range of 4,000-500 cm−1. 3. Results and discussion 3.1. Effects of pretreatments on chemical composition As one of the main components of plant cell wall, the lignin provides a physical barrier which limits accessibility of cellulases to their substrate (Várnai et al., 2010). To expose the highly ordered crystalline structure of cellulose and facilitate substrate access by hydrolytic enzymes, reducing the lignin content of the biomass is expected
(Sun et al., 2002). After various pretreatments, the chemical composition in residues was shown in Fig.1. The content of cellulose, hemicellulose and lignin was 51.7%, 20.4% and 12.7% in untreated RS, respectively, while was 57.2%, 24.2% and 6.1% in LD-1 treated RS (Fig. 1A). As can be seen from the results, significant decrease of lignin had happened in the step of bacterial treatment. In other words, the bacterium Sphingobacterium sp. LD-1 showed the capacity of delignification in RS pretreatment. Over the years, fungi especially white rot fungi were thought to the most promising organisms that can efficiently metabolize lignin in various of lignocellulosic materials (Hatakka, 1983; Yu et al., 2010). However, the long processing cycle (more than 30 d in most cases) of fungal treatment was a big obstacle for industrial application. In recent years, because of the rapid growth and easy genetic manipulation, bacteria are increasingly being taken as the emerging role in lignin degradation (Chen et al., 2012a; Chen et al., 2012b; Salvachua et al., 2015). The results in Fig.1B showed that the chemical composition of NU treated RS had changed significantly as compared with the untreated RS. The content of lignin reduced to 5.1%-11.8% from 12.7%, whereas the cellulose content increased to 56.8% - 69.2% from 51.7%. In recent years, Zhang’s research group found that sodium hydroxide with urea (7%/12% NU) at -20 °C can dissolve cellulose completely to produce regenerated cellulose. This method was also used to enhance enzymatic hydrolysis by cellulose dissolution pretreatment (Kuo et al., 2009; Li et al., 2010). During the NU pretreatment,
the sodium hydroxide destroyed inter- and intra-hydrogen bonds between cellulose molecules. Simultaneously, urea hydrates function as hydrogen bonds donor and receptor between solvent molecules and prevented the reassociation of cellulose molecules, causing the cellulose depolymerization and dissolution. Accordingly, NU treatment showed the potential of application in lignocellulose pretreatment. However, this method required demanding conditions (such as freezing-thawing and stirring violently), making large-scale pretreatment of lignocellulose difficult. In this study, improved NU treatment without freezing-thawing and stirring was employed for the lignocellulose pretreatment. Removal of lignin, reduction of cellulose polymerization and disruption of the lignocellulose structure were all the purposes of lignocellulose pretreatment. In this sense, the favorable effects were obtained under milder conditions. As the results shown in Fig. 1B, the cellulose content increased and the lignin content decreased, whether the RS was treated with lower NU concentration (2%/3%) or at higher temperature (20 °C). Furthermore, the combination pretreatment of bacterial treatment with NU treatment was carried out in this study and the chemical composition of co-treated RS was shown in Fig.1C. The content of cellulose and hemicellulose in co-treated RS were 62.7%-73% and 10.6%-20%, respectively. Compared to sole bacterial or NU pretreatment, an increase trend in cellulose content and a decline trend in hemicellulose content had appeared, but no obvious change in lignin content. The results indicated that the effects of co-pretreatment with LD-1 and NU were helpful for the saccharification
of RS. 3.2. Effects of pretreatment on enzymatic hydrolysis To evaluate the efficiency of various pretreatments, the RS residues were subsequently hydrolyzed with commercially available cellulase for 72 h and the results were shown in Fig. 2 and Fig. 3. The enzymatic hydrolysis was inefficient without pretreatment, and the yield of reducing sugar and glucose was only 1.73 and 1.09 mg ml-1, respectively. Even after LD-1 treatment for 4 d, the yield of reducing sugar and glucose slightly increased to 1.93 and 1.14 mg ml-1, respectively, indicating that saccharification of RS had barely enhanced by sole LD-1 treatment. These results can be attributed to the unmodified crystalline structure of cellulose and hindrance of lignin (Chandra et al., 2007). As a consequence, other treatment combined with LD-1 treatment should be introduced for a better enzymatic hydrolysis. With regard to the sole NU treatment, the reducing sugar yield was in a range from 4.87 to 8.51 mg ml-1 as well as from 3.18 to 6.12 mg ml-1 for glucose, showing a significant enhancement of enzymatic hydrolysis (Fig. 2A). In addition, the saccharification was improved with the increase of NU concentration, and the similar results were obtained as the treatment temperature decreased. The maximum production of sugars was obtained with 7%/12% NU treatment at -20 °C and the yield of reducing sugar and glucose increased 4.92 and 5.61-fold, respectively, compared to untreated sample. These increases of sugar yields had proved that the NU treatment could be used
