Influence of Oxygen and Mediators on Laccase-Catalyzed

Research Article pubs.acs.org/journal/ascecg

Influence of Oxygen and Mediators on Laccase-Catalyzed Polymerization of Lignosulfonate Daniela Huber,† Andreas Ortner,† Andreas Daxbacher,† Gibson S. Nyanhongo,*,†,‡ Wolfgang Bauer,*,§ and Georg M. Guebitz†,∥ †

Institute of Environmental Biotechnology, IFA Tulln, University of Natural Resources and Life Sciences Vienna, Konrad Lorenz Straße 20, 3430 Tulln, Austria ‡ Botswana International University of Science and Technology, Private Bag 16, Palapye, Botswana § Institute of Paper, Pulp, and Fiber Technology, Graz University of Technology, Inffeldgasse 23, 8010 Graz, Austria ∥ Austrian Centre of Industrial Biotechnology GmbH, Konrad Lorenz Straße 20, 3430 Tulln, Austria S Supporting Information *

ABSTRACT: Industrial utilization of lignin is of high interest since it represents around 30% of all nonfossil-based carbon sources worldwide. For various applications of lignosulfonates such as for dispersants or adhesives a larger molecular weight is essential. Here, we investigated laccase-catalyzed polymerization of lignosulfonate directly from the pulp and paper industry in the presence and absence of natural and synthetic mediators with and without oxygen supply. For example, laccase-mediated polymerization in the presence of a 2.5 mM TEMPO as mediator with a 10 cm3 min−1 oxygen flow rate led to a 12-fold increase of the molecular weight, while without TEMPO a 13-fold increase was achieved. In contrast, without an external oxygen supply, only a 7-fold increase in molecular weight was achieved compared to a 4-fold increase for the TEMPO−laccase system. Fluorescence intensity, phenol content, and size exclusion chromatography measurements indicate that generally in the presence of high concentrations of mediators, such as TEMPO, vanillin, HBT, and 2,6-dimethoxyphenol, oxidation of other structural units in lignosulfonates may counteract desired polymerization reactions. In summary, for laccase-catalyzed polymerization of lignosulfonates, an external oxygen supply was found to be much more beneficial than the presence of laccase mediators. KEYWORDS: Lignosulfonate, Laccase, Oxidation, Polymerization, Natural and synthetic mediators, Continuous oxygen supply

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additives, adhesives, etc.2 To achieve maximum exploitation of these lignins, a number of chemical, enzymatic, and chemoenzymatic methods4−6 are being developed. For example, polymerization of lignin makes it interesting for use in binders or adhesives. Many chemo-enzymatic or enzymatic-based polymerization systems using mainly laccase are increasingly being investigated.4,5,7 For various applications of lignosulfonates such as for dispersants, a larger molecular weight is beneficial and was achieved by using laccases.8 Laccases (benzenediol:oxygen oxidoreductases, EC.1.10.3.2) are multicopper enzymes catalyzing the oxidation of various phenolic molecules to phenoxy radicals while reducing molecular oxygen to water9,10 and are extensively used to improve lignin exploitation for various applications. Laccases are widely distributed in plants and fungi; they play an important role in the biosynthesis of lignin as well as in its

INTRODUCTION Lignin represents 20%−30% of plant biomass and is next to cellulose and hemicellulose a major component in lignocellulosic materials, where it functions as a cell wall constituent to provide strength to the cell wall and resistance to microbial degradation.1 During chemical pulp production as estimated in 2010, around 50 million tons of lignin was extracted, while most of it was burnt. Therefore, industrial exploitation of lignin, which in fact represents 30% of all nonfossil-based carbon sources worldwide,1,2 is receiving tremendous scientific and industrial attention. Depending on the botanical source and also the extraction method, various types of lignin (lignosulfonate, kraft lignin, organosolv lignin, etc.) are generated. The heterogeneity of lignin is high, which might be the reason why presently only 2% of the lignin produced by the pulp and paper industry is used for industrial applications.2,3 Lignosulfonates result from the sulfite process which depending on the applied base is operating at a pH of 1−5 and 150 °C. They contain sulfonate groups on the aliphatic side chain which makes them water soluble although they are highly polydisperse. Lignosulfonates are receiving a lot of interest for many industrial applications including binders, dispersing agents, cement © XXXX American Chemical Society

Special Issue: Lignin Refining, Functionalization, and Utilization Received: April 5, 2016 Revised: May 24, 2016

