The screening of new potential microbes for the selective degradation of lignin makes agricultural straws available to achieve the complete conversion to biofuel. Because of the capability of laccase to oxidize phenolic compounds and to reduce the molecular oxygen of water, laccase has attracted much interest in recent years for its industrial applications. In this study, a fungal strain with a relatively high laccase activity was isolated from corn farm residue and identified as Trametes KS-2. The maximum laccase activity was 631 U/L for Trametes KS-2 when glucose/corn stalk (1/1, w/w) and peptone were used as the carbon source and nitrogen source, with 0.09 mmol of Cu2+ at pH 5.5 and 28 °C for 10 days. Laccase activity of Trametes KS-2 was relatively stable in the presence of salt and an ionic liquid. Scanning electron microscopy analysis indicated the morphological alteration of lignocelluloses via Trametes KS-2 treatment. Remarkable degradation of lignin in corn stalk was achieved with Trametes KS-2. After 15 days, the lignin was noticeably reduced to 76 mg, and the degradation rate was increased to 65.4%. Trametes KS-2 could be potentially utilized in the microbial degradation of lignin for lignocellulosic biomass and the industrial production of laccase.
Efficient Lignin Degradation of Corn Stalk by Trametes with High Laccase Activity and Enzymatic Stability in Salt and Ionic Liquid
Shi-Jia Dong,a,1 Bi-Xian Zhang,b,1 Fu-Ling Wang,a Liu Xin,a Yun-Fei Gao,b Wei Ding,a,* Xin-Miao He,b,c Di Liu,b,c,* and Xiao-Mei Hu a,*
The screening of new potential microbes for the selective degradation of lignin makes agricultural straws available to achieve the complete conversion to biofuel. Because of the capability of laccase to oxidize phenolic compounds and to reduce the molecular oxygen of water, laccase has attracted much interest in recent years for its industrial applications. In this study, a fungal strain with a relatively high laccase activity was isolated from corn farm residue and identified as Trametes KS-2. The maximum laccase activity was 631 U/L for Trametes KS-2 when glucose/corn stalk (1/1, w/w) and peptone were used as the carbon source and nitrogen source, with 0.09 mmol of Cu2+ at pH 5.5 and 28 °C for 10 days. Laccase activity of TrametesKS-2 was relatively stable in the presence of salt and an ionic liquid. Scanning electron microscopy analysis indicated the morphological alteration of lignocelluloses via Trametes KS-2 treatment. Remarkable degradation of lignin in corn stalk was achieved with Trametes KS-2. After 15 days, the lignin was noticeably reduced to 76 mg, and the degradation rate was increased to 65.4%. Trametes KS-2 could be potentially utilized in the microbial degradation of lignin for lignocellulosic biomass and the industrial production of laccase.
Keywords: Lignin degradation; Trametes; Laccase; Corn stalk
Contact information: a: Northeast Agricultural University, Harbin, 150030, China; b: Key Laboratory of Combining Farming and Animal Husbandry, Ministry of Agricultural and Rural Affairs, 150086, P.R. China; c: Heilongjiang Academy of Agricultural Sciences, Harbin, 150086,China; 1These authors contributed equally to this work; *Corresponding author: firstname.lastname@example.org;
Lignocellulosic biomass is a potentially valuable resource for biofuels and bioproducts. Agricultural straws are the most available lignocellulosic biomass and non-food resource. An annual total of 700 million tons of agricultural straws are produced in China, accounting for 20% to 30% of the world’s production.
Lignocellulosic biomass consists mainly of three polymeric components, which are lignin, hemicelluloses, and cellulose. Lignin is a three-dimensional aromatic polymer in the form of the phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). Lignin is considered to be a physical barrier to the biological and chemical hydrolysis of cellulose, making lignocellulose inherently recalcitrant (Himmel et al. 2007). Lignin can be depolymerized by thermochemical methods such as pyrolysis, hydrogenolysis, gasification, hydrolysis, and chemical oxidation under supercritical conditions (Pandey and Kim 2011). However, these pretreatment technologies require significant energy inputs, environmentally harmful processes, or produce hazardous waste. Biodegradation serves as an attractive energy-saving and environmentally friendly option. The use of microbial degradation of lignin makes the lignocellulose available to achieve the complete conversion to biofuel.
Microbial degradation of lignin has been primarily studied with the use of white-rot fungi, such as Phanerochaete chrysosporium, Ganoderma lucidum, Ceriporiopsis subvermispora,Lentinus edodes, Pleurotus eryngii, and Pleurotus ostreatus (Potumarthi et al. 2013; Cianchetta et al. 2014; Ma and Ruan 2015), which are capable of producing several extracellular ligninolytic enzymes including laccase, manganese peroxidase (MnPs), lignin peroxidase (LiPs), and versatile peroxidase (Xu et al. 2017). Moreover, laccases have attracted much interest in recent years because of their low substrate specificity and good oxidative abilities for their industrial applications, such as dye decolorization (Abadulla et al. 2000), pulp biobleaching in the paper industry (Arias et al. 2003), the production of valuable compounds from lignin, soil bioremediation, biodegradation of environmental phenolic pollutants, and removal of endocrine disruptors (Fukuda et al. 2001; Kidwai et al. 2012; Sole et al. 2012; Divya et al. 2013a).