to enhance the enzymatic hydrolysis for lignocellulose. When it went to combination pretreatments, the reducing sugar and glucose yields of co-treated RS with LD-1 and NU were 5.49-9.30 mg ml-1 and 4.13-6.12 mg ml-1, respectively, higher than that of sole NU treatment (Fig. 2B). An obvious increase of the sugar yields was obtained under low concentration of NU (2%/3% and 4%/6%), and a slight increase was obtained under high concentration of NU (7%/12%). The results suggested that the synergy of combination pretreatment was greater at moderate condition. The increase of saccharification of combination pretreatment can be attributed to the synergistic effect of biological and NU treatment. The reason might be the LD-1 degraded and modified lignin in RS, which made the accessibility of NU to lignin easier (Ma et al., 2010). But the intense chemical pretreatment conditions might cover up the effect of the biological pretreatment, the similar conclusions were obtained Ma’s (Ma et al., 2010) and Yu’s researches (Yu et al., 2009). The yield of reducing sugar and glucose was 9.25 and 5.98 mg ml-1 respectively under co-treated with LD-1 and 4%/6% NU at -10 °C, showing almost the same result under 7%/12% NU at -20 °C. However, in sole NU treatment, the result of 4%/6% NU at -10°C was obviously lower than that of 7%/12% NU -20 °C. Therefore, the first step of LD-1 treatment could reduce the requirements of the second step of NU treatment in the combination pretreatment process. After LD-1 treatment, the concentration of NU solution decreased from 7%/12% to 4%/6% and the temperature increased from -20 °C to -10 °C. The results suggested that the moderate conditions for NU treatment could be
obtained after LD-1 treatment, making the enzymatic conversion more promising. The temperature of -10 °C was easy to obtain without a lot of energy consumption. Even in most areas of northern China, the temperature below -10 °C usually occurs in winter. Considering the industrial application, the optimum conditions for combination pretreatment would be 4%/6% NU at -10 °C in this study. The time course of enzymatic hydrolysis of RS after various pretreatments can be seen in Fig. 3, showing that the enzymatic hydrolysis of RS was limited without treatment and a slight enhancement after LD-1 treatment. Meanwhile, in contrast with LD-1 pretreatment, the NU pretreatment showed a more effective saccharification. Furthermore, saccharification of RS co-treated with LD-1 and 4%/6% NU at -10 °C resulted in a significant increase of sugar yields (1.396-fold and 1.372-fold of reducing sugar and glucose, respectively) than that of sole NU treatment. These results indicated a synergistic effect existed in the stepwise pretreatment of combination of LD-1 treatment with NU treatment. As an economical and comparative environment-friendly process, many investigations concerning biological treatment or combination of biological treatment with chemical treatment have been reported (Wan et al., 2011; Wang et al., 2012; Yu et al., 2010; Yu et al., 2009). Table 1 summarized the recent reports of combined biological and chemical pretreatments similar with this work. Compared to the previous results in Table 1, in general, the combination of LD-1 pretreatment with NU pretreatment in this work demonstrated comparatively promising results. Notably, in the NU treatment step of this work, there was no agitation equipment
and it was cooled just in a common fridge, suggesting this process is easy to follow. In addition, the bacterial pretreatment in this work required much less time than the fungal pretreatments in Table 1. Therefore, the new design of combination pretreatment performed well at simplifying the requirements of equipment and required a short biological treatment time, which resulted in significant improvement in lowering cost and/or increasing efficiency. 3.3. TOC and UV-Vis analysis In an effective pretreatment process, partial substances from the biomass were dissolved out inevitably. Therefore, the lixivium TOC content could partly reflect the effects of pretreatment. After various treatments, the TOC content of collected supernatant was measured. The TOC content of control was only 33.98 mg l-1, indicating that only small amounts of the substances were dissolved out. The TOC content was 76.91 and 125 mg l-1, showing a significant increase after LD-1 treatment and NU treatment, respectively. And then a maximum TOC content of 188.5 mg l-1 was obtained by the combination of LD-1 with 4%/6% NU at -10 °C. Since the RS was composed primarily of cellulose, hemicelluloses and lignin, the increase of TOC in supernatant probably was attributed to the disruption of cellulose, hemicelluloses or lignin. In addition, the UV-Vis absorption spectra of supernatant were analyzed. The supernatant of control had no obvious absorption at the wavelength from 600-250 nm as well as slight absorption from 250-200 nm. However, there was significant increase in
the absorption from 450-200 nm after pretreatments and the maximum absorption was obtained by the combination of LD-1 with 4%/6% NU at -10 °C. Besides, a remarkable absorption peak near 280 nm appeared. It was well accepted that the absorption at 280 nm was the characteristic absorption peak of benzene rings. There was only lignin containing benzene ring structures in the components of RS, hence, the absorption peak at 280 nm of the supernatant indicated that the lignin was disrupted by pretreatments. The breakdown of plant lignocellulose to glucose monomers is the basis for second-generation cellulosic bio-ethanol production, but the lignin provides a physical barrier which limits accessibility of cellulases or hemicellulases to their substrate. Removal of the lignin or disruption of structure and its linkages with the rest of the biomass was the important effects of the pretreatment. In this work, the stepwise pretreatment of combination of LD-1 with NU had a remarkable effect of lignin disruption. 3.4. SEM analysis The untreated and treated RS were analyzed by SEM to observe the microstructure and surface morphology. The untreated RS had a regular and tough structure, as well as a smooth and compact surface. That was why RS needed a pretreatment for the saccharification. Compared to the untreated RS samples, much larger changes had taken place in the microstructure and surface morphology after combination pretreatment. Instead of the straight and rhabdoid structure, it became curly and irregular. Besides, the smooth, compact surface was broken completely and became very porous and rough.
Plant biomass was composed primarily of cellulose, hemicelluloses and lignin and they were intimately associated to form the structural framework of the plant, making plant biomass very recalcitrant to enzymatic hydrolysis or degradation. Therefore, disruption of structure and increase of porosity in biomass substrate were effective ways to improve enzymatic hydrolysis (Jørgensen et al., 2007; Van Dyk et al., 2012). In this regard, it was very effective after a two-step pretreatment in this work. 3.5. FTIR analysis. The component modification and changes of functional groups were further evaluated by FTIR spectroscopy. From the FT-IR spectrum of RS samples, one peak disappeared in the combination pretreatment RS and there were some differences in parts of peak transmittance (Table 2). The peak at 1729 cm-1 which corresponded the presence of the carboxylic ester (C=O) in lignin and/or hemicelluloses (Qian et al., 2014), disappeared in the combination pretreatment RS. The intensity of 1515 cm-1, the aromatic skeleton vibration and ring breathing in the C-O stretching in lignin (Irfan et al., 2014), was weaker in combination pretreatment RS than untreated RS. Moreover, the peak at 1246 cm-1 (Wang et al., 2004), the indicator of aromatic C=O stretching out of lignin,
decreased
in
intensity
in
the
combination
pretreatment
RS.
The
carbohydrate-related peaks at 1161 cm-1 was assigned to the C-O-C vibration in cellulose and hemicellulose (Irfan et al., 2014), which also decreased in intensity after combination pretreatment, These results indicated that the lignin, cellulose and/or hemicellulose in RS were modified and even degraded by combination pretreatment.
The alteration of the functional groups of lignocelluloses was confirmed by the results of composition analysis (Fig. 1). Moreover, the changes of functional groups further proved that decomposition of the stable chemical structures of raw RS was extensive, resulting a significant enhancement of saccharification by combination pretreatment in this study.
4. Conclusions A novel design of combined Sphingobacterium sp. LD-1 pretreatment with NU pretreatment was conducted. An obvious enhancement on enzymatic hydrolysis was achieved by combination pretreatment, indicating a synergistic effect existed in the stepwise pretreatment for RS saccharification. In the combination pretreatment process, a rapid bacterial treatment by LD-1 reduced the requirements of the subsequent NU treatment. Furthermore, combination pretreatment with LD-1 and 4%/6% NU at -10 °C resulted in 1.396-fold and 1.372-fold increase of reducing sugar and glucose yield respectively than that of sole NU treatment. The novel stepwise pretreatment presents more promising for NU treatment on industrial application. Acknowledgements This work was supported by the Research Foundation of Education Bureau of Hunan Province, China (No. 14C1085) and Opening Foundation of the Chinese National Engineering Research Center for Control and Treatment of Heavy metal Pollution, Changsha, 410083, China (No. 2015CNERC-CTHMP-).
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Spectroscopy, biological and chemical analyses. Landbauforschung. Volk. 54, 163-169. 31 Yu, H.B., Du, W.Q., Zhang, J., Ma, F.Y., Zhang, X.Y., Zhong, W.X., 2010. Fungal treatment of cornstalks enhances the delignification and xylan loss during mild alkaline pretreatment and enzymatic digestibility of glucan. Bioresour. Technol. 101, 6728-6734. 32 Yu, J., Zhang, J.B., He, J., Liu, Z.D., Yu, Z.N., 2009. Combinations of mild physical or chemical pretreatment with biological pretreatment for enzymatic hydrolysis of rice hull. Bioresour. Technol. 100, 903-908. 33 Zhang, L.N., Ruan, D., Gao, S.J., 2002. Dissolution and regeneration of cellulose in NaOH/thiourea aqueous solution. J. Polym. Sci. Pol. Phys. 40, 1521-1529. 34 Zhou, J.P., Zhang, L.N., Cai, J., 2004. Behavior of cellulose in NaOH/Urea aqueous solution characterized by light scattering and viscometry. J. Polym. Sci. Pol. Phys. 42, 347-353. 35 Zhu, L., O'Dwyer, J.P., Chang, V.S., Granda, C.B., Holtzapple, M.T., 2010. Multiple linear regression model for predicting biomass digestibility from structural features. Bioresour. Technol. 101, 4971-4979.
Figure captions Fig. 1. Changes in percentage composition of RS after various pretreatments. A: The sole
biological treatment (4 d). B: The sole NU treatments at different conditions of NU concentration and temperature (4 h). C: The combination pretreatments at different conditions of NU concentration and temperature (4 h). Components: Cel, cellulose; Hcel, hemicellulose; Lig, lignin. Fig. 2. The reducing sugar and glucose yield with 300 U/g enzyme loading after 72 h saccharification. A: The sole NU treatments at different conditions of NU concentration and temperature. B: The combination treatments at different conditions of NU concentration and temperature. R: reducing sugar; G: glucose. Fig. 3. Time course of enzymatic hydrolysis of RS after various pretreatments. A: reducing sugar yield; B: glucose yield.
Table 1 Comparison of combination pretreatments of different substrates and the increase of sugar yield as compared with the untreated substrate. Substrat
The pretreatment process
The es increase of the sugar yield Rice hull H2O2 (2%, 48 h) + P. ostreatus (18 d) and 3.4-fold Ultrasonic (250 W, 30 min)+ P. ostreatus and (18 d) 2.7-fold Cornstal I. lacteus (15 d) + NaOH (1.5%, 60 °C, 120 4.7-fold ks min) Wheat Hot water extraction (85 °C, 10 min) + 2-fold straw C. subvermispora (18 d) Soybean Liquid hot water (170 °C, 40-50 min, 1.3-fold straw 400rpm) + C. subvermispora (18 d) Populou L. betulina C5617 (28 d) + liquid hot water 8.9-fold s tomentosa (200 °C, 30 min) Rice Sphingobacterium sp. LD-1 (4 d) + NU 5.4-fold straw (4%/6%, -10°C, 4 h)
References
Yu et al, 2009 Yu et al., 2010 Wan et al., 2011 Wan et al., 2011 Wang et al., 2012 This study
Table 2 Assignment of FT-IR spectra from untreated and combination treatment (LD-1+4%/6% NU; at -10 °C) RS samples. Assignment
C=O stretching vibration of lignin and/or hemicellulose aromatic skeleton vibration and ring breathing in the C-O stretching of lignin aromatic C=O stretching out of lignin C-O-C vibration in cellulose and hemicellulose
Untreated (cm-1)
Combination pretreated (cm-1)
1729.28 1515.67
1515.01
1246.54
1246.15
1160.61
1161.52
Highlights
1. A novel pretreatment combined S. sp. LD-1 with NU was conducted. 2. Rapid bacterial treatment reduced the requirements of NU treatment. 3. Synergistic effect existed in the stepwise pretreatment for RS saccharification. 4. Combination pretreatment resulted in significant increase of sugar yields.
S0960-8524(15)01370-X http://dx.doi.org/10.1016/j.biortech.2015.09.091 BITE 15600
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
2 August 2015 22 September 2015 23 September 2015
Please cite this article as: Dai, Y., Si, M., Chen, Y., Zhang, N., Zhou, M., Liao, Q., Shi, D., Liu, Y., Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.09.091
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Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw
Youzhi Dai a, Mengying Si a, Yuehui Chen a,∗, Nianlei Zhang a, Mo Zhou a, Qi Liao b, c Deqiang Shi d, Yine Liu a
a Department of Environmental Science and Engineering, Xiangtan University, Xiangtan City 411105, China. b Department of Environmental Engineering, School of Metallurgy and Environment, Central South University, Changsha, 410083, China. c Chinese National Engineering Research Centre for Control and Treatment of Heavy Metal Pollution, Changsha, 410083, China. d Guangxi Academy of Sciences, Nanning City, Guangxi Zhuang Autonomous Region 530007, China.
∗
Corresponding author
Tel.: +86 731 58292231; fax: +86 731 58292231 E-mail address: [email protected]
1
Abstract A stepwise pretreatment of combination of bacterial treatment with NaOH/Urea (NU) treatment was conducted to enhance enzymatic hydrolysis of rice straw (RS). The results showed that the composition of RS changed significantly, the lignin and hemicellulose decreased while cellulose increased. The biological treatment with a bacterium Sphingobacterium sp. LD-1 achieved mild conditions for the sequential NU treatment, reducing the concentration of the NU solution from 7%/12% to 4%/6% and increasing the temperature from -20 °C to -10 °C. The saccharification of rice straw co-treated with bacterium Sphingobacterium sp. LD-1 and 4%/6% NU at -10 °C resulted in 1.396-fold and 1.372-fold increase of reducing sugar and glucose yield respectively than that of sole NU treatment. Keywords: Lignocellulose pretreatment, Sphingobacterium sp. LD-1, NaOH/Urea, Rice straw, Enzymatic hydrolysis
1. Introduction Recent years, to reduce the reliance on fossil fuels, governments have initiated the extensive research into the renewable energies (Dyk et al., 2012). The breakdown of plant lignocellulose (such as corn stover, bagasse, rice straw, and dedicated energy crops) to glucose monomers is the basis for second-generation cellulosic bio-ethanol production (Pickett et al., 2008). Among this rice straw is to be considered as the largest available biomass in the world which is about 731 million tons of dry rice straw annually and Asia is the largest region in the world which is responsible for 90% of the annual global production (Karimi et al., 2006). Since rice straw is high in cellulose (~45% w/w) content and hemicellulose (~25% w/w) content (Binod et al., 2010), it has the potential to be converted to more fermentable sugars by enzymatic saccharification. Conversion of lignocelluloses to ethanol employs three major steps: (1) pretreatment, to break down the recalcitrant structures of lignocellulose; (2) enzymatic hydrolysis, to hydrolyze polysaccharides (e.g., cellulose, hemicellulose) into fermentable sugars; and (3) fermentation, to convert sugars into ethanol (Huang et al., 2011). Nevertheless, pretreatment is essential to make biomass digestible by altering biomass structural features since structural modifications of lignocelluloses are highly dependent on the type of pretreatment employed and have a great effect on enzymatic hydrolysis (Zhu et al., 2010). Furthermore, the pretreatment constitutes more than one-third of the total production costs, which makes it to be a major barrier in the process of bio-ethanol conversion. Therefore, a number of different technologies have
been developed for pretreatment of lignocellulose. The typical pretreatment methods, such as acid, alkali, and steam explosion, have some disadvantages in the commercialization of lignocelluloses conversion, including inefficiency, special equipment requirements and a lot of energy consumption (Mosier et al., 2005). Microbial pretreatment, as an environmental friendly and low cost method, is attracting increasing attention in recent years (Jin et al., 2009; Salame et al., 2012). To date, the biological pretreatments mainly focused on white rot fungi which showed ability to break down and mineralize lignin (Wang et al., 2012). However, relative low efficiency, considerable loss of carbohydrates and long residence periods are the three major disadvantages for the fungal pretreatment (Yu et al., 2009). New strategies should be adopted to overcome these feeble sides. In recent years, a number of bacteria that were able to break down lignin were reported, and the lignin-degrading bacteria seemed to play an emerging role in decomposing lignin because of the rapider growth and easier genetic manipulation as compared with fungi (Bugg et al., 2011; Bugg et al., 2015). However, bacterial pretreatment in conversion of lignocellulose was rarely reported. In our previous study, a bacterial strain Sphingobacterium sp. LD-1 (CGMCC No.10920) was isolated from the effluent sludge of a pulp paper mill, which showed high activity in lignocellulose degradation. In addition, sodium hydroxide with urea was employed to dissolve cellulose completely to produce regenerated cellulose by Zhang’s group (Cai et al., 2005; Zhang et al., 2002; Zhou et al., 2004). These reports showed that
NaOH/Urea pretreatment had a great potential in conversion of lignocellulosic biomass to ethanol. To obtain a pretreatment method with short biological pretreatment period, low energy consumption and common equipment requirements, a stepwise pretreatment of combination of bacterial treatment with NaOH/Urea (NU) treatment was employed in this work. The RS was dealt with by Sphingobacterium sp. LD-1, and then treated with NU. To evaluate the effects of the combined pretreatment, changes in the components of the RS were investigated. Sugar yields of enzymatic hydrolysis of the pretreated RS were also tested. To gain insight into the mechanism of the combined pretreatment, Total organic carbon (TOC) and Ultraviolet-visible spectroscopy (UV-Vis) analysis of supernatant were measured. And scanning electron microscopy (SEM) and the Fourier transform infrared spectroscopy (FTIR) were used to observe the structural change of the RS after the pretreatments.
2. Methods 2.1. Microorganism and Rice straw The degradation of alkali lignin, sodium carboxymethylcellulose and xylan by Sphingobacterium sp. LD-1 was all more than 40% in 5 days (data unpublished), which showed high potentiality for lignocellulose pretreatment. The bacterium LD-1 was cultured on a rotary shaker at 30 °C with a speed of 125 rpm in Luria-Bertani broth medium for 18 h. The Luria-Bertani broth grown bacterial culture was used as seed culture for biological pretreatment.
RS from countryside of Harbin was ground into powder and air-dried. The dried RS powder was then sifted by a 40-mesh griddle and used as the feedstock for the pretreatment. 2.2 Pretreatment All experiments were performed in duplicate for result confirmatory purposes. All analyses were carried out in triplicates and the average values were presented in this text. 2.2.1. Biological pretreatment 100 ml of the seed culture was centrifuged to harvest bacterial cells. The collected cells were then inoculated into 1000 ml sterile mineral salt medium containing 3 g RS. The composition of mineral salt medium was as follows (g l-1 de-ionized water): (NH4)2SO4 2, K2HPO4 1, MgSO4 0.2, CaCl2 0.1, FeSO4 0.05, MnSO4 0.02, KH2PO4 1, pH 7.0. The biological pretreatment was carried on a rotary shaker at 30 °C with a speed of 125 rpm. After 4 d incubation, the bio-treated RS sample was collected and washed with distilled water thoroughly, and then dried at 55 °C for further experiments. 2.2.2. NU pretreatment Series of condition experiments of NU concentration and temperature for NU pretreatment were done. 1 g untreated RS was soaked in a 20 ml solution of different concentration of NU (2%/3%, 4%/6%, 7%/12%) and then treated statically at different temperatures (-20 °C, -10 °C, 4 °C, 20 °C) for 4 h. Likewise, the bio-treated RS was performed aforementioned experiments.
Subsequently, the sole NU treated and co-treated RS samples were washed with distilled water thoroughly and then dried at 55 °C for further analysis. 2.3. Chemical composition measurement of RS The chemical composition of the RS were measured according to the laboratory analytical procedures of the National Renewable Energy Laboratory (Sluiter, 2008). 2.4. Enzymatic hydrolysis and measurement of sugar yield Commercial cellulase produced by Trichoderma viride was purchased from Sinopharm Chemical Reagent. A typical hydrolysis mixture consisted of 0.5 g RS sample, 20 ml of 50 Mm citric acid buffer (pH 4.8) supplemented with antibiotic (40 µg ml-1) and cycloheximide (30 µg ml-1) to prevent microbial contamination, and 300 U g-1 substrate of cellulase. The mixture was incubated at 47.5 °C in a rotary shaker at 100 rpm for 72 h. After incubation, samples were collected and centrifuged for sugar analysis. The reducing sugar and glucose were measured by the DNS assay (Miller, 1959) and enzyme-colorimetric method (the Standard of the People’s Republic of China, GB/T 16285-2008), respectively. 2.5. Analytical methods The washed and air-dried RS samples were used for SEM and FTIR analysis. The lixivium were used for TOC and UV-Vis analysis. 2.5.1. TOC and UV-Vis analysis of supernatant After LD-1 treatment, NU treatment and combination treatment, respectively, the samples were centrifuged and supernatant was collected for TOC and UV-Vis analysis.
The supernatant from untreated sample (0.5 g RS was soaked in 20 ml distilled water at room temperature for 4 h) was used as control. 1 ml sample was diluted with water to 10 ml then filtered with 0.45 µm membrane filters and the concentration of TOC and the UV-Vis were measured by TOC (Shimadzu TOC-VCPH) analyzer and Ultraviolet-visible spectroscopy (Shimadzu UV-2550), respectively. 2.5.2. SEM observation Some samples obtained after various pretreatments coated with gold using a sputter coater were observed under a SEM (JEOL JSM-6360LV) observation. 2.5.3. FTIR analysis. 2 mg RS sample was prepared by mixing with 120 mg of spectroscopic grade KBr then pressed in a standard device to produce 13 mm diameter pellets. The background spectrum of pure potassium bromide was subtracted from that of the sample spectrum. FTIR spectra of samples were obtained with analysis performed on a Thermo Scientific Nicolet-380 FTIR spectrometer in the wave number range of 4,000-500 cm−1. 3. Results and discussion 3.1. Effects of pretreatments on chemical composition As one of the main components of plant cell wall, the lignin provides a physical barrier which limits accessibility of cellulases to their substrate (Várnai et al., 2010). To expose the highly ordered crystalline structure of cellulose and facilitate substrate access by hydrolytic enzymes, reducing the lignin content of the biomass is expected
(Sun et al., 2002). After various pretreatments, the chemical composition in residues was shown in Fig.1. The content of cellulose, hemicellulose and lignin was 51.7%, 20.4% and 12.7% in untreated RS, respectively, while was 57.2%, 24.2% and 6.1% in LD-1 treated RS (Fig. 1A). As can be seen from the results, significant decrease of lignin had happened in the step of bacterial treatment. In other words, the bacterium Sphingobacterium sp. LD-1 showed the capacity of delignification in RS pretreatment. Over the years, fungi especially white rot fungi were thought to the most promising organisms that can efficiently metabolize lignin in various of lignocellulosic materials (Hatakka, 1983; Yu et al., 2010). However, the long processing cycle (more than 30 d in most cases) of fungal treatment was a big obstacle for industrial application. In recent years, because of the rapid growth and easy genetic manipulation, bacteria are increasingly being taken as the emerging role in lignin degradation (Chen et al., 2012a; Chen et al., 2012b; Salvachua et al., 2015). The results in Fig.1B showed that the chemical composition of NU treated RS had changed significantly as compared with the untreated RS. The content of lignin reduced to 5.1%-11.8% from 12.7%, whereas the cellulose content increased to 56.8% - 69.2% from 51.7%. In recent years, Zhang’s research group found that sodium hydroxide with urea (7%/12% NU) at -20 °C can dissolve cellulose completely to produce regenerated cellulose. This method was also used to enhance enzymatic hydrolysis by cellulose dissolution pretreatment (Kuo et al., 2009; Li et al., 2010). During the NU pretreatment,
the sodium hydroxide destroyed inter- and intra-hydrogen bonds between cellulose molecules. Simultaneously, urea hydrates function as hydrogen bonds donor and receptor between solvent molecules and prevented the reassociation of cellulose molecules, causing the cellulose depolymerization and dissolution. Accordingly, NU treatment showed the potential of application in lignocellulose pretreatment. However, this method required demanding conditions (such as freezing-thawing and stirring violently), making large-scale pretreatment of lignocellulose difficult. In this study, improved NU treatment without freezing-thawing and stirring was employed for the lignocellulose pretreatment. Removal of lignin, reduction of cellulose polymerization and disruption of the lignocellulose structure were all the purposes of lignocellulose pretreatment. In this sense, the favorable effects were obtained under milder conditions. As the results shown in Fig. 1B, the cellulose content increased and the lignin content decreased, whether the RS was treated with lower NU concentration (2%/3%) or at higher temperature (20 °C). Furthermore, the combination pretreatment of bacterial treatment with NU treatment was carried out in this study and the chemical composition of co-treated RS was shown in Fig.1C. The content of cellulose and hemicellulose in co-treated RS were 62.7%-73% and 10.6%-20%, respectively. Compared to sole bacterial or NU pretreatment, an increase trend in cellulose content and a decline trend in hemicellulose content had appeared, but no obvious change in lignin content. The results indicated that the effects of co-pretreatment with LD-1 and NU were helpful for the saccharification
of RS. 3.2. Effects of pretreatment on enzymatic hydrolysis To evaluate the efficiency of various pretreatments, the RS residues were subsequently hydrolyzed with commercially available cellulase for 72 h and the results were shown in Fig. 2 and Fig. 3. The enzymatic hydrolysis was inefficient without pretreatment, and the yield of reducing sugar and glucose was only 1.73 and 1.09 mg ml-1, respectively. Even after LD-1 treatment for 4 d, the yield of reducing sugar and glucose slightly increased to 1.93 and 1.14 mg ml-1, respectively, indicating that saccharification of RS had barely enhanced by sole LD-1 treatment. These results can be attributed to the unmodified crystalline structure of cellulose and hindrance of lignin (Chandra et al., 2007). As a consequence, other treatment combined with LD-1 treatment should be introduced for a better enzymatic hydrolysis. With regard to the sole NU treatment, the reducing sugar yield was in a range from 4.87 to 8.51 mg ml-1 as well as from 3.18 to 6.12 mg ml-1 for glucose, showing a significant enhancement of enzymatic hydrolysis (Fig. 2A). In addition, the saccharification was improved with the increase of NU concentration, and the similar results were obtained as the treatment temperature decreased. The maximum production of sugars was obtained with 7%/12% NU treatment at -20 °C and the yield of reducing sugar and glucose increased 4.92 and 5.61-fold, respectively, compared to untreated sample. These increases of sugar yields had proved that the NU treatment could be used
to enhance the enzymatic hydrolysis for lignocellulose. When it went to combination pretreatments, the reducing sugar and glucose yields of co-treated RS with LD-1 and NU were 5.49-9.30 mg ml-1 and 4.13-6.12 mg ml-1, respectively, higher than that of sole NU treatment (Fig. 2B). An obvious increase of the sugar yields was obtained under low concentration of NU (2%/3% and 4%/6%), and a slight increase was obtained under high concentration of NU (7%/12%). The results suggested that the synergy of combination pretreatment was greater at moderate condition. The increase of saccharification of combination pretreatment can be attributed to the synergistic effect of biological and NU treatment. The reason might be the LD-1 degraded and modified lignin in RS, which made the accessibility of NU to lignin easier (Ma et al., 2010). But the intense chemical pretreatment conditions might cover up the effect of the biological pretreatment, the similar conclusions were obtained Ma’s (Ma et al., 2010) and Yu’s researches (Yu et al., 2009). The yield of reducing sugar and glucose was 9.25 and 5.98 mg ml-1 respectively under co-treated with LD-1 and 4%/6% NU at -10 °C, showing almost the same result under 7%/12% NU at -20 °C. However, in sole NU treatment, the result of 4%/6% NU at -10°C was obviously lower than that of 7%/12% NU -20 °C. Therefore, the first step of LD-1 treatment could reduce the requirements of the second step of NU treatment in the combination pretreatment process. After LD-1 treatment, the concentration of NU solution decreased from 7%/12% to 4%/6% and the temperature increased from -20 °C to -10 °C. The results suggested that the moderate conditions for NU treatment could be
obtained after LD-1 treatment, making the enzymatic conversion more promising. The temperature of -10 °C was easy to obtain without a lot of energy consumption. Even in most areas of northern China, the temperature below -10 °C usually occurs in winter. Considering the industrial application, the optimum conditions for combination pretreatment would be 4%/6% NU at -10 °C in this study. The time course of enzymatic hydrolysis of RS after various pretreatments can be seen in Fig. 3, showing that the enzymatic hydrolysis of RS was limited without treatment and a slight enhancement after LD-1 treatment. Meanwhile, in contrast with LD-1 pretreatment, the NU pretreatment showed a more effective saccharification. Furthermore, saccharification of RS co-treated with LD-1 and 4%/6% NU at -10 °C resulted in a significant increase of sugar yields (1.396-fold and 1.372-fold of reducing sugar and glucose, respectively) than that of sole NU treatment. These results indicated a synergistic effect existed in the stepwise pretreatment of combination of LD-1 treatment with NU treatment. As an economical and comparative environment-friendly process, many investigations concerning biological treatment or combination of biological treatment with chemical treatment have been reported (Wan et al., 2011; Wang et al., 2012; Yu et al., 2010; Yu et al., 2009). Table 1 summarized the recent reports of combined biological and chemical pretreatments similar with this work. Compared to the previous results in Table 1, in general, the combination of LD-1 pretreatment with NU pretreatment in this work demonstrated comparatively promising results. Notably, in the NU treatment step of this work, there was no agitation equipment
and it was cooled just in a common fridge, suggesting this process is easy to follow. In addition, the bacterial pretreatment in this work required much less time than the fungal pretreatments in Table 1. Therefore, the new design of combination pretreatment performed well at simplifying the requirements of equipment and required a short biological treatment time, which resulted in significant improvement in lowering cost and/or increasing efficiency. 3.3. TOC and UV-Vis analysis In an effective pretreatment process, partial substances from the biomass were dissolved out inevitably. Therefore, the lixivium TOC content could partly reflect the effects of pretreatment. After various treatments, the TOC content of collected supernatant was measured. The TOC content of control was only 33.98 mg l-1, indicating that only small amounts of the substances were dissolved out. The TOC content was 76.91 and 125 mg l-1, showing a significant increase after LD-1 treatment and NU treatment, respectively. And then a maximum TOC content of 188.5 mg l-1 was obtained by the combination of LD-1 with 4%/6% NU at -10 °C. Since the RS was composed primarily of cellulose, hemicelluloses and lignin, the increase of TOC in supernatant probably was attributed to the disruption of cellulose, hemicelluloses or lignin. In addition, the UV-Vis absorption spectra of supernatant were analyzed. The supernatant of control had no obvious absorption at the wavelength from 600-250 nm as well as slight absorption from 250-200 nm. However, there was significant increase in
the absorption from 450-200 nm after pretreatments and the maximum absorption was obtained by the combination of LD-1 with 4%/6% NU at -10 °C. Besides, a remarkable absorption peak near 280 nm appeared. It was well accepted that the absorption at 280 nm was the characteristic absorption peak of benzene rings. There was only lignin containing benzene ring structures in the components of RS, hence, the absorption peak at 280 nm of the supernatant indicated that the lignin was disrupted by pretreatments. The breakdown of plant lignocellulose to glucose monomers is the basis for second-generation cellulosic bio-ethanol production, but the lignin provides a physical barrier which limits accessibility of cellulases or hemicellulases to their substrate. Removal of the lignin or disruption of structure and its linkages with the rest of the biomass was the important effects of the pretreatment. In this work, the stepwise pretreatment of combination of LD-1 with NU had a remarkable effect of lignin disruption. 3.4. SEM analysis The untreated and treated RS were analyzed by SEM to observe the microstructure and surface morphology. The untreated RS had a regular and tough structure, as well as a smooth and compact surface. That was why RS needed a pretreatment for the saccharification. Compared to the untreated RS samples, much larger changes had taken place in the microstructure and surface morphology after combination pretreatment. Instead of the straight and rhabdoid structure, it became curly and irregular. Besides, the smooth, compact surface was broken completely and became very porous and rough.
Plant biomass was composed primarily of cellulose, hemicelluloses and lignin and they were intimately associated to form the structural framework of the plant, making plant biomass very recalcitrant to enzymatic hydrolysis or degradation. Therefore, disruption of structure and increase of porosity in biomass substrate were effective ways to improve enzymatic hydrolysis (Jørgensen et al., 2007; Van Dyk et al., 2012). In this regard, it was very effective after a two-step pretreatment in this work. 3.5. FTIR analysis. The component modification and changes of functional groups were further evaluated by FTIR spectroscopy. From the FT-IR spectrum of RS samples, one peak disappeared in the combination pretreatment RS and there were some differences in parts of peak transmittance (Table 2). The peak at 1729 cm-1 which corresponded the presence of the carboxylic ester (C=O) in lignin and/or hemicelluloses (Qian et al., 2014), disappeared in the combination pretreatment RS. The intensity of 1515 cm-1, the aromatic skeleton vibration and ring breathing in the C-O stretching in lignin (Irfan et al., 2014), was weaker in combination pretreatment RS than untreated RS. Moreover, the peak at 1246 cm-1 (Wang et al., 2004), the indicator of aromatic C=O stretching out of lignin,
decreased
in
intensity
in
the
combination
pretreatment
RS.
The
carbohydrate-related peaks at 1161 cm-1 was assigned to the C-O-C vibration in cellulose and hemicellulose (Irfan et al., 2014), which also decreased in intensity after combination pretreatment, These results indicated that the lignin, cellulose and/or hemicellulose in RS were modified and even degraded by combination pretreatment.
The alteration of the functional groups of lignocelluloses was confirmed by the results of composition analysis (Fig. 1). Moreover, the changes of functional groups further proved that decomposition of the stable chemical structures of raw RS was extensive, resulting a significant enhancement of saccharification by combination pretreatment in this study.
4. Conclusions A novel design of combined Sphingobacterium sp. LD-1 pretreatment with NU pretreatment was conducted. An obvious enhancement on enzymatic hydrolysis was achieved by combination pretreatment, indicating a synergistic effect existed in the stepwise pretreatment for RS saccharification. In the combination pretreatment process, a rapid bacterial treatment by LD-1 reduced the requirements of the subsequent NU treatment. Furthermore, combination pretreatment with LD-1 and 4%/6% NU at -10 °C resulted in 1.396-fold and 1.372-fold increase of reducing sugar and glucose yield respectively than that of sole NU treatment. The novel stepwise pretreatment presents more promising for NU treatment on industrial application. Acknowledgements This work was supported by the Research Foundation of Education Bureau of Hunan Province, China (No. 14C1085) and Opening Foundation of the Chinese National Engineering Research Center for Control and Treatment of Heavy metal Pollution, Changsha, 410083, China (No. 2015CNERC-CTHMP-).
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Figure captions Fig. 1. Changes in percentage composition of RS after various pretreatments. A: The sole
biological treatment (4 d). B: The sole NU treatments at different conditions of NU concentration and temperature (4 h). C: The combination pretreatments at different conditions of NU concentration and temperature (4 h). Components: Cel, cellulose; Hcel, hemicellulose; Lig, lignin. Fig. 2. The reducing sugar and glucose yield with 300 U/g enzyme loading after 72 h saccharification. A: The sole NU treatments at different conditions of NU concentration and temperature. B: The combination treatments at different conditions of NU concentration and temperature. R: reducing sugar; G: glucose. Fig. 3. Time course of enzymatic hydrolysis of RS after various pretreatments. A: reducing sugar yield; B: glucose yield.
Table 1 Comparison of combination pretreatments of different substrates and the increase of sugar yield as compared with the untreated substrate. Substrat
The pretreatment process
The es increase of the sugar yield Rice hull H2O2 (2%, 48 h) + P. ostreatus (18 d) and 3.4-fold Ultrasonic (250 W, 30 min)+ P. ostreatus and (18 d) 2.7-fold Cornstal I. lacteus (15 d) + NaOH (1.5%, 60 °C, 120 4.7-fold ks min) Wheat Hot water extraction (85 °C, 10 min) + 2-fold straw C. subvermispora (18 d) Soybean Liquid hot water (170 °C, 40-50 min, 1.3-fold straw 400rpm) + C. subvermispora (18 d) Populou L. betulina C5617 (28 d) + liquid hot water 8.9-fold s tomentosa (200 °C, 30 min) Rice Sphingobacterium sp. LD-1 (4 d) + NU 5.4-fold straw (4%/6%, -10°C, 4 h)
References
Yu et al, 2009 Yu et al., 2010 Wan et al., 2011 Wan et al., 2011 Wang et al., 2012 This study
Table 2 Assignment of FT-IR spectra from untreated and combination treatment (LD-1+4%/6% NU; at -10 °C) RS samples. Assignment
C=O stretching vibration of lignin and/or hemicellulose aromatic skeleton vibration and ring breathing in the C-O stretching of lignin aromatic C=O stretching out of lignin C-O-C vibration in cellulose and hemicellulose
Untreated (cm-1)
Combination pretreated (cm-1)
1729.28 1515.67
1515.01
1246.54
1246.15
1160.61
1161.52
Highlights
1. A novel pretreatment combined S. sp. LD-1 with NU was conducted. 2. Rapid bacterial treatment reduced the requirements of NU treatment. 3. Synergistic effect existed in the stepwise pretreatment for RS saccharification. 4. Combination pretreatment resulted in significant increase of sugar yields.