Degradation of ionic liquids by a UV/H2O2 process and CMCase from novel ionic liquid-tolerant alkaliphilic Nocardiopsis sp. SSC4

ABSTRACT We demonstrated the degradation of two ionic liquids (1-butyl-3-methylimidazolium chloride, [BMIM]Cl, and 1-ethylpyridinium bromide, [EtPy]Br) that are useful for the solubilization of wood components. [BMIM]+ and [EtPy]+ were detected by thin-layer chromatography (TLC) and electrospray ionization–mass spectrometry (ESI-MS). [BMIM]+ was harder to degrade than [EtPy]+. Ultraviolet (UV) irradiation with 0.2% (v/v) H2O2 for 16 h degraded 1 mmol/L [BMIM]+, whereas UV irradiation alone degraded 1 mmol/L [EtPy]+. Additionally, we isolated an ionic liquid-tolerant alkaliphilic actinomycete, Nocardiopsis sp. SSC4. Strain SSC4 produced carboxymethylcellulase (CMCase) in the presence of 1.0% (v/v, 48.1 mmol/L) 1-ethyl-3-methylimidazolium trifluoromethanesulphonate ([EMIM]CF3SO3), which is useful for the extraction of cellulose-rich materials from wood. In the case of strain SSC4, CMCase was inducibly synthesized by more than 0.5% CMC. The addition of 0%–1.0% tryptone or 0%–2.0% yeast extract decreased the CMCase activity in a concentration-dependent manner. After cultivation of strain SSC4 with 1.0% (w/v) CMC medium (pH 9.0) for 48 h at 37 °C, the culture supernatant exhibited CMCase activity at 0.03 U/mg. The optimum reaction temperature of CMCase was 45 °C. CMCase was stable up to 37 °C for 20 h incubation. The degradation characteristics of [BMIM]+ and [EtPy]+ and the activity of CMCase in the presence of [EMIM]CF3SO3 may be useful for the development of a bioconversion system for biomass resources.


Introduction
Ionic liquids are a great variety of molten organic salts that are composed of a bulky asymmetric cation and a small anion. Cation and anion combinations can be modified, and hydrophilic and hydrophobic ionic liquids can be prepared [1,2]. Unlike conventional organic solvents applied for biocatalytic reactions, ionic liquids dissolve many compounds, remain liquid over a wide range of temperatures, and possess no vapour pressure. Ionic liquids have prominent physical properties for use as reaction solvents, and we have developed effective biocatalytic reactions using ionic liquids [3,4]. To solve the problems of growth inhibition of microorganisms and denaturation of enzymes by ionic liquids, we isolated the ionic liquid-tolerant Bacillus amyloliquefaciens CMW1 and demonstrated the ionic liquid-tolerant protease BapIL [5,6]. Although organic solvents are indispensable as reaction solvents, in the development of bioconversion systems, ionic liquids are focused on as potential 'green' replacements for organic solvents.
In recent years, biomass resources have attracted much attention as a substitute for fossil resources. Wood contains cellulose, hemicellulose, and lignin, and is considered a promising biomass source of energy [7,8]. Treatment of wood with ionic liquids is under consideration. For instance, [BMIM]Cl (1-butyl-3-methylimidazolium chloride; Figure 1(A)) and [EtPy]Br (1-ethylpyridinium bromide; Figure 1(B)) have been found to liquefy these wood components [9,10]. [EMIM]CF 3 SO 3 (1-ethyl-3-methylimidazolium trifluoromethanesulphonate; Figure 1(C)) has been employed to dissolve lignin for the extraction of cellulose from wood [11]. These findings are expected to be applied to wood-processing technology in consideration of the effects of ionic liquids on the environment. Although the degradation of some ionic liquids by the ultraviolet (UV)/H 2 O 2 process has been analysed with the aim of practical use of ionic liquids [12,13], little information is available about the degradation of 1-butyl-3-methylimidazolium cation ([BMIM] + ) and 1-ethylpyridinium cation ([EtPy] + ). Additionally, in order to develop a process of bioconversion of the cellulose-rich material extracted from wood with [EMIM]CF 3 SO 3 to fermentable reducing sugars, acquisition of a novel [EMIM]CF 3 SO 3 -tolerant bacterium exhibiting cellulase activity is expected. An environmentally friendly cellulolytic reaction is expected to be carried out at around room temperature.
In this study, we evaluated the degradation of [BMIM] Cl and [EtPy]Br by the UV/H 2 O 2 process. We already reported that an alkali-tolerant protease (BapIL) effectively functions in the presence of various ionic liquids [6]. Using 1.0% (v/v) [EMIM]CF 3 SO 3 under a weakly alkaline condition (pH 9.0) and using carboxymethylcellulose (CMC) as the carbon source, we explored a novel [EMIM]CF 3 SO 3 -tolerant bacterium producing carboxymethylcellulase (CMCase). Additionally, the effect of nutrient concentration on the production of CMCase was evaluated and the effect of temperature on CMCase activity and stability was examined.

Degradation of [BMIM] + and [EtPy] + by UV/H 2 O 2 process
The photocatalytic degradation of [BMIM] + and [EtPy] + was examined in the presence of H 2 O 2 under UV illumination. The reaction mixture (5 mL) containing 0.2% (v/v) H 2 O 2 (Nacalai Tesque, Kyoto, Japan) and 1 mmol/L each of an ionic liquid, namely [BMIM]Cl (Kanto Chemical, Tokyo, Japan) or [EtPy]Br (Tokyo Chemical Industry, Tokyo, Japan), was prepared with ultra-pure water (Wako Pure Chemical, Osaka, Japan). For photodegradation experiments, illumination was provided by a germicidal UVC lamp (254 nm, GL-15, Hitachi, Tokyo, Japan). The distance from the light source to the reaction mixture was 85 mm, and the reaction was carried out for 16 h at room temperature, while the reaction mixture was stirred with a magnetic stirrer.

Detection and quantification of [BMIM] + and [EtPy] +
After the UV/H 2 O 2 treatment, [BMIM] + and [EtPy] + in the reaction mixture were detected by thin-layer chromatography (TLC) analysis and quantified by electrospray ionization-mass spectrometry (ESI-MS) analysis. For TLC analysis, 10 mL of each reaction mixture was developed using acetonitrile:50 mmol/L ammonium acetate (pH 5.

Phylogenetic analysis of bacterial isolate
The bacterial isolate strain SSC4, which exhibited 1.0% (v/v, 48.1 mmol/L) [EMIM]CF 3 SO 3 tolerance and high CMC activity, was evaluated. The DNA fraction was extracted from a bacterial cell pellet of strain SSC4 using an Illustra bacteria genomic Prep Mini Spin Kit (GE Healthcare, Munich, Germany). To determine the phylogenetic classification, 1520 bp of the 16S rRNA gene fragment was amplified from the DNA fraction by PCR with eubacterial primers 27f and 1525r and Blend Taq (Toyobo Co. Ltd., Osaka, Japan). The DNA sequencing was carried out by FASMAC DNA Sequencing Services (Kanagawa, Japan). Nucleotide substitution rates (Knuc) were determined, and a distance matrix tree was constructed by using the neighbour-joining method with the CLUSTAL ç X program [14][15][16]. Alignment gaps and unidentified base positions were not taken into consideration for the calculations. The topology of the phylogenetic tree was evaluated by performing a bootstrap analysis with 1000 replications.

Evaluation of CMCase activity with culture supernatant
Strain SSC4 was grown in 1.0% (w/v) CMC medium (1.0% (w/v) CMC (Nacalai Tesque), 0.25% Bacto tryptone (Difco), 1.0% Bacto yeast extract (Difco), 1.0% MgSO 4 ・ 7H 2 O, 0.5% KCl and 1.0% NaHCO 3 , pH 9.0) at 37 C for 48 h to obtain 25 mL of the culture supernatant by the centrifugation at 4 C at 20 000 g for 15 min. CMCase activity in the culture supernatant was measured by the release of reducing sugars from CMC by the Somogyi-Nelson method using a glucose standard [17]. The standard assay mixture (500 mL) contained 0.5% (w/v) CMC (Nacalai Tesque), 10 mmol/L glycine-NaOH buffer (pH 9.0) and 10 mL of culture supernatant. After incubation for 18 h at 37 C, 100 mL of the reaction mixture was used for the quantification of reducing sugars by the Somogyi-Nelson method. One unit (1 U) of CMCase activity was defined as the enzyme necessary to catalyse the production of 1 mmol of glucose equivalent per min at 37 C and pH 9.0. To determine the optimum temperature for hydrolysis of CMC, the activity of CMCase was evaluated. The culture supernatant (2.1 mg) was added to 0.5% (w/v) CMC in 10 mmol/L glycine-NaOH buffer (pH 9.0). The reaction was done for 18 h at 30-60 8C. To assess the thermal stability of CMCase, the culture supernatant (2.1 mg) was preincubated for 20 h at 4-60 8C in 10 mmol/L glycine-NaOH buffer (pH 9.0). Heat treatment was stopped by cooling on ice. The residual activity was measured at 37 C for 18 h.

Protein concentration
The protein concentration was determined with a protein assay kit (Nacalai Tesque) according to the manufacturer's instructions.