Biological decomposition is a more acceptable, feasible, and economical process in the production of biofuels. However, white rot fungi that are suitable for biodelignification of wood may not be suitable for biodelignification of agricultural straws. More efforts on selecting the most effective strain for different lignocelluloses are necessary. In addition, the selective degradation of lignin should be improved due to the ability of some white rot fungi to degrade the lignin simultaneously with cellulose. Furthermore, most of the corn straw has not been well utilized in China, and straw burning is still used, which causes environment pollution and haze weather. In this study, a strain with selective degradation of lignin in corn stalk was isolated and identified. The culture conditions for the enzyme production of the strain including carbon and nitrogen sources, incubation time and temperature, Cu2+ concentration, and initial pH were optimized. The enzyme activity in the presence of salt and ionic liquids was determined. The degradation of lignin from corn stalk by this strain was also investigated.
The surface soil (0 to 5 cm) was collected from a corn farm of Keshan County in the Heilongjiang province in China. A total of 10 g of the soil sample was added into 90 mL of enrichment medium (sucrose 30 g, CuSO4 0.5 g, K2HPO4 1.0 g, MgSO4 0.5 g, NaNO3 2 g, KCl 0.5 g, FeSO4 0.01 g, and water 1000 mL). The medium was incubated at 150 rpm and 28 °C for 3 days. Following that, 1 mL of the suspension was mixed with 9 mL of deionized water and the suspension was diluted into 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6 (mL/mL). Then, 200 μL of dilution was applied on the potato dextrose agar (PDA) medium (potato 200 g, glucose 20 g, agar 15 g, and water 1000 mL) and incubated at 28 °C for 7 days. The single colony of fungi was isolated on the PDA medium.
Next, the single colony was incubated on the PDA medium at 28 °C for 7 days. Then, 1 cm of agar piece was removed and transferred into 30 mL of PDA medium with the addition of guaiacol (0.1%, w/w) or aniline blue (0.1%, w/w). The solutions were cultured at 28 °C for 7 days with shaking at 150 rpm. Each sample was cultured three times. The strains with reddish brown zones were selected as potential microbes for producing laccase. The strains with color fading zones were selected as potential microbes for producing manganese peroxidase or lignin peroxidase.
A total of 1 cm of agar piece was added into 100 mL of fermentation medium (glucose 10 g, ammonium tartrate 0.2 g, KH2PO4 1 g, MgSO4 0.5 g, CaCl2 0.1 g, vitamin B1 100 mL [0.1 g/L], CuSO4 0.007 g, MnSO4 0.035 g, ZnSO4 0.006 g, FeSO4 0.005 g, CoCl2 0.001 g, Tween 80 (Aladdin, Shanghai, China) 1 mL, acetic acid/sodium acetate buffer 100 mL [0.1 mol/L], water 1000 mL) at 28 °C with an inoculation size of 3% at 150 rpm (Shaker: ZQLY-108S; Shanghai Zhichu Instrument Co., Shanghai, China) for an appropriate time. The resulting solution was then centrifuged at 10,000 rpm (Allegra TMX-22R; Beckman Coulter Inc., Brea, CA, USA) for 5 min at 4 °C to give the crude enzyme solution. Enzyme assay was performed in 4 mL of the solution containing 0.5 mL ABTS (2,2′-azino-bis[3-ethylbenzthiazoline-6-sulfonate], 0.5 mmom/L) (Wang et al. 2013), the crude enzyme (200 μL), and acetic acid/sodium acetate buffer (3.3 mL, 0.1 mol/L) at 30 °C for 3 min. The absorbance was measured at 420 nm on a UV-mini-1240 spectrometer (Shimadzu, Kyoto, Japan). Three replicates were performed. One unit (U) of enzyme activity was defined as the amount of the enzyme that oxidized 1 μmol of ABTS/min under the conditions indicated.
A DNA kit (Omega, Georgia, USA) was used to extract the DNA of the fungi. Fungal analysis was performed with the universal primers ITS1 (5’-TCCGTAGGT-GAACCT GCGG-3’) and ITS4 (5’-TCCTCGCCTTATTGATATGC-3’). The sequence was amplified via polymerase chain reaction (PCR) with 50 μL of the mixture containing template DNA (2 μL), forward primer (2 μL), reverse primer (2 μL), ddH2O (19 μL), and 2× Taq PCR MasterMix (25 μL, Tiangen, Beijing, China). The PCR products were sequenced by the Huada Company (Beijing, China). The bioinformatics tools were available online via BLAST and the MEGA program (Molecular Evolutionary Genetics Analysis Company, version 5.0, USA) with the neighbor-joining (NJ) algorithm, were employed to build phylogenetic trees (Felsenstein 1985). The ITS sequence data for the fungal samples were deposited into the National Center for Biotechnology Information (NCBI) GenBank database.
Optimization of the conditions for laccase production
Individual carbon sources, including starch, glucose, sucrose, lactose, xylose, corn stalk powder, poplar powder, and bamboo powder, at a concentration of 1% (w/w) were measured with peptone as the nitrogen source with inoculation size of 3% (w/w) after incubating at 150 rpm and 28 °C for 7 days on the basis of fermentation medium. Nitrogen sources, including peptone, ammonium sulfate, ammonium chloride, urea, and ammonium nitrate, at a concentration of 0.2% (w/w) were measured with glucose/corn stalk (1/1, w/w) as the carbon source with inoculation size of 3% (w/w) after incubating at 150 rpm and 28 °C for 7 days. In addition, pH (pH 3.0, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, and pH 6.5), Cu2+ concentration (0 mmol/L, 0.03 mmol/L, 0.06 mmol/L, 0.09 mmol/L, 0.12 mmol/L, and 0.20 mmol/L), and incubation temerature (18 °C to 38 °C) with 5 intervals were investigated with glucose/corn stalk (1/1, w/w) as the carbon source and peptone as the nitrogen source for 7 days at 28 °C.
Determination of laccase activity in the presence of salt and ionic liquid
1-Ethyl-3-methylimidazolium acetate ([EMIM]CH3COOH) was prepared using a published method in the literature (Wasserscheid and Welton 2003). The laccase activity of the crude enzyme was determined under different concentrations of NaCl and ionic liquid in the presence of 0%, 2.5%, 5.0%, and 7.5% (w/v) of NaCl and 0%, 2.5%, 5.0%, and 7.5% (w/v) of ionic liquid after 1 h.
Scanning electron microscopy (SEM) analysis was conducted on a Hitachi S-3400N microscope (Hitachi, Tokyo, Japan). Prior to acquiring images, the samples were mounted with double sided carbon tape on corn stalk sample stubs and sputter coated (Hitachi, Tokyo, Japan) with approximately 30 angstroms of Au/Pd with a 5 kV accelerating voltage.
The corn stalk powder was incubated with Trametes KS-2 at 28 °C and pH 5.5 with glucose/corn stalk (1/1, w/w) and peptone for 5 days, 10 days, and 15 days. The remaining solid was filtered and dried under vacuum at 60 °C for 24 h. The components of corn stalk was determined according to National Renewable Energy Laboratory standard procedure (NREL) (Sluiter et al. 2011; Zhang et al. 2015). Lignin degradation was determined as the ratio of the division of the lignin (mg) before and after Trametes KS-2 treatment to the lignin (mg) in corn stalk according to Eq. 1:
RESULTS AND DISCUSSION
Isolation and Identification of the Microbes
If microbes had the ability to degrade lignin, a reddish brown zone could be produced in the medium containing guaiacol due to the oxidation of guaiacol by this strain (Wang 2013). If the diameter of the zone was larger and the red color was deeper, the capability of the strain to produce laccase was stronger. The strains that produced a reddish brown zone were isolated as the positive microbes, indicating their ability to secrete laccase. The enzyme activities of these microbes were then determined.
The supernatant of the microbes became visibly green following exposure to ABTS (2,2′-azino-bis[3-ethylbenzthiazoline-6-sulfonate]) and a linear increase in the absorbance at 420 nm was observed. Laccase activity was measured based on the ABTS oxidation at 420 nm. As shown in Fig. 1, the laccase activity noticeably increased as time increased from 4 days to 10 days. A high laccase activity (107 U/L) was observed with glucose and peptone at 28 °C after 10 days. After that, the enzyme activity was reduced. Initially, nutrients were efficiently utilized by microbes, which resulted in a high yield of laccase, and then the laccase production was decreased due to the depletion of nutrients in the fermentation medium.
Fig. 1. Effect of fermentation time on the laccase production
Molecular Identification of Microbes
The strain was determined based on its ITS sequence of DNA. A phylogenetic tree is shown in Fig. 2. The ITS sequence of KS-2 showed 99% sequence similarity with Trametes hirsuta SYBC-L8(HQ891292), Trametes hirsuta 5154(EF546240), Trametes hirsuta A19(KC414249), Trametes hirsuta NBRC 6477(AB733168), Trametes hirsuta SICAU SDT36(KJ028001), and Trametes hirsuta XSD-65(EU326211) (Fig. 2). Therefore, the strain was identified as Trametes KS-2.The obtained nucleotide sequence has been submitted to the NCBI GenBank under the accession number MK182788. Laccase production occurs in various fungi over a wide range, for example, white rot fungi such as Lentinus tigrinus (Ferraroni et al. 2007), Pleurotus ostreatus D1 (Pozdniakova et al. 2006), Trametes sp. Strain AH28-2 (Xiao et al. 2003), Trametes pubescens (Shleev et al. 2007), and Trametes versicolor (Arora and Gill 2000). Trametes KS-2 might be a new effective strain for laccase production.
Fig. 2. Phylogenetic relationship of KS-2 strain
Optimization of the Conditions for Laccase Production
Effects of carbon sources
Generally, an appropriate carbon source is important for the successful production of laccase. In this work, the favorable carbon sources, including starch, glucose, sucrose, lactose, and xylose, and the natural lignocellulosic materials, including corn stalk powder, poplar powder, and bamboo powder, were investigated at 30 °C and pH 7.2 with peptone at 150 rpm for 10 days. The results are shown in Fig. 3.
Both corn stalk and glucose were the most effective carbon sources, while starch slightly reduced laccase production. Glucose is a widely used carbon source for most microbes, and a good enzyme yield was obtained at 107 U/L with glucose in this study. In addition, lignin was required by Trametes KS-2; thus, higher laccase activity was observed at 118 U/L in the presence of corn stalk as the sole carbon source. The chemical composition, the chemical or physical associations, and the accessibility of lignin and other valuable components in corn stalk increased the enzyme hydrolysis by Trametes KS-2. However, a low activity was observed when poplar and bamboo powder were used, as their chemical constitutions and fiber characteristics are different from agricultural straws (Scurlock et al. 2000). A selective degradation of corn stalk was found for Trametes KS-2.
Furthermore, the laccase activity was increased in the glucose-containing medium as compared to those produced in the glucose-free medium. The maximum laccase activity (321 U/L) was achieved when glucose and corn stalk were added at the ratio of 1/1 (w/w). This result was in agreement with earlier reports that the presence of glucose as a readily metabolizable substrate could enhance laccase activity (Couto and Herrera 2006; Mechichi et al. 2006). Laccase production was dependent on the nature of the carbon source used in the culture medium. Both corn stalk and glucose provided the appropriate nutrients for the fermentation process of Trametes KS-2.
Fig. 3. Effects of carbon sources on the laccase production
Effects of nitrogen sources
The investigation of nitrogen sources was performed at 30 °C and pH 7.2 with corn stalk/glucose (1/1, w/w) at 150 rpm for 5 days. As shown in Fig. 4, peptone was the suitable organic nitrogen source for Trametes KS-2, which supported the high level of laccase activity up to 574 U/L. Good enzyme yield was obtained when ammonium sulfate was used as the inorganic nitrogen at 485 U/L.
Laccase production occurs during the secondary metabolic phase of the fungi and is often triggered by nitrogen depletion. Thus, variation in the nitrogen source has a considerable effect on the metabolic processes of Trametes KS-2. Peptone as the effective nitrogen source supported high enzyme yield for Trametes KS-2.
Fig. 4. Effects of nitrogen sources on the laccase production
Effect of Cu2+ concentration
Laccases are oxidoreductases that contain copper ions at the catalytic center and play a crucial role in lignin degradation (Kiiskinen et al. 2002). The investigation of Cu2+ concentration was performed at 30 °C and pH 4.0 for 10 days. As shown in Fig. 5, a high laccase activity of Trametes KS-2 of 598 U/L was obtained with Cu2+ concentration at 0.09 mmol/L.
Fig. 5. Effect of Cu2+ concentration on laccase production
The investigation of Cu2+ concentration was performed at 30 °C and pH 4.0 for 10 days. As shown in Fig. 5, a high laccase activity of Trametes KS-2 of 598 U/L was obtained with Cu2+concentration at 0.09 mmol/L. Laccase activity was noticeably inhibited at concentrations above 0.2 mmol/L. There is no significant difference in laccase activity under the Cu2+concentration of 0.03-0.09 mmol/L.
Effect of initial pH
Analysis of pH was completed at 30 °C for 5 days. As shown in Fig. 6, the laccase activity exhibited good stability in the range of pH 4.5 to 5.5. The optimal pH of Trametes KS-2 appeared at pH 5.5 and the laccase activity was 626 U/L.
The initial pH of the medium is one of the important factors that affects microbe growth, enzyme production, and transport of various components across the cell membrane. A pH value that is lower or higher than the optimum affects the metabolic activities of the organism. It also influences the stability of the enzyme and may lead to protein denaturation (Kalra and Sandhu 1986).
The optimum pH for the microbes with high laccase production was reported to be between pH 4.5 and 6.0 (D’Souza et al. 2006; Sadhasivam et al. 2008). The optimum pH for TrametesKS-2 was obtained in the range of pH 4.5 to 5.5.
Fig. 6. Effect of pH on laccase production
Effect of incubation temperature
As presented in Fig. 7, the laccase activity was increased from 18 °C to 28 °C and decreased from 28 °C to 38 °C. High enzyme production was found at 28 °C for 10 days at pH 5.5 and the resulting laccase activity was 631 U/L.
Incubation temperature is a major process parameter of a fermentation system due to alterations in microbial protein structure and properties with temperature variations. At lower or higher temperatures, metabolic activities are reduced with consequent inhibition in growth and enzyme synthesis, resulting in the reduction of enzyme production.
Therefore, the maximum laccase activity was 631 U/L for Trametes KS-2 in the optimum conditions with glucose/corn stalk (1/1, w/w) and peptone for 10 days at 28 °C at pH 5.5. The culture conditions of a particular strain were well optimized for the maximum production of laccase and subsequent industrial implementation.
Fig. 7. Effect of temperature on the laccase production
Laccase Activity of Trametes KS-2 in the Presence of Salt
Laccase activity of Trametes KS-2 was evaluated under different NaCl concentrations of 0% to 7.5% (w/v). Maximum activity was achieved at 2.5% (w/v) NaCl concentration (Fig. 8); 687 U/L of original laccase activity was observed. The enzyme was found to be stable at NaCl concentrations up to 5% (w/v), and 96% of its original activity was retained. A slight reduction in laccase stability was observed at NaCl concentrations up to 7.5% (w/v).
In high salt concentrations, proteins contain an excessive number of negatively charged acidic amino acids on their surface, possibly to form a hydrated ion network or to prevent the protein aggregation through electrostatic repulsive charges at the protein surface to keep the protein soluble (Zhang et al. 2011).
Salt stability is one of the preferred characteristics for enzymes. Laccase from Trametes KS-2 has better prospects for application purposes.
Fig. 8. Laccase activity of Trametes KS-2 in the presence of salt
Laccase Activity of Trametes KS-2 in the Presence of ILs
Ionic liquid (IL)-assisted pretreatment of lignocellulosic biomass has been extensively studied and [EMIM]CH3COOH was widely reported as an effective pretreated solvent. However, because the fungal enzymes were inhibited by ILs (Turner et al. 2003; Kamiya et al. 2008), extensive water washing was required to remove any residue from ILs, which increased the difficulty of IL recovery and its ability to be reused. It is important to identify the relatively stable enzymes in the presence of ILs to decrease the amount of washing and subsequently reduce the extra cost.
Laccase activity of Trametes KS-2 was investigated under different concentrations of 1-ethyl-3-methylimidazolium acetate ([EMIM]CH3COOH) in the range of 0% to 7.5% (w/v). As shown in Fig. 9, laccase activity was 580U/L in the medium containing 2.5% (w/v) of [EMIM]CH3COOH. As the concentration of [EMIM]CH3COOH was increased to 5% (w/v), 377 U/L of laccase activity was retained.
Fig. 9. Laccase activity of Trametes KS-2 in the presence of ILs
The morphological modification of the corn stalk that was treated by Trametes KS-2 for an appropriate time was analyzed. The original corn stalk showed the compact ordered and intact structures (Fig. 10). A relatively smooth surface was observed. After the treatment with Trametes KS-2, it became cracked and porous. Some holes appeared in the corn stalk. The morphological alteration indicated the disruption of linkages in corn stalk and the remarkable reduction of lignin content after fungal treatment.
Lignin Degradation of Corn Stalk
The results of lignin degradation of corn stalk are shown in Table 1. Corn stalk consist 42.8% cellulose, 18.7% hemicellulose, 20.8% lignin and 0.7% ash. Initially, lignin was measured as 100% (208 mg) in the original corn stalk according to a National Renewable Energy Lab (NREL) method (2011). After 5 days, the lignin content was reduced to 136 mg and lignin degradation was 40.1%. As the fermentation time was increased, the lignin degradation was increased. After 10 days, the lignin was decreased to 97 mg and the degradation was 53.3%.
Fig. 10. SEM analysis: (A) original corn stalk (100 μm), (B) after 5 days (100 μm), (C) after 10 days (500 μm), and (D) after 15 days (500 μm)
After 15 days, the lignin was reduced to 76 mg and the degradation was increased to 65.4%.
It was reported that 28.3% lignin degradation was achieved with Phanerochaete chrysosporium, and 34.7% was obtained with Fusarium moniliforme after 10 days fermentation with rice straw as the carbon substrate. Cellulase activities were also observed for Phanerochaete chrysosporium (Chang et al. 2012). Myrothecium verrucaria was reported as an effective fungus for selectively removing lignin and led to lignin degradation that reached 45.50 ± 2.12% in birch sawdust (Wang et al. 2017). In this study, high lignin degradation (65.4%) was observed for Trametes KS-2 with high laccase production. The cellulase activities were further studied. No filter paper cellulase (FPase), endoglucanase (CMCase), or β-glucosidase was obtained.
Table 1. Lignin Degradation of Corn Stalk
Lignin, the most recalcitrant component of lignocellulosic material, acts as a barrier and prevents access of any lignocellulolytic enzymes to the interior lignocellulosic structure. Microbes are increasingly reported to be responsible for the efficient lignin degradation in lignocelluloses, primarily by producing lignin peroxidase (Lip) and manganese peroxidas (MnP). Phanerochaete chrysosporium ME-446 was described to be able to effectively produce Lip and MnP in the medium containing wheat straw (Kapich et al. 2004). Fusarium moniliformewas found capable of highly selective lignin degradation in rice straw by producing Lip and MnP (Chang et al. 2012). Fusarium concolor selectively delignified wheat straw based on the production of Lip, MnP, and laccase (Li et al. 2008). In this study, laccase was obtained from Trametes KS-2, while neither lignin peroxidase nor manganese peroxidase activities were detected, indicating that the degradation of lignin was specifically dependent on laccase activity. It was reported that the maximum laccase activity for Pleurotus ostreatus strains was up to 168.8 U/L (An et al. 2018). Laccase activity was obtained at a high production (631 U/L) for Trametes KS-2.
In addition, selective degradation on lignin was preferred for the production of biofuels because cellulose and hemicelluloses could be further utilized by cellulase. However, a reduction of hemicellulose and cellulose was observed for some microbes with high laccase activity. Ma et al. (2015) described a type of bacteria named P. ananatis Sd-1 with 2.59 U/mL of Lip activity and 0.61 U/mL of laccase activity. Lignin, cellulose, and hemicelluloses of rice straw were reduced 35.6%, 75.2%, and 78.8%, respectively, after 6 days by P. ananatis Sd-1 (Ma 2016). The ideal pretreatment for enzymatic saccharification should minimize the content of lignin and maximize the retention of cellulose (Ding et al. 2012). In this study, high lignin degradation (65.4%) was observed for Trametes KS-2. No cellulase activity was detected and no noticeable reduction of holocellulose was observed. A selective degradation of lignin in corn stalk was favorable for Trametes KS-2.
Furthermore, salt stability is one of the preferred properties for enzymes for industrial application purposes. Good laccase activity of Trametes KS-2 was observed at 2.5% to 5% (w/v) NaCl concentration. A relatively stable capability in the presence of 2.5% of ionic liquid was also found, which would decrease the amount of washing, reduce extra costs, and improve recovery and reuse of ILs.
- In this study, a fungal strain was newly isolated and identified as Trametes KS-2. Based on the optimal conditions, the maximum laccase activity was 631 U/L when glucose/corn stalk (1/1, w/w) and peptone were used at pH 5.5 and 28 °C for 10 days.
- Laccase activity of Trametes KS-2 was relatively stable in the presence of NaCl (2.5% to 5%). A slight reduction of activity was observed in the presence of an ionic liquid.
- The SEM analysis indicated the morphological alteration of lignocellulose. Noticeable degradation of lignin in corn stalk was achieved by Trametes KS-2. After 15 days, the lignin was reduced to 76 mg and the degradation increased to 65.4%.
- Trametes KS-2 could be attractive for potential biotechnological applications in selectively removing lignin from corn stalks for biofuels. The resulting high laccase production could be utilized in dye decolorization, paper pulp bleaching, or phenolic chemical degradation.
This work was supported financially by the General Financial Grant from the 54th China Postdoctoral Science Foundation (2013M540308), the Heilongjiang Postdoctoral Foundation (LBH-Z12233), and the Harbin Science and Technology Innovative Talents Project (RC2012QN002143).
Abadulla, E., Tzanov, T., Costa, S., Robra, K. H., Cavaco-Paulo, A., and Gubitz, G. M. (2000). “Decolorization and detoxification of textile dyes with a laccase from Trametes hirsute,” Applied and Environmental Microbiology 66(8), 3357-3362. DOI: 10.1128/aem.66.8.3357-3362.2000
An, Q., Ma, H.-F., Han, M.-L., Si, J., and Dai, Y.-C. (2018). “Effects of different induction media as inducers on laccase activities of Pleurotus ostreatus strains in submerged fermentation,” BioResources 13(1), 1143-1156. DOI: 10.15376/biores.13.1.1143-1156
Arias, M. E., Arenas, M., Rodriguez, J., Soliveri, J., Ball, A. S., and Hernandez, M. (2003). “Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335,” Applied and Environmental Microbiology 69(4), 1953-1958. DOI: 10.1128/aem.69.4.1953-1958.2003
Arora, D. S., and Gill, P. K. (2000). “Laccase production by some white rot fungi under different nutritional conditions,” Bioresource Technology 73(3), 283-285. DOI: 10.1016/s 09608524(99)00141-8
Chang, A. J., Fan, J., and Wen, X. (2012). “Screening of fungi capable of highly selective degradation of lignin in rice straw,” International Biodeterioration & Biodegradation 72, 26-30. DOI: 10.1016/j.ibiod.2012.04.013
Cianchetta, S., Di Maggio, B., Burzi, P. L., and Galletti, S. (2014). “Evaluation of selected white-rot fungal isolates for improving the sugar yield from wheat straw,” Applied Biochemistry and Biotechnology 173(2), 609-623. DOI: 10.1007/s12010-014-0869-3
Couto, S. R., and Herrera, J. L. T. (2006). “Industrial and biotechnological applications of laccases: A review,” Biotechnology Advances 24(5), 500-513. DOI: 10.1016/j.biotechadv.2006.04.003
Ding, S.-Y., Liu, Y.-S., Zeng, Y., Himmel, M. E., Baker, J. O., and Bayer, E. A. (2012). “How does plant cell wall nanoscale architecture correlate with enzymatic digestibility?,” Science338(6110), 1055-1060. DOI: 10.1126/science.1227491
Divya, L. M., Prasanth, G. K., and Sadasivan, C. (2013a). “Elimination of estrogenic activity of thermal paper using laccase from Trichoderma sp NFCCI-2745,” Applied Biochemistry and Biotechnology 169(4), 1126-1133. DOI: 10.1007/s12010-012-0016-y
Divya, L. M., Prasanth, G. K., and Sadasivan, C. (2013b). “Isolation of a salt tolerant laccase secreting strain of Trichoderma sp NFCCI-2745 and optimization of culture conditions and assessing its effectiveness in treating saline phenolic effluents,” Journal of Environmental Sciences 25(12), 2410-2416. DOI: 10.1016/s1001-0742(12)60321-0
D’Souza, D. T., Tiwari, R., Sah, A. K., and Raghukumar, C. (2006). “Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes,” Enzyme and Microbial Technology 38(3-4), 504-511. DOI: 10.1016/j.enzmictec.2005.07.005
Felsenstein, J. (1985). “Confidence limits on phylogenies: An approach using the bootstrap,” Evolution; International Journal of Organic Evolution 39(4), 783-791. DOI: 10.1111/j.1558-5646.1985.tb00420.x
Ferraroni, M., Myasoedova, N. M., Schmatchenko, V., Leontievsky, A. A., Golovleva, L. A., Scozzafava, A., and Briganti, F. (2007). “Crystal structure of a blue laccase from Lentinus tigrinus: Evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases,” BMC Structural Biology 7, 60-72. DOI: 10.1186/1472-6807-7-60
Fukuda, T., Uchida, H., Takashima, Y., Uwajima, T., Kawabata, T., and Suzuki, M. (2001). “Degradation of bisphenol A by purified laccase from Trametes villosa,” Biochemical and Biophysical Research Communications 284(3), 704-706. DOI: 10.1006/bbrc.2001.5021
Himmel, M. E., Ding, S. Y., Johnson, D. K., Adney, W. S., Nimlos, M. R., Brady, J. W., and Foust, T. D. (2007). “Biomass recalcitrance: Engineering plants and enzymes for biofuels production,” Science 315(5813), 804-807. DOI: 10.1126/science.1137016
Kalra, M. K., and Sandhu, D. K. (1986). “Optimal production of cellulolytic enzymes and their location in Trichoderma pseudokoningii,” Engineering in Life Sciences 6(2), 161-166. DOI: 10.1002/abio.370060214
Kamiya, N., Matsushita, Y., Hanaki, M., Nakashima, K., Narita, M., Goto, M., and Takahashi, H. (2008). “Enzymatic in situ saccharification of cellulose in aqueous-ionic liquid media,” Biotechnology Letters 30, 1037-1040. DOI: 10.1007/s10529-008-9638-0
Kapich, A. N., Prior, B. A., Botha, A., Galkin, S., Lundell, T., and Hatakka, A. (2004). “Effect of lignocellulose-containing substrates on production of ligninolytic peroxidases in submerged cultures of Phanerochaete chrysosporium ME-446,” Enzyme and Microbial Technology 34(2), 187-195. DOI: 10.1016/j.enzmictec.2003.10.004
Kidwai, M., Jain, A., Sharma, A., and Kuhad, R. C. (2012). “Ecofriendly approach for detection of phenols in water using laccase from different fungi,” Water Science and Technology66(2), 385-393. DOI: 10.2166/wst.2012.198
Kiiskinen, L. L., Viikari, L., and Kruus, K. (2002). “Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces,” Applied Microbiology and Biotechnology 59(2-3), 198-204. DOI: 10.1007/s00253-002-1012-x
Li, L., Li, X. Z., Tang, W. Z., Zhao, J., and Qu, Y. B. (2008). “Screening of a fungus capable of powerful and selective delignification on wheat straw,” Letters in Applied Microbiology47(5), 415-420. DOI: 10.1111/j.1472-765X.2008.02447.x
Ma, J. S. (2016). Analysis of the Lignocellulose Degradation System in Endophytic Bacterium Pantoea ananatis Sd-1, Master’s Thesis, Hunan University, ChangSha, China.
