Investigating Laccase and Titanium Dioxide for Lignin Degradation

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Investigating Laccase and Titanium Dioxide for Lignin Degradation Khanita Kamwilaisak†,‡ and Phillip C. Wright*,† †

Department of Chemical Engineering, Faculty of Engineering, Khon Kaen University, Khon Kaen 40002, Thailand ChELSI Institute, Chemical and Biological Engineering, The University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom

‡

S Supporting Information *

ABSTRACT: We assess whether biocatalytic and photocatalytic processes separately or in combination are successful for lignin degradation. Laccase from Trametes versicolor served as the biocatalyst, and TiO2 served as the photocatalyst. The catalysts were used in single- and dual-step configurations. For comparison, lignin degradation by laccase and titania alone were studied. Operational conditions were 50 ± 1 °C, pH 5.0, and with a lignin concentration (molecular weight of 16 000−175 000) of 1.0 g/ L. H2O2 was used as a mediator to increase laccase and TiO2 degradation ability. The results show that H2O2 plays a significant role in improving lignin degradation by TiO2 and that 100% decolorization and delignification was achieved. Gas chromatography−mass spectrometry analysis confirmed the presence of organic acids being a prominent compound class in TiO2/H2O2 processes. We show that not only can laccase and TiO2 completely degrade lignin but the process also yields highly desirable byproduct, such as succinic and malonic acids. Biorefinery opportunities for the processes demonstrated here are discussed. (TEMPO)4 can extend the substrate range of laccase to nonphenolic subunits of lignin. However, these synthetic mediators are expensive and some contain subunits, such as N−(OH)−, that can produce toxic residual compounds.6 It has been suggested7−10 that one can improve enzyme biodegradation efficiency by applying photocatalytic processes involving the illumination of semiconductor materials that have large band gaps. For instance, a significant enhancement of degradation efficiency of trinitrotoluene (TNT) by laccase from Phanerochaete chrysosporium was observed by application of titanium dioxide (TiO2)-assisted photocatalytic pretreatment.7 Furthermore, Paralta-Zarnora et al.8 determined how the decolorization efficiency of immobilized laccase using TiO2 photocatalytic pretreatment is affected by (1) support materials, (2) mediators, and (3) colors. TiO2 or titania is a promising photocatalyst and has particular potential for lignin degradation. There has been increasing interest in the environmental applications of titania because it is an inexpensive, strongly oxidizing stable chemical. It is also capable of the photo-oxidative destruction of most organic pollutants,11,12 and the degradation of organic substrates is initiated when TiO2 absorbs ultraviolet (UV) light. We detail what we believe to be the first published account of a combined process that uses both photocatalytic processes (TiO2 and UV light) and biocatalytic processes (laccase) together to degrade high-molecular-weight lignin, with the hypothesis being that there would be more degradation from a combined process. We conduct multiple-replicated experiments (single and dual stage) to make this assessment. For comparison, experiments using only one of the test materials, either TiO2 or laccase, are also conducted, and the role of H2O2

1. INTRODUCTION Lignin is naturally abundant in trees, plants, and agricultural crops and is one of the three major constituents of plant matter, along with cellulose and hemicellulose.1 Lignin is a randomly branded polyphenol, comprising of phenylpropane (C9) units.2 Lignin is difficult to eliminate or breakdown, and because of its brown coloration, it has long been considered a waste product in the pulp and paper industry, which, when manufacturing white-colored paper, has traditionally used chlorine or chlorinebased compounds as bleaching agents to remove the lignin. Such compounds lead to chlorine waste products, such as chlorophenols and chloroguaiacols,3 which require detoxification to avoid environmental damage. The residue that remains consists of cellulose, hemicelluloses, and other carbohydrates. Although often considered a waste product, lignin contains carbon, hydrogen, and oxygen, all highly useful elements. With the right processes, lignin could be used as a biofuel feedstock. However, lignin is heterogeneous and chemically complex; therefore, it needs to be degraded to improve the availability of its chemical components. Lignin biodegradation has been studied using an extracellular enzyme from the white rot fungus Trametes versicolor.4 This enzyme, the multicopper-containing laccase (EC 1.10.3.1), is mild and environmentally friendly. Laccase catalyzes the oxidation of the substrate by accepting electrons from the copper irons and then reducing molecular oxygen to water.5 Laccase has been widely investigated for biobleaching Kraft pulp (a wood pulp that is almost pure cellulose)2 because of its ability to delignify.3,5 However, laccase alone has limited bioremediation potential because of its specificity for the phenolic compounds in lignin, to the exclusion of other compounds. It has been reported that the inclusion of a mediator, such as 1-hydroxybenzotriazole (HBT),3 2,2azonobis(3-ethylbenzthiazoline-6-sulfonate (ABTS), violuric acid (VLA), and 2,2′,6,6′-tetramethyl-piperidine-N-oxyl © 2012 American Chemical Society

Received: January 10, 2012 Revised: March 20, 2012 Published: March 21, 2012 2400

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biocatalytic (laccase) lignin degradation process, the total volume of each bioreactor solution was 10 mL and it contained 2.5 units/mL laccase and 1 g/L lignin in a sodium acetate buffer (0.05 M at pH 5.0). The process was carried out in the dark to avoid photocatalytic reactions. For the photocatalytic (titania) process, the sample contained titania at a concentration of 3 g/L and lignin at a concentration of 1 g/L in a sodium acetate buffer (0.05 M at pH 5.0). The reaction was carried out under UV irradiation (5.30 ± 0.10 μE cm−2 s−1) by being housed in the UV box containing six UV tubes (Philips TL-4W/08, Holland) described earlier. The single-step reaction process was carried out in the same manner as the photocatalytic reaction experiment in terms of equipment, quantities, setup, and conditions but with 2.5 units/mL laccase added at the beginning of the reaction process with titania. For the dual-step reaction process, the reaction was initiated by the photocatalytic reaction experiment. After 24 h of UV irradiation at 50 °C, the UV light was switched off and then 2.5 units/mL laccase was added to the system to continue the reaction at 50 ± 1 °C in the dark for a further 24 h. All experiments were repeated in the presence of 5.55 g/L H2O2. H2O2 was always added at the beginning of the reaction, except for the dual-step processes, when it was added with the laccase at the beginning of the second step. 2.4. Analytical Methods. The color measurement followed the CPPA standard methodology (Standard H.5P), as reported by Archibald et al.14 The pH of the sample was adjusted to 7.6 by adding 2 M NaOH. The absorbance was detected at 465 nm against buffer and nonbuffer solutions by an Ultraspec 2100 pro UV−vis spectrophotometer (U.K.). The absorbance was converted into color units (CU) using the equation: CU = 500(As/Ap), where As is the absorbance of the effluent sample and Ap is the absorbance of a 500 CU platinum−cobalt standard solution (0.132 in this case). The percentage of delignification of lignin was measured at 280 nm2 by an Ultraspec 2100 pro UV−vis spectrophotometer (U.K.) and calculated using the following equation: percent delignification = (ABSlignin − ABStreated lignin) × 100/ABSlignin, where ABSlignin and ABStreated lignin are the absorbance of untreated and treated lignin, respectively. Sample identification and the quantification of low-molecular-weight aromatic compounds were carried out using Finnigan Trace GC ultra (ThermoElectron, U.K.) gas chromatography−mass spectrometry (GC−MS) interfaced to a Trace DSQ MS 2140 mass selective detector. The samples were filtered post-reaction through a 0.45 μm membrane filter to remove the titania powder. The filtered sample (1 mL) was acidified to pH 2 using HCl (1 N), extracted with ethyl acetate (5 mL), and then dried overnight by a rotary evaporator (Eppendorf, U.K.). To prepare the trimethylsilyl (TMS) derivatives, 100 μL of dioxane, 50 μL of BSTFA, and 10 μL of pyridine were added to the organic extract and then maintained at 35 °C for 2 h. Veratraldehyde was used as an internal standard to quantify low-molecular-weight aromatic compounds by adding 0.1% (w/v) to the dioxane.15 The internal standard was veratraldehyde (C9H10O3), used for its inert property. It was not produced in the experiment, and its peak was at a retention time (RT) of 12.48 min. The analytical column connected to the system was a Rxi-5 ms capillary column, 30 m long, with a 0.25 mm internal diameter, and 0.25 μm film thicknesses. Helium at 1 mL/min was used as the carrier gas. A total of 1 μL of sample was injected into the column. The column temperature program was 80 °C for 2 min and then ramping at 8 °C/min up to 280 °C. The hold time was 5 min. The identification of any low-molecular-weight aromatic compounds as TMS derivatives produced from the experiments was performed by both comparing their mass spectra to those of the National Institute of Science and Technology (NIST) library (binary version 05,190,825 EI mass spectra of 163 198 compounds with 163 195 chemical structures, U.S.) and comparing the retention time to those of available authentic compounds. The quantification of intermediate compounds was performed by comparing their peak area to the calibration curve of the peak area of the veratraldehyde internal standard (0−0.1%, w/w). The amount of Cu in the sample was determined by atomic absorption analysis (model AAnalyst 200, Perkin-Elmer Instruments, U.K.). The sample was centrifuged at

is examined. Of further interest is the analysis of intermediate compounds resulting from lignin breakdown, because these may have potential biorefinery applications. Overall, we detail which configurations offer opportunities for laccase and/or titania in providing an environmentally friendly alternative to chlorine and mild operating conditions or as an upstream process in a biorefinery chain.

2. EXPERIMENTAL SECTION 2.1. Chemical Reagents. Laccase from Trametes versicolor (0.72 unit/mg), catechol (C6H4-1,2-(OH)2), hydrogen peroxide solution [30% (w/w) H2O2], Kraft lignin (alkali, carboxylated molecular weight of 16 000−175 000), TiO2 (99.9%), veratraldehyde (C9H10O3), sulfuric acid (H2SO4), sodium acetate trihydrate (CH3COONa·3H2O), acetic acid (CH3COOH), and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (U.K.). N,OBis(trimethylsilyl)trifluoroacetamide (BSTFA, 99% for GC), dioxine, and pyridine were bought from Fisher Scientific (U.K.). All of these chemicals were of analytical grade and used without further purification. Distilled water was deionized (18 mΩ) using a Milli-Q system from Millipore (U.K.). 2.2. Other Equipment. Syringes (BD Plastipak, 5 mL) and a syringe-driven filter unit (Millex-LCR, 0.45 μm) were purchased from Fisher Scientific (U.K.). The photoreaction was carried out in an UV chamber equipped with 6 × 4 W (Philips TL-5W/08, Holland) UV tubes. This black light lamp emits near UV rays at 315−400 nm with strong photochemical and fluorescent effects.13 The UV box and the controller set were built in house (Workshop, Department of Chemical and Biological Engineering, The University of Sheffield). The temperature was controlled constantly throughout the reaction time by a controller set composed of a hot plate (self-adhesive heater mat, RS Components, U.K.), oil bath (mineral oil in a Pyrex bath), and fan (Bisonic, model 12p-230MB, Tubeaxal Fan, Bisonic Technology Corp., U.K.). The UV-light intensity (scalar irradiance) was measured by a Quantum Scalar Laboratory (QSL) lab radiometer (QSL-2100, Biospherical Instruments, Inc., U.K.) and set to 5.30 ± 0.10 μE cm−2 s−1. A schematic diagram of the UV box used is shown in Figure S1 of the Supporting Information. 2.3. Lignin Degradation Procedures. Experimental work programs (each with at least two replicates) were conducted to compare the ability of laccase and titania to degrade lignin and to quantify the catalytic effects of hydrogen peroxide on the reaction. These experiments can be detailed as follows: (1) lignin under ultraviolet light (control), (2) lignin in the presence of hydrogen peroxide (control), (3) laccase alone (biocatalytic reaction), (4) titania alone (photocatalytic reaction), (5) laccase in the presence of hydrogen peroxide, (6) titania in the presence of hydrogen peroxide, (7) titania and laccase together in a single-step reaction, (8) titania then laccase in a dual-step reaction, (9) titania and laccase together in a single-step reaction in the presence of hydrogen peroxide, and (10) titania alone followed by laccase in the presence of hydrogen peroxide in a dual-step reaction process. Control experiments were carried out to study the effect of UV light, H2O2, temperature, and pH on biocatalyst and photocatalyst reactions (these data are depicted in Figures S2−S18 of the Supporting Information). The results of laccase reactions in the absence and presence of H2O2 show that UV light had no significant effect on delignification. The percentage delignification differs by approximately 2−3 and 1−2% in the absence and presence of H2O2, respectively. The effects of the temperature and pH were investigated in range of 30−70 ± 1 °C and 3−11, respectively. A temperature of 50 ± 1 °C was the best condition for the laccase reaction, while a temperature above 40 °C was a little different for the photoreaction process. Further, a pH in the range of 5−7 had no significant effect on delignification by the laccase reaction (see the Supporting Information). Thus, the comparison of both reactions was conducted at 50 ± 1 °C and pH 5.0 for 24 h, in a closed system with stirring at 110 rpm. Each reaction was carried out in a 25 mL Erlenmeyer flask with a 10 mL total volume of each reactor solution. In the case of the 2401

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5000g for 10 min. Nitric acid (HNO3) was added to the supernatant to make a 2% (w/w) final concentration prior to analysis.

3. RESULTS AND DISCUSSION 3.1. Decolorization. Decolorization results depicted in Figure 1 show that H2O2 increases the individual decolorization

Figure 2. Possible catalytic cycles of laccase in the presence of H2O2.

esque. It seems probable that H2O2 in this reaction not only allowed for a complete laccase reaction but also produced •OH radicals, strongly oxidizing reagents capable of decomposing lignin. This mechanism was proposed by Gugg et al.24 and suggests that a hydroxyl radical is generated by Fenton oxidation chemistry.

Figure 1. Percentage of lignin decolorization in the absence and presence of H2O2 for various experiment configurations. The operating conditions were 50 °C at pH 5.0 (sodium acetate buffer solution) for 24 h (n = 2).

abilities of laccase and titania, as well as in the dual-step process, yet it inhibits performance when laccase and titania are combined in a single-step process. The control experiment shows that lignin under UV light alone is decolorized by ca. 2%, with this increasing to 17% in the presence of the H2O2 mediator. The benefit of H2O2 addition is particularly conspicuous when highlighted by its absence in the case of the laccase alone condition, because the colorization actually increased by approximately 13%. This may be the effect of a lack of oxygen molecules in laccase oxidation; therefore, laccase could not complete the reaction. Furthermore, the yellowish-brown color in the lignin sample could be ascribed to the production of quinones. Generally, some quinones (e.g., quinomethides) are very unstable and may easily recover their hydroxyl groups or polymerize. However, they are more stable as part of the lignin macromolecule. The increase of the lignin color could be caused by these structures, because phenyl propanoid units absorb predominantly in the middle and near UV ranges (300− 400 nm). Also, the bonding of such carbonyl groups coupled with the benzene ring by a double bond in lignin molecules could lead to the absorbance increase.16 However, laccase in the presence of H2O2, even under dark anaerobic conditions, increased the decolorization efficiency. It decolorized lignin by 60%, because the catalytic cycle of laccase was completed in the presence of H2O2. The laccase catalytic reaction process is initiated by electrons that are oxidized from the lignin molecule (phenols and aromatics or aliphatic amines).17,18 The electron is transferred to the T2/T3 site, where one molecule of oxygen is reduced to be two molecules of water.19,20 These reactive intermediates can then produce dimers, oligomers, and polymers (Figure 2). Under our conditions, the CuI ions can be oxidized in fully reduced laccase as follows:21 1 Cu+ + H 2O2 + H+ = Cu 2 + + H 2O (1) 2 I Furthermore, it is possible that Cu ions can produce highly reactive hydroxyl radicals in the presence of hydrogen peroxide. Fenton22 discovered that several metals have a special oxygentransfer property, which improves the use of hydrogen peroxide,23 and the reaction of Cu+ seen in eq 2 is Fenton-

Cu+ + H 2O2 → Cu 2 + + •OH + OH−

(2)

In the photocatalytic reaction, hydrogen peroxide can form OH radicals when it is exposed to UV light in the range of 200−280 nm.25 The ionization process is seen in eqs 3−5. •

H 2O2 + h υ → 2OH H 2O2 +

e−CB •

(3)

•

−

•

−

→ OH + OH

(4)

H 2O2 + O2 → OH + OH + O2

(5)

•

The likely increased number of OH radicals meant that the photocatalytic reaction increased remarkably. This led to a 97% decolorization efficiency by TiO2/H2O2/UV. This was much higher than the 55% decolorization achieved by TiO2/UV alone. In the single- and dual-step processes without H2O2, the color was reduced by 25 and 30%, respectively. The percentage decolorization of the dual-step process was 5% higher than that in the single-step process because of the CuI release, as explained above. However, a more dramatic difference in decolorization occurred when compared to the single- and dual-step processes in the presence of H2O2: whereas the single-step process decolorized by 20%, the dual-step process achieved 100% decolorization. In the single-step laccase process, TiO2, UV light, and H2O2 were all added when the reaction started. The OH− ions were generated by this combination bound with type 2 (T2) and type 3 (T3) copper, which reduced O2 to water. This process could simultaneously inhibit laccase activity and deplete the photocatalytic reaction. This effect of OH− inhibition of laccase activity is detailed elsewhere.26 However, in the dual-step process, the lignin reacts first with TiO2 under UV light and then laccase with the H2O2 mediator. The first step, the photocatalytic reaction, decolorized lignin by ca. 55%, and then the second step (laccase/H2O2/dark) completely removed the entire remaining lignin color. 3.2. Delignification. The lignin concentration was measured by UV−vis spectroscopy at 280 nm. Figure 3 shows the percentage of delignification obtained with the various experiment configurations in the absence and presence 2402

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observed here, TiO2/UV yielded some organic acids, such as succinic, palmitic, and acetic acids. This implies that the phenolic compounds in the lignin molecule were decomposed in this reaction. These organic acids were also found by Hasegawa et al.,34 who used a hydrothermal oxidation process to degrade alkali lignin. However, in their case, the hydrothermal reaction was carried out at a higher temperature and the alkali lignin that they used had a lower molecular weight than the lignin tested in this work. In contrast, the compounds that we identified from the degradation by laccase were substantially different from those observed from lignin and photocatalytic lignin degradation. For example, everal polysaccharides were detected in the extracted samples sourced from the biocatalytic reaction, as well as from the single- and dual-step processes. The peaks obtained were D,L-arabinose, methyl α-D-glucopyranoside, hexose (D-galactose), hexopyranose, D-glucose, and glucopyranose. These monosaccharides could be carbohydrate derivatives from the laccase molecule. The carbohydrate moiety of the majority of laccases consists of mannose, N-acetylglucoamine, and galactose35 at about 10−20% for fungal laccases.35,36 In comparison of the concentrations of intermediate compounds in lignin with laccase (standard), it was noted that the amount of palmitic acids significantly decreased for the biocatalytic and dual-step process but were the same concentrations as found in the single-step process. Furthermore, the amount of stearic acid that considerably decreased in the single- and dual-step processes was unchanged in the biocatalytic process. These compounds were also found in Kraft lignin degradation mediated by Bacillus sp. (accession number AY 952465), as reported elsewhere.31 The occurrence of these compounds indicates that lignin was degraded. Figure 4 shows the amount

Figure 3. Effect of H2O2 on the delignification performance for various configurations. The operating conditions were 50 °C at pH 5.0 (sodium acetate buffer solution) for 24 h (n = 2).

of H2O2. These results show similar tendencies to those observed for decolorization. Lignin under UV light was reduced by 7%, increasing slightly about 9% in the presence of H2O2. Laccase was 45% more efficient in the presence of H2O2, showing the benefit of using H2O2. H2O2 not only completed the reaction but also behaved like a mediator. The reduction of CuI into CuII in the presence of H2O2, thus producing •OH radicals, causes the decomposition of lignin. Some other groups have used laccase in the presence of a mediator to degrade lignin. For example, Bourbonnaiss et al.27 delignified ca. 23.4 and 22.5% in the presence of ABTS and HBT (0.05 M acetate buffer solution at pH 5.0 for 24 h), respectively. Also, Oudia et al.28 achieved ca. 55% delignification by laccase in the presence of violuric acid. The photocatalytic reaction (TiO2/UV/H2O2) increased the lignin elimination by about 27% above that attainable using TiO2/UV alone. This is due to an increase in the amount of •OH and •O2− radicals. Similarly, in the dualstep process, H2O2 was added at the start of the second step, which can conduct hydroxide ions as a source of hydroxyl radicals in photocatalysis.29,30 This caused a 30% increase in delignification over the same dual-step configuration without H2O2. Conversely, the percentage of delignification of the singlestep process in the presence of H2O2 was 15% lower than in the absence of H2O2 because of the effect of OH− ions. These not only inhibit laccase activity26 but also deplete the photocatalytic reaction. OH− binds with CuI, causing a reduction of •OH radicals. 3.3. Identification and Quantification of Low-Molecular-Weight Compounds. GC−MS was used for the identification and quantification of the intermediate compounds in lignin, as widely demonstrated in the literature.31,32 Figures S19 and S20 of the Supporting Information show the total ion chromatogram (TIC) and compounds identified from ethyl-acetate-extractable products obtained from Kraft lignin, lignin degradation by titania, laccase, and titania with laccase in the single- and dual-step processes in the absence of H2O2. Similar main intermediate compounds between lignin and lignin degraded by photocatalytic reactions were observed, but higher peak intensities were seen in the cases of lignin degradation. The main identified compounds were organic acids: acetic acid, malonic acid, succinic acid, butylated hydroxytoluene, vanillin, veratric acid, and palmitic acid. The photodegradation of phenolic compounds by TiO2 begins with • OH radical attack on the phenyl rings, producing catechol, resorcinol, and hydroquinone. Subsequently, the phenyl rings in these compounds break up first to give malonic acid and then short-chain organic acids, such as maleic, oxalic, acetic, and formic acids, before finally yielding CO2.29,33 In the reactions

Figure 4. Amount of intermediate compounds from lignin by category from experiments conducted in the absence of H2O2.

of identified intermediate compounds from lignin in each category from experiments conducted in the absence of H2O2. The results show that there was a similar amount of fatty acid in all treated lignin samples. It was ca. 15%, while it was only ca. 10% in the lignin standard samples. The maximum amount of carbohydrate was found in the biocatalytic reaction sample at 20%, while it was negligible in the photocatalytic reaction process. The percentage of carbohydrate contents in single- and dual-step reaction samples was ca. 10 and 5%, respectively. This may comprise carbohydrate moieties from the laccase, as explained above.35 The highest amount of organic acids was obtained in the photoreaction process at ca. 20%. As explained previously, the photoreaction can produce short-chain organic acids before finally yielding CO2.29,33 We found industrially useful organic acids, including succinic, acetic, and lactic acids. Figures S21 and S22 and Table S2 of the Supporting Information show the total ion chromatograph and identified compounds of ethyl-acetate-extractable products obtained from 2403

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Figure 5. Amount of intermediate compounds from lignin in each category from experiments conducted in the presence of H2O2.

Figure 6. Mechanism for the oxidative degradation of lignin proposed by Hasegawa et al.34 (modified).

enzyme becoming denatured because of the removal of the carbohydrates. Thus, deglycosylation of the protein appears to have a detrimental effect on activity. This hypothesis has been underlined by reports that suggest that the carbohydrate moiety would protect the enzyme against proteolysis and inactivation by free radicals.35 Figure 5 depicts the amount of identified product compounds derived from lignin in each category from experiments conducted in the presence of H2O2 (see Figures S21 and S22 and Table S2 of the Supporting Information for more detail). The maximum amount of fatty acids and carbohydrates was found in the dual-step reaction process, at ca. 25 and 20%, respectively, while the lowest amount of fatty acids and carbohydrates was obtained in the bioreaction at 10% and was negligible for the photoreaction process, respectively. The photoreaction process, in contrast, produces the maximum amount of organic acids at 23%. The majority of the organic acids was malonic, succinic, acetic, and lactic acids, which can be very useful for the chemical industry and as a feedstock for biofuel processes. Furthermore, the lignin degradation mechanism by TiO2/UV is in agreement with the proposed mechanism by Hasegawa et al.34 However, we did not find formic acid in our breakdown samples (Figure 6). This could be due to different reaction conditions, because our work was undertaken at a lower temperature (50 °C). The exact mechanism of lignin degradation carried out by laccase is still unclear. However, the initial laccase reaction could be benign, as mentioned in section 3.1.

untreated Kraft lignin, lignin degradation by laccase, TiO2, and TiO2 with laccase in the single- and dual-step processes in the presence of H2O2. Higher peak intensities were observed in samples obtained from degradation catalyzed by laccase and in the single- and dual-step processes. In the case of the photocatalytic reaction using TiO2 alone, the low peak areas were possibly due to the lignin being completely decomposed. This result can be confirmed by the significant increase in acetic acid and succinic acid formation, both of which are short-chain organic acids, followed finally by decomposition to CO2.37 Several polysaccharides obviously decreased in the biocatalytic and single-step reaction in the presence of H2O2. This may be the effect of H2O2 that could degrade the carbohydrate compounds in the laccase molecules themselves during the reaction process. It has been reported38 that many sugars are lost during oxidative pretreatment because of non-selective oxidation. However, the amount of hexopyronase and glucopyronase increased remarkably in the dual-step process. The polysaccharides in lignin molecules were decomposed in the first step (photoreaction) because we could not find those compounds in the TiO2/UV-treated samples. Therefore, it may be the case that carbohydrate moieties from laccase molecules might be broken down by the H2O2 reaction. The loss of laccase activity is strong evidence that this in fact occurred. The percentage of laccase activity remaining in this reaction was negligible (data not shown here). This effect has been detailed in the literature,39 where it was suggested that, when carbohydrate moieties in the laccase molecules are damaged, the overall enzyme would lose activity. This is likely due to the 2404

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ACKNOWLEDGMENTS This research was supported by U.K.’s Engineering and Physical Sciences Research Council (EPSRC, Grant EP/ E036252/1) and Sustainable Liquid Biofuels from Biomass Biorefining (SUNLIBB, EU Framework VII Grant Agreement 251132).

Of the intermediate compounds, succinic acid (also known as butanedioic acid and historically as “spirit of amber”) is of note. Because of its composition (C4H6O4), this substance has potential for applications in biorefineries.40 It can be used to make a range of useful chemicals, including γ-butyrolactone, 1,4-butanediol, 1,4-diaminobutane, succinimide, succinonitrile, dibasic ester, n-methyl pyrrolidinone, 2-pyrrolidone, and tetrahydrofuran (THF).40 Currently, succinic acid is normally produced by a chemical process using fossil resources as a raw material, but the drawback of using a chemical-based process, such as this, is the long-term limitation of petroleum reserves and increasing (and volatile) oil price.41 Furthermore, the hydrothermal oxidation process reported in the literature was used to degrade alkali lignin that produced many organic acids, including succinic and acetic acids.34 However, this hydrothermal oxidation process required high-temperature conditions (200 °C for 2 min), while the photoreaction process that we report here employed 50 °C for 24 h. This is because the Kraft lignin used in this work has a 30% higher molecular weight (ca. 99 500) than the molecular weight of 70 000 tested by Hasegawa et al.34 Biotechnological routes are also used to produce succinic acid. These bioprocesses can employ agricultural waste as a raw material, but it takes a long time for this bioreaction route (ca. 36−72 h).42 Here, succinic acid can be produced via our photocatalytic process in 24 h, using a waste product (lignin) as a raw material. Further optimizations of the experimental configurations used here may produce a design suitable for larger scale production of this desirable byproduct.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S22 and Tables S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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4. CONCLUSION This work presents an experimental process that combines TiO2 and laccase to degrade lignin in single- and dual-step processes. The results satisfy the hypothesis that there would be increased lignin degradation using both processes rather than using only process. The results also show that this combination of photocatalytic and biocatalytic reactions is best performed as a two-stage process, with TiO2 first and laccase second, because this configuration was much more successful than a single-step process. The investigation also shows that laccase requires a supply of oxygen to produce high degradation success rates. Primarily for practical reasons, H2O2 was chosen. In addition to these main conclusions, GC−MS analyses identified and quantified the lignin breakdown products. Of these data, perhaps the most interesting was that TiO2 used in the presence of H2O2 yielded a significant quantity of succinic and malonic acids. These byproducts of the main reaction have the potential to be used to produce a variety of useful chemicals.

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*Telephone: +44(0)114-2227577. Fax: +44(0)114-2227501. E-mail: p.c.wright@sheffield.ac.uk. Notes

The authors declare no competing financial interest. 2405

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