Degradation of [BMIM]Cl and [EtPy]Br by UV/H 2 O 2 process
Ionic liquids, being polar solvents with very low vapour pressure and low flammability, are often considered as promising 'green' substitutes for volatile organic solvents. However, in order to convincingly demonstrate the 'green' nature of ionic liquids, the degradation process must be investigated. After 16 h reaction, [BMIM] + and [EtPy] + were completely degraded by the UV/H 2 O 2 process (Figure 2(A)). The degradation of [BMIM] + required the addition of 0.2% (v/v) H 2 O 2 and UV irradiation (254 nm), while the degradation of [EtPy] + was achieved by UV irradiation alone. As shown in Figure 2 (B), [BMIM] + was harder to degrade than [EtPy] + . Although it was reported that more than 55% of [BMIM] + is degraded after 6 h of UV irradiation alone with a 1000 W Xenon arc lamp [13], we demonstrated that the addition of H 2 O 2 was necessary for the degradation of [BMIM] + .
Biodegradation intermediates and pathways for 1octyl-3-methylimidazolium chloride and 1-butyl-3-methylpyridinium bromide with activated sludge have been reported [18,19]. In each degradation pathway, the much further broken structures of methylimidazolium and methylpyridinium were not measured. We examined the biodegradation of [BMIM] + and [EtPy] + using 27 samples (e.g. activated sludges, soils, and seawater) as sources for microorganisms. Although reaction mixtures (5 mL) containing 0.1% (w/v) of each sample, 0.0001%-0.5% (v/v) of each ionic liquid and microbial nutrients were incubated with aerobic shaking for 2 - (Figure 2), this process was shown to be suitable for the degradation of cations. medium, good growth (OD 660 6.5) was confirmed and the culture supernatant was prepared. As shown in Figure 3, the halo of the 1.0% (w/v) CMC degradation was detected by using the culture supernatant containing 1.0% (v/v, 48.1 mmol/L) [EMIM]CF 3 SO 3 .

Isolation and phylogenetic analysis of bacterial isolate SSC4
The 16S rRNA gene of strain SSC4 consisted of 1520 nucleotides. The results from the phylogenetic analysis based on the 16S rRNA gene sequence data are shown in Figure 4. Strain SSC4 clearly falls within the genus Nocardiopsis and belongs to actinomycetes. The nucleotide sequence of the 16S rRNA gene of strain SSC4 was most closely related to those of Nocardiopsis alba PCM2702 (JQ277723) and Nocardiopsis valliformis rsk9 (KX832933) with similarities of 99% for both. The next closest level of similarity was 98% similarity with the nucleotide sequences of Nocardiopsis oceani 10A08A T (NR ç 137 417) and Nocardiopsis nanhaiensis 10A08B T (KF270095). The 16S rRNA sequence of strain SSC4 was submitted to the NCBI GenBank (Accession No. LC205716).

Effect of nutrient concentration on production of CMCase
To determine the optimum conditions for production of CMCase by strain SSC4, the effects of nutrient concentration on cell growth and CMCase activity were evaluated. Using 0.1%-1.0% (w/v) CMC or 0%-1.0% (w/v) tryptone, growth to OD 660 of about 5.0-6.5 was detected ( Figure 5  (A,B)). Using 0%-2.0% (w/v) yeast extract, the growth increased from OD 660 2.3 to 7.3 in a yeast extract concentration-dependent manner ( Figure 5(C)). Detection of the increase in CMCase activity after the addition of 0.5%-1.0% (w/v) CMC revealed that this enzyme was inducibly produced with the addition of CMC ( Figure 5(A)). In the related Nocardiopsis actinomycetes [25,26], we clearly  showed the induction of CMCase activity by the addition of CMC for the first time.
It was also revealed that the CMCase activity decreased as the concentrations of tryptone and yeast extract increased ( Figure 5(B,C)). In the case of strain SSC4, the poor nutritional conditions were thought to be suitable for CMCase production. Using the 0.5% (w/v) CMC medium containing 0.1% yeast extract without tryptone, strain SSC4 was grown and the culture supernatant was prepared. When 0.5% (w/v) CMC medium was used, the total activity of CMCase in the culture supernatant decreased to 25% of that when 1.0% (w/v) CMC medium was used.

Effect of temperature on activity and stability of CMCase
To determine the optimum temperature for hydrolysis of CMC, the activity and stability of CMCase were evaluated at different temperatures with culture supernatant. The culture supernatant was prepared with 1.0% (w/v) CMC medium by the cultivation of strain SSC4 for 3 d at 37 C. As shown in Figure 6(A), the optimal temperature for CMCase activity was around 45 C at pH 9.0 in 10 mmol/ L glycine-NaOH buffer. To assess the thermal stability of CMCase, the enzyme was incubated at various temperatures for 20 h in 10 mmol/L glycine-NaOH buffer (pH 9.0), and residual activity was measured (Figure 6(B)). The enzyme was stable up to about 37 C. By using the standard assay, 0.03 U/mg of CMCase activity was detected in the culture supernatant of Nocardiopsis sp. SSC4.
Nocardiopsis sp. KNU and Nocardiopsis sp. SES28 produce thermo-tolