Characterization of a novel laccase-producing Bacillus sp. A4 and its application in Miscanthus degradation

Abstract

Full Article

Characterization of a Novel Laccase-producing Bacillus sp. A4 and its Application in Miscanthus Degradation

Haipeng Guo,^a,b Chaoyang Lin,^a,c Shihui Wang,^a,d Dean Jiang,^b Bingsong Zheng,^e Yun Liu,^d and Wensheng Qin ^a,*

Bacillus sp. A4 exhibiting laccase production was isolated from forest soil. Its laccase secreted into a LB medium exhibited a maximum activity of 3.9 U mg^-1 protein at the optimal temperature (37 °C) and pH (6.0). The purified laccase of Bacillus sp. A4 demonstrated a low molecular mass of 33 kDa, and its optimal temperature and pH were 40 °C and 4.6, respectively, when using ABTS as a substrate. The activity of the purified laccase was significantly increased in the presence of Cu²⁺, methanol, and ethanol, but it was totally inhibited by L-cysteine. The laccase production of this strain was markedly stimulated when the strain was incubated with 0.5% different lignocellulosic biomasses. The highest activity of laccase (22.6 U mg^-1 protein) was obtained in using algal biomass. This new strain efficiently decreased the lignin content of lignocellulose biomasses after 9 d of incubation at 37 °C, especially lignin from grasses. Further analysis showed that, compared to that of all tested biomasses, the new strain was a more efficient decomposer of the lignin of Miscanthus, which exhibited much more lignin loss and cell wall structure destruction in a short span of time. Therefore, the potential use of this strain could be advantageous for using lignin in Miscanthus for industrial processes.

Keywords: Laccase; Bacillus sp.; Miscanthus; Lignin; Scanning electron microscope

Contact information: a: Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1 Canada; b: College of Life Sciences, Zhejiang University, Hangzhou, ZJ 310058 China; c: State Key Laboratory of Rice Biology and Institute of Insect Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, ZJ 310058 China; d:Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029 China; e: State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Linan, ZJ 311300 China;

* Corresponding author: wqin@lakeheadu.ca

INTRODUCTION

Laccases (EC 1.10.3.2) are a group of copper-containing oxidases that play an important role in oxidizing a range of aromatic compounds in nature, such as phenolic units and amine compounds in lignin (Leonowicz et al. 2001; Majeau et al. 2010). The characteristics of laccase have been widely studied in fungi, especially white rot fungi, which have been regarded as the most efficient laccase producers and are used in industrial processes (Senthivelan et al. 2016). However, fungi often grow slowly and require a long time to produce laccase; also, most of their laccases have the drawbacks of low thermostability and a limited pH-activity range (pH 4.0 to 6.0) (Majeau et al. 2010; Mukhopadhyay et al. 2013). The majority of fungal laccases usually rapidly drop their activity at temperatures above 60 °C and the half-life range of their activities at 60 °C was reported as 0.3 to 2 h (Hildén et al. 2009). To overcome the obstacles inherent in fungal laccases, more and more researchers have focused on bacterial laccases due to their rapid production, superior stability at high temperatures and pH values, and easy genetic manipulation, although the laccase production yields of bacteria are lower than those of fungi (Martins et al. 2002; Muthukumarasamy and Murugan 2014).

To date, bacterial laccases have been widely reported and applied to lignocellulosic degradation and bioremediation. For example, Saritha and Lata (2012) isolated a Streptomyces griseusfrom leaf litter and found that it selectively decreased the Klason lignin content of hardwood and softwood and produced high levels of cellulose complex when growing on wood substrates. A recombinant protein CotA from Bacillus licheniformis showed spectroscopic properties and effectively oxidized syringic acid to 2,6-demethoxy-1,4-benzoquinone (Koschorreck et al. 2008). A newly discovered Bacillus licheniformis LS04, exhibiting high laccase activity, was isolated from forest soil and was shown to be able to decolorize various chemical dyes (Lu et al. 2012). Lončar et al. (2013) screened a laccase production strain and identified it as Bacillus amyloliquefaciens, which showed wastewater decolorization ability. Furthermore, it was reported that bacterial laccases showed superior tolerance to various harsh environments. Fang et al. (2011) isolated a bacterial laccase from a marine microbial metagenome that exhibited chloride tolerance and dye decolorization ability. The spore laccase of Bacillus licheniformis LS04 was found to be quite stable at high temperatures, showed no loss of laccase activity after 10 days of incubation at pH 9.0, and demonstrated organic solvent tolerance (Lu et al. 2012).

Lignocellulosic biomass consisting of carbohydrate polymers (cellulose and hemicellulose) and an aromatic polymer (lignin) is thought to be the most abundantly sustainable raw material on Earth that can be used for the production of biofuels and chemicals. Among various biomass sources, the Miscanthus species are promising crops for bioethanol production due to their high biomass yield and remarkable adaptability to different environments (Brosse et al. 2012). However, biomass recalcitrance has posed a significant challenge in biofuel production. This is due to the fact that lignin-carbohydrate complexes formed by lignin are intimately interlaced with carbohydrates through ester and ether bonds, preventing the enzymatic hydrolysis of fermentable sugars (Zhao et al. 2012; De Souza et al. 2015). Among the various techniques developed for the removal of biomass recalcitrance from lignocellulosic biomass, the engineering of microbial fermentation has been proposed as a cost-efficient, eco-friendly, and sustainable development strategy to reduce the risk to the environment and improve the efficiency of biofuel production (Maki et al. 2009; Weber et al. 2010). In this study, the authors isolated a novel laccase-producing Bacillus sp. A4 strain that showed excellent growth and laccase production characteristics. Furthermore, the strain was used to evaluate its degradative capability using various biomasses as carbon sources.

EXPERIMENTAL

Materials

Isolation and identification of laccase-producing strain Bacillus sp. A4

Bacillus sp. A4 was isolated from forest soil (Thunder Bay, Ontario, Canada). A soil sample (5.0 g) were suspended in 50 mL of sterile distilled water; the mixture was shaken at 200 rpm and 37 °C for 30 min. Then, the isolate was spread via the standard serial dilution plate method using a Luria-Bertani (LB) medium that contained 0.5 mM guaiacol. The plates were incubated at 37 °C for 3 to 5 days. The laccase-producing strains were visualized via the formation of reddish brown zones in the medium (Coll et al. 1993). All the above experiments were performed in a super clean bench under sterile conditions. The A4 strain was picked up and purified for further study through single-colony isolation because it showed the largest reddish brown zone and the highest laccase activity. To identify the A4 strain, its genomic DNA was extracted with a Bacteria DNA kit (Bio Basic, Markham, Ontario, Canada). The internal transcribed spacer (ITS) region was amplified using the universal primers HAD-1 (5’-GACTCCTACGGGAGGCAGC AT-3’) and E1115R (5’-AGGGTTGCGCTCGTTGCGGG-3’). The polymerase chain reaction (PCR) products were cloned into the pUCM-T vector and sequenced. Then, the sequence was retrieved by basic local alignment search tool (BLAST) searches on the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) website, aligned using ClustalX 1.83 (Thompson et al. 1997), and subjected to phylogenetic analysis using the neighbor-joining method with MEGA6 (MEGA Inc., Englewood, NJ) using 1,000 bootstraps (Lin et al. 2015).

Growth characterization of Bacillus sp. A4

In all experimental conditions, the A4 strain was incubated in a 250-mL Erlenmeyer flask containing 50 mL of the LB medium with agitation at 200 rpm. To determine the point of maximal strain growth and laccase production, the optical density at 600 nm (OD₆₀₀) and laccase activities in the supernatant and cells were determined on each day over a 9-day period, starting from the first day of inoculation. The OD₆₀₀ cells were measured as a growth parameter. To investigate laccase activity, 1 mL of bacterial culture was harvested and centrifuged for 3 min at 12,000 g. The activities of the supernatant were immediately measured. The cells were re-suspended in 0.1 M potassium phosphate buffer (pH 7.4) that contained 50 mM KCl and were sonicated at a resonance of 40 kHz and 4 °C for 2 min using the “duty cycle” control, which consisted of 10 s of working time and 10 s of stop time. The cell extract was obtained viacentrifugation at 12,000 g for 3 min as crude intracellular enzyme and kept at 4 °C until activity measurement. To investigate laccase production under different temperature and pH conditions, laccase activity was measured after culturing the strain at various temperatures (20 °C to 42 °C) and various pH values (4.4 to 8.7). The effect of the initial inoculum concentration on laccase production was investigated by inoculating the strain at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% (v/v) concentrations.

Laccase activity and protein content assay

The laccase activity was measured according to the method of Guo et al. (2017), with some modifications. Briefly, a total of 200 μL of reaction mixture containing 20 μL of diluted crude enzyme and 20 μL of 20 mM 2 2′-Azino-bis [3-ethylbenzothiazoline-6-sulfonate] (ABTS; Sigma, St. Louis, MO, USA) in a 0.1 M citrate buffer (pH 4.6) was incubated at 40 °C for 3 min. The absorbance was then determined at 420 nm (ɛ₄₂₀ = 36,000 M^-1 cm^-1) using a Microplate Spectrophotometer (Epoch, Bio Tek Instruments, Inc., Winooski, VT, USA). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of substrate per min. The protein content was measured by the Bradford Protein Assay Kit (Bio Basic Canada Inc., Markhan, Ontario, Canada) according to the manufacturer’s instructions.

Biological Characterization of Laccase

On the third day, the crude intracellular enzyme was obtained via the above-described method and purified according to the method of Wang et al. (2016). Then, the purified laccase was used in the following experiments.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis

To determine the molecular weight of the laccase from the A4 strain, the purified enzyme was run, along with standard protein marker, using a 12% (W/V) polyacrylamide gel with an 8% stacking gel according to the method of Lin et al. (2015). After electrophoresis, the gel was divided into two parts. One part of the gel was stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich, Oakville, Canada) to visualize the protein. The other part of gel was immersed in 1% Triton-X 100 for 30 min to remove the SDS. Following this, the protein bands with laccase activity were visualized by soaking the gel in 0.1 M citrate buffer (pH 4.6) containing 1.0 mM ABTS.

The effects of pH and temperature on laccase activity

The activity of purified laccase was assayed in a pH range of 3.8 to 8.2 at 40 °C. The buffer solutions used were as follows: pH 3.8 to 6.6 in 0.1 M citrate buffer and pH 7.0 to 8.2 in 0.1 M Tris-HCl buffer. The optimum temperature for the purified laccase was determined over the temperature range of 30 °C to 80 °C, using ABTS as the substrate, in 0.1 M citrate buffer (pH 4.6).

The effects of metal ions, organic solvents, and inhibitors on laccase activity

The effects of various metal ions on laccase activity were determined in the presence of 1.0 mM Li⁺, Cd²⁺, Zn²⁺, Cu²⁺, Mg²⁺, Ca²⁺, Ba²⁺, Al³⁺, and Co²⁺ and 5.0 mM of the same metals. To determine the optimum Cu²⁺ concentration, laccase activity was measured at different Cu²⁺ concentrations from 0 mM to 10 mM. The effects of organic solvents, methanol, ethanol, acetone, and dimethyl sulphoxide on laccase activity were measured by adding 5%, 10%, and 20% (v/v) of these into the reaction mixture. The influences of SDS, L-cysteine, and ethylenediaminetetraacetic acid (EDTA) on laccase were examined after 30 min of incubation with the enzymes at 40 °C. To determine which concentration of L-cysteine could completely inhibit laccase activity, the activity of laccase was measured in the presence of 0.1 mM to 1.0 mM L-cysteine.

Lignin degradation of various biomasses using Bacillus sp. A4

To measure the degradation of lignin in various biomasses using Bacillus sp. A4, the strain was grown overnight in a LB broth medium. Then, 1 mL of the above-described bacterial suspension was transferred to 50 mL of a minimal salt medium containing 1.0 g L^-1 sodium nitrate (NaNO₃), 1.0 g L^-1 dipotassium phosphate (K₂HPO₄), 1.0 g L^-1 potassium chloride (KCl), 0.5 g L^-1 magnesium sulfate (MgSO₄), 0.5 g L^-1 yeast extract, and 5% algae, agave, wild rice husk, 2% NaOH-pretreated Miscanthus sacchariflorus (M. sacchariflorus) [incubated M. sacchariflorus with 2% sodium hydroxide (NaOH) for 3 h at 50 °C], wheat straw, non-pretreatment M. sacchariflorus, wood dust, or pine in 250-mL Erlenmeyer flasks. The flasks were placed in an incubator shaker at 37 °C with agitation at 200 rpm for 9 d. The fermentation broth was harvested every day and centrifuged at 12,000 g for 3 min. The supernatants were used to complete the laccase activity measurements.

After 9 d of fermentation, the pretreated biomass, after being separated through filtration, was dried at 80 °C for 72 h and used for the lignin content analysis. The lignin content levels of various biomasses were determined via an acetyl bromide procedure (Johnson et al. 1961; Iiyama and Wallis 1988). Briefly, the samples (1 mg) were placed in 15-mL glass reaction bottles with solutions of 25% (w/w) acetyl bromide in acetic acid (1 mL). The bottles were sealed with polytetrafluoroethylene-coated silicone caps and incubated at 70 °C for 2 h. Then, the mixture was diluted to 10 mL with acetic acid that contained 2 M sodium hydroxide and 7.5 M hydroxylamine hydrochloride. The lignin content was measured by examining the absorbance at 280 nm, using lignin absorptivities of 23.3 g^-1 L cm^-1 for algae, agave, wild rice husk, M. sacchariflorus, and wheat straw and 23.6 g^-1 L cm^-1 for wood dust and pine.

Methods

Scanning electron microscope (SEM) observation

After 16 d of incubation, the remaining residues from the bacteria-treated non-pretreatment M. sacchariflorus were collected through filtration and washed with distilled water three times. Then, the samples were dried at 40 °C, sputter-coated with gold (Ion sputter E-1030; Hitachi Ltd., Tokyo, Japan), and observed via scanning electron microscopy (SEM Hitachi AU-70, Tokyo, Japan).

Statistical analysis

All of the experiments were performed in triplicate, and the results are shown as means ± SDs. The statistical analysis was performed via a one-way analysis of variance using SPSS (SPSS Inc., Chicago, USA, Version 13.0).

RESULTS AND DISCUSSION

Isolation and Identification of A4 Strain

A total of 12 isolates showed a brown color around the margin of the colonies on the LB medium plate that contained 0.5 mM guaiacol.

Fig. 1. Phylogenetic tree of 16S rDNA gene sequences between strain A4 and other related Bacillus species; 16S rDNA gene sequences were retrieved by BLAST searches in NCBI and subjected to phylogenetic analysis using the neighbor-joining method with MEGA6 using 1,000 bootstraps

One isolate, A4, which could oxidize ABTS in 1 min and thus exhibited the highest level of laccase activity, was selected for further study. The strain showed colonies of approximately 1 mm in diameter that were white in color and Gram-positive on the LB medium plate without guaiacol. Furthermore, the A4 strain also exhibited cellulose activity in the carboxymethyl cellulose agar plate assay (data not shown). To identify the strain, the 16S rDNA gene sequence of A4 was amplified and sequenced. The blast analysis of the sequence in NCBI and the phylogenetic tree showed that this strain belonged to the Bacillus genus (Fig. 1). Therefore, the A4 strain was finally identified as Bacillus sp. according to the morphological and phylogenetic characteristics of this strain, as well as the comparative analysis of the 16S rDNA sequence.

Strain Growth Characteristic and Laccase Production

The effects of incubation time, temperature, pH, and inoculum concentration on A4 strain growth and laccase production were measured by recording the OD₆₀₀ and relative laccase activity of the fermentation supernatant under various conditions (Figs. 2 and 3). The strain growth and laccase production of the supernatant and cells of Bacillus sp. A4 significantly increased initially and then decreased with increased incubation time (Fig. 2). The OD₆₀₀ of the A4 strain reached a maximum of 2.55 after 2 d of incubation, at which time the laccase activity in the supernatant was first detected, and then reached a peak after 5 d with a value of 3.9 U mg^-1 protein. Thereafter, the laccase activity level decreased and remained relatively constant until Day 8.

The intracellular laccase activity of the A4 strain increased significantly with incubation time up to 3 d, at which point it reached a value of 5.4 U mg^-1 protein, and then decreased with additional incubation time (Fig. 2). Furthermore, the optimal temperature, pH, and inoculum concentration for laccase production were 37 °C, 6.0, and 2.0%, respectively (Fig. 3).

Fig. 2. Time course of OD₆₀₀ and laccase production in supernatant and cells at 37 °C; Data shown are means ± SDs (n = 3)

Fig. 3. Effects of culture temperature (a), pH (b), and incubation concentration (c) on OD₆₀₀ and laccase activity in LB medium after 5 d co-cultivation; Data shown are means ± SDs (n = 3)

SDS-PAGE and Zymogram Analysis

The purified laccase was confirmed via SDS-PAGE and zymogram analysis, in which appeared a single protein band (Fig. 4). The molecular weight of the enzyme was 33 kDa, as measured by SDS-PAGE (Fig. 4), and the activity at that size was shown via a zymogram analysis (Fig. 4).

Fig. 4. Electrophoretic analysis of the laccase purified by crude intracellular enzyme, (a) SDS-PAGE was stained by Coomassie Brilliant Blue R-250; (b) Zymogram was strained by 1 mM ABTS; Lane M: protein marker; Lane 1: Purified laccase; the protein size is about 33 kDa

Effects of Temperature, pH, Metal Ions, Organic Solvents, and Inhibitors on Laccase Activity

The laccase activity significantly differed with changing incubation temperatures and pH values (Fig. 5). The purified laccase showed activity over a broad range of temperatures (30 °C to 80 °C), with the optimal activity at 40 °C. The relative enzyme activity levels at 30 °C, 50 °C, 60 °C, 70 °C, and 80 °C were 89%, 85%, 81%, 65%, and 51% of that seen at 40 °C, respectively (Fig. 5a). The enzyme showed more than 50% relative activity in the 3.8 to 6.2 pH range, with the optimum pH being 4.6. Beyond a pH 4.6, laccase activity declined rapidly, and the relative activity decreased 90% at pH 8.2 (Fig. 5b).

Fig. 5. Effects of temperature (a) and pH (b) on the activity of the laccase enzyme from Bacillus sp. A4.; Data shown are means ± SDs (n = 3)

The effects of metal ions, organic solvents, and inhibitors on laccase activity were measured by adding these into the reaction mixture (Fig. 6). The metal ions had almost no effect on laccase activity, but Cu²⁺ significantly increased activity, especially at concentrations of 0.5 mM and 1.0 mM, which increased the activity 45% and 80%, respectively (Figs. 6a and 7a). Moreover, laccase activity was also induced by ≤ 10% (v/v) methanol, ethanol, and acetone. Enzyme activity increased 62%, 30%, and 25% in the presence of 10% methanol, ethanol, and acetone but was inhibited by 20% concentrations of these and all concentrations of dimethyl sulfoxide (Fig. 6b). The laccase was almost totally inhibited with increased L-cysteine concentration, and the reductions were 50%, 78%, and 96% in 0.1 mM, 0.5 mM, and 1.0 mM L-cysteine, respectively (Figs. 6c and 7b). Also, SDS and EDTA strongly restrained the activity of laccase, which decreased 59% and 52% in the presence of these at 1.0 mM, respectively (Fig. 6c).

Fig. 6. Effects of metal ions (a), organic solvents (b), and inhibitors (c) on the activity of the laccase enzyme from Bacillus sp. A4; CK means the control without adding any ions in (a); Data shown are means ± SDs (n = 3)

Fig. 7. Effects of Cu²⁺ concentration (a) and L-cysteine concentration (b) on the activity of the laccase enzyme from Bacillus sp. A4; Data shown are means ± SDs (n = 3)

Laccase Production and its Effects on Biomass Degradation

The laccase production of the Bacillus sp. A4 strain using various biomasses significantly differed with increased incubation time (Fig. 8). Laccase activity was strongly induced by algae, 2% NaOH-pretreated M. sacchariflorus, and non-pretreated M. sacchariflorus biomass; it was rapidly increased after incubation using algae and 2% NaOH-pretreated M. sacchariflorusand reached values of 18.0 U mg^-1 protein and 14.9 U mg^-1 protein, respectively, on Day 3. Then, it increased gradually and reached a maximum (22.6 U mg^-1 protein) after 6 d of incubation using algae, while it decreased with increased incubation time using 2% NaOH-pretreated M. sacchariflorus. However, the laccase production of non-pretreated M. sacchariflorus was induced gradually and reached a peak (13.5 U mg^-1 protein) after 7 d of incubation (Fig. 8). Moreover, laccase activity increased significantly when supplemented with agave, wild rice husk, wheat straw, wood dust, and pine biomass, and the corresponded maximum activities were 4.2 U mg^-1 protein, 4.1 U mg^-1 protein, 4.1 U mg^-1 protein, 2.5 U mg^-1protein, and 5.3 U mg^-1 protein, respectively (Fig. 8).

Fig. 8. The effects of various biomasses on the laccase production of Bacillus sp. A4. Mis: non-pretreatment M. sacchariflorus; Mis (2% NaOH): incubated M. sacchariflorus with 2% sodium hydroxide (NaOH) for 3 h at 50 °C; Data shown are means ± SDs (n = 3)

To determine whether the different cell wall components of these biomasses contributed to laccase production, the lignin content of biomasses treated with Bacillus sp. A4 was measured, as compared to that of biomasses left untreated. The lignin content levels were significantly different (p < 0.01) when the strain was incubated with algae, agave, 2% NaOH-pretreated M. sacchariflorus, and non-pretreated M. sacchariflorus as compared to those of biomasses untreated with Bacillus. The lignin content levels of algae, agave, 2% NaOH-pretreated M. sacchariflorus, and non-pretreated M. sacchariflorus were 1.9%, 5.6%, 11.7%, and 22.8% of dry weight biomass before incubation with the A4 strain, respectively, and they were 0.5%, 2.7%, 4.4%, and 11.5% of dry weight biomass after incubation, respectively (Fig. 9). Furthermore, the lignin content levels of wild rice husk, wheat straw, wood dust, and pine were also significantly degraded (p < 0.05) by the A4 strain; they decreased by 28%, 26%, 11%, and 15%, respectively (Fig. 9).

Fig. 9. The lignin content levels of untreated biomass and biomass treated with Bacillus sp. A4 for 9 d; Data shown are means ± SDs (n = 3); the * symbol indicates a significant difference at p < 0.05, and the ** symbol indicates a very significant difference at p < 0.01

To further confirm the degradation of M. sacchariflorus by the A4 strain, the cell wall structures of untreated and bacteria-treated non-pretreated M. sacchariflorus were observed over an incubation period of 16 d. The bacteria-untreated non-pretreated M. sacchariflorus’s surface was smooth and tight after 16 d of incubation, which indicated no degradation of the cell wall (Figs. 10 a and b). In contrast, the samples treated with bacteria showed a rough, fragile, and paralyzed surface that indicated degradation after 16 d of incubation (Figs. 10 c and d).

In the present study, a highly laccase-producing strain was isolated on a LB medium plate that contained guaiacol. The laccase produced by this strain showed the ability to oxidize ABTS. Sheikhi et al. (2012) reported that the laccase produced by Bacillus subtilis WPI was more sensitive to ABTS than guaiacol as a substrate due to the laccase inactivation of guaiacol reaction products (Robles et al. 2000). The strain was identified as Bacillus sp. A4 according to its morphological and biochemical characteristics (Fig. 1) and the 16S rDNA sequence, which has been widely used to identify bacteria generally and construct phylogenetic trees due to the slow evolutionary rate of bacteria (Goto et al. 2000; Yarza et al. 2014). A time course for the intracellular and supernatant laccase activity of Bacillus sp. A4 was assayed at the optimal temperature (37 °C) and pH (6.0) with a 2% inoculum concentration. Intracellular laccase could be detected at every measuring time and reached a peak at Day 3, but the laccase activity of the supernatant was first detected at Day 2 of incubation and reached a peak at Day 5 (Fig. 2). This indicated that this enzyme could be slowly released into the medium with increased incubation time. This was also found to be true for Trametes versicolor, which produced laccase in medium after 2 d of incubation, reached a peak after 6 d, and then decreased slightly and remained relatively constant up to Day 15 (Collins and Dobson 1997). Lu et al. (2013) reported that the laccase activity of the culture supernatant reached a maximum of 227.9 U L^-1 on Day 7 and then decreased gradually and that this decrease in laccase production might have been caused by the activation of some proteases at a lower pH.

Fig. 10. The scanning electron microscopy (SEM) of non-pretreated M. sacchariflorus after co-cultivation with Bacillus sp. A4 for 16 d. (a-b) Samples not treated with Bacillus sp. A4; (c-d) samples treated with Bacillus sp. A4; the arrows indicate the degradation of M. sacchariflorus by Bacillus sp. A4

The molecular weights of most bacterial and fungal laccase proteins range from 43 kDa to 110 kDa (Yaropolov et al. 1994; Madhavi and Lele 2009). In this study, the molecular weight of laccase was approximately 33 kDa (Fig. 4), which was different from most of the reported laccases of bacteria and fungi, though several isoforms of laccases from bacteria have been reported to range from 31 kDa to 40 kDa in size. For example, Sondhi et al. (2014) found a novel extracellular thermo-alkali-stable laccase of Bacillus tequilensis SN4 that produced a 32 kDa monomeric protein. The molecular weight of laccase from Streptomyces coelicolor was around 32 kDa and able to rapidly decolourize dye in the presence of syringaldehyde, which acted as a redox mediator (Dubé et al. 2008). The laccase exhibited maximal activity at 40 °C, and retained more than 50% of its activity at 80 °C (Fig. 5a). The optimum temperature for laccase was lower than those seen in some other bacterial strains, such as 60 °C in Bacillus licheniformis LS04 (Lu et al. 2012), 65 °C in Bacillus amyloliquefaciens 12B (Lončar et al.2013), and 85 °C in Bacillus vallismortis fmb-103, and was similar to those of Bacillus subtilis (Hullo et al. 2001) and Bacillus sp. ADR (Telke et al. 2011). The optimal pH value for purified laccase in terms of oxidizing ABTS was 4.6 (Fig. 5b), like most bacterial laccase (Hullo et al. 2001; Dubé et al. 2008; Lu et al. 2012).

Furthermore, the effects of metal ions, organic solvents, and inhibitors were measured. Among the metal ions tested, laccase activity was highly induced by < 5.0 mM Cu²⁺, but it was insensitive to other heavy metals (Figs. 6a and 7a). Murugesan et al. (2009) reported that Ca²⁺, Co²⁺, Cu²⁺, and Zn²⁺ enhanced the laccase activity of Ganoderma lucidum up to a 1.0 mM concentration and that chemical dyes were decolorized during 1 h of incubation in the presence of 1.0 mM Cu²⁺. Lorenzo et al. (2005) reported that the addition of Cu²⁺ to the reaction mixture stimulated laccase activity at concentrations lower than 1.0 mM and that Mn²⁺ and Zn²⁺ did not affect enzyme activity at any of the concentrations tested (0.5 mM to 80 mM). The filling of the Type I Copper binding site by Cu²⁺ could be the main cause of this increased laccase activity (Shekher et al. 2011; Sondhi et al. 2014). Purified laccase activity was induced 5% to 10% methanol and 10% to 20% ethanol (Fig. 6b). Previous studies have shown that bacterial laccases from Bacillus licheniformis LS04 (Lu et al. 2012) and Bacillus pumilus W3 (Guan et al. 2014) were highly induced by organic solvents, which allow access to some insoluble substrates in the reaction mixtures and help to detoxify several stable organic pollutants (Torres et al. 2003). At a 1.0 mM concentration, the inhibitors SDS and EDTA could partly inactivate the laccase activity of Bacillus sp. A4, but laccase activity was completely inhibited by L-cysteine at the same concentration (Fig. 6c and 7b). Partial inactivation by a low concentration of SDS and EDTA was also found in the laccase of Bacillus licheniformis (Lu et al.2013) and Bacillus pumilus (Guan et al. 2014). The total inactivation in the presence of 1.0 mM L-cysteine was similar to that of the recombinant laccase from Bacillus licheniformis(Baldrian et al. 2004; Lu et al. 2013).

Lignin carbohydrate complexes are intimately interlaced with carbohydrates through ester and ether bonds, and this is a primary obstacle to the production of biofuels from lignocellulosic biomass (Studer et al. 2011). The lignin content of biomass is usually in the range of 15% to 35% (Azadi et al. 2013). The microbial degradation of lignin has been well-studied in fungi, such as white-rot and brown-rot fungi (Leonowicz et al. 1999; Guillén et al. 2005), but little is known about the degradation of lignin in bacteria. The present study showed that the novel laccase-producing Bacillus sp. A4 produced a great deal of laccase and decreased lignin content after incubation with various biomass (Fig. 8). Laccase activity was maximally induced by algae, which contained only 1.9% lignin content and indicated that the highest level of induction was mostly caused by the algal phytochrome, which could be also used as the oxidation substrate of laccase (Shekher et al. 2011). Laccase has been commonly induced by the biomass of grasses (agave, wild rice husk, wheat straw, and Miscanthus), but induction was difficult in the presence of wood biomass (wood dusk and pine) (Figs. 8 and 9). The various levels of induction and degradation caused by the lignin from grasses and woods could be attributed to their different structural compositions (Demirbaş 2005). Antai and Crawford (1982) reported that two Streptomyces strains substantially degraded both the lignin and the carbohydrate components of softwood, hardwood, and grass lignocelluloses but that they were more efficient decomposers of grass lignin than of wood lignin. The authors were interested in the fact that Miscanthus biomass, both pretreatment and non-pretreatment, significantly stimulated laccase production, which resulted in a significant decrease (p < 0.01) in lignin content and the breakage of the cell wall structure in Miscanthus (Figs. 8 through 10). The high level of laccase induction could be partially due to the high cellulose/lignin ration and total ester-bound phenolics content compared to other bioenergy crops, such as switchgrass and reed (Lygin et al. 2011). The different types of lignin and extractives in the structures of various species of lignocellulosic materials may also lead to different levels of degradation by Bacillus sp. A4 (Demirbaş 2001). Furthermore, the efficiency of lignin degradation in Bacillus sp. A4 was much higher than that in white rot fungus (Wan and Li 2010; Liu et al. 2013). The white rot fungus Ceriporiopsis subvermispora degraded 39.2% lignin of corn stover after 42 d of cultivation (Wan and Li 2010). The lignin content of switchgrass decreased by 30% after 36 d of incubation with fungus Pycnoporus sp. SYBC-13 (Liu et al. 2013), while the Bacillus sp. A4 removed 11 to 74% lignin of the tested biomass after only 9 d of cultivation (Fig. 9). Electron microscopic and biochemical studies of lignocellulose degradation by most fungi and bacteria have shown that enzymes, such as lignin peroxidases, laccase, and cellulases, are too large to penetrate undegraded secondary wood cell walls (Evans et al. 1994; Blanchette 1995; Tuor et al. 1995). Therefore, the high lignin degradation ability of Bacillus sp. A4 may be due to the low molecular mass of the laccase, which helped it to penetrate the cell wall tissue and then degrade the lignin of plants (Ten Have and Teunissen 2001).

CONCLUSIONS

1. A novel laccase-producing strain was isolated from forest soil and identified as Bacillus sp. A4. The purified laccase retained relatively high levels of activity over a wide temperature range (30 °C to 70 °C). It had a low molecular weight (33 kDa) and was significantly induced by Cu²⁺ and some organic solvents.
2. The laccase production of the strain was markedly stimulated when incubated with various lignocellulosic biomasses. The novel strain could efficiently decrease lignin content of lignocellulosic biomasses after 9 d of incubation, especially the lignin of grasses.
3. The new strain A4 was a more efficient decomposer of lignin in Miscanthus, which demonstrated much more lignin loss and cell wall structure destruction in a short span of time.

ACKNOWLEDGEMENTS

The authors wish to thank the support of BioFuelNet Canada and the China Scholarship Council (Grant No. 201506320044).

REFERENCES CITED

Antai, S. P., and Crawford, D. L. (1982). “Degradation of extractive-free lignocelluloses by Coriolus versicolor and Poria placenta,” European Journal of Applied Microbiology and Biotechnology 14(3), 165-168. DOI: 10.1007/BF00497894

Azadi, P., Inderwildi, O. R., Farnood, R., and King, D. A. (2013). “Liquid fuels, hydrogen and chemicals from lignin: A critical review,” Renewable and Sustainable Energy Reviews 21, 506-523. DOI: 10.1016/j.rser.2012.12.022

Baldrian, P. (2004). “Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme,” Applied Microbiology and Biotechnology 63(5), 560-563. DOI: 10.1007/s00253-003-1434-0

Blanchette, R. A. (1995). “Degradation of the lignocellulose complex in wood,” Canadian Journal of Botany-revue Canadienne de Botaniqu 73, 999-1010. DOI: 10.1139/b95-350

Brosse, N., Dufour, A., Meng, X., Sun, Q., and Ragauskas, A. (2012). “Miscanthus: A fast-growing crop for biofuels and chemicals production,” Biofuels, Bioproducts and Biorefining6(5), 580-598. DOI: 10.1002/bbb.1353

Coll, P. M., Fernandez-Abalos, J. M., Villanueva, J. R., Santamaria, R., and Perez, P. (1993). “Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971),” Applied and Environmental Microbiology 59(8), 2607-2613.

Collins, P. J., and Dobson, A. (1997). “Regulation of laccase gene transcription in Trametes versicolor,” Applied and Environmental Microbiology 63(9), 3444-3450.

De Souza, A. P., Kamei, C. L. A., Torres, A. F., Pattathil, S., Hahn, M. G., Trindade, L. M., and Buckeridge, M. S. (2015). “How cell wall complexity influences saccharification efficiency in Miscanthus sinensis,” Journal of Experimental Botany 66(14), 4351-4365. DOI: 10.1093/jxb/erv183

Demirbaş, A. (2001). “Relationships between lignin contents and heating values of biomass,” Energy Conversion and Management 42(2), 183-188. DOI: 10.1016/S0196-8904(00)00050-9

Demirbaş, A. (2005). “Estimating of structural composition of wood and non-wood biomass samples,” Energy Sources 27(8), 761-767. DOI: 10.1080/00908310490450971

Dubé, E., Shareck, F., Hurtubise, Y., Daneault, C., and Beauregard, M. (2008). “Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye,” Applied Microbiology and Biotechnology 79(4), 597-603. DOI: 10.1007/s00253-008-1475-5

Evans, C. S., Dutton, M. V., Guillén, F., and Veness, R. G. (1994). “Enzymes and small molecular mass agents involved with lignocellulose degradation,” FEMS Microbiology Review 13, 235-239. DOI: 10.1111/j.1574-6976.1994.tb00044.x

Fang, Z., Li, T., Wang, Q., Zhang, X., Peng, H., Fang, W., Hong, Y., Ge, H., and Xiao, Y. (2011). “A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability,” Applied Microbiology and Biotechnology 89(4), 1103-1110. DOI: 10.1007/s00253-010-2934-3

Goto, K., Omura, T., Hara, Y., and Sadaie, Y. (2000). “Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus,” The Journal of General and Applied Microbiology 46(1), 1-8.

Guan, Z. B., Song, C. M., Zhang, N., Zhou, W., Xu, C. W., Zhou, L. X., Zhao, H., Cai, Y. J., and Liao, X. R. (2014). “Overexpression, characterization, and dye-decolorizing ability of a thermostable, pH-stable, and organic solvent-tolerant laccase from Bacillus pumilus W3,” Journal of Molecular Catalysis B- Enzymatic 101, 1-6. DOI: 10.1016/j.molcatb.2013.11.009

Guillén, F., Martínez, M. J., Gutiérrez, A., and Del Rio, J. C. (2005). “Biodegradation of lignocellulosics: Microbial, chemical, and enzymatic aspects of the fungal attack of lignin,” International Microbiology 8, 195-204.

Guo, H. P., Chen, H. M., Fan, L., Linklater, A., Zheng, B. S., Jiang, D. A., and Qin, W. S. (2017). “Enzymes produced by biomass-degrading bacteria can efficiently hydro-lyze algal cell walls and facilitate lipid extraction,” Renewable Energy 109, 195-201.

Hildén, K., Hakala, T. K., and Lundell, T. (2009). “Thermotolerant and thermostable laccases,” Biotechnology letters 31, 1117-1128.

Hullo, M. F., Moszer, I., Danchin, A., and Martin-Verstraete, I. (2001). “CotA of Bacillus subtilis is a copper-dependent laccase,” Journal of Bacteriology 183(18), 5426-5430. DOI: 10.1128/JB.183.18.5426-5430.2001

Iiyama, K., and Wallis, A. F. A. (1988). “An improved acetyl bromide procedure for determining lignin in woods and wood pulps,” Wood Science and Technology 22(3), 271-280. DOI: 10.1007/BF00386022

Johnson, D. B., Moore, W. E., and Zank, L. C. (1961). “The spectrophotometric determination of lignin in small wood samples,” Tappi 44(11), 793-798.

Koschorreck, K., Richter, S. M., Ene, A. B., Roduner, E., Schmid, R. D., and Urlacher, V. B. (2008). “Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids,” Applied Microbiology and Biotechnology 79(2), 217-224. DOI: 10.1007/s00253-008-1417-2

Leonowicz, A., Matuszewska, A., Luterek, J., Ziegenhagen, D., Wojtaś-Wasilewska, M., Cho, N. S., Hofrichter, M., and Rogalski, J. (1999). “Biodegradation of lignin by white rot fungi,” Fungal Genetics and Biology 27(2), 175-185. DOI: 10.1006/fgbi.1999.1150

Leonowicz, A., Cho, N., Luterek, J., Wilkolazka, A., Wojtas-Wasilewska, M., Matuszewska, A., Hofrichter, M., Wesenberg, D., and Rogalski, J. (2001). “Fungal laccase: Properties and activity on lignin,” Journal of Basic Microbiology 41(3-4), 185-227. DOI: 10.1002/1521-4028(200107)41:3/4<185::AID-JOBM185>3.0.CO;2-T

Lin, C., Shen, Z., Zhu, T., and Qin, W. (2015). “Newly isolated Penicillium ramulosum N1 is excellent for producing protease-pesistant acidophilic xylanase,” Journal of Molecular Microbiology and Biotechnology 25(5), 320-326. DOI: 10.1159/000439170

Liu, J., Wang, M. L., Tonnis, B., Habteselassie, M., Liao, X., and Huang, Q. (2013). “Fungal pretreatment of switchgrass for improved saccharification and simultaneous enzyme production,” Bioresource Technology 135, 39-45. DOI: 10.1016/j.biortech.2012.10.095

Lončar, N., Božić, N., Lopez-Santin, J., and Vujčić, Z. (2013). “Bacillus amyloliquefaciens laccase–from soil bacteria to recombinant enzyme for wastewater decolorization,” Bioresource Technology 147, 177-183. DOI: 10.1016/j.biortech.2013.08.056

Lorenzo, M., Moldes, D., Couto, S. R., and Sanromán, M. A. A. (2005). “Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds,” Chemosphere 60(8), 1124-1128. DOI: 10.1016/j.chemosphere.2004.12.051

Lu, L., Zhao, M., Wang, T. N., Zhao, L. Y., Du, M. H., Li, T. L., and Li, D. B. (2012). “Characterization and dye decolorization ability of an alkaline resistant and organic solvents tolerant laccase from Bacillus licheniformis LS04,” Bioresource Technology 115, 35-40. DOI: 10.1016/j.biortech.2011.07.111

Lu, L., Wang, T. N., Xu, T. F., Wang, J. Y., Wang, C. L., and Zhao, M. (2013). “Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization,” Bioresource Technology 134, 81-86. DOI: 10.1016/j.biortech.2013.02.015

Lygin, A. V., Upton, J., Dohleman, F. G., Juvik, J., Zabotina, O. A., Widholm, J. M., and Lozovaya, V. V. (2011). “Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop–Miscanthus,” GCB Bioenergy 3(4), 333-345. DOI: 10.1111/j.1757-1707.2011.01091.x

Madhavi, V., and Lele, S. S. (2009). “Laccase: Properties and applications,” BioResources 4(4), 1694-1717. DOI: 10.15376/biores.4.4.1694-1717

Majeau, J. A., Brar, S. K., and Tyagi, R. D. (2010). “Laccases for removal of recalcitrant and emerging pollutants,” Bioresource Technology 101(7), 2331-2350. DOI: 10.1016/j.biortech.2009.10.087

Maki, M., Leung, K. T., and Qin, W. (2009). “The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass,” International Journal of Biological Sciences5(5), 500-516. DOI: 10.7150/ijbs.5.500

Martins, L. O., Soares, C. M., Pereira, M. M., Teixeira, M., Costa, T., Jones, G. H., and Henriques, A.O. (2002). “Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat,” Journal of Biological Chemistry 277(21), 18849-18859. DOI: 10.1074/jbc.M200827200

Mukhopadhyay, A., Dasgupta, A. K., and Chakrabarti, K. (2013). “Thermostability, pH stability and dye degrading activity of a bacterial laccase are enhanced in the presence of Cu₂O nanoparticles,” Bioresource Technology 127, 25-36. DOI: 10.1016/j.biortech.2012.09.087

Murugesan, K., Kim, Y. M., Jeon, J. R., and Chang, Y. S. (2009). “Effect of metal ions on reactive dye decolorization by laccase from Ganoderma lucidum,” Journal of Hazardous Materials 168(1), 523-529. DOI: 10.1016/j.jhazmat.2009.02.075

Muthukumarasamy, N. P., and Murugan, S. (2014). “Production, purification and application of bacterial laccase: A review,” Biotechnology 13(5), 196-205. DOI: 10.3923/biotech.2014.196.205

Robles, A., Lucas, R., de Cienfuegos, G. A., and Gálvez, A. (2000). “Phenol-oxidase (laccase) activity in strains of the hyphomycete Chalara paradoxa isolated from olive mill wastewater disposal ponds,” Enzyme and Microbial Technology 26(7), 484-490. DOI: 10.1016/S0141-0229(99)00197-0

Saritha, M., and Lata, A. A. (2012). “Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility,” Indian Journal of Microbiology 52(2), 122-130. DOI: 10.1007/s12088-011-0199-x

Senthivelan, T., Kanagaraj, J., and Panda, R. C. (2016). “Recent trends in fungal laccase for various industrial applications: An eco-friendly approach – A review,” Biotechnology and Bioprocess Engineering 21(1), 19-38. DOI: 10.1007/s12257-015-0278-7

Sheikhi, F., Ardakani, M. R., Enayatizamir, N., and Rodriguez-Couto, S. (2012). “The determination of assay for laccase of Bacillus subtilis WPI with two classes of chemical compounds as substrates,” Indian Journal of Microbiology 52(4), 701-707. DOI: 10.1007/s12088-012-0298-3

Shekher, R., Sehgal, S., Kamthania, M., and Kumar, A. (2011). “Laccase: Microbial sources, production, purification, and potential biotechnological applications,” Enzyme research2011(1), 1-11. DOI: 10.4061/2011/217861

Sondhi, S., Sharma, P., Saini, S., Puri, N., and Gupta, N. (2014). “Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensisSN4,” PloS One 9(5), 1-10. DOI: 10.1371/journal.pone.0096951

Studer, M. H., DeMartini, J. D., Davis, M. F., Sykes, R. W., Davison, B., Keller, M., Tuskan, G. A., and Wyman, C. E. (2011). “Lignin content in natural Populus variants affects sugar release,” Proceedings of the National Academy of Sciences 108(15), 6300-6305. DOI: 10.1073/pnas.1009252108

Telke, A. A., Ghodake, G. S., Kalyani, D. C., Dhanve, R. S., and Govindwar, S. P. (2011). “Biochemical characteristics of a textile dye degrading extracellular laccase from a Bacillus sp. ADR,” Bioresource Technology 102(2), 1752-1756. DOI: 10.1016/j.biortech.2010.08.086

Ten Have, R., and Teunissen, P. J. (2001). “Oxidative mechanisms involved in lignin degradation by white-rot fungi,” Chemical Reviews 101(11), 3397-3414. DOI: 10.1021/cr000115l

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997). “The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools,” Nucleic Acids Research 25(24), 4876-4882. DOI: 10.1093/nar/25.24.4876

Torres, E., Bustos-Jaimes, I., and Le Borgne, S. (2003). “Potential use of oxidative enzymes for the detoxification of organic pollutants,” Applied Catalysis B- Environmental 46(1), 1-15. DOI: 10.1016/S0926-3373(03)00228-5

Tuor, U., Winterhalter, K., and Fiechter, A. (1995). “Enzymes of white-rot fungi involved in lignin degradation and ecological determinants for wood decay,” Journal of Biotechnology41(1), 1-17. DOI: 10.1016/0168-1656(95)00042-O

Wan, C., and Li, Y. (2010). “Microbial delignification of corn stover by Ceriporiopsis subvermispora for improving cellulose digestibility,” Enzyme and Microbial Technology 47(1), 31-36. DOI: 10.1016/j.enzmictec.2010.04.001

Wang, S., Jeyaseelan, J., Liu, Y., and Qin, W. (2016). “Characterization and optimization of amylase production in WangLB, a high amylase-producing strain of Bacillus,” Applied Biochemistry and Biotechnology 180(1), 136-151. DOI: 10.1007/s12010-016-2089-5

Weber, C., Farwick, A., Benisch, F., Brat, D., Dietz, H., Subtil, T., and Boles, E. (2010). “Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels,” Applied Microbiology and Biotechnology 87(4), 1303-1315. DOI: 10.1007/s00253-010-2707-z

Yaropolov, A. I., Skorobogat’Ko, O. V., Vartanov, S. S., and Varfolomeyev, S. D. (1994). “Laccase,” Applied Biochemistry and Biotechnology 49(3), 257-280. DOI: 10.1007/BF02783061

Yarza, P., Yilmaz, P., Pruesse, E., Glöckner, F. O., Ludwig, W., Schleifer, K. H., Whitman, W. B., Euzéby, J., Amann, R., and Rosselló-Móra, R. (2014). “Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences,” Nature Reviews Microbiology 12(9), 635-645. DOI: 10.1038/nrmicro3330

Zhao, X., Zhang, L., and Liu, D. (2012). “Biomass recalcitrance. Part I: The chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose,” Biofuels, Bioproducts and Biorefining 6(4), 465-482. DOI: 10.1002/bbb.1331

Article submitted: February 20, 2017; Peer review completed: April 13, 2017; Revised version received: April 23, 2017; Accepted: May 1, 2017; Published: May 15, 2017.

DOI: 10.15376/biores.12.3.4776-4794