Bioresource Technology 156 (2014) 368–371
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Natural laccase mediators separated from water-washed solution of steam exploded corn straw by nanofiltration and organic solvent fractionation Weihua Qiu, Wenyan Zhang, Hongzhang Chen ⇑ State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
h i g h l i g h t s Nanofiltration can enrich the phenolic substances in the WWS of SECS. Successively organic solvent extraction can separate materials in WWS effectively. Organic solvent extractive of WWS could act as natural mediator for laccase. Laccase/EE can decolorize crystal violet more efficiently than laccase/ABTS. This work would benefit for reducing the cost of laccase-mediator system.
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online 23 January 2014 Keywords: Natural laccase mediator Water-washed solution Steam exploded corn straw Nanofiltration Successive organic solvent extraction
a b s t r a c t Artificially synthetic mediators of laccase had the limitation of high cost and possible toxicity. The separation of natural laccase mediators from water-washed solution (WWS) of steam exploded corn straw (SECS) was studied using nano-filtration and successive organic solvents extraction. Results indicated that the UV absorption intensity of nano-filtrated WWS was significantly enhanced. The UV absorption intensity of each extractive from WWS could be ranked as ether extractive (EE) > ethyl acetate extractive (EAE) > chloroform extractive (CE). Decoloration of crystal violet catalyzed by laccase/EE was higher than that by laccase/ABTS, which was 66.95% and 61.9% at 8 h, respectively. All the decoloration rates of malachite green at 60 min using EE, EAE and ABTS as mediator were both more than 80%. This research would benefit for broaden the source of laccase mediator and reduce the using cost of laccase/mediator system. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Laccase is multicopper-containing oxidase with phenoloxidase activity, which widely distributes in plant, fungus and bacteria (Cañas and Camarero, 2010). It can catalyze the oxidation of substituted phenols and anilines using oxygen as the electron acceptor and producing water as by-product (Majeau et al., 2010). Originally, due to the relative low redox potential (60.8 V), laccase cannot directly oxidize non-phenolic substrates with a high redox potential (above 1.3 V). This limitation has been overcame after discovering some synthetic compounds severed as redox mediators, such as 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), violuric acid, 1-hydroxybenzotriazole (HBT), N-hydroxyphthalimide (HPI), and N-hydroxyacetanilide (NHA). However, the application of mediator is hindered by the economic ⇑ Corresponding author. Tel./fax: +86 10 82627071. E-mail address: [email protected] (H. Chen). http://dx.doi.org/10.1016/j.biortech.2014.01.044 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
cost and the generation of possible toxic substance (Cañas and Camarero, 2010). Therefore, many researchers focused on searching alternative mediators which should be environmental-friendly and available economically. Presently, several studies have shown that some lignin-related natural mediators were similar to or even better than the best synthetic mediators. For example, compared with HBT, acetosyringone and syringaldehyde (both dimethoxy substituted phenols derived from syringyl lignin units) exhibited higher decolorization rate, even under low mediator concentration (Camarero et al., 2005; Moldes et al., 2008). Meanwhile, the lignin-related phenols can be easily obtained from plant materials (Fillat et al., 2010). It had also been proved that natural mediators for laccase can be readily obtained from pulping liquors and phenolic metabolites, such as 4-hydroxybenzoic acid, 4-hydroxybenzylic alcohol (Gutiérrez et al., 2007), which should reduce the capital cost. Actually, steam explosion hydrolyzate of lignocellulosic biomass would be an efficiently resource for separation of natural
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mediators for laccase. Steam explosion is one of the most popular and effective methods for the hydrolysis of lignocellulosic materials (Chen and Qiu, 2010; Liu et al., 2013). It indicated that large amount of ferulic acid and p-hydroxybenzoic acid and certain amount of phenolic acids, vanillic acid, and p-coumaric acid would be generated during the steam explosion process due to the cleavage of ester bond and guaiacyl bond in lignocellulose (Chen and Liu, 2007). Some of these compounds have been proven to be natural laccase mediator, such as syringaldehyde, acetosyringone and p-coumaric acid (Fillat et al., 2010). However, the lignin-related phenols were considered as the undesired materials due to the inhibition on the activity of microbe and enzyme (Ludwig et al., 2013; Wang and Chen, 2011). There was almost no report on the separation of natural mediators of laccase from steam explosion pretreated liquor of lignocellulosic biomass. Present study highlighted the separation of natural mediators of laccase from water-washed solution (WWS) of steam exploded corn straw (SECS). The WWS of SECS was fractionated by integrated nanofiltration and subsequent organic solvent extraction. The composition of each fractionation was assayed using UV and HPLC. Meanwhile, the decoloration of laccase taking each fractionation as natural mediator was also systematically studied.
2. Methods 2.1. Raw material and steam-explosion pretreatment Corn straw (CS) was obtained from Beijing, China. The CS was manually cut into pieces with the average size of 5.0 cm and washed with deionized water to remove soil and dust. The chopped and washed CS (200 g by dry weight, 50% of water content) was soaked at 1:1 (w/w) solid-to-water ratio for 20 min and then put into a 4.5 L steam explosion reactor (Weihai Automatic Control Co. Ltd., Weihai, China). Steam exploded corn straw (SECS) was conducted following the same protocols reported by Chen and Liu (2007) with 1.2 MPa saturated steam for 5 min.
2.2. Enzymes
2.4. Successive organic solvent fractionation for the WWS of SECS One hundred milliliter of nano-cutoff WWS and nano-filtrated WWS was successively extracted with petroleum ether, ether, ethyl acetate, and chloroform, respectively. The extraction processes were conducted as follows. Mixed WWS with 1:1 (v/v) of petroleum ether in separatory funnel at room temperature and collected the organic phase after being statically mixed for 2 h. The extraction of ether, ethyl acetate and chloroform were then conducted successively by analogy. All the organic phases were collected for further use. The nano-cutoff WWS and nano-filtrated WWS without organic solvents extraction were used as control group. 2.5. Decoloration by laccase with different mediator Taking crystal violet and malachite green as the research object, the decolorizing mixture consisted of 0.5 ml aqueous solution of dye (0.5 g l1), 3.3 ml of enzyme solution (8 U ml1), and 0.025 mg mediator. The total volume was then set to 5 ml with distilled water. In the experimental group, ether extractive (EE) and ethyl acetate extractive (EAE) from nano-filtrated WWS of SECS were used as the mediators for laccase. In the control group, ABTS was used as the mediator for laccase. All reactions were incubated at 50 °C statically. Dye decoloration, which was associated with its oxidation, was determined by monitoring the decrease in the absorbance peak at 582 nm for crystal violet and 616 nm for malachite green, respectively, using UVmini-1240 spectrophotometer (Shiamdzu Spectrophotometer, Japan). The decoloration rate was calculated using the following formula.
Decoloration rate ð%Þ ¼
A0 A1 100% A0
ð1Þ
where, A0 is the initial absorption of reaction system with deactivated laccase; A1 is the absorption of sample after being reacted with laccase for a certain time. All the experiments were run in triplicate and the mean (±SEM) was calculated. 2.6. Analytical methods
Laccase from Trametes versicolor was purchase from Sigma Chemicals (USA) with activity P0.5 U/mg. The activity was measured by oxidation of 0.5 mM 2,20 -amino-di(2-ethyl-benzothiazoline sulphonic acid-6) ammonium salt (ABTS, e530 = 3.6 104 M1 cm1) in 0.2 M sodium acetate (pH 4.8) at 30 °C. The enzyme unit (U) was defined as the amount of enzyme required to oxidize 1 lmol of ABTS per minute (Kalyani et al., 2012).
The total phenolic content was determined by Folin–Ciocalteau method (Zhao et al., 2013). UVmini-1240 Spectrophotometer (Shiamdzu, Japan) was used for the all band ultraviolet scanning. The material composition of WWS was analyzed using HPLC (Agilent 1200, USA) equipped with a diode array detector (Agilent, USA). YMC-Triart C18 (250 mm 4.6 mm) column (YMC separation technology, Japan) was used at 30 °C with 2:3 (v/v) methanol: 1% acetic solution as mobile phase at a flow rate of 0.6 ml min1.
2.3. Preparation for nano-filtrated WWS of SECS
3. Results and discussion
Mixed SECS with distilled water and soaked for 2 h at 30 °C, and then washed SECS repeatedly with three times. The washing liquid was combined to set the finally 1:10 (w/w) washing water to the dry weight of SECS ratio. The washing liquid mixture was filtrated to remove the small impurities, and the supernatant was centrifuged at 1800g for 10 min to get the WWS of SECS. The WWS of SECS were then nanofiltrated with NF-1812 roll polyamide composite membrane (Shanghai Filter Co., Ltd, China). The molecular weight cutoff (MWCO) was 1000 Da. The filtrated fraction (named nano-filtrated WWS) and cutoff fraction (named nano-cutoff WWS) were collected respectively and concentrated to the same volume under vacuum at 65 °C.
3.1. UV analysis of fractionations from WWS Steam explosion could deform the lignin with the increases of coumaric acid, vanillic acid and ferulic acid due to the cleavage of ester bonds of the lignin (Alvira et al., 2010). In the hydrolysate of steam exploded straw, many phenolic substances, such as 4-hydroxybenzaldehyde, catechol, syringaldehyde, syringic acid, vanillin, vanillic acid, ferulic acid, coumaric acid, etc., were observed (Sun et al., 2005). During the steam explosion, hemicellulose in lignocellulosic biomass was significantly hydrolyzed into mono- or oligo-saccharides, while the lignin was partially degraded and the Mw of degraded lignin was between 3 103 and 4 103 g mol1 with polydispersity of 1.4–2.0 (Chanda and
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Nagani, 2010). In this article, the WWS of SECS was separated into nano-filtrated fraction and nano-cutoff fraction by membrane with MWCO of 1000 Da. Petroleum ether extractive (PEE) from nano-cutoff WWS gave almost no UV absorption. The substances in nano-filtrated WWS with the wavelengths of 305 nm, 290 nm, and 262 nm were extracted by petroleum ether. According to the similar compatible principle, the PEE mainly contained strong lipophilic components, such as volatile oil, fat, sterol and so on, which almost gave no UV absorption. The polar substances in WWS of SECS were extracted by ether, chloroform and ethyl acetate successively. Owing to the different partition coefficient in each solvent, the organic solvent extractives contained different type and content of substances. The UV absorption of each extractive can be ranked from high to low in the following order: ether extractive (EE) > ethyl acetate extractive (EAE) > chloroform extractive (CE). Meanwhile, the UV absorption of each extractive from nano-filtrated WWS was higher than that from nano-cutoff WWS. As for nano-cutoff WWS, both of EE and CE had the major UV absorption peak at 305 nm and 280 nm, but only 280 nm for EAE. The major UV absorption peaks of EAE extracted from nano-filtrated WWS were 305 nm and 280 nm, but only 280 nm for CE. After the sequential extraction of organic solvents, the substances with UV absorption in the alkaline methane solution were almost completely extracted. In order to further clarify the difference between substances in organic solvent extractives, HPLC was used to illustrate the composition of each extractive.
3.3. Decoloration of laccase-mediator system In general, triphenylmethane dyes is difficult to be degraded by microorganisms due to its complicated chemical structure. Thus, look for new biological treatment methods are getting more attention. Laccase-based decolorization treatments are more potentially advantageous than white-rot decolorization due to that the enzyme is produced in larger amounts and requires less fastidious induction conditions than either lignin peroxidase (LiP) and Mndependent peroxidase (MnP) (Asgher et al., 2008; Pointing and Vrijmoed, 2000). Therefore, laccase becomes an attractive option for biobleaching (Hadibarata et al., 2012; Hao et al., 2013). In present study, taking crystal violet and malachite green as example of triphenylmethane dyes, their decolorations catalyzed by laccase with different mediators were studied. At first 30 min, crystal violet was rapidly decolorized by laccase/EE (Fig. 1A). The decoloration of crystal violet at 20 min catalyzed by laccase/EE was 21.5% but only 3.28% for laccase/ABTS. The decolorization of crystal violet catalyzed by laccase/EAE was faster than that by laccase/ ABTS at first 60 min. However, the decolorization efficiency catalyzed by laccase/EAE proceeded significantly slowly after 60 min. The 8 h decoloration of crystal violet catalyzed by laccase with EE, EAE and ABTS as mediator was 66.95%, 42.36%, and 58.49%, respectively (Fig. 1A). The decoloration efficiency of malachite green by laccase/mediator system was even higher than crystal violet. There was 69.4%,
3.2. HPLC analysis of fractionations from nano-filtrated WWS When detected using 280 nm, PEE of nano-filtrated WWS almost gave no absorption under the HPLC condition. In this article, Rt T was used to define the substance with the special retention time (min) detected by HPLC. Substances of Rt 3.8418, Rt 6.8672, Rt 21.6416, and Rt 31.156 were the major substance in ether extractive (EE). The strong polar substances of Rt 3.8418 and Rt 6.8672 were extracted by chloroform, while substances of Rt 3.8418, Rt 6.0077, and Rt 17.7482 were dominated in EAE. As for nano-cutoff WWS, PEE also gave almost no UV absorption. Major substances in EE were Rt 3.9879, Rt 7.1597, Rt 20.7048, and Rt 34.9721. And major substances in EAE were similar to substances that extracted from nano-filtrated WWS by EAE. There were much more abundant substances with 305 nm absorption in nano-filtrated WWS than in nano-cutoff WWS. In the EE of nano-filtrated WWS, substance of Rt 31.156 had strong absorption, but Rt 34.9721 had weak absorption. CE of nano-filtrated WWS, including the substances of Rt 6.8672, Rt 29.8345, and Rt 33.3476, was pretty complicated than other organic solvent extractives. The dominant substance in EAE of nano-filtrated WWS was Rt 17.8943 with strong 305 nm absorption. As for nano-cutoff WWS, there were few substances with 305 nm absorption. The substances in EE of nano-cutoff WWS included Rt 34.9721 and Rt 39.8328, which had strong absorption and weak absorption, respectively. Substance of Rt 17.8943 was dominant in EAE. However, almost no substance with 305 nm absorption was detected in strong polar region. In the nano-filtrated WWS, substances in EAE with 260 nm UV absorption were Rt 6.07 and Rt 10.8401. The major substances in EE were Rt 15.6102 and Rt 21.6416. While the substances with 260 nm absorption were mainly existed in EE with retention time of 13.0094 min and 20.7523 min in the nano-cutoff WWS. Therefore, results showed that different organic solvent could fractionate weakly polar substances with high separation degree from WWS of SECS. It laid a good foundation for the subsequent structural analysis and identification of substances in WWS.
Fig. 1. Decolorization of dyes by laccase (25 IU) with different mediator at 50 °C. Dye concentration: 0.25 mg crystal violet (A); 0.25 mg malachite green (B). Mediator concentration: 0.025 ± 0.005 mg. (j) Ether extractive (EE) from nanofiltrated water washed solution (WWS) of steam exploded corn straw (SECS), (d) ethyl acetate extractive (EAE) from WWS of SECS, (N) ABTS.
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68.57%, and 81.52% of malachite green was decolorized by laccase/ EE, laccase/EAE and laccase/ABTS at 10 min, respectively. However, the decolorization speed of malachite green slowed down obviously after 10 min. After being decolorized by 60 min, more than 80% of decolorization was obtained for all mediators (Fig. 1B). Therefore, laccase combined with different mediators separated from WWS of SECS had a good performance for decoloration of triphenylmethane dyes, which should be the suitable methods for enhancing the decoloration performance of other solutions. 4. Conclusion Searching natural laccase mediators from the hydrolyzate of lignocellulosic biomass is an efficient approach to decrease the cost of laccase catalytic system and reduce the possible risk on environment. The substances with UV absorption in the nano-filtrated water washed solution (WWS) of steam exploded corn straw (SECS) were enriched by nanofiltration. The successively organic solvents extraction could separate the WWS of SECS into fractionations with different substances. The natural laccase mediators were effective for laccase to catalyze the decolorization of crystal violet and malachite green. In the further research, the substance in each organic solvent fractionation should be identified. Acknowledgements Financial support to this study was provided by the National Natural Science Foundation of China (Grant No. 21206176), the National Basic Research Program of China (No. 2011CB707401), the National Key Project of Scientific and Technical Supporting Program of China (No. 2011BAD22B02). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 01.044. References Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861.
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Asgher, M., Bhatti, H.N., Ashraf, M., Legge, R.L., 2008. Recent developments in biodegradation of industrial pollutants by white rot fungi and their enzyme system. Biodegradation 19, 771–783. Camarero, S., Ibarra, D., Martínez, M.J., Martínez, Á.T., 2005. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl. Environ. Microbiol. 71, 1775–1784. Cañas, A.I., Camarero, S., 2010. Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes. Biotechnol. Adv. 28, 694–705. Chanda, S.V., Nagani, K.V., 2010. Antioxidant capacity of Manilkara zapota L. Leaves extracts evaluated by four in vitro methods. Nat. Sci. 8, 260–266. Chen, H.Z., Liu, L.Y., 2007. Unpolluted fractionation of wheat straw by steam explosion and ethanol extraction. Bioresour. Technol. 98, 666–676. Chen, H.Z., Qiu, W.H., 2010. Key technologies for bioethanol production from lignocellulose. Biotechnol. Adv. 28, 556–562. Fillat, A., Colom, J.F., Vidal, T., 2010. A new approach to the biobleaching of flax pulp with laccase using natural mediators. Bioresour. Technol. 101, 4104–4110. Gutiérrez, A., Rencoret, J., Ibarra, D., Molina, S., Camarero, S., Romero, J., Del Río, J.C., Martínez, Á.T., 2007. Removal of lipophilic extractives from paper pulp by laccase and lignin-derived phenols as natural mediators. Environ. Sci. Technol. 41, 4124–4129. Hadibarata, T., Yusoff, A.R.M., Aris, A., Hidayat, T., Kristanti, R.A., 2012. Decolorization of azo, triphenylmethane and anthraquinone dyes by laccase of a newly isolated Armillaria sp. F022. Water Air Soil Pollut. 223, 1045–1054. Hao, L.Y., Wang, R., Liu, J.Q., Fang, K.J., 2013. The influence of auxiliary chemicals on the decolorization of reactive blue dye by laccase. Adv. Mater. Res. 709, 53–57. Kalyani, D., Dhiman, S.S., Kim, H., Jeya, M., Kim, I.W., Lee, J.K., 2012. Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass. Process Biochem. 47, 671–678. Liu, Z.H., Qin, L., Jin, M.J., Pang, F., Li, B.Z., Kang, Y., Dale, B.E., Yuan, Y.J., 2013. Evaluation of storage methods for the conversion of corn stover biomass to sugars based on steam explosion pretreatment. Bioresour. Technol. 132, 5–15. Ludwig, D., Amann, M., Hirth, T., Rupp, S., Zibek, S., 2013. Development and optimization of single and combined detoxification processes to improve the fermentability of lignocellulose hydrolyzates. Bioresour. Technol. 133, 455–461. Majeau, J.A., Brar, S.K., Tyagi, R.D., 2010. Laccases for removal of recalcitrant and emerging pollutants. Bioresour. Technol. 101, 2331–2350. Moldes, D., Díaz, M., Tzanov, T., Vidal, T., 2008. Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus Kraft pulp. Bioresour. Technol. 99, 7959–7965. Pointing, S., Vrijmoed, L., 2000. Decolorization of azo and triphenylmethane dyes by Pycnoporus sanguineus producing laccase as the sole phenoloxidase. World J. Microbiol. Biotechnol. 16, 317–318. Sun, X.F., Xu, F., Sun, R.C., Geng, Z.C., Fowler, P., Baird, M.S., 2005. Characteristics of degraded hemicellulosic polymers obtained from steam exploded wheat straw. Carbohyd. Polym. 60, 15–26. Wang, L., Chen, H.Z., 2011. Increased fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production by removal of fermentation inhibitors. Process Biochem. 46, 604–607. Zhao, X., Li, M., Huang, G., Chen, Y., 2013. Determination of total polyphenols in apple vinegar drink by Folin–Ciocalteu method. Chinese Food Sci. 34, 31–34.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Natural laccase mediators separated from water-washed solution of steam exploded corn straw by nanofiltration and organic solvent fractionation Weihua Qiu, Wenyan Zhang, Hongzhang Chen ⇑ State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
h i g h l i g h t s Nanofiltration can enrich the phenolic substances in the WWS of SECS. Successively organic solvent extraction can separate materials in WWS effectively. Organic solvent extractive of WWS could act as natural mediator for laccase. Laccase/EE can decolorize crystal violet more efficiently than laccase/ABTS. This work would benefit for reducing the cost of laccase-mediator system.
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online 23 January 2014 Keywords: Natural laccase mediator Water-washed solution Steam exploded corn straw Nanofiltration Successive organic solvent extraction
a b s t r a c t Artificially synthetic mediators of laccase had the limitation of high cost and possible toxicity. The separation of natural laccase mediators from water-washed solution (WWS) of steam exploded corn straw (SECS) was studied using nano-filtration and successive organic solvents extraction. Results indicated that the UV absorption intensity of nano-filtrated WWS was significantly enhanced. The UV absorption intensity of each extractive from WWS could be ranked as ether extractive (EE) > ethyl acetate extractive (EAE) > chloroform extractive (CE). Decoloration of crystal violet catalyzed by laccase/EE was higher than that by laccase/ABTS, which was 66.95% and 61.9% at 8 h, respectively. All the decoloration rates of malachite green at 60 min using EE, EAE and ABTS as mediator were both more than 80%. This research would benefit for broaden the source of laccase mediator and reduce the using cost of laccase/mediator system. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Laccase is multicopper-containing oxidase with phenoloxidase activity, which widely distributes in plant, fungus and bacteria (Cañas and Camarero, 2010). It can catalyze the oxidation of substituted phenols and anilines using oxygen as the electron acceptor and producing water as by-product (Majeau et al., 2010). Originally, due to the relative low redox potential (60.8 V), laccase cannot directly oxidize non-phenolic substrates with a high redox potential (above 1.3 V). This limitation has been overcame after discovering some synthetic compounds severed as redox mediators, such as 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), violuric acid, 1-hydroxybenzotriazole (HBT), N-hydroxyphthalimide (HPI), and N-hydroxyacetanilide (NHA). However, the application of mediator is hindered by the economic ⇑ Corresponding author. Tel./fax: +86 10 82627071. E-mail address: [email protected] (H. Chen). http://dx.doi.org/10.1016/j.biortech.2014.01.044 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
cost and the generation of possible toxic substance (Cañas and Camarero, 2010). Therefore, many researchers focused on searching alternative mediators which should be environmental-friendly and available economically. Presently, several studies have shown that some lignin-related natural mediators were similar to or even better than the best synthetic mediators. For example, compared with HBT, acetosyringone and syringaldehyde (both dimethoxy substituted phenols derived from syringyl lignin units) exhibited higher decolorization rate, even under low mediator concentration (Camarero et al., 2005; Moldes et al., 2008). Meanwhile, the lignin-related phenols can be easily obtained from plant materials (Fillat et al., 2010). It had also been proved that natural mediators for laccase can be readily obtained from pulping liquors and phenolic metabolites, such as 4-hydroxybenzoic acid, 4-hydroxybenzylic alcohol (Gutiérrez et al., 2007), which should reduce the capital cost. Actually, steam explosion hydrolyzate of lignocellulosic biomass would be an efficiently resource for separation of natural
369
W. Qiu et al. / Bioresource Technology 156 (2014) 368–371
mediators for laccase. Steam explosion is one of the most popular and effective methods for the hydrolysis of lignocellulosic materials (Chen and Qiu, 2010; Liu et al., 2013). It indicated that large amount of ferulic acid and p-hydroxybenzoic acid and certain amount of phenolic acids, vanillic acid, and p-coumaric acid would be generated during the steam explosion process due to the cleavage of ester bond and guaiacyl bond in lignocellulose (Chen and Liu, 2007). Some of these compounds have been proven to be natural laccase mediator, such as syringaldehyde, acetosyringone and p-coumaric acid (Fillat et al., 2010). However, the lignin-related phenols were considered as the undesired materials due to the inhibition on the activity of microbe and enzyme (Ludwig et al., 2013; Wang and Chen, 2011). There was almost no report on the separation of natural mediators of laccase from steam explosion pretreated liquor of lignocellulosic biomass. Present study highlighted the separation of natural mediators of laccase from water-washed solution (WWS) of steam exploded corn straw (SECS). The WWS of SECS was fractionated by integrated nanofiltration and subsequent organic solvent extraction. The composition of each fractionation was assayed using UV and HPLC. Meanwhile, the decoloration of laccase taking each fractionation as natural mediator was also systematically studied.
2. Methods 2.1. Raw material and steam-explosion pretreatment Corn straw (CS) was obtained from Beijing, China. The CS was manually cut into pieces with the average size of 5.0 cm and washed with deionized water to remove soil and dust. The chopped and washed CS (200 g by dry weight, 50% of water content) was soaked at 1:1 (w/w) solid-to-water ratio for 20 min and then put into a 4.5 L steam explosion reactor (Weihai Automatic Control Co. Ltd., Weihai, China). Steam exploded corn straw (SECS) was conducted following the same protocols reported by Chen and Liu (2007) with 1.2 MPa saturated steam for 5 min.
2.2. Enzymes
2.4. Successive organic solvent fractionation for the WWS of SECS One hundred milliliter of nano-cutoff WWS and nano-filtrated WWS was successively extracted with petroleum ether, ether, ethyl acetate, and chloroform, respectively. The extraction processes were conducted as follows. Mixed WWS with 1:1 (v/v) of petroleum ether in separatory funnel at room temperature and collected the organic phase after being statically mixed for 2 h. The extraction of ether, ethyl acetate and chloroform were then conducted successively by analogy. All the organic phases were collected for further use. The nano-cutoff WWS and nano-filtrated WWS without organic solvents extraction were used as control group. 2.5. Decoloration by laccase with different mediator Taking crystal violet and malachite green as the research object, the decolorizing mixture consisted of 0.5 ml aqueous solution of dye (0.5 g l1), 3.3 ml of enzyme solution (8 U ml1), and 0.025 mg mediator. The total volume was then set to 5 ml with distilled water. In the experimental group, ether extractive (EE) and ethyl acetate extractive (EAE) from nano-filtrated WWS of SECS were used as the mediators for laccase. In the control group, ABTS was used as the mediator for laccase. All reactions were incubated at 50 °C statically. Dye decoloration, which was associated with its oxidation, was determined by monitoring the decrease in the absorbance peak at 582 nm for crystal violet and 616 nm for malachite green, respectively, using UVmini-1240 spectrophotometer (Shiamdzu Spectrophotometer, Japan). The decoloration rate was calculated using the following formula.
Decoloration rate ð%Þ ¼
A0 A1 100% A0
ð1Þ
where, A0 is the initial absorption of reaction system with deactivated laccase; A1 is the absorption of sample after being reacted with laccase for a certain time. All the experiments were run in triplicate and the mean (±SEM) was calculated. 2.6. Analytical methods
Laccase from Trametes versicolor was purchase from Sigma Chemicals (USA) with activity P0.5 U/mg. The activity was measured by oxidation of 0.5 mM 2,20 -amino-di(2-ethyl-benzothiazoline sulphonic acid-6) ammonium salt (ABTS, e530 = 3.6 104 M1 cm1) in 0.2 M sodium acetate (pH 4.8) at 30 °C. The enzyme unit (U) was defined as the amount of enzyme required to oxidize 1 lmol of ABTS per minute (Kalyani et al., 2012).
The total phenolic content was determined by Folin–Ciocalteau method (Zhao et al., 2013). UVmini-1240 Spectrophotometer (Shiamdzu, Japan) was used for the all band ultraviolet scanning. The material composition of WWS was analyzed using HPLC (Agilent 1200, USA) equipped with a diode array detector (Agilent, USA). YMC-Triart C18 (250 mm 4.6 mm) column (YMC separation technology, Japan) was used at 30 °C with 2:3 (v/v) methanol: 1% acetic solution as mobile phase at a flow rate of 0.6 ml min1.
2.3. Preparation for nano-filtrated WWS of SECS
3. Results and discussion
Mixed SECS with distilled water and soaked for 2 h at 30 °C, and then washed SECS repeatedly with three times. The washing liquid was combined to set the finally 1:10 (w/w) washing water to the dry weight of SECS ratio. The washing liquid mixture was filtrated to remove the small impurities, and the supernatant was centrifuged at 1800g for 10 min to get the WWS of SECS. The WWS of SECS were then nanofiltrated with NF-1812 roll polyamide composite membrane (Shanghai Filter Co., Ltd, China). The molecular weight cutoff (MWCO) was 1000 Da. The filtrated fraction (named nano-filtrated WWS) and cutoff fraction (named nano-cutoff WWS) were collected respectively and concentrated to the same volume under vacuum at 65 °C.
3.1. UV analysis of fractionations from WWS Steam explosion could deform the lignin with the increases of coumaric acid, vanillic acid and ferulic acid due to the cleavage of ester bonds of the lignin (Alvira et al., 2010). In the hydrolysate of steam exploded straw, many phenolic substances, such as 4-hydroxybenzaldehyde, catechol, syringaldehyde, syringic acid, vanillin, vanillic acid, ferulic acid, coumaric acid, etc., were observed (Sun et al., 2005). During the steam explosion, hemicellulose in lignocellulosic biomass was significantly hydrolyzed into mono- or oligo-saccharides, while the lignin was partially degraded and the Mw of degraded lignin was between 3 103 and 4 103 g mol1 with polydispersity of 1.4–2.0 (Chanda and
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Nagani, 2010). In this article, the WWS of SECS was separated into nano-filtrated fraction and nano-cutoff fraction by membrane with MWCO of 1000 Da. Petroleum ether extractive (PEE) from nano-cutoff WWS gave almost no UV absorption. The substances in nano-filtrated WWS with the wavelengths of 305 nm, 290 nm, and 262 nm were extracted by petroleum ether. According to the similar compatible principle, the PEE mainly contained strong lipophilic components, such as volatile oil, fat, sterol and so on, which almost gave no UV absorption. The polar substances in WWS of SECS were extracted by ether, chloroform and ethyl acetate successively. Owing to the different partition coefficient in each solvent, the organic solvent extractives contained different type and content of substances. The UV absorption of each extractive can be ranked from high to low in the following order: ether extractive (EE) > ethyl acetate extractive (EAE) > chloroform extractive (CE). Meanwhile, the UV absorption of each extractive from nano-filtrated WWS was higher than that from nano-cutoff WWS. As for nano-cutoff WWS, both of EE and CE had the major UV absorption peak at 305 nm and 280 nm, but only 280 nm for EAE. The major UV absorption peaks of EAE extracted from nano-filtrated WWS were 305 nm and 280 nm, but only 280 nm for CE. After the sequential extraction of organic solvents, the substances with UV absorption in the alkaline methane solution were almost completely extracted. In order to further clarify the difference between substances in organic solvent extractives, HPLC was used to illustrate the composition of each extractive.
3.3. Decoloration of laccase-mediator system In general, triphenylmethane dyes is difficult to be degraded by microorganisms due to its complicated chemical structure. Thus, look for new biological treatment methods are getting more attention. Laccase-based decolorization treatments are more potentially advantageous than white-rot decolorization due to that the enzyme is produced in larger amounts and requires less fastidious induction conditions than either lignin peroxidase (LiP) and Mndependent peroxidase (MnP) (Asgher et al., 2008; Pointing and Vrijmoed, 2000). Therefore, laccase becomes an attractive option for biobleaching (Hadibarata et al., 2012; Hao et al., 2013). In present study, taking crystal violet and malachite green as example of triphenylmethane dyes, their decolorations catalyzed by laccase with different mediators were studied. At first 30 min, crystal violet was rapidly decolorized by laccase/EE (Fig. 1A). The decoloration of crystal violet at 20 min catalyzed by laccase/EE was 21.5% but only 3.28% for laccase/ABTS. The decolorization of crystal violet catalyzed by laccase/EAE was faster than that by laccase/ ABTS at first 60 min. However, the decolorization efficiency catalyzed by laccase/EAE proceeded significantly slowly after 60 min. The 8 h decoloration of crystal violet catalyzed by laccase with EE, EAE and ABTS as mediator was 66.95%, 42.36%, and 58.49%, respectively (Fig. 1A). The decoloration efficiency of malachite green by laccase/mediator system was even higher than crystal violet. There was 69.4%,
3.2. HPLC analysis of fractionations from nano-filtrated WWS When detected using 280 nm, PEE of nano-filtrated WWS almost gave no absorption under the HPLC condition. In this article, Rt T was used to define the substance with the special retention time (min) detected by HPLC. Substances of Rt 3.8418, Rt 6.8672, Rt 21.6416, and Rt 31.156 were the major substance in ether extractive (EE). The strong polar substances of Rt 3.8418 and Rt 6.8672 were extracted by chloroform, while substances of Rt 3.8418, Rt 6.0077, and Rt 17.7482 were dominated in EAE. As for nano-cutoff WWS, PEE also gave almost no UV absorption. Major substances in EE were Rt 3.9879, Rt 7.1597, Rt 20.7048, and Rt 34.9721. And major substances in EAE were similar to substances that extracted from nano-filtrated WWS by EAE. There were much more abundant substances with 305 nm absorption in nano-filtrated WWS than in nano-cutoff WWS. In the EE of nano-filtrated WWS, substance of Rt 31.156 had strong absorption, but Rt 34.9721 had weak absorption. CE of nano-filtrated WWS, including the substances of Rt 6.8672, Rt 29.8345, and Rt 33.3476, was pretty complicated than other organic solvent extractives. The dominant substance in EAE of nano-filtrated WWS was Rt 17.8943 with strong 305 nm absorption. As for nano-cutoff WWS, there were few substances with 305 nm absorption. The substances in EE of nano-cutoff WWS included Rt 34.9721 and Rt 39.8328, which had strong absorption and weak absorption, respectively. Substance of Rt 17.8943 was dominant in EAE. However, almost no substance with 305 nm absorption was detected in strong polar region. In the nano-filtrated WWS, substances in EAE with 260 nm UV absorption were Rt 6.07 and Rt 10.8401. The major substances in EE were Rt 15.6102 and Rt 21.6416. While the substances with 260 nm absorption were mainly existed in EE with retention time of 13.0094 min and 20.7523 min in the nano-cutoff WWS. Therefore, results showed that different organic solvent could fractionate weakly polar substances with high separation degree from WWS of SECS. It laid a good foundation for the subsequent structural analysis and identification of substances in WWS.
Fig. 1. Decolorization of dyes by laccase (25 IU) with different mediator at 50 °C. Dye concentration: 0.25 mg crystal violet (A); 0.25 mg malachite green (B). Mediator concentration: 0.025 ± 0.005 mg. (j) Ether extractive (EE) from nanofiltrated water washed solution (WWS) of steam exploded corn straw (SECS), (d) ethyl acetate extractive (EAE) from WWS of SECS, (N) ABTS.
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68.57%, and 81.52% of malachite green was decolorized by laccase/ EE, laccase/EAE and laccase/ABTS at 10 min, respectively. However, the decolorization speed of malachite green slowed down obviously after 10 min. After being decolorized by 60 min, more than 80% of decolorization was obtained for all mediators (Fig. 1B). Therefore, laccase combined with different mediators separated from WWS of SECS had a good performance for decoloration of triphenylmethane dyes, which should be the suitable methods for enhancing the decoloration performance of other solutions. 4. Conclusion Searching natural laccase mediators from the hydrolyzate of lignocellulosic biomass is an efficient approach to decrease the cost of laccase catalytic system and reduce the possible risk on environment. The substances with UV absorption in the nano-filtrated water washed solution (WWS) of steam exploded corn straw (SECS) were enriched by nanofiltration. The successively organic solvents extraction could separate the WWS of SECS into fractionations with different substances. The natural laccase mediators were effective for laccase to catalyze the decolorization of crystal violet and malachite green. In the further research, the substance in each organic solvent fractionation should be identified. Acknowledgements Financial support to this study was provided by the National Natural Science Foundation of China (Grant No. 21206176), the National Basic Research Program of China (No. 2011CB707401), the National Key Project of Scientific and Technical Supporting Program of China (No. 2011BAD22B02). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 01.044. References Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861.
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Asgher, M., Bhatti, H.N., Ashraf, M., Legge, R.L., 2008. Recent developments in biodegradation of industrial pollutants by white rot fungi and their enzyme system. Biodegradation 19, 771–783. Camarero, S., Ibarra, D., Martínez, M.J., Martínez, Á.T., 2005. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl. Environ. Microbiol. 71, 1775–1784. Cañas, A.I., Camarero, S., 2010. Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes. Biotechnol. Adv. 28, 694–705. Chanda, S.V., Nagani, K.V., 2010. Antioxidant capacity of Manilkara zapota L. Leaves extracts evaluated by four in vitro methods. Nat. Sci. 8, 260–266. Chen, H.Z., Liu, L.Y., 2007. Unpolluted fractionation of wheat straw by steam explosion and ethanol extraction. Bioresour. Technol. 98, 666–676. Chen, H.Z., Qiu, W.H., 2010. Key technologies for bioethanol production from lignocellulose. Biotechnol. Adv. 28, 556–562. Fillat, A., Colom, J.F., Vidal, T., 2010. A new approach to the biobleaching of flax pulp with laccase using natural mediators. Bioresour. Technol. 101, 4104–4110. Gutiérrez, A., Rencoret, J., Ibarra, D., Molina, S., Camarero, S., Romero, J., Del Río, J.C., Martínez, Á.T., 2007. Removal of lipophilic extractives from paper pulp by laccase and lignin-derived phenols as natural mediators. Environ. Sci. Technol. 41, 4124–4129. Hadibarata, T., Yusoff, A.R.M., Aris, A., Hidayat, T., Kristanti, R.A., 2012. Decolorization of azo, triphenylmethane and anthraquinone dyes by laccase of a newly isolated Armillaria sp. F022. Water Air Soil Pollut. 223, 1045–1054. Hao, L.Y., Wang, R., Liu, J.Q., Fang, K.J., 2013. The influence of auxiliary chemicals on the decolorization of reactive blue dye by laccase. Adv. Mater. Res. 709, 53–57. Kalyani, D., Dhiman, S.S., Kim, H., Jeya, M., Kim, I.W., Lee, J.K., 2012. Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass. Process Biochem. 47, 671–678. Liu, Z.H., Qin, L., Jin, M.J., Pang, F., Li, B.Z., Kang, Y., Dale, B.E., Yuan, Y.J., 2013. Evaluation of storage methods for the conversion of corn stover biomass to sugars based on steam explosion pretreatment. Bioresour. Technol. 132, 5–15. Ludwig, D., Amann, M., Hirth, T., Rupp, S., Zibek, S., 2013. Development and optimization of single and combined detoxification processes to improve the fermentability of lignocellulose hydrolyzates. Bioresour. Technol. 133, 455–461. Majeau, J.A., Brar, S.K., Tyagi, R.D., 2010. Laccases for removal of recalcitrant and emerging pollutants. Bioresour. Technol. 101, 2331–2350. Moldes, D., Díaz, M., Tzanov, T., Vidal, T., 2008. Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus Kraft pulp. Bioresour. Technol. 99, 7959–7965. Pointing, S., Vrijmoed, L., 2000. Decolorization of azo and triphenylmethane dyes by Pycnoporus sanguineus producing laccase as the sole phenoloxidase. World J. Microbiol. Biotechnol. 16, 317–318. Sun, X.F., Xu, F., Sun, R.C., Geng, Z.C., Fowler, P., Baird, M.S., 2005. Characteristics of degraded hemicellulosic polymers obtained from steam exploded wheat straw. Carbohyd. Polym. 60, 15–26. Wang, L., Chen, H.Z., 2011. Increased fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production by removal of fermentation inhibitors. Process Biochem. 46, 604–607. Zhao, X., Li, M., Huang, G., Chen, Y., 2013. Determination of total polyphenols in apple vinegar drink by Folin–Ciocalteu method. Chinese Food Sci. 34, 31–34.