Ma, K., and Ruan, Z. (2015). “Production of a lignocellulolytic enzyme system for simultaneous bio-delignification and saccharification of corn stover employing co-culture of fungi,” Bioresource Technology 175, 586-593. DOI: 10.1016/j.biortech.2014.10.161
Mechichi, T., Mhiri, N., and Sayadi, S. (2006). “Remazol Brilliant Blue R decolourization by the laccase from Trametes trogii,” Chemosphere 64(6), 998-1005. DOI: 10.1016/j.chemosphere.2005.12.061
Palmieri, G., Giardina, P., Bianco, C., Scaloni, A., Capasso, A., and Sannia, G. (1997). “A novel white laccase from Pleurotus ostreatus,” Journal of Biological Chemistry 272, 31301-31307. DOI: 10.1074/jbc.272.50.31301
Pandey, M. P., and Kim, C. S. (2011). “Lignin depolymerization and conversion: A review of thermochemical methods,” Chemical Engineering & Technology 34(1), 29-41. DOI: 10.1002/ceat.201000270
Potumarthi, R., Baadhe, R. R., Nayak, P., and Jetty, A. (2013). “Simultaneous pretreatment and sacchariffication of rice husk by Phanerochete chrysosporium for improved production of reducing sugars,” Bioresource Technology 128, 113-117. DOI: 10.1016/j.biortech.2012.10.030
Pozdniakova, N. N., Turkovskaia, O. V., Iudina, E. N., and Rodakiewicz-Nowak, Y. (2006). “Yellow laccase from the fungus Pleurotus ostreatus D1: Purification and characterization,” Applied Biochemistry and Microbiology 42(1), 56-61. DOI: 10.1134/s000368380601008x
Sadhasivam, S., Savitha, S., Swaminathan, K., and Lin, F.-H. (2008). “Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1,” Process Biochemistry 43(7), 736-742. DOI: 10.1016/j.procbio.2008.02.017
Scurlock, J. M. O., Dayton, D. C., and Hames, B. (2000). “Bamboo: An overlooked biomass resource?,” Biomass and Bioenergy 19(4), 229-244. DOI: 10.1016/s0961-9534(00)00038-6
Shleev, S., Nikitina, O., Christenson, A., Reimann, C. T., Yaropolov, A. I., Ruzgas, T., and Gorton, L. (2007). “Characterization of two new multiforms of Trametes pubescens laccase,” Bioorganic Chemistry 35(1), 35-49. DOI: 10.1016/j.bioorg.2006.08.001
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., and Crocker, D. (2011). Determination of Structural Carbohydrates and Lignin in Biomass (NREL/TP-510-42628), National Renewable Energy Laboratory, Golden, CO.
Sole, M., Mueller, I., Pecyna, M. J., Fetzer, I., Harms, H., and Schlosser, D. (2012). “Differential regulation by organic compounds and heavy metals of multiple laccase genes in the aquatic Hyphomycete Clavariopsis aquatic,” Applied and Environmental Microbiology 78(13), 4732-4739. DOI: 10.1128/aem.00635-12
Turner, M. B., Spear, S. K., Huddleston, J. G., Holbrey, J. D., and Rogers, R. D. (2003). “Ionic liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei,” Green Chemistry 5(4), 443-447. DOI: 10.1039/b302570e
Wang, Q.-F., Niu, L.-L., Jiao, J., Guo, N., Zang, Y.-P., Gai, Q.-Y., and Fu, Y.-J. (2017). “Degradation of lignin in birch sawdust treated by a novel Myrothecium verrucaria coupled with ultrasound assistance,” Bioresource Technology 244(Part 1), 969-974. DOI: 10.1016/j.biortech.2017.07.164
Wang, M. C. (2013). Screening and Identification of Lignin Degrading Fungi and Related Enzymes Activities Research, Southwest University, ChongQing, China.
Wasserscheid, P., and Welton, T. (2003). Ionic Liquids in Synthesis, Wiley-VCH, Weinheim, Germany.
Xiao, Y. Z., Tu, X. M., Wang, J., Zhang, M., Cheng, Q., Zeng, W. Y., and Shi, Y. Y. (2003). “Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp strain AH28-2,” Applied Microbiology and Biotechnology 60(6), 700-707. DOI: 10.1007/s00253-002-1169-3
Xu, X., Xu, Z., Shi, S., and Lin, M. (2017). “Lignocellulose degradation patterns, structural changes, and enzyme secretion by Inonotus obliquus on straw biomass under submerged fermentation,” Bioresource Technology 241, 415-423. DOI: 10.1016/j.biortech.2017.05.087
Zhang, P., Dong, S. J., Ma, H. H., Zhang, B. X., Wang, Y. F., and Hu, X. M. (2015). “Fractionation of corn stover into celluose, hemicellulose and lignin using a series of ionic liquids,” Industrial Crops and Products 76, 688-696. DOI: 10.1016/j.indcrop.2015.07.037
Zhang, T., Datta, S., Eichler, J., Ivanova, N., Axen, S. D., Kerfeld, C. A., Chen, F., Kyrpides, N., Hugenholtz, P., Cheng, J.-F., et al. (2011). “Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance,” Green Chemistry 13(8), 2083-2090. DOI: 10.1039/c1gc15193b
Article submitted: March 1, 2019; Peer review completed: May 4, 2019; Revised version received: May 8, 2019; Accepted: May 9, 2019; Published: