- Email: [email protected]
Evolution and stabilization of environmental persistent free radicals during the decomposition of lignin by laccase
Chemosphere 248 (2020) 125931
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Evolution and stabilization of environmental persistent free radicals during the decomposition of lignin by laccase Yafang Shi a, Kecheng Zhu a, Yunchao Dai a, Chi Zhang a, Hanzhong Jia a, b, * a
College of Natural Resources and Environment, Northwest A & F University, Yangling, 712100, China State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau, Institute of Soil and Water Conservation, Northwest A & F University, Yangling, 712100, China
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The mechanism of interaction between lignin and laccase from the perspective of EPFRs was described. EPFRs and ROS were detected during the decomposition of lignin by laccase. ROS concentration had a strong correlation with the EPFRs concentration. Laccase induced the demethylation and oxidation of lignin.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 December 2019 Received in revised form 9 January 2020 Accepted 14 January 2020 Available online 21 January 2020
Soil microbial enzymes may induce lignin decomposition, accompanied by generation of free radicals. The evolution of environmentally persistent free radicals (EPFRs) and reactive oxygen species (ROS) during laccase-catalyzed lignin decomposition remains unclear. Characterization by electron paramagnetic resonance spectroscopy revealed gradually increased concentration of EPFRs, with maximum levels within 6 h that remained constant, accompanied by the increase in g-factor from 2.0037 to 2.0041. The results suggested the generation of oxygen-centered radicals on lignin. The EPFRs produced on solid samples slowly decreased by 17.2% over 17 d. ROS were also detected to have a similar trend as that of the evolution of EPFRs. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, gel permeation chromatography and nuclear magnetic resonance analyses suggested the demethylation and oxidation of lignin. We clarify the biogeochemical transformation of lignin and potential contributions to the generation of EPFRs and ROS in soil. © 2020 Elsevier Ltd. All rights reserved.
Handling Editor: T Cutright Keywords: Lignin Laccase Decomposition Environmentally persistent free radicals Reactive oxygen species
1. Introduction Lignin is an amorphous, three-dimensional, and highly branched polyphenolic macromolecule with a complex structure
* Corresponding author. College of Natural Resources and Environment, Northwest A & F University, Yangling, 712100, China. E-mail address: [email protected] (H. Jia). https://doi.org/10.1016/j.chemosphere.2020.125931 0045-6535/© 2020 Elsevier Ltd. All rights reserved.
consisting of three types of aromatic units, including syringyl, guaiacyl, and p-hydroxyphenyl (Li et al., 2015). Wood and herbaceous plants are the main sources of lignin, which finally present in natural phases, especially soil and sediments (Mishra et al., 2016). After entering the soil, lignin is inevitably decomposed. Soil microbial communities play a significant role in this process (Crawford et al., 1977). As reported previously, approximately 53.75% of lignin in tobacco stalks could be degraded by
2
Y. Shi et al. / Chemosphere 248 (2020) 125931
Phanerochaete chrysosporium in 15 d (Su et al., 2016). Fungi may induce the complete decomposition of lignin during the same length of time (Davis and Sello, 2010). Microbe-mediated depolymerization of lignin is a complex process involving various types of enzymes, which include lignin peroxidase, manganese peroxidase, and laccase (Higuchi, 2004). These enzymes can directly attack lignin, cellulose, and hemicellulose of the plant cell wall, which often results in the structural modification of lignin (Orpin, 1984). Laccase is a type of oxidases that may promote cleavages in lignin molecules, including opening of the aromatic ring, alkyl-aryl disruption, and increase in phenolic hydroxyl group (Kawai et al., 1988a). During these processes, bond cleavage and redox reaction via electron transfer always participate in lignin decomposition. As a consequence, a large variety of free radical species may be produced within the lignin matrix (Kudanga et al., 2011). The polymer-associated free organic radicals may be stable and detected by electron paramagnetic resonance (EPR) spectroscopy under natural condition, and have been termed environmentally persistent free radicals (EPFRs) (Perna et al., 2019). EPFRs have attracted a lot of attentions recently due to their unique properties, which include the potential to induce the electron transfer reaction (Jia and Wang, 2013) and promote the generation of ROS (Jia et al., 2018), such as peroxide, superoxide, and hydroxyl radicals, which have benefits and drawbacks concerning the environmental effects of EPFRs (Khachatryan et al., 2011). On the one hand, EPFRs-induced ROS may eliminate the organic contaminants in natural phases (Chen et al., 2017; Jia et al., 2015; Wang et al., 2015). On the other hand, the potential formation of ROS may have a negative effect on human health by the oxidative stress (Ray et al., 2012; Zhang et al., 2019). Much work has been performed to understand the formation and fate of EPFRs associated with various matrices, such as fly ash, carbonaceous materials, and superfund sites contaminated by organic contaminants (Cruz et al., 2011; Liao et al., 2014; Zhao et al., 2019). Only a few studies have been conducted to examine the evolution and potential generation of ROS of lignin-EPFRs induced by enzymatic treatment (Silva et al., 2011). The objectives of this work were (1) to probe the evolution of EPFRs during the decomposition of lignin by laccase, (2) to observe the generated ROS in the lignin/laccase system using the chemical spin trap, and (3) to clarify the underlying mechanisms for the generation of lignin-EPFRs using scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC). The findings clarify the biogeochemical transformations of lignin in the soil environment, and provide a new insight into the environmental significance of the generation of EPFRs and ROS during the decomposition of lignin by laccase.
2.2. Enzymatic treatment of lignin with laccase Batch experiments were conducted in 10 mL brown glass bottles. Specifically, 0.05 g of lignin samples and 5 mL 0.08 g L1 of laccase in HAc-NaAc buffer solution were successively added into the reactors, which were then incubated on a shaker at 120 rpm at room temperature (approximately 25 C) in the dark. At the preselected reaction times of 1, 2, 3, and 24 h, the reacted samples were collected and freeze-dried for 24 h in a model LGJ-10C freezedryer (Karaltay Instruments Co., Ltd., Beijing, China). The freezedried samples were characterized by EPR and other techniques. A control experiment was carried out without the addition of laccase under the same conditions. Each treatment was repeated three times. 2.3. EPR measurement of EPFRs EPR was applied to determine the type and concentration of EPFRs on lignin samples before and after laccase treatment. Briefly, 0.02 g of solid samples were placed into an EPR tube and measured by an EMX micro spectrometer (Bruker, Karlsruhe, Germany) at room temperature. To investigate the decay of lignin-EPFRs, the lignin samples treatment for 24 h were collected and freeze-dried. And then evolution of EPFRs of samples was monitored in the dark. The specific operation parameters were presented in Text S1. 2.4. Determination of ROS ROS generated during the treatment of lignin by laccase were determined with the chemical spin-trapping method and EPR spectroscopy (Cao et al., 2008). Specifically, 0.05 g of lignin samples were suspended in 5 mL HAc-NaAc buffer solution (pH 5). Then, PBN and laccase were successively added to the mixture and stirred at 120 rpm at room temperature (approximately 25 C) in the dark. At the pre-selected reaction time, ethyl acetate was added to the reactor and the samples were sonicated for 5 min. The suspension was centrifuged for 5 min at 7500 rpm. The organic phase was separated and filtered using a 0.45 mm membrane syringe filter. The extraction solution was immediately transferred to an EPR capillary tube and then was detected. The measurement parameters of EPR were described in Text S1. 2.5. Characterizations of lignin The lignin samples before and after treatment by laccase were characterized by SEM, ATR-FTIR, 13C NMR spectra, two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR spectra and GPC. The details about these characterizations can be found in Text S2. 2.6. Determination of phenolic hydroxyl content
2. Materials and methods 2.1. Chemicals and materials Alkaline lignin and potassium bromide (KBr, 99.0%) were obtained from J&K Scientific Ltd. (Beijing, China). Laccase (activity: 0.5 U/mg, derived from Trametes versicolor), N-tert-butyl-a-phenylnitrone (PBN, 98.0%) and dimethyl sulfoxide-d6 (DMSO-d6, 99.8%) were provided by SigmaeAldrich (Beijing, China). Sodium acetate (NaAc, 99.0%), sodium carbonate (Na2CO3, 99.8%), Folin Cioulteau (FC, 99.0%) reagent, acetic acid (HAc, 99.5%), and pyrogallic acid (PA, 99.5%) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
The contents of phenolic hydroxyl (PhOH) in lignin samples were determined by the Folin Cioulteau method as previously reported (Ainsworth and Gillespie, 2007). Briefly, 0.5 mg mL1 lignin suspension was mixed with 1 N Folin Cioulteau reagent and then reacted for 5 min at the room temperature. Subsequently, 2 mL 20% (w/v) of Na2CO3 and 45 mL of deionized water were added in sequence. The reaction was conducted at room temperature for 2 h. Then the samples were measured by ultravioletevisible (UVeVis) spectroscopy using a model 201 spectrophotometer (Thermo Evolution, Boston, MA, USA) at 760 nm. All measurements were carried out in triplicate. Pyrogallic acid was determined in the same way as the standard curve.
Y. Shi et al. / Chemosphere 248 (2020) 125931
3
3. Results and discussion 3.1. Formation and evolution of EPFRs on lignin samples To explore the formation of EPFRs, the lignin samples treated by laccase for different time were monitored by EPR. As shown in Fig. 1a, all EPR spectra presented a single and symmetric line without any hyperfine splitting. The intensity of EPR signal for the samples treated by laccase was higher than the original lignin. Thus, the detected EPR signal could be mainly contributed by the decomposition of lignin by laccase. The g-factor of the EPR signal ranged from 2.0037 to 2.0041, which were assigned to organic radicals (Jia et al., 2016). With the elapsed reaction time, the yields of EPFRs increased rapidly in the first 6 h, and then gradually reached a plateau. The highest yields of EPFRs on lignin reached approximately 4.7 1016 spins g1 after 24 h (Fig. 1b). In the control experiment, spin densities of EPFRs on lignin samples remained constant during the reaction (Fig. S1). Thus, the detected EPR signal could be mainly contributed by the decomposition of lignin by laccase. In general, the characteristic of EPR spectrum such as g-factor can be used to identify the type of free radicals (Borrowman et al., 2015). The radicals with g-factor < 2.0030 can be assigned to carbon-centered radicals and the radicals with g-factor > 2.0040 are designated as oxygen-centered radicals (Christoforidis et al., 2007). When the g-factor is in the range of 2.0030e2.0040, it indicates that the system contains not only carbon-centered but also oxygen-centered radicals (Jezierski et al., 2008). Thus, the EPFRs on original lignin sample can be assigned as the mixture of carboncentered radicals and oxygen-centered radicals. With increased reaction time, the g-factor of the treated lignin was gradually increased, indicating that more oxygen-centered radicals, such as semiquinone radicals, were formed on the lignin. The g-factor of EPFRs produced on the lignin samples was relatively high, which was probably attributed to the longer lifetime of oxygenic radicals (Jia et al., 2016). A previous study on laccase-catalyzed oxidation of beech wood fibers illustrated the formation of EPFRs (Felby et al., 1997). Radicals are always reactive and readily react with other chemicals (Samanta et al., 1989). In the present work, we recorded the evolution of EPFRs on laccase-treated lignin samples with the elapsed reaction time. These results are displayed in Fig. 2. In general, the spin densities of EPFRs on lignin gradually decreased with the increase of time. After 17 d, the concentration of EPFRs decreased by 15% of the initial spin densities of lignin. The decay of
Fig. 2. Evolution of g-factor and spin density of EPR spectra on lignin after treatment for 24 h by laccase in the dark.
radicals was most likely due to the radical-radical combination and/ or reaction between free organic radicals and some other small molecules, which led to the generation of non-radical products (Munk et al., 2017). Most likely, the electron transfer reactions between EPFRs and O2/H2O induced the consumption of EPFRs and formation of ROS. 3.2. Formation of ROS on the lignin samples The ROS generated during the decomposition of lignin by laccase were determined by spin-trapping method and EPR spectroscopy. As shown in Fig. 3a, variable EPR spectral features were observed as a function of reaction time, suggesting that various abundances of ROS were produced. The observed signals could be fit by a triplet peak at g ¼ 2.005 and aN ¼ 15 G, which is a characteristic feature of PBN-OH spin adducts (Zhou et al., 2009). No EPR signal of PBNeOH spin adducts was detected in the laccasefree control experiment (Fig. S2). The obtained results indicated that the formation of OH during the decomposition of lignin by the laccase. More importantly, the amount of OH increased rapidly in the first 6 h of the reaction and then gradually reached a plateau, which was consistent with the trend of the evolution of EPFRs. The highest accumulation of OH reached approximately 1.3 1014
Fig. 1. Evolution of (a) EPR spectra and (b) their g-factor and spin density as a function of reaction time in the reaction system of lignin and laccase.
4
Y. Shi et al. / Chemosphere 248 (2020) 125931
Fig. 3. EPR spectra (a) and concentration of OH (b) as a function of reaction time in the reaction system of lignin and laccase.
spins g1 after 24 h (Fig. 3b). As discussed above, the formation of the ROS could be due to the lignin-bound EPFRs donating the electron to the oxygen molecules (Chen et al., 2017). The treated lignin containing EPFRs readily reacts with O2 to form superoxide radicals, which likely induce the generation of H2O2 upon interaction with H2O (Campos-Martin et al., 2006). The H2O2 that forms may be decomposed to OH by dismutation and Fenton-like reactions via activation by ligninassociated quinone moieties, such as semiquinone-type radicals (Lavrent et al., 2014). In this process, free electrons on lignin moieties participate in the redox-cycling reaction, which is crucial to promote ROS formation (Yuan et al., 2004). To further certify this process, the laccase-treated lignin was freeze-dried and enzyme was inactivated. As shown in Fig. S3, the EPR signal of OH was still detected in the mixture of PBN and dried lignin samples. The concentration of OH also showed a trend of a rapid increase followed by the gradual attainment of a plateau, suggesting that the generation of ROS was mainly due to the change of lignin characteristics, such as the newly-produced EPFRs and other physicochemical properties, during the enzymatic treatment
(Arangio et al., 2016). As shown in Fig. 4, the generation of OH positively correlated with the spin densities of lignin samples (r2 ¼ 0.86, p < 0.01). These results indicated that the laccaseinduced free organic radicals readily provided electrons to O2 and induced the formation of ROS. Based on these results, ligninassociated EPFRs could be classified into two types of radicals, i.e., original one that inactive to oxygen molecules, and newly-formed ones with relatively high activity for ROS generation. In the original lignin sample, the detected EPFRs might be confined heterogeneously in lignin polymer and were protected by hydrophobic microenvironment in the lignin matrix. Therefore, the small molecules, such as O2 and H2O, not easily access with the structural free organic radicals, exhibiting lower activity and higher stability than new-produced free radicals, which is consistent with previous studies (Paul et al., 2006; Zhao et al., 2019). 3.3. Characterization of lignin samples Formation of EPFRs during the decomposition of lignin by laccase might be accompanied by changes of physicochemical properties of lignin. In our study, lignin samples were systematically characterized by following methods. 3.3.1. SEM SEM was adopted to observe the potential change of surface structures of lignin during the enzymatic treatment. As shown in Fig. S4, the pristine lignin samples were spherical particles with smooth surface. However, after 24 h of treatment with laccase, the lignin displayed an obvious morphology change, and fissures with rugged texture were observed on the surface. Similar morphological changes were detected in previous works, which was related to the chain scission processes (Ramalingam et al., 2017).
Fig. 4. Correlation between the formation of EPFRs and OH on lignin samples.
3.3.2. GPC The weight-average (Mw), number-average (Mn) molecular weights, and polydispersity (PDI, Mw/Mn) of lignin samples before and after enzymatic treatment are listed in Table S1. Mw and Mn of the treated lignin samples were obviously lower than the values of the original lignin sample. These results indicated that the lignin samples were degraded after treatment with laccase (Alejandro et al., 2014). Fig. S5 displays the molecular weight distribution curves of lignin samples. Compared to the original lignin, the GPC peaks of
Y. Shi et al. / Chemosphere 248 (2020) 125931
5
the treated lignin samples shifted toward higher retention times and lower refractive indices, which suggested that lower molecular weight of lignin after the enzymatic treatment. These results were consistent with the changes of Mw and Mn. The increased PDI after laccase treatment indicated the structural heterogeneity of the lignin. Similar results were also obtained during the degradation of lignin in jute fiber by laccase (Zhang et al., 2014). These collective findings demonstrated the partial depolymerization of lignin during treatment with laccase. 3.3.3. ATR-FTIR The absorption spectra of the lignin samples were recorded in the range of 400e4000 cm1 (Fig. S6). These spectra indicate an appreciable change for lignin samples following treatment. The band at 3423 cm1, which could be attributed to OeH stretching vibration of hydroxyl groups (Guo et al., 2018), displayed a higher absorption intensity for the treated sample than that of the original sample. This could be attributed to the oxidation and degradation of lignin during the enzymatic treatment. The bands at 2920 and 3000 cm1 were assigned to CeH stretching vibration of eCH3, eCH2 and CH3O groups (Tejado et al., 2007). The absorptions of these bands for the treated lignin samples were substantially lower than that of the original one. It is conceivable that some methoxy groups were removed from the aromatic ring during the treatment with laccase. The band at 1696 cm1 could be attributed to the C]O stretching of the aromatic skeleton. Increased absorption at 1696 cm1 in treated samples indicated an overall increase in ketones and carboxylic acids. The intensity at 1567 cm1, which was assigned to the benzene skeleton vibration, was also decreased. These results indicated that the structure of the lignin samples was significantly changed, and more oxygenic groups were formed in the process of enzymatic treatment (Longe et al., 2018). 3.3.4. 13C NMR spectra 13 C NMR spectra were also used to identify the type of chemical moieties in lignin samples. As shown in Fig. S7, the aliphatic CeC (from 0 to 50 ppm) was formed and the decreasing of methoxy groups (from 50 to 60 ppm) was observed. Most of the aliphatic CeO signals (from 60 to 95 ppm) and aromatic region (from 95 to 157 ppm) nearly disappeared. Formation of the carboxyl group (from 157 to 215 ppm) was also observed. The
Fig. 6. Ph-OH content of the lignin after treatment for different time by laccase.
finding indicated that the aromatic rings were easily broken up by laccase and transformed to non-aromatic moieties. 3.3.5. 2D HSQC NMR spectra Further insights in the characteristic change of lignin were obtained by 2D-NMR HSQC measurement. The observed peaks could be assigned to aliphatics, methoxy groups, oxygenated aliphatics, and aromatics, respectively (Fig. 5). During the enzymatic treatment, the peaks in the aromatic region decreased, while the peaks in the oxygenated aliphatic region increased, indicating the opening of aromatic ring. The decrease of methoxy group was also observed for the treated sample, which was consistent with 13C NMR, indicating that demethylation may occur in the process. Prasetyo et al. (2010) also observed the decrease in the aromatic region in 2D HSQC NMR spectra of lignosulfonates after treatment with laccase. 3.3.6. Ph-OH content Contents of Ph-OH in lignin samples were further determined.
Fig. 5. 2D HSQC NMR spectra of lignin samples after treatement for 0 h and 24 h by laccase.
6
Y. Shi et al. / Chemosphere 248 (2020) 125931
Fig. 7. Possible formation pathway of EPFRs during the decomposition of lignin by laccase.
Compared with the original lignin, the treated lignin samples possessed higher Ph-OH content. Meanwhile, the Ph-OH content increased as the reaction time increased (Fig. 6), which were consistent with previous reports (Li et al., 2018). The collective findings of these analyses indicated that lignin experienced chain cleavages such as demethylation or/and hydroxylation, leading to the formation of the polyphenol structure during the decomposition by laccase. 3.4. Possible formation pathway of EPFRs The foregoing findings indicated that chain cleavage, opening of the benzene ring, and hydroxylation reactions might occur and were responsible for EPFRs formation on lignin. A proposed pathway is presented in Fig. 7. According to previous studies, when the lignin samples came into contact with laccase, they were oxidized to form phenoxy radicals (Radical 1) (Kawai et al., 1988b). These radicals are very unstable and may lead to further reaction. Pathway A involved electron transfer and resulted in carboncentered radicals (Radical 2). The oxygen-substituted carbon of the aromatic ring was easily to react with O2, forming ring-opening ^do et al., 2019). As shown in pathway B, the CeO products (Figueire bond of methoxyl groups were cleaved, inducing the formation of phenol hydroxyl-rich products (Filley et al., 2002), which was consistent with 2D-NMR HSQC and phenolic hydroxyl content analyses. In the proposed pathway, laccase induced the cleavage of benzene ring and formation of low molecular products and small mobile radicals, leading to formation of lower molecular weight of lignin, which were confirmed by NMR and GPC analyses. The demethylation and depolymerization accounted for the increase of PhOH contents and decreased Mw in lignin (Munk et al., 2015). Intermediates of this oxidation, such as phenoxy radicals, led to spontaneous generation of ROS and degradation of the lignin. 4. Conclusions Our results clearly demonstrate the generation of EPFRs during the decomposition of lignin by laccase. However, the spin density of the generated EPFRs can gradually decay in the dark. A scheme for the decomposition of lignin is proposed. The scheme includes the formation of EPFRs and oxidized products. EPFRs formed on the surfaces of lignin can be transformed to ROS. The findings increase our understanding about the biogeochemical transformations of lignin and potential contributions to the natural generation of EPFRs and ROS in the soil environment.
CRediT authorship contribution statement Yafang Shi: Investigation, Formal analysis, Writing - original draft. Kecheng Zhu: Validation, Data curation. Yunchao Dai: Project administration. Chi Zhang: Writing - review & editing. Hanzhong Jia: Supervision, Resources, Funding acquisition. Acknowledgment This study was supported by the National Natural Science Foundation of China (Grants No. 41571446 & 41877126), the National Key R&D Program of China (Grant No. 2018YFC1802004), Shaanxi Key R&D Program of China (Grant No. 2019ZDLNY01-0201), the “One Hundred Talents” program of Shaanxi Province (SXBR9171), the CAS Youth Innovation Promotion Association (2016380), and the Shaanxi Science Fund for Distinguished Young Scholars (Grant No. 2019JC-18). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2020.125931. References Ainsworth, E.A., Gillespie, K.M., 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using FolineCiocalteu reagent. Nat. Protoc. 2, 875e877. Alejandro, R., Jorge, R., Río, J.C., Del, Martínez, A.T., Ana, G., 2014. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol. Biofuels 7, 14. € schl, U., Shiraiwa, M., 2016. Quantification of Arangio, A.M., Tong, H., Socorro, J., Po environmentally persistent free radicals and reactive oxygen species in atmospheric aerosol particles. Atmos. Chem. Phys. 16, 13105e13119. Borrowman, C.K., Zhou, S., Burrow, T.E., Abbatt, J.P., 2015. Formation of environmentally persistent free radicals from the heterogeneous reaction of ozone and polycyclic aromatic compounds. Phys. Chem. Chem. Phys. 18, 205e212. Campos-Martin, J.M., Blanco-Brieva, G., Fierro, J.L.G., 2006. Hydrogen peroxide synthesis: an outlook beyond the anthraquinone process. ChemInform 45, 6962e6984. Cao, Y.J., Duan, X.F., Cao, Y.L., Lue, J.X., Zhu, J.Q., Zhou, G.W., Zhao, B.L., 2008. ESR study in reactive oxygen species free radical production of Pinus kesiya var. langbinensis Heartwood treated with laccase. Appl. Magn. Reson. 35, 205e211. Chen, N., Huang, Y., Hou, X., Ai, Z., Zhang, L., 2017. Photochemistry of hydrochar: reactive oxygen species generation and sulfadimidine degradation. Environ. Sci. Technol. 51, 11278e11287. Christoforidis, K.C., Un, S., Deligiannakis, Y., 2007. High-Field 285 GHz electron paramagnetic resonance study of indigenous radicals of humic acids. J. Phys. Chem. A 111, 11860e11866. Crawford, D.L., Floyd, S., Pometto, A.L., Crawford, R.L., 1977. Degradation of natural and Kraft lignins by the microflora of soil and water. Can. J. Microbiol. 23, 434e440. Cruz, A.L.N., Dela, William, G., Slawomir, L., Robert, C., Barry, D., 2011. Detection of
Y. Shi et al. / Chemosphere 248 (2020) 125931 environmentally persistent free radicals at a superfund wood treating site. Environ. Sci. Technol. 45, 6356e6365. Davis, J.R., Sello, J.K., 2010. Regulation of genes in Streptomyces bacteria required for catabolism of lignin-derived aromatic compounds. Appl. Microbiol. Biotechnol. 86, 921e929. Felby, C., Nielsen, B.R., Olesen, P.O., Skibsted, L.H., 1997. Identification and quantification of radical reaction intermediates by electron spin resonance spectrometry of laccase-catalyzed oxidation of wood fibers from beech (Fagus sylvatica). Appl. Microbiol. Biotechnol. 48, 459e464. ^do, M.B., Deuss, P.J., Venderbosch, R.H., Heeres, H.J., 2019. Valorization of Figueire pyrolysis liquids: ozonation of the pyrolytic lignin fraction and model components. ACS Sustain. Chem. Eng. 7, 4755e4765. Filley, T.R., Cody, G.D., Goodell, B., Jellison, J., Noser, C., Ostrofsky, A., 2002. Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi. Org. Geochem. 33, 111e124. Guo, X., Pang, J., Chen, S., Jia, H., 2018. Sorption properties of tylosin on four different microplastics. Chemosphere 209, 240e245. Higuchi, T., 2004. Microbial degradation of lignin: role of lignin peroxidase, manganese peroxidase, and laccase. P. JPN. ACAD. B-PHYS. 80, 204e214. Jezierski, A., Skrzypek, G., Jezierski, P., Paul, D., Jedrysek, M.O., 2008. Electron paramagnetic resonance (EPR) and stable isotope records of paleoenvironmental conditions during peat formation. Acta Part A Molec. Biomolec. Spectrosc. 69, 1311e1316. Jia, H., Chen, H., Nulaji, G., Li, X., Wang, C., 2015. Effect of low-molecular-weight organic acids on photo-degradation of phenanthrene catalyzed by Fe(III)smectite under visible light. Chemosphere 138, 266e271. Jia, H., Nulaji, G., Gao, H., Wang, F., Zhu, Y., Wang, C., 2016. Formation and stabilization of environmentally persistent free radicals induced by the interaction of anthracene with Fe(III)-modified clays. Environ. Sci. Technol. 50, 6310e6319. Jia, H., Wang, C., 2013. Comparative studies on montmorillonite-supported zerovalent iron nanoparticles produced by different methods: reactivity and stability. Environ. Technol. 34, 25e33. Jia, H., Zhao, S., Shi, Y., Zhu, L., Wang, C., Sharma, V.K., 2018. Transformation of polycyclic aromatic hydrocarbons and formation of environmentally persistent free radicals on modified montmorillonite: the role of surface metal ions and polycyclic aromatic hydrocarbon molecular properties. Environ. Sci. Technol. 52, 5725e5733. Kawai, S., Umezawa, T., Higuchi, T., 1988a. Degradation mechanisms of phenolic beta-1 lignin substructure model compounds by laccase of Coriolus versicolor. Arch. Biochem. Biophys. 262, 99e110. Kawai, S., Umezawa, T., Shimada, M., Higuchi, T., 1988b. Aromatic ring cleavage of 4,6-di(tert-butyl)guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor. FEBS Lett. 236, 309e311. Khachatryan, L., Vejerano, E., Lomnicki, S., Dellinger, B., 2011. Environmentally persistent free radicals (EPFRs). 1. Generation of reactive oxygen species in aqueous solutions. Environ. Sci. Technol. 45, 8559e8566. Kudanga, T., Nyanhongo, G.S., Guebitz, G.M., Burton, S., 2011. Potential applications of laccase-mediated coupling and grafting reactions: a review. Enzym. Microb. Technol. 48, 195e208. Lavrent, K., Mcferrin, C.A., Hall, R.W., Barry, D., 2014. Environmentally persistent free radicals (EPFRs). 3. Free versus bound hydroxyl radicals in EPFR aqueous solutions. Environ. Sci. Technol. 48, 9220e9226. Li, Z.L., Zhang, J.B., Qn, L., Ge, Y.Y., 2018. Enhancing antioxidant performance of lignin by enzymatic treatment with laccase. ACS Sustain. Chem. Eng. 6, 2591e2595. Li, Z., Yan, K., Ge, Y., 2015. Synthesis of porous lignin xanthate resin for Pb 2þ removal from aqueous solution. Chem. Eng. J. 270, 229e234. Liao, S., Pan, B., Li, H., Zhang, D., Xing, B., 2014. Detecting free radicals in biochars and determining their ability to inhibit the germination and growth of corn, wheat and rice seedlings. Environ. Sci. Technol. 48, 8581e8587. Longe, L.F., Couvreur, J., Grandchamp, M.L., Garnier, G., Allais, F., Saito, K., 2018.
7
Importance of mediators for lignin degradation by fungal laccase. ACS Sustain. Chem. Eng. 6, 10097e10107. Mishra, P.K., Wimmer, R., Gregor, T., 2016. Utilising Brewer’s spent grain as a source of cellulose nanofibres following separation of protein-based biomass. Bioresources 12, 107e116. Munk, L., Punt, A.M., Kabel, M.A., Meyer, A.S., 2017. Laccase catalyzed grafting of eNeOH type mediators to lignin via radicaleradical. RSC Adv. 7, 3358e3368. Munk, L., Sitarz, A.K., Kalyani, D.C., Mikkelsen, J.D., Meyer, A.S., 2015. Can laccases catalyze bond cleavage in lignin? Biotechnol. Adv. 33, 13e24. Orpin, C.G., 1984. Microbial attack on lignocellulose in the rumen. Appl. Biochem. Biotechnol. 9, 327e328. Paul, A., Stosser, R., Zehl, A., Zwirnmann, E., Vogt, R.D., 2006. Steinberg, C. E. W., Nature and abundance of organic radicals in natural organic matter: effect of pH and irradiation. Environ. Sci. Technol. 40, 5897e5903. Perna, V., Agger, J.W., Andersen, M.L., Holck, J., Meyer, A.S., 2019. Laccase induced lignin radical formation kinetics evaluated by electron paramagnetic resonance spectroscopy. ACS Sustain. Chem. Eng. 7, 10425e10434. rrez, A., Río, J.C.D., Prasetyo, E.N., Kudanga, T., Østergaard, L., Rencoret, J., Gutie nez-Barbero, J., Martínez, A.T., 2010. Polymerization of Santos, J.I., Nieto, L., Jime lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 101, 5054e5062. Ramalingam, B., Sana, B., Seayad, J., Ghadessy, F.J., Sullivan, M.B., 2017. Towards understanding of laccase-catalysed oxidative oligomerisation of dimeric lignin model compounds. RSC Adv. 7, 11951e11958. Ray, P.D., Bo-Wen, H., Yoshiaki, T., 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell. Signal. 24, 981e990. Samanta, A., Bhattacharyya, K., Das, P.K., Kamat, P.V., Hug, G.L., 1989. Quenching of excited doublet states of organic radicals by stable radicals. J. Phys. Chem. 93, 3651e3656. , T., Kim, S.Y., Padra ~o, J., Prasetyo, E.N., Kudanga, T., Silva, C., Matama Nyanhongo, G.S., Guebitz, G.M., Casal, M., Cavaco-Paulo, A., 2011. Antimicrobial and antioxidant linen via laccase-assisted grafting. React. Funct. Polym. 71, 713e720. Su, Y., Xian, H., Shi, S., Zhang, C., Manik, S.M.N., Mao, J., Zhang, G., Liao, W., Wang, Q., Liu, H., 2016. Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk. BMC Biotechnol. 16, 1e9. Tejado, A., Pena, C., Labidi, J., Echeverria, J.M., Mondragon, I., 2007. Physico-chemical characterization of lignins from different sources for use in phenolformaldehyde resin synthesis. Bioresour. Technol. 98, 1655e1663. Wang, Z., Zang, L., Fan, X., Jia, H., Li, L., Deng, W., Wang, C., 2015. Defect-mediated of Cu@TiO2 coreeshell nanoparticles with oxygen vacancies for photocatalytic degradation 2,4-DCP under visible light irradiation. Appl. Surf. Sci. 358, 479e484. Yuan, X., Cong, Y., Hao, Shan, Y., Zhao, Z., Wang, S., Chen, J., 2004. Regulation of LIP level and ROS formation through interaction of H-ferritin with G-CSF receptor. J. Mol. Biol. 339, 131e144. Zhang, Y., Guo, X., Si, X.H., Yang, R.X., Zhou, J.T., Quan, X., 2019. Environmentally persistent free radical generation on contaminated soil and their potential biotoxicity to luminous bacteria. Sci. Total Environ. 687, 348e354. Zhang, Y., Wang, Q., Fan, X., Yuan, J., 2014. Structural changes of lignin in the jute fiber treated by laccase and mediator system. J. Mol. Catal. B Enzym. 101, 133e136. Zhao, S., Gao, P., Miao, D., Wu, L., Qian, Y., Chen, S., Sharma, V.K., Jia, H., 2019. Formation and evolution of solvent-extracted and nonextractable environmentally persistent free radicals in fly ash of municipal solid waste incinerators. Environ. Sci. Technol. 53, 10120e10130. Zhou, G., Li, J., Chen, Y., Zhao, B., Cao, Y., Duan, X., Cao, Y., 2009. Determination of reactive oxygen species generated in laccase catalyzed oxidation of wood fibers from Chinese fir (Cunninghamia lanceolata) by electron spin resonance spectrometry. Bioresour. Technol. 100, 505e508.
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Evolution and stabilization of environmental persistent free radicals during the decomposition of lignin by laccase Yafang Shi a, Kecheng Zhu a, Yunchao Dai a, Chi Zhang a, Hanzhong Jia a, b, * a
College of Natural Resources and Environment, Northwest A & F University, Yangling, 712100, China State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau, Institute of Soil and Water Conservation, Northwest A & F University, Yangling, 712100, China
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The mechanism of interaction between lignin and laccase from the perspective of EPFRs was described. EPFRs and ROS were detected during the decomposition of lignin by laccase. ROS concentration had a strong correlation with the EPFRs concentration. Laccase induced the demethylation and oxidation of lignin.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 December 2019 Received in revised form 9 January 2020 Accepted 14 January 2020 Available online 21 January 2020
Soil microbial enzymes may induce lignin decomposition, accompanied by generation of free radicals. The evolution of environmentally persistent free radicals (EPFRs) and reactive oxygen species (ROS) during laccase-catalyzed lignin decomposition remains unclear. Characterization by electron paramagnetic resonance spectroscopy revealed gradually increased concentration of EPFRs, with maximum levels within 6 h that remained constant, accompanied by the increase in g-factor from 2.0037 to 2.0041. The results suggested the generation of oxygen-centered radicals on lignin. The EPFRs produced on solid samples slowly decreased by 17.2% over 17 d. ROS were also detected to have a similar trend as that of the evolution of EPFRs. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, gel permeation chromatography and nuclear magnetic resonance analyses suggested the demethylation and oxidation of lignin. We clarify the biogeochemical transformation of lignin and potential contributions to the generation of EPFRs and ROS in soil. © 2020 Elsevier Ltd. All rights reserved.
Handling Editor: T Cutright Keywords: Lignin Laccase Decomposition Environmentally persistent free radicals Reactive oxygen species
1. Introduction Lignin is an amorphous, three-dimensional, and highly branched polyphenolic macromolecule with a complex structure
* Corresponding author. College of Natural Resources and Environment, Northwest A & F University, Yangling, 712100, China. E-mail address: [email protected] (H. Jia). https://doi.org/10.1016/j.chemosphere.2020.125931 0045-6535/© 2020 Elsevier Ltd. All rights reserved.
consisting of three types of aromatic units, including syringyl, guaiacyl, and p-hydroxyphenyl (Li et al., 2015). Wood and herbaceous plants are the main sources of lignin, which finally present in natural phases, especially soil and sediments (Mishra et al., 2016). After entering the soil, lignin is inevitably decomposed. Soil microbial communities play a significant role in this process (Crawford et al., 1977). As reported previously, approximately 53.75% of lignin in tobacco stalks could be degraded by
2
Y. Shi et al. / Chemosphere 248 (2020) 125931
Phanerochaete chrysosporium in 15 d (Su et al., 2016). Fungi may induce the complete decomposition of lignin during the same length of time (Davis and Sello, 2010). Microbe-mediated depolymerization of lignin is a complex process involving various types of enzymes, which include lignin peroxidase, manganese peroxidase, and laccase (Higuchi, 2004). These enzymes can directly attack lignin, cellulose, and hemicellulose of the plant cell wall, which often results in the structural modification of lignin (Orpin, 1984). Laccase is a type of oxidases that may promote cleavages in lignin molecules, including opening of the aromatic ring, alkyl-aryl disruption, and increase in phenolic hydroxyl group (Kawai et al., 1988a). During these processes, bond cleavage and redox reaction via electron transfer always participate in lignin decomposition. As a consequence, a large variety of free radical species may be produced within the lignin matrix (Kudanga et al., 2011). The polymer-associated free organic radicals may be stable and detected by electron paramagnetic resonance (EPR) spectroscopy under natural condition, and have been termed environmentally persistent free radicals (EPFRs) (Perna et al., 2019). EPFRs have attracted a lot of attentions recently due to their unique properties, which include the potential to induce the electron transfer reaction (Jia and Wang, 2013) and promote the generation of ROS (Jia et al., 2018), such as peroxide, superoxide, and hydroxyl radicals, which have benefits and drawbacks concerning the environmental effects of EPFRs (Khachatryan et al., 2011). On the one hand, EPFRs-induced ROS may eliminate the organic contaminants in natural phases (Chen et al., 2017; Jia et al., 2015; Wang et al., 2015). On the other hand, the potential formation of ROS may have a negative effect on human health by the oxidative stress (Ray et al., 2012; Zhang et al., 2019). Much work has been performed to understand the formation and fate of EPFRs associated with various matrices, such as fly ash, carbonaceous materials, and superfund sites contaminated by organic contaminants (Cruz et al., 2011; Liao et al., 2014; Zhao et al., 2019). Only a few studies have been conducted to examine the evolution and potential generation of ROS of lignin-EPFRs induced by enzymatic treatment (Silva et al., 2011). The objectives of this work were (1) to probe the evolution of EPFRs during the decomposition of lignin by laccase, (2) to observe the generated ROS in the lignin/laccase system using the chemical spin trap, and (3) to clarify the underlying mechanisms for the generation of lignin-EPFRs using scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC). The findings clarify the biogeochemical transformations of lignin in the soil environment, and provide a new insight into the environmental significance of the generation of EPFRs and ROS during the decomposition of lignin by laccase.
2.2. Enzymatic treatment of lignin with laccase Batch experiments were conducted in 10 mL brown glass bottles. Specifically, 0.05 g of lignin samples and 5 mL 0.08 g L1 of laccase in HAc-NaAc buffer solution were successively added into the reactors, which were then incubated on a shaker at 120 rpm at room temperature (approximately 25 C) in the dark. At the preselected reaction times of 1, 2, 3, and 24 h, the reacted samples were collected and freeze-dried for 24 h in a model LGJ-10C freezedryer (Karaltay Instruments Co., Ltd., Beijing, China). The freezedried samples were characterized by EPR and other techniques. A control experiment was carried out without the addition of laccase under the same conditions. Each treatment was repeated three times. 2.3. EPR measurement of EPFRs EPR was applied to determine the type and concentration of EPFRs on lignin samples before and after laccase treatment. Briefly, 0.02 g of solid samples were placed into an EPR tube and measured by an EMX micro spectrometer (Bruker, Karlsruhe, Germany) at room temperature. To investigate the decay of lignin-EPFRs, the lignin samples treatment for 24 h were collected and freeze-dried. And then evolution of EPFRs of samples was monitored in the dark. The specific operation parameters were presented in Text S1. 2.4. Determination of ROS ROS generated during the treatment of lignin by laccase were determined with the chemical spin-trapping method and EPR spectroscopy (Cao et al., 2008). Specifically, 0.05 g of lignin samples were suspended in 5 mL HAc-NaAc buffer solution (pH 5). Then, PBN and laccase were successively added to the mixture and stirred at 120 rpm at room temperature (approximately 25 C) in the dark. At the pre-selected reaction time, ethyl acetate was added to the reactor and the samples were sonicated for 5 min. The suspension was centrifuged for 5 min at 7500 rpm. The organic phase was separated and filtered using a 0.45 mm membrane syringe filter. The extraction solution was immediately transferred to an EPR capillary tube and then was detected. The measurement parameters of EPR were described in Text S1. 2.5. Characterizations of lignin The lignin samples before and after treatment by laccase were characterized by SEM, ATR-FTIR, 13C NMR spectra, two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR spectra and GPC. The details about these characterizations can be found in Text S2. 2.6. Determination of phenolic hydroxyl content
2. Materials and methods 2.1. Chemicals and materials Alkaline lignin and potassium bromide (KBr, 99.0%) were obtained from J&K Scientific Ltd. (Beijing, China). Laccase (activity: 0.5 U/mg, derived from Trametes versicolor), N-tert-butyl-a-phenylnitrone (PBN, 98.0%) and dimethyl sulfoxide-d6 (DMSO-d6, 99.8%) were provided by SigmaeAldrich (Beijing, China). Sodium acetate (NaAc, 99.0%), sodium carbonate (Na2CO3, 99.8%), Folin Cioulteau (FC, 99.0%) reagent, acetic acid (HAc, 99.5%), and pyrogallic acid (PA, 99.5%) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
The contents of phenolic hydroxyl (PhOH) in lignin samples were determined by the Folin Cioulteau method as previously reported (Ainsworth and Gillespie, 2007). Briefly, 0.5 mg mL1 lignin suspension was mixed with 1 N Folin Cioulteau reagent and then reacted for 5 min at the room temperature. Subsequently, 2 mL 20% (w/v) of Na2CO3 and 45 mL of deionized water were added in sequence. The reaction was conducted at room temperature for 2 h. Then the samples were measured by ultravioletevisible (UVeVis) spectroscopy using a model 201 spectrophotometer (Thermo Evolution, Boston, MA, USA) at 760 nm. All measurements were carried out in triplicate. Pyrogallic acid was determined in the same way as the standard curve.
Y. Shi et al. / Chemosphere 248 (2020) 125931
3
3. Results and discussion 3.1. Formation and evolution of EPFRs on lignin samples To explore the formation of EPFRs, the lignin samples treated by laccase for different time were monitored by EPR. As shown in Fig. 1a, all EPR spectra presented a single and symmetric line without any hyperfine splitting. The intensity of EPR signal for the samples treated by laccase was higher than the original lignin. Thus, the detected EPR signal could be mainly contributed by the decomposition of lignin by laccase. The g-factor of the EPR signal ranged from 2.0037 to 2.0041, which were assigned to organic radicals (Jia et al., 2016). With the elapsed reaction time, the yields of EPFRs increased rapidly in the first 6 h, and then gradually reached a plateau. The highest yields of EPFRs on lignin reached approximately 4.7 1016 spins g1 after 24 h (Fig. 1b). In the control experiment, spin densities of EPFRs on lignin samples remained constant during the reaction (Fig. S1). Thus, the detected EPR signal could be mainly contributed by the decomposition of lignin by laccase. In general, the characteristic of EPR spectrum such as g-factor can be used to identify the type of free radicals (Borrowman et al., 2015). The radicals with g-factor < 2.0030 can be assigned to carbon-centered radicals and the radicals with g-factor > 2.0040 are designated as oxygen-centered radicals (Christoforidis et al., 2007). When the g-factor is in the range of 2.0030e2.0040, it indicates that the system contains not only carbon-centered but also oxygen-centered radicals (Jezierski et al., 2008). Thus, the EPFRs on original lignin sample can be assigned as the mixture of carboncentered radicals and oxygen-centered radicals. With increased reaction time, the g-factor of the treated lignin was gradually increased, indicating that more oxygen-centered radicals, such as semiquinone radicals, were formed on the lignin. The g-factor of EPFRs produced on the lignin samples was relatively high, which was probably attributed to the longer lifetime of oxygenic radicals (Jia et al., 2016). A previous study on laccase-catalyzed oxidation of beech wood fibers illustrated the formation of EPFRs (Felby et al., 1997). Radicals are always reactive and readily react with other chemicals (Samanta et al., 1989). In the present work, we recorded the evolution of EPFRs on laccase-treated lignin samples with the elapsed reaction time. These results are displayed in Fig. 2. In general, the spin densities of EPFRs on lignin gradually decreased with the increase of time. After 17 d, the concentration of EPFRs decreased by 15% of the initial spin densities of lignin. The decay of
Fig. 2. Evolution of g-factor and spin density of EPR spectra on lignin after treatment for 24 h by laccase in the dark.
radicals was most likely due to the radical-radical combination and/ or reaction between free organic radicals and some other small molecules, which led to the generation of non-radical products (Munk et al., 2017). Most likely, the electron transfer reactions between EPFRs and O2/H2O induced the consumption of EPFRs and formation of ROS. 3.2. Formation of ROS on the lignin samples The ROS generated during the decomposition of lignin by laccase were determined by spin-trapping method and EPR spectroscopy. As shown in Fig. 3a, variable EPR spectral features were observed as a function of reaction time, suggesting that various abundances of ROS were produced. The observed signals could be fit by a triplet peak at g ¼ 2.005 and aN ¼ 15 G, which is a characteristic feature of PBN-OH spin adducts (Zhou et al., 2009). No EPR signal of PBNeOH spin adducts was detected in the laccasefree control experiment (Fig. S2). The obtained results indicated that the formation of OH during the decomposition of lignin by the laccase. More importantly, the amount of OH increased rapidly in the first 6 h of the reaction and then gradually reached a plateau, which was consistent with the trend of the evolution of EPFRs. The highest accumulation of OH reached approximately 1.3 1014
Fig. 1. Evolution of (a) EPR spectra and (b) their g-factor and spin density as a function of reaction time in the reaction system of lignin and laccase.
4
Y. Shi et al. / Chemosphere 248 (2020) 125931
Fig. 3. EPR spectra (a) and concentration of OH (b) as a function of reaction time in the reaction system of lignin and laccase.
spins g1 after 24 h (Fig. 3b). As discussed above, the formation of the ROS could be due to the lignin-bound EPFRs donating the electron to the oxygen molecules (Chen et al., 2017). The treated lignin containing EPFRs readily reacts with O2 to form superoxide radicals, which likely induce the generation of H2O2 upon interaction with H2O (Campos-Martin et al., 2006). The H2O2 that forms may be decomposed to OH by dismutation and Fenton-like reactions via activation by ligninassociated quinone moieties, such as semiquinone-type radicals (Lavrent et al., 2014). In this process, free electrons on lignin moieties participate in the redox-cycling reaction, which is crucial to promote ROS formation (Yuan et al., 2004). To further certify this process, the laccase-treated lignin was freeze-dried and enzyme was inactivated. As shown in Fig. S3, the EPR signal of OH was still detected in the mixture of PBN and dried lignin samples. The concentration of OH also showed a trend of a rapid increase followed by the gradual attainment of a plateau, suggesting that the generation of ROS was mainly due to the change of lignin characteristics, such as the newly-produced EPFRs and other physicochemical properties, during the enzymatic treatment
(Arangio et al., 2016). As shown in Fig. 4, the generation of OH positively correlated with the spin densities of lignin samples (r2 ¼ 0.86, p < 0.01). These results indicated that the laccaseinduced free organic radicals readily provided electrons to O2 and induced the formation of ROS. Based on these results, ligninassociated EPFRs could be classified into two types of radicals, i.e., original one that inactive to oxygen molecules, and newly-formed ones with relatively high activity for ROS generation. In the original lignin sample, the detected EPFRs might be confined heterogeneously in lignin polymer and were protected by hydrophobic microenvironment in the lignin matrix. Therefore, the small molecules, such as O2 and H2O, not easily access with the structural free organic radicals, exhibiting lower activity and higher stability than new-produced free radicals, which is consistent with previous studies (Paul et al., 2006; Zhao et al., 2019). 3.3. Characterization of lignin samples Formation of EPFRs during the decomposition of lignin by laccase might be accompanied by changes of physicochemical properties of lignin. In our study, lignin samples were systematically characterized by following methods. 3.3.1. SEM SEM was adopted to observe the potential change of surface structures of lignin during the enzymatic treatment. As shown in Fig. S4, the pristine lignin samples were spherical particles with smooth surface. However, after 24 h of treatment with laccase, the lignin displayed an obvious morphology change, and fissures with rugged texture were observed on the surface. Similar morphological changes were detected in previous works, which was related to the chain scission processes (Ramalingam et al., 2017).
Fig. 4. Correlation between the formation of EPFRs and OH on lignin samples.
3.3.2. GPC The weight-average (Mw), number-average (Mn) molecular weights, and polydispersity (PDI, Mw/Mn) of lignin samples before and after enzymatic treatment are listed in Table S1. Mw and Mn of the treated lignin samples were obviously lower than the values of the original lignin sample. These results indicated that the lignin samples were degraded after treatment with laccase (Alejandro et al., 2014). Fig. S5 displays the molecular weight distribution curves of lignin samples. Compared to the original lignin, the GPC peaks of
Y. Shi et al. / Chemosphere 248 (2020) 125931
5
the treated lignin samples shifted toward higher retention times and lower refractive indices, which suggested that lower molecular weight of lignin after the enzymatic treatment. These results were consistent with the changes of Mw and Mn. The increased PDI after laccase treatment indicated the structural heterogeneity of the lignin. Similar results were also obtained during the degradation of lignin in jute fiber by laccase (Zhang et al., 2014). These collective findings demonstrated the partial depolymerization of lignin during treatment with laccase. 3.3.3. ATR-FTIR The absorption spectra of the lignin samples were recorded in the range of 400e4000 cm1 (Fig. S6). These spectra indicate an appreciable change for lignin samples following treatment. The band at 3423 cm1, which could be attributed to OeH stretching vibration of hydroxyl groups (Guo et al., 2018), displayed a higher absorption intensity for the treated sample than that of the original sample. This could be attributed to the oxidation and degradation of lignin during the enzymatic treatment. The bands at 2920 and 3000 cm1 were assigned to CeH stretching vibration of eCH3, eCH2 and CH3O groups (Tejado et al., 2007). The absorptions of these bands for the treated lignin samples were substantially lower than that of the original one. It is conceivable that some methoxy groups were removed from the aromatic ring during the treatment with laccase. The band at 1696 cm1 could be attributed to the C]O stretching of the aromatic skeleton. Increased absorption at 1696 cm1 in treated samples indicated an overall increase in ketones and carboxylic acids. The intensity at 1567 cm1, which was assigned to the benzene skeleton vibration, was also decreased. These results indicated that the structure of the lignin samples was significantly changed, and more oxygenic groups were formed in the process of enzymatic treatment (Longe et al., 2018). 3.3.4. 13C NMR spectra 13 C NMR spectra were also used to identify the type of chemical moieties in lignin samples. As shown in Fig. S7, the aliphatic CeC (from 0 to 50 ppm) was formed and the decreasing of methoxy groups (from 50 to 60 ppm) was observed. Most of the aliphatic CeO signals (from 60 to 95 ppm) and aromatic region (from 95 to 157 ppm) nearly disappeared. Formation of the carboxyl group (from 157 to 215 ppm) was also observed. The
Fig. 6. Ph-OH content of the lignin after treatment for different time by laccase.
finding indicated that the aromatic rings were easily broken up by laccase and transformed to non-aromatic moieties. 3.3.5. 2D HSQC NMR spectra Further insights in the characteristic change of lignin were obtained by 2D-NMR HSQC measurement. The observed peaks could be assigned to aliphatics, methoxy groups, oxygenated aliphatics, and aromatics, respectively (Fig. 5). During the enzymatic treatment, the peaks in the aromatic region decreased, while the peaks in the oxygenated aliphatic region increased, indicating the opening of aromatic ring. The decrease of methoxy group was also observed for the treated sample, which was consistent with 13C NMR, indicating that demethylation may occur in the process. Prasetyo et al. (2010) also observed the decrease in the aromatic region in 2D HSQC NMR spectra of lignosulfonates after treatment with laccase. 3.3.6. Ph-OH content Contents of Ph-OH in lignin samples were further determined.
Fig. 5. 2D HSQC NMR spectra of lignin samples after treatement for 0 h and 24 h by laccase.
6
Y. Shi et al. / Chemosphere 248 (2020) 125931
Fig. 7. Possible formation pathway of EPFRs during the decomposition of lignin by laccase.
Compared with the original lignin, the treated lignin samples possessed higher Ph-OH content. Meanwhile, the Ph-OH content increased as the reaction time increased (Fig. 6), which were consistent with previous reports (Li et al., 2018). The collective findings of these analyses indicated that lignin experienced chain cleavages such as demethylation or/and hydroxylation, leading to the formation of the polyphenol structure during the decomposition by laccase. 3.4. Possible formation pathway of EPFRs The foregoing findings indicated that chain cleavage, opening of the benzene ring, and hydroxylation reactions might occur and were responsible for EPFRs formation on lignin. A proposed pathway is presented in Fig. 7. According to previous studies, when the lignin samples came into contact with laccase, they were oxidized to form phenoxy radicals (Radical 1) (Kawai et al., 1988b). These radicals are very unstable and may lead to further reaction. Pathway A involved electron transfer and resulted in carboncentered radicals (Radical 2). The oxygen-substituted carbon of the aromatic ring was easily to react with O2, forming ring-opening ^do et al., 2019). As shown in pathway B, the CeO products (Figueire bond of methoxyl groups were cleaved, inducing the formation of phenol hydroxyl-rich products (Filley et al., 2002), which was consistent with 2D-NMR HSQC and phenolic hydroxyl content analyses. In the proposed pathway, laccase induced the cleavage of benzene ring and formation of low molecular products and small mobile radicals, leading to formation of lower molecular weight of lignin, which were confirmed by NMR and GPC analyses. The demethylation and depolymerization accounted for the increase of PhOH contents and decreased Mw in lignin (Munk et al., 2015). Intermediates of this oxidation, such as phenoxy radicals, led to spontaneous generation of ROS and degradation of the lignin. 4. Conclusions Our results clearly demonstrate the generation of EPFRs during the decomposition of lignin by laccase. However, the spin density of the generated EPFRs can gradually decay in the dark. A scheme for the decomposition of lignin is proposed. The scheme includes the formation of EPFRs and oxidized products. EPFRs formed on the surfaces of lignin can be transformed to ROS. The findings increase our understanding about the biogeochemical transformations of lignin and potential contributions to the natural generation of EPFRs and ROS in the soil environment.
CRediT authorship contribution statement Yafang Shi: Investigation, Formal analysis, Writing - original draft. Kecheng Zhu: Validation, Data curation. Yunchao Dai: Project administration. Chi Zhang: Writing - review & editing. Hanzhong Jia: Supervision, Resources, Funding acquisition. Acknowledgment This study was supported by the National Natural Science Foundation of China (Grants No. 41571446 & 41877126), the National Key R&D Program of China (Grant No. 2018YFC1802004), Shaanxi Key R&D Program of China (Grant No. 2019ZDLNY01-0201), the “One Hundred Talents” program of Shaanxi Province (SXBR9171), the CAS Youth Innovation Promotion Association (2016380), and the Shaanxi Science Fund for Distinguished Young Scholars (Grant No. 2019JC-18). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2020.125931. References Ainsworth, E.A., Gillespie, K.M., 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using FolineCiocalteu reagent. Nat. Protoc. 2, 875e877. Alejandro, R., Jorge, R., Río, J.C., Del, Martínez, A.T., Ana, G., 2014. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol. Biofuels 7, 14. € schl, U., Shiraiwa, M., 2016. Quantification of Arangio, A.M., Tong, H., Socorro, J., Po environmentally persistent free radicals and reactive oxygen species in atmospheric aerosol particles. Atmos. Chem. Phys. 16, 13105e13119. Borrowman, C.K., Zhou, S., Burrow, T.E., Abbatt, J.P., 2015. Formation of environmentally persistent free radicals from the heterogeneous reaction of ozone and polycyclic aromatic compounds. Phys. Chem. Chem. Phys. 18, 205e212. Campos-Martin, J.M., Blanco-Brieva, G., Fierro, J.L.G., 2006. Hydrogen peroxide synthesis: an outlook beyond the anthraquinone process. ChemInform 45, 6962e6984. Cao, Y.J., Duan, X.F., Cao, Y.L., Lue, J.X., Zhu, J.Q., Zhou, G.W., Zhao, B.L., 2008. ESR study in reactive oxygen species free radical production of Pinus kesiya var. langbinensis Heartwood treated with laccase. Appl. Magn. Reson. 35, 205e211. Chen, N., Huang, Y., Hou, X., Ai, Z., Zhang, L., 2017. Photochemistry of hydrochar: reactive oxygen species generation and sulfadimidine degradation. Environ. Sci. Technol. 51, 11278e11287. Christoforidis, K.C., Un, S., Deligiannakis, Y., 2007. High-Field 285 GHz electron paramagnetic resonance study of indigenous radicals of humic acids. J. Phys. Chem. A 111, 11860e11866. Crawford, D.L., Floyd, S., Pometto, A.L., Crawford, R.L., 1977. Degradation of natural and Kraft lignins by the microflora of soil and water. Can. J. Microbiol. 23, 434e440. Cruz, A.L.N., Dela, William, G., Slawomir, L., Robert, C., Barry, D., 2011. Detection of
Y. Shi et al. / Chemosphere 248 (2020) 125931 environmentally persistent free radicals at a superfund wood treating site. Environ. Sci. Technol. 45, 6356e6365. Davis, J.R., Sello, J.K., 2010. Regulation of genes in Streptomyces bacteria required for catabolism of lignin-derived aromatic compounds. Appl. Microbiol. Biotechnol. 86, 921e929. Felby, C., Nielsen, B.R., Olesen, P.O., Skibsted, L.H., 1997. Identification and quantification of radical reaction intermediates by electron spin resonance spectrometry of laccase-catalyzed oxidation of wood fibers from beech (Fagus sylvatica). Appl. Microbiol. Biotechnol. 48, 459e464. ^do, M.B., Deuss, P.J., Venderbosch, R.H., Heeres, H.J., 2019. Valorization of Figueire pyrolysis liquids: ozonation of the pyrolytic lignin fraction and model components. ACS Sustain. Chem. Eng. 7, 4755e4765. Filley, T.R., Cody, G.D., Goodell, B., Jellison, J., Noser, C., Ostrofsky, A., 2002. Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi. Org. Geochem. 33, 111e124. Guo, X., Pang, J., Chen, S., Jia, H., 2018. Sorption properties of tylosin on four different microplastics. Chemosphere 209, 240e245. Higuchi, T., 2004. Microbial degradation of lignin: role of lignin peroxidase, manganese peroxidase, and laccase. P. JPN. ACAD. B-PHYS. 80, 204e214. Jezierski, A., Skrzypek, G., Jezierski, P., Paul, D., Jedrysek, M.O., 2008. Electron paramagnetic resonance (EPR) and stable isotope records of paleoenvironmental conditions during peat formation. Acta Part A Molec. Biomolec. Spectrosc. 69, 1311e1316. Jia, H., Chen, H., Nulaji, G., Li, X., Wang, C., 2015. Effect of low-molecular-weight organic acids on photo-degradation of phenanthrene catalyzed by Fe(III)smectite under visible light. Chemosphere 138, 266e271. Jia, H., Nulaji, G., Gao, H., Wang, F., Zhu, Y., Wang, C., 2016. Formation and stabilization of environmentally persistent free radicals induced by the interaction of anthracene with Fe(III)-modified clays. Environ. Sci. Technol. 50, 6310e6319. Jia, H., Wang, C., 2013. Comparative studies on montmorillonite-supported zerovalent iron nanoparticles produced by different methods: reactivity and stability. Environ. Technol. 34, 25e33. Jia, H., Zhao, S., Shi, Y., Zhu, L., Wang, C., Sharma, V.K., 2018. Transformation of polycyclic aromatic hydrocarbons and formation of environmentally persistent free radicals on modified montmorillonite: the role of surface metal ions and polycyclic aromatic hydrocarbon molecular properties. Environ. Sci. Technol. 52, 5725e5733. Kawai, S., Umezawa, T., Higuchi, T., 1988a. Degradation mechanisms of phenolic beta-1 lignin substructure model compounds by laccase of Coriolus versicolor. Arch. Biochem. Biophys. 262, 99e110. Kawai, S., Umezawa, T., Shimada, M., Higuchi, T., 1988b. Aromatic ring cleavage of 4,6-di(tert-butyl)guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor. FEBS Lett. 236, 309e311. Khachatryan, L., Vejerano, E., Lomnicki, S., Dellinger, B., 2011. Environmentally persistent free radicals (EPFRs). 1. Generation of reactive oxygen species in aqueous solutions. Environ. Sci. Technol. 45, 8559e8566. Kudanga, T., Nyanhongo, G.S., Guebitz, G.M., Burton, S., 2011. Potential applications of laccase-mediated coupling and grafting reactions: a review. Enzym. Microb. Technol. 48, 195e208. Lavrent, K., Mcferrin, C.A., Hall, R.W., Barry, D., 2014. Environmentally persistent free radicals (EPFRs). 3. Free versus bound hydroxyl radicals in EPFR aqueous solutions. Environ. Sci. Technol. 48, 9220e9226. Li, Z.L., Zhang, J.B., Qn, L., Ge, Y.Y., 2018. Enhancing antioxidant performance of lignin by enzymatic treatment with laccase. ACS Sustain. Chem. Eng. 6, 2591e2595. Li, Z., Yan, K., Ge, Y., 2015. Synthesis of porous lignin xanthate resin for Pb 2þ removal from aqueous solution. Chem. Eng. J. 270, 229e234. Liao, S., Pan, B., Li, H., Zhang, D., Xing, B., 2014. Detecting free radicals in biochars and determining their ability to inhibit the germination and growth of corn, wheat and rice seedlings. Environ. Sci. Technol. 48, 8581e8587. Longe, L.F., Couvreur, J., Grandchamp, M.L., Garnier, G., Allais, F., Saito, K., 2018.
7
Importance of mediators for lignin degradation by fungal laccase. ACS Sustain. Chem. Eng. 6, 10097e10107. Mishra, P.K., Wimmer, R., Gregor, T., 2016. Utilising Brewer’s spent grain as a source of cellulose nanofibres following separation of protein-based biomass. Bioresources 12, 107e116. Munk, L., Punt, A.M., Kabel, M.A., Meyer, A.S., 2017. Laccase catalyzed grafting of eNeOH type mediators to lignin via radicaleradical. RSC Adv. 7, 3358e3368. Munk, L., Sitarz, A.K., Kalyani, D.C., Mikkelsen, J.D., Meyer, A.S., 2015. Can laccases catalyze bond cleavage in lignin? Biotechnol. Adv. 33, 13e24. Orpin, C.G., 1984. Microbial attack on lignocellulose in the rumen. Appl. Biochem. Biotechnol. 9, 327e328. Paul, A., Stosser, R., Zehl, A., Zwirnmann, E., Vogt, R.D., 2006. Steinberg, C. E. W., Nature and abundance of organic radicals in natural organic matter: effect of pH and irradiation. Environ. Sci. Technol. 40, 5897e5903. Perna, V., Agger, J.W., Andersen, M.L., Holck, J., Meyer, A.S., 2019. Laccase induced lignin radical formation kinetics evaluated by electron paramagnetic resonance spectroscopy. ACS Sustain. Chem. Eng. 7, 10425e10434. rrez, A., Río, J.C.D., Prasetyo, E.N., Kudanga, T., Østergaard, L., Rencoret, J., Gutie nez-Barbero, J., Martínez, A.T., 2010. Polymerization of Santos, J.I., Nieto, L., Jime lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 101, 5054e5062. Ramalingam, B., Sana, B., Seayad, J., Ghadessy, F.J., Sullivan, M.B., 2017. Towards understanding of laccase-catalysed oxidative oligomerisation of dimeric lignin model compounds. RSC Adv. 7, 11951e11958. Ray, P.D., Bo-Wen, H., Yoshiaki, T., 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell. Signal. 24, 981e990. Samanta, A., Bhattacharyya, K., Das, P.K., Kamat, P.V., Hug, G.L., 1989. Quenching of excited doublet states of organic radicals by stable radicals. J. Phys. Chem. 93, 3651e3656. , T., Kim, S.Y., Padra ~o, J., Prasetyo, E.N., Kudanga, T., Silva, C., Matama Nyanhongo, G.S., Guebitz, G.M., Casal, M., Cavaco-Paulo, A., 2011. Antimicrobial and antioxidant linen via laccase-assisted grafting. React. Funct. Polym. 71, 713e720. Su, Y., Xian, H., Shi, S., Zhang, C., Manik, S.M.N., Mao, J., Zhang, G., Liao, W., Wang, Q., Liu, H., 2016. Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk. BMC Biotechnol. 16, 1e9. Tejado, A., Pena, C., Labidi, J., Echeverria, J.M., Mondragon, I., 2007. Physico-chemical characterization of lignins from different sources for use in phenolformaldehyde resin synthesis. Bioresour. Technol. 98, 1655e1663. Wang, Z., Zang, L., Fan, X., Jia, H., Li, L., Deng, W., Wang, C., 2015. Defect-mediated of Cu@TiO2 coreeshell nanoparticles with oxygen vacancies for photocatalytic degradation 2,4-DCP under visible light irradiation. Appl. Surf. Sci. 358, 479e484. Yuan, X., Cong, Y., Hao, Shan, Y., Zhao, Z., Wang, S., Chen, J., 2004. Regulation of LIP level and ROS formation through interaction of H-ferritin with G-CSF receptor. J. Mol. Biol. 339, 131e144. Zhang, Y., Guo, X., Si, X.H., Yang, R.X., Zhou, J.T., Quan, X., 2019. Environmentally persistent free radical generation on contaminated soil and their potential biotoxicity to luminous bacteria. Sci. Total Environ. 687, 348e354. Zhang, Y., Wang, Q., Fan, X., Yuan, J., 2014. Structural changes of lignin in the jute fiber treated by laccase and mediator system. J. Mol. Catal. B Enzym. 101, 133e136. Zhao, S., Gao, P., Miao, D., Wu, L., Qian, Y., Chen, S., Sharma, V.K., Jia, H., 2019. Formation and evolution of solvent-extracted and nonextractable environmentally persistent free radicals in fly ash of municipal solid waste incinerators. Environ. Sci. Technol. 53, 10120e10130. Zhou, G., Li, J., Chen, Y., Zhao, B., Cao, Y., Duan, X., Cao, Y., 2009. Determination of reactive oxygen species generated in laccase catalyzed oxidation of wood fibers from Chinese fir (Cunninghamia lanceolata) by electron spin resonance spectrometry. Bioresour. Technol. 100, 505e508.