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DOI: 10.1021/acssuschemeng.6b00692 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering degradation.11,12 Many laccase-based reaction systems have been developed including the use of mediators to broaden the range of substrates that can be directly and indirectly modified by laccase. Laccase alone or in combination with mediators are used for grafting phenolic-based molecules onto lignocellulosic materials to improve their properties, some of which were summarized in Widsten et al.13 Among the common mediators used are natural phenolic-based molecules like syringaldehyde, vanillin, p-coumaric acid, acetosyringone, etc., and synthetic mediators like 2,2′-azino-di(3-ethylbenzthiazolin-6-sulfonic acid) (ABTS), N,N′-bis(1H-tetrazol-5-yl)-hydrazine (HBT), violuric acid, etc., with the disadvantage that they are very cost intensive.13 Recent studies demonstrated that oxygen was a limiting factor in studies aimed at improving lignin properties using laccase-based reaction systems.12,14,15 However, the role of laccase mediators has not yet been investigated in this regard,16 although it is well known that laccase-mediator systems (LMS) can oxidize nonphenolic groups of lignin leading to cleavage reactions17 and thus counteracting enzymatic polymerization. On the other hand, we have previously shown that laccase-catalyzed polymerization of lignosulfonates may initiate via condensation reactions between the phenoxy radicals formed, leading to new ether and C−C aryl−aryl or aryl−alkyl linkages.8 It is also interesting to mention that previous studies by Collins et al. in 1996 noted that during laccase oxidation studies, oxygen was directly incorporated in the aromatic ring of polyaromatic ring compounds.18 This raises the possibility that by adding oxygen, more oxygen-centered lignin reactive species are created which in turn lead to an increase in lignin reactivity and/or subsequent polymerization of lignin. Hence, in this study, the effect of oxygen in laccase-based lignin polymerization in the presence and absence of mediators was investigated.

zoline-6-sulfonic acid) (ABTS) 2,6-dimethoxyphenol, and vanillin were added to a final concentration of 2.5 and 25 mM. After addition of the laccase with a final enzyme activity of 167 nkat mL−1 and a stirring of 600 rpm, the reaction was started. Additionally, pure oxygen (10 or 20 cm3 min−1 as indicated below) was added with an air stone to the 100 mL reaction vessel containing 60 mL of the incubation mixture. In parallel, controls without oxygen were also setup. Additionally, a lignosulfonate blank was performed where no mediator was added. Here, pure oxygen was also added with a volume of 10 and 20 cm3 min−1 and also a sample with no oxygen was provided. Samples were withdrawn for analysis after 1, 3, and 6 h. Monitoring the Oxygen Content during the Polymerization Process. The oxygen content of the laccase-mediated polymerization of lignosulfonates was monitored with the Firesting device from PyroScience GmbH (Germany, Aachen) during the whole procedure. The Firesting device consists of an indicator dye immobilized in the reaction vessel. The oxygen was measured by an excitation of 620 nm and at an emission of 760 nm. The oxygen signal is detected by quenching the luminescence of the oxygen indicator. The system was calibrated to 100% oxygen with ambient air and to 0% oxygen with pure nitrogen.20−22 Fluorescence Intensity Measurements. Fluorescence intensity was measured as already described in Ortner et al.15 One hundred microliters of lignosulfonate taken at the times described above were incubated with 120 μL of a methoxyethanol:water (2:1 v/v) mixture, and the absorbance was measured at an excitation of 355 nm and an emission of 400 nm using a spectrophotometer (Infinite M200, Tecan, Switzerland). Decrease in Phenol Content after Laccase Mediated Oxidation of Lignosulfonate. The concentration of phenolic groups was measured using the Folin Ciocalteu (FC) method as described by Blainski et al.23 The FC assay is a colorimetric assay to measure the total concentration of phenolic hydroxyl groups in the plant extracts that reacts with the FC reagent to form a blue complex by reduction of a phosphowolframate− phosphomolybdate complex that is determined spectrophotometrically.24 For the measurement, 20 μL of lignosulfonate was mixed with 60 μL of the FC reagent and incubated for 5 min at ambient temperature. Subsequently, 200 μL of MQ-water and 120 μL of a 20% (w/v) sodium carbonate solution was added. After incubation for 2 h at 800 rpm, the samples were measured spectrophotometrically at 760 nm using a plate reader (Infinite M200, Tecan, Switzerland). The concentration of the phenol was estimated using vanillin standard curve in g L−1. Aqueous Size Exclusion Chromatography (SEC) of Lignosulfonates. Molecular weight of the lignin samples was measured with an Agilent 1290 Infinity system. The HPLC system was equipped with a degasser, a binary pump, and a refracting index (RI) detector (Agilent Technologies, U.S.A.). The samples were separated with an Agilent PL aquagel−OH MIXED-H 8 μm, 7.5 mm × 300 mm, and the appropriate Agilent guard column (PL aquagel−OH Guard 8 μm, 7.5mm × 50 mm). The mobile phase was 50 mM sodium nitrate with 0.8 mM sodium azide. Calibration of the SEC system was performed with poly(styrenesulfonic acid sodium salts) with the following molecular weight: 208, 4230, 6520, 14,900, 29,100, 75,600, 148,000, 505,100, 829,500, 1,188,400, and 2,260,000 g mol−1. The data were analyzed with Cirrus Addon

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MATERIALS AND METHODS Materials. Industrial Mg-lignosulfonate originating from the evaporation plant with a dry content of 30% was used. The purity of the lignosulfonate was around 76%, and the impurities consisted of 4.5% sugars (cellobiose, glucose, mannose, etc.), 14% salts, around 3% organic acids, and alcohols. Lignosulfonate was taken untreated for the following reactions. Folin Cioulteau (FC) reagent, N,N′-bis(1H-tetrazol-5-yl)-hydrazine (HBT), (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol, and vanillin were purchased from Sigma-Aldrich (St. Louis, U.S.A.). Laccase from Myceliophthora thermophila was obtained from Novozyme (Denmark). Poly(styrenesulfonic acid sodium salt) as well as all other used chemicals were supplied in analytical grade by Sigma-Aldrich (U.S.A.). Enzyme Activity Assay. Activity of laccase from Myceliophthora thermophila was measured according to the procedure of Prasetyo et al.19 with slight modifications. The oxidation of ABTS to its cation radical at an absorbance of 420 nm was monitored for 1 min. The activity was measured at a pH of 7 in 0.1 M sodium phosphate buffer. Laccase activity was expressed in katal (kat), which is the amount of enzyme needed to oxidize on mole substrate per second. Oxidation of Lignosulfonate. Lignosulfonate was diluted to a final content of 10% (w/v) with distilled water, and the pH was adjusted to 7 using sodium hydroxide solution. N,N′Bis(1H-tetrazol-5-yl)-hydrazine (HBT), (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), 2,2′-azino-bis(3-ethylbenzthiaB


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Figure 1. (A) Oxygen profile of lignosulfonate oxidation with no mediator added in the presence of MTL and an oxygen flow rate of 10 and 20 cm3 min−1 and without O2. (B) Oxygen profile of lignosulfonate oxidation with 2.5 mM TEMPO in the presence of MTL and a continuous oxygen flow rate of 10 and 20 cm3 min−1 and without O2. The system was calibrated with ambient air to 100% oxygen, and pure oxygen was supplied in excess.

an excess of oxygen during the polymerization process of 6 h. The oxygen concentration was way above 100% because the oxygen supply was in excess, and the system was calibrated to 100% with ambient air. In contrast, in the reaction vessel without external oxygen supply, the oxygen saturation was still at 0% after 6 h of incubation indicating that polymerization of the lignosulfonate was not finished. In the next step, the effect of laccase mediators on lignin polymerization was assessed. By definition, mediators regenerate during the oxidation process, while several molecules termed mediators have previously been shown to be incorporated into oxidation products.29,30 When a 2.5 mM TEMPO−laccase system was investigated, quite expectedly oxygen was consumed indicated by a sharp fall of the oxygen concentration to almost 0% in some seconds (Figure 1B), which was the same as when no mediator is present. When compared to the system without mediator, it is most likely that highly reactive intermediates are formed that can react with the nonphenolic moieties of the lignosulfonate29,30 which are not oxidized in the absence of mediators. Furthermore, highly reactive species may undergo several coupling reactions as well as rearrangement, polymerization, or/and cleavage reactions,11,17,31,32 which would either lead to a decrease or increase in the molecular weight of the lignin depending on the initiated reaction. The oxygen drop after addition of the laccase was the same for all used synthetic and natural mediators in the present study, which was also reported by Johannes et al. for ABTS.33 The oxygen profiles were determined for all LMS, but are not all shown here due to similar oxygen consumption profiles as presented for a 2.5 mM TEMPO−laccase system (presented in Figure 1B). Phenol Content and Fluorescence Intensity Measurements during Laccase-Mediated Polymerization of Lignosulfonate. Fluorescence intensity and phenol content

(B.04.03) for Chemstation 32 B.04.03 [108] (Agilent Technologies, U.S.A.).

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RESULTS AND DISCUSSION Oxygen Consumption during Polymerization of Lignosulfonates. Laccase-catalyzed polymerization of lignosulfonates has been demonstrated to be important for application such as in dispersants.8 However, it is well known that in the presence of mediators laccases can catalyze lignin (de)polymerization.17 On the other hand, oxygen is a limiting factor in laccase-catalyzed lignin oxidation systems.15,25 Thus, the present study investigates the effect of oxygen and natural/ synthetic mediators on polymerization of lignosulfonates since continuous oxygen supply was never considered as a critical limiting factor in previous studies.26 Hence, in this study, the role of mediators during laccase-catalyzed polymerization of lignosulfonates was investigated. The reaction was started by adding lignosulfonate to the reaction vessel, and the polymerization process was initiated after addition of laccase, leading to a rapid decrease in the oxygen concentration to almost 0% within a few seconds (Figure 1A). This shows that laccase immediately used the oxygen as the electron acceptor during the oxidation process24,27,25 and that polymerization of the lignosulfonate started immediately. Although the incubation of laccase with lignosulfonate and lignosulfonate and mediator led to a sharp decrease in the oxygen concentration in seconds, the incubation in the absence of laccase caused a slow decrease over several hours, indicating some auto-oxidation of the lignosulfonate. This was not unexpected since oxygen is widely applied for lignin oxidation for bleaching purposes, although at much higher pH, temperature, and pressure.28 After adding pure oxygen at a flow rate of 10 and 20 cm3 min−1 with an air stone, the oxygen saturation in the reaction solution increased due to C


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FTIR, and Py-GC/MS analysis, we have previously shown that laccase-catalyzed polymerization of lignosulfonates may initiate via condensation reactions between the phenoxy radicals formed, leading to new ether and C−C aryl−aryl or aryl− alkyl linkages.8 The phenol concentration profiles in the presence of a 2.5 mM vanillin−laccase system during the 6 h polymerization are presented in Figure 2B. Interestingly, when compared to the reactions without mediator (Figure 2A), for all oxygen concentrations, a higher phenol group content was observed. This indicates less oxidation of phenolic moieties, which confirms the fact that in the presence of mediators other structural elements of lignin are oxidized. In contrast, in the presence of the mediator HBT, a faster decrease in phenolic groups during lignin oxidation in comparison to only laccasemediated oxidation was observed previously35 during the polymerization process. There, the conversion of aromatic compounds was faster with the LMS system, which can be due to (re)polymerization35 of lignin, which was not the case in our study due to the fact that phenol concentrations in the absence of HBT were lower, so the polymerization was more efficient. However, in this previous study, continuous external oxygen supply was not studied. In the case of the synthetic mediator ABTS, a 2.5 mM concentration led to a more pronounced decrease in the phenol groups compared to lignosulfonate polymerization without mediator. Hence, the higher concentration of the mediator with 25 mM in comparison to 2.5 mM ABTS led to a lower phenol content with a value of 0.92 g L−1 after 6 h with 25 mM ABTS compared to 1.80 g L−1 for 2.5 mM ABTS at an oxygen flow rate of 10 cm3 min−1. This does not generally apply for all synthetic and natural mediators tested. For TEMPO and vanillin, the higher mediator concentration led to increased phenol group concentrations after 6 h of polymerization compared to a lower mediator concentration. For the mediators HBT and 2,6-dimethoxyphenol, the phenol content for 2.5 and 25 mM mediator concentrations exhibited an insignificant difference (Table 1). In Table S1 of the Supporting Information, a list of all the phenolic contents and fluorescence intensity as well as the results from the size exclusion chromatography is presented. Additionally, all used mediators resulted in a decrease in the phenol content for both concentrations of 2.5 and 25 mM with the oxygenated samples. For all nonoxygenated LMS samples, a lower decrease in comparison to the oxygenated samples was seen, indicating the importance of external oxygen supply. For all the used synthetic as well as natural mediators, the oxygen flow rate had no significant influence on the phenol concentration, so the flow rate of 10 cm3 min−1 demonstrated similar results as 20 cm3 min−1 (Table 1; data for 20 cm3 min−1 in the Supporting Information). This phenomenon was also mentioned for laccase-mediated polymerization of lignosulfonate without the help of mediators. Oxidation of lignosulfonate with MTL and different oxygen flow rates demonstrated that after 6 h of incubation the fluorescence intensity decreased for an oxygen flow rate of 10 and 20 cm3 min−1 O2 with 2174.3 and 2116.3 RFU, respectively, in comparison to untreated lignosulfonate (Figure 2 C,D and Table 1). Additionally, the fluorescence intensity without an external oxygen supply was significantly higher with 3936.1 RFU compared to an oxygen flow rate of 10 cm3 min−1 with 2174.3 RFU (Figure 2 C,D). This also applies for the mediator vanillin as presented in Figure 2 C,D and for all the

are two different methods to determine the phenol content of the polymerized and unpolymerized lignosulfonate samples. In comparison to the fluorescence intensity, the phenol content is a quantitative method, and both methods were used to show the clear trend during the laccase-mediated polymerization of lignosulfonate samples. During polymerization of lignosulfonate by laccase, a decrease in the phenol content (FC method34) was observed (shown in Figure 2A,B) at different time points in

Figure 2. Phenol content and fluorescence intensity for vanillin and vanillin−MTL-treated lignosulfonate. Phenol content [g L−1] for lignosulfonate polymerized with MTL (A) and with 2.5 mM vanillin− MTL (B) and fluorescence intensity [RFU] with MTL (C) and with 2.5 mM vanillin-MTL (D) are presented with an oxygen flow rate of 10 and 20 cm3 min−1 and without oxygen supply over 6 h.

the polymerization process (1, 3, and 6 h). For lignosulfonate treatment without mediators, oxygen was supplied with a flow rate of 10 and 20 cm3 min−1. After 1 h of MTL-mediated polymerization of lignosulfonate and an oxygen flow rate of 10 cm3 min−1, the phenol concentration was higher with 5.13 g L−1 in comparison to an oxygen flow rate of 20 cm3 min−1 with 4.93 g L−1 (Figure 2A); however, the difference is not significant considering the standard deviation. At the end of the polymerization process (6 h), the final values were 3.44 and 3.37 g L−1 for 10 and 20 cm3 min−1, respectively. Hence, there was no significant distinction between the oxygen flow rates of 10 and 20 cm3 min−1 between all measured time points, which means that the higher oxygen flow rate is not necessary for a more efficient polymerization (presented in Figure 2A). The decrease in the phenolic content during the laccase oxidation of lignin was also reported by Areskogh et al.26 In the laccasemediated oxidation process, phenols are oxidized, and reactive radicals are generated that cause self-coupling reactions by forming C−C and C−O bonds to form dimers. Later on, the dimers undergo oligo- and polymerization.8,27 So, there is a strong relationship between the consumption of phenol groups and the rising molecular weight during the laccase-mediated polymerization process of lignosulfonates, and therefore, the phenolic group content is one of the major characteristics for creating high molecular weight lignins.26 Using 13C NMR, D


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Table 1. Phenol Content, Fluorescence Intensity, and Mw (determined by SEC) of Laccase-Treated Lignosulfonate Samples after 6 h of Polymerizationa treatment of lignosulfonate untreated No mediator, 10 cm3 min−1 O2 no O2 ABTS 2.5 mM + 10 cm3 min−1 O2 2.5 mM, no O2 25 mM + 10 cm3 min−1 O2, 25 mM, no O2 TEMPO 2.5 mM, 10 cm3 min−1 O2 2.5 mM, no O2 25 mM, 10 cm3 min−1 O2 25 mM, no O2 HBT 2.5 mM, 10 cm3 min−1 O2 2.5 mM, no O2 25 mM, 10 cm3 min−1 O2 25 mM, no O2 Vanillin 2.5 mM, 10 cm3 min−1 O2 2.5 mM, no O2 25 mM, 10 cm3 min−1 O2 25 mM, no O2 2,6-dimethoxyphenol 2.5 mM, 10 cm3 min−1 O2 2.5 mM, no O2 25 mM, 10 cm3 min−1 O2 25 mM, no O2 a

phenol content [g L−1] 9.07 3.44 4.79 1.80 3.24 0.92 4.65 2.84 3.96 3.29 4.64 4.65 5.63 4.16 5.03 3.82 8.23 4.39 10.15 3.15 6.64 3.35 5.36

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

fluorescence Intensity [RFU]

0.07 0.14 0.10 0.08 0.04 0.02 0.11 0.04 0.11 0.08 0.26 0.23 0.15 0.06 0.52 0.34 0.21 0.06 0.11 0.05 0.11 0.02 0.08

40535.0 2174.3 3936.0 729.7 2226.3 1332.3 1157.3 2089.7 3481.7 1928.0 2199.7 3144.7 5178.7 1789.3 2237.3 2367.0 7938.0 1370.0 5277.0 2066.0 4649.1 1676.2 5322.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

172.5 11.0 128.2 8.3 15.8 9.1 21.5 69.2 75.4 10.4 12.7 111.5 77.2 13.6 26.5 127.0 381.0 72.0 299.0 95.0 301.0 80.0 337.2

Mw [Da]

PD

fold untreated (Mw)

1693 21,481 10,967 24,817 6064 13,044 5576 19,517 7376 16,970 9480 16,918 9349 11,480 10,029 14,629 4544 4892 3775 18,863 9560 24,987 5078

2.5 24.9 13.7 21.9 6.6 11.4 6.1 19.6 8.3 14.6 9.3 16.0 9.7 11.4 10.6 23.0 7.9 9.1 6.9 20.9 11.3 23.2 5.9

12.7 6.5 14.7 3.6 7.7 3.3 11.5 4.4 10.0 5.6 10.0 5.5 6.8 5.9 8.6 2.7 2.9 2.2 11.1 5.6 14.8 3.0

Different treatments of lignosulfonate solutions are listed with no O2 and 10 cm3 min−1 O2 (data for 20 cm3 min−1 in Supporting Information).

Figure 3. Mw of lignosulfonate oxidized by laccase in the presence of different flow rates of oxygen and the mediator vanillin. Mw of lignosulfonate treated with MTL (A) with 2.5 mM vanillin−MTL (B) and 25 mM vanillin−MTL (C) under various oxygen supplies.

Size Exclusion Chromatography (SEC) of LaccaseTreated Lignosulfonate. To study the extent of laccasecatalyzed polymerization of the lignosulfonate in the presence/ absence of mediators and/or oxygen supply, aqueous SEC was performed. For an oxygen flow rate of 10 cm3 min−1, the weight-average molecular weight (Mw) of lignosulfonate increased by 12.7-fold and for 20 cm3 min−1 by 12.3-fold in comparison to unpolymerized lignosulfonate (Figure 3A and Table 1 and Table S1). The Mw after polymerization of

other mediators (Table 1 and Table S1). Measuring the fluorescence intensity confirmed results on the phenol concentration during incubation of lignosulfonate with laccase as previously reported by Prasetyo et al. in 2010.8 The changes/ decrease in fluorescence shows modifications in conjugated biphenyl, phenylcoumarins, carbonyl, and stilbene groups.36 The difference in the fluorescence intensity as well in the phenol concentration between the oxygen flow rate of 10 and 20 cm3 min−1 O2 (data not shown) was not very relevant. E


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Figure 4. Molecular weight distribution by SEC analysis of lignosulfonates polymerized by laccase in the presence of different amounts of oxygen and mediator. The graph is presenting logM in Da, which is corresponding to retention time vs differential mass weight fraction distribution (dw/d logM). (A) Lignosulfonate treated for 6 h with MTL with 10 cm3 min−1 O2 (4, yellow), 20 cm3 min−1 O2 (3, green), and without external O2 supply (2, red). (B) Lignosulfonate sample treated with MTL and 2.5 mM HBT with 10 cm3 min−1 O2 (4, yellow), 20 cm3 min−1 O2 (3, green), without O2 (2, red) after 6 h. The untreated lignosulfonate is presented as line 1 (black line) in (A) and (B).

polymerizes phenolic substrates. Depending on the time of the polymerization process, LMS can also degrade the formed polymers which was shown by a time-dependent measurement.38 Findings in the present study indicate that a higher concentration of mediators in an LMS polymerization of lignosulfonate led to a lower overall polymerization except for the mediator 2,6-dimethoxyphenol (data for no oxygen supply and 10 cm3 min−1 O2 are shown in Table 1). The oxygen flow rate had an essential influence on the polymerization of the lignosulfonate with the LMS system (Table 1 and Table S1). In comparison to the untreated lignosulfonate sample, an oxygen flow rate of 10 cm3 min−1 led to a 12.7-fold increase in the Mw. When no oxygen was supplied, the increase in the Mw was only 6.5 fold. Obviously oxygen is essential as an electron acceptor for the laccase reaction affecting polymerization of lignosulfonates. Additionally, the increase in Mw in LMS was only given when oxygen was supplied. LMS alone without oxygen supply led to a lower increase in Mw than in the presence of O2. Whether oxygen may have an additional effect by, for example, being incorporated into the reaction products like in oxidation of laccase of polycyclic aromatic hydrocarbons (PAHs),18 remains to be investigated. LMS depend on the individual mediator molecules. The synthetic mediator ABTS is oxidized by laccase to the cationic radical (ABTS·+), which is then slowly oxidized to the dication

lignosulfonate without an oxygen supply was increased by 6.5fold. Thus, in agreement with previous studies, the oxygen supply plays an essential role in the polymerization process.15 Laccase generates reactive species in lignosulfonate that subsequently results in radical−radical/radical quinone or semiquinones coupling, forming new phenyl ether−carbon and/or carbon−carbon bonds, which was previously demonstrated by our group and by others and results in an increase in the Mw.8,26 In the next step, the increase in Mw in the presence of mediators was studied. The Mw after 6 h polymerization for the 2.5 mM vanillin−MTL system was increased by 8.6-fold with 10 cm3 min−1 O2 in comparison to only 2.9-fold for the 25 mM vanillin−laccase (Figure 3B,C, Table 1, and Table S1). Hence, depolymerization reaction at a higher vanillin concentration may counteract polymerization. Similar results were found for the laccase mediator ABTS, which has previously been described to enhance lignin oxidation11,29 and also for the mediators HBT and TEMPO. In the presence of an external oxygen supply, the Mw increase was by far higher than in absence of oxygen (Figure 3). As described by Wang et al. in 2016, the oxidation of lignin with LMS can lead to a degradation of the lignin by the fact that Cβ of the β-O-4 functional groups decreased.11,37 Additionally, a study by Potthast et al. in 1999 demonstrated the fact that LMS F



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(ABTS2+). The oxidation is taking place via an electron transfer route.39 HBT is belonging to the >N−OH mediator family, which are very efficient due to the creation of a highly reactive nitroxyl radical (>N−O·). An enzymatic removal of an electron followed by a proton released is the mechanism here.39 In comparison to HBT, TEMPO is forming an oxoammonium ion after laccase oxidation that follows a nonradical-ionic mechanism. Natural mediators, like vanillin and 2,6-dimethoxyphenol, are fungal metabolites or products derived from their own biodegradation process that acts as redox mediators for ligninolytic oxidreductases, like laccase. They are creating phenoxy radicals following a hydrogen atom transfer (HAT) oxidation mechanism. Phenolic mediators are similar to >N− OH mediators like HBT is.39−41 As presented in Table 1 the influence of the mediators on the polymerization process is presented. The natural mediators (vanillin and 2,6-dimethoxyphenol) are less effective in the polymerization process than ABTS and TEMPO. ABTS and TEMPO were already mentioned in the literature to be one of the most effective mediators.29,39 HBT was one of the less effective synthetic mediators with a marked decrease in polymerization at higher HBT concentration. 2,6-Dimethoxyphenol was more effective in the polymerization than vanillin, which could be due to the fact that para- and meta-substituted compounds are less effective substrates.29 Overall, the most effective mediators were ABTS, followed by the natural mediator 2,6-dimethoxyphenol. In the end, a higher polymerization degree, in comparison to no mediator present, was only achieved by ABTS and only in the presence with an excess of oxygen. The Mw distribution of laccase-polymerized lignosulfonates depending on the oxygen flow rate is presented in Figure 4A. It is clearly visible that the Mw for an oxygen flow rate of 10 and 20 cm3 min−1 O2 is extremely increased compared to samples polymerized without external oxygen supply. Additionally, there is no visible difference between the Mw distribution for 10 and 20 cm3 min−1 O2. There, the degree of polymerization is nearly the same with 10.0- and 10.1-fold for 10 and 20 cm3 min−1, respectively, for MTL polymerization and 12.7- and 12.2-fold for 10 and 20 cm3 min−1 for HBT−laccase polymerization. The Mw was also increased with the HBT−laccase system as presented in Figure 4B for 2.5 mM HBT where a more pronounced increase in Mw was seen in the presence of external oxygen supply. The presence of mediators is not resulting in a shortened polymerization process. This only applies when oxygen supply is present in the polymerization system (in the presence or absence of mediators).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b00692. Detailed experimental information about fluorescence intensity, phenol content, and size exclusion chromatography with all time points. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (G.S.N.). *E-mail: [email protected] (W.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support in the framework of the Ph.D. school DokIn’Holz funded by the Austrian Federal Ministry of Science, Research and Economy and the Austrian Centre of Industrial Biotechnology ACIB GmbH and by the Austrian Research Promotion Agency (FFG) through the K-project “Future Lignin and Pulp Processing Research” (FLIPPR°) (Grant 3038341).

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ABBREVIATIONS HBT, N,N′-bis(1H-tetrazol-5-yl)-hydrazine; TEMPO, (2,2,6,6tetramethyl-piperidin-1-yl)oxyl; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); MTL, Myceliophthora thermophila; FC, Folin Cioulteau; Mn, number-average molecular weight; Mz, higher average molecular weights; Mw, molecular weight; PD, polydispersity; LMS, laccase mediator system; SEC, size exclusion chromatography; O2, oxygen; Py-GCMS, pyrolysis-gas chromatography−mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; FTIR, Fourier transform infrared spectroscopy; Da, Dalton; Na, sodium

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REFERENCES

(1) Tuomela, M. Biodegradation of lignin in a compost environment: a review. Bioresour. Technol. 2000, 72 (2), 169−183. (2) Laurichesse, S.; Avérous, L. Chemical modification of lignins: Towards biobased polymers. Prog. Polym. Sci. 2014, 39 (7), 1266− 1290. (3) Vázquez, G.; Antorrena, G.; González, J.; Freire, S. The Influence of Pulping Conditions on the Structure of Acetosolv Eucalyptus Lignins. J. Wood Chem. Technol. 1997, 17 (1−2), 147−162. (4) Hüttermann, A.; Mai, C.; Kharazipour, A. Modification of lignin for the production of new compounded materials. Appl. Microbiol. Biotechnol. 2001, 55 (4), 387−384. (5) Dashtban, M.; Schraft, H.; Syed, T. A.; Qin, W. Fungal biodegradation and enzymatic modification of lignin. Int. J. Biochem. Mol. Biol. 2010, 1 (1), 36−50. (6) Hüttermann, A.; MilsteinN, O.; Nicklas, B.; Trojanowski, J.; Haars, A.; Kharazipour, A. Enzymatic modification of lignin for technical use: strategies and results. ACS Symp. Ser. 1988, 397, 361− 370. (7) Leonowicz, A.; Cho, N. S.; Luterek, J.; Wilkolazka, A.; WojtasWasilewska, M.; Matuszewska, A.; Hofrichter, M.; Wesenberg, D.; Rogalski, J. Fungal laccase: properties and activity on lignin. J. Basic Microbiol. 2001, 41 (3−4), 185−227. (8) Nugroho Prasetyo, E.; Kudanga, T.; Østergaard, L.; Rencoret, J.; Gutiérrez, A.; del Río, J. C.; Ignacio Santos, J.; Nieto, L.; JiménezBarbero, J.; Martínez, A. T.; et al. Polymerization of lignosulfonates by

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CONCLUSION The results of this study demonstrate that continuous external oxygen supply is important for laccase-catalyzed polymerization of lignosulfonates. The use of toxic and expensive natural and synthetic mediators does not significantly improve the polymerization process of lignosulfonates since the molecular weight is only increasing significantly when oxygen is supplied. Rather, oxidation of nonphenolic lignin moieties in the presence of mediators may counteract polymerization. In general, the higher the mediator concentration was, the lower was the molecular weight achieved. In contrast, the influence of a continuous external oxygen supply was by far more pronounced as indicated by phenol content, fluorescence intensity, and molecular weight changes. G


Research Article


(29) Morozova, O. V.; Shumakovich, G. P.; Shleev, S. V.; Yaropolov, Y. I. Laccase-mediator systems and their applications: A review. Appl. Biochem. Microbiol. 2007, 43 (5), 523−535. (30) Shumakovich, G. P.; Shleev, S. V.; Morozova, O. V.; Khohlov, P. S.; Gazaryan, I. G.; Yaropolov, A. I. Electrochemistry and kinetics of fungal laccase mediators. Bioelectrochemistry 2006, 69 (1), 16−24. (31) Crestini, C.; Jurasek, L.; Argyropoulos, D. S. On the mechanism of the laccase-mediator system in the oxidation of lignin. Chem. - Eur. J. 2003, 9 (21), 5371−5378. (32) Baiocco, P.; Barreca, A. M.; Fabbrini, M.; Galli, C.; Gentili, P. Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase???mediator systems. Org. Biomol. Chem. 2003, 1 (1), 191−197. (33) Johannes, C.; Majcherczyk, A. Laccase activity tests and laccase inhibitors. J. Biotechnol. 2000, 78 (2), 193−199. (34) Faustino, H.; Gil, N.; Baptista, C.; Duarte, A. P. Antioxidant activity of lignin phenolic compounds extracted from kraft and sulphite black liquors. Molecules 2010, 15 (12), 9308−9322. (35) Euring, M.; Rühl, M.; Ritter, N.; Kües, U.; Kharazipour, A. Laccase mediator systems for eco-friendly production of mediumdensity fiberboard (MDF) on a pilot scale: Physicochemical analysis of the reaction mechanism. Biotechnol. J. 2011, 6 (10), 1253−1261. (36) Albinsson, B.; Li, S.; Lundquist, K.; Stomberg, R. The origin of lignin fluorescence. J. Mol. Struct. 1999, 508 (1−3), 19−27. (37) Wang, H.; Liu, Y.; Wang, Z.; Yang, G.; Lucia, L. A. Structural Analysis of Fast-Growing Aspen Alkaline Peroxide Mechanical Pulp Lignin: A Post-Enzymatic Treatment. BioResources 2015, 11 (1), 2723−2733. (38) Potthast, A.; Rosenau, T.; Koch, H.; Fischer, K. The Reaction of Phenolic Model Compounds in the Laccase-Mediator System (LMS) Investigations by Matrix Assisted Laser Desorption Ionization Timeof-Flight Mass Spectrometry (MALDI-TOF-MS). Holzforschung 1999, 53 (2), 175−180. (39) Cañas, A. I.; Camarero, S. Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes. Biotechnol. Adv. 2010, 28 (6), 694−705. (40) d’Acunzo, F.; Galli, C. First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models. Eur. J. Biochem. 2003, 270 (17), 3634−3640. (41) Moldes, D.; Díaz, M.; Tzanov, T.; Vidal, T. Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp. Bioresour. Technol. 2008, 99 (17), 7959−7965.

the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 2010, 101 (14), 5054−5062. (9) Nyanhongo, G. S.; Nugroho Prasetyo, E.; Herrero Acero, E.; Guebitz, G. M. Engineering Strategies for Successful Development of Functional Polymers Using Oxidative Enzymes. Chem. Eng. Technol. 2012, 35 (8), 1359−1372. (10) Rodríguez Couto, S.; Toca Herrera, J. L. Industrial and biotechnological applications of laccases: a review. Biotechnol. Adv. 2006, 24 (5), 500−513. (11) Bourbonnais, R.; Paice, M.; Reid, I.; Lanthier, P.; Yaguchi, M. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. Appl. Environ. Microbiol. 1995, 61 (5), 1876−1880. (12) Rittstieg, K.; Suurnakki, A.; Suortti, T.; Kruus, K.; Guebitz, G.; Buchert, J. Investigations on the laccase-catalyzed polymerization of lignin model compounds using size-exclusion HPLC. Enzyme Microb. Technol. 2002, 31 (4), 403−410. (13) Widsten, P.; Kandelbauer, A. Laccase applications in the forest products industry: A review. Enzyme Microb. Technol. 2008, 42 (4), 293−307. (14) Gouveia, S.; Fernández-Costas, C.; Sanromán, M. A.; Moldes, D. Enzymatic polymerisation and effect of fractionation of dissolved lignin from Eucalyptus globulus Kraft liquor. Bioresour. Technol. 2012, 121, 131−138. (15) Ortner, A.; Huber, D.; Haske-Cornelius, O.; Weber, H. K.; Hofer, K.; Bauer, W.; Nyanhongo, G. S.; Guebitz, G. M. Laccase mediated oxidation of industrial lignins: Is oxygen limiting? Process Biochem. 2015, 50 (8), 1277−1283. (16) Kües, U. Fungal enzymes for environmental management. Curr. Opin. Biotechnol. 2015, 33, 268−278. (17) Fabbrini, M.; Galli, C.; Gentili, P. Radical or electron-transfer mechanism of oxidation with some laccase/mediator systems. J. Mol. Catal. B: Enzym. 2002, 18 (1−3), 169−171. (18) Collins, P.; Kotterman, M.; Field, J.; Dobson, A. Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor. Appl. Environ. Microbiol. 1996, 62 (12), 4563−4567. (19) Nugroho Prasetyo, E.; Kudanga, T.; Steiner, W.; Murkovic, M.; Nyanhongo, G. S.; Guebitz, G. M. Antioxidant activity assay based on laccase-generated radicals. Anal. Bioanal. Chem. 2009, 393 (2), 679− 687. (20) Greimel, K. J.; Perz, V.; Koren, K.; Feola, R.; Temel, A.; Sohar, C.; Herrero Acero, E.; Klimant, I.; Guebitz, G. M. Banning toxic heavymetal catalysts from paints: enzymatic cross-linking of alkyd resins. Green Chem. 2013, 15 (2), 381. (21) Wolfbeis, O. S. Materials for fluorescence-based optical chemical sensors. J. Mater. Chem. 2005, 15 (27−28), 2657. (22) Schäferling, M. The art of fluorescence imaging with chemical sensors. Angew. Chem., Int. Ed. 2012, 51 (15), 3532−3554. (23) Blainski, A.; Lopes, G. C.; de Mello, J. C. P. Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from Limonium brasiliense L. Molecules 2013, 18 (6), 6852−6865. (24) Schofield, P.; Mbugua, D.; Pell, A. Analysis of condensed tannins: a review. Anim. Feed Sci. Technol. 2001, 91 (1−2), 21−40. (25) Mattinen, M.-L.; Suortti, T.; Gosselink, R.; Argyropoulos, D. S.; Evtuguin, D.; Suurnäkki, A.; Jong, E. de; Tamminen, T. Polymerization of different lignins by laccase. BioResources 2008, 549−565. (26) Areskogh, D.; Li, J.; Gellerstedt, G.; Henriksson, G. Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis. Biomacromolecules 2010, 11 (4), 904−910. (27) Riva, S. Laccases: blue enzymes for green chemistry. Trends Biotechnol. 2006, 24 (5), 219−226. (28) Moodley, B.; Mulholland, D.; Brookes, H. The chemical oxidation of lignin found in Sappi Saiccor dissolving pulp mill effluent. Water SA 2012, 38 (1), 1−8. H


Influence of Oxygen and Mediators on Laccase-Catalyzed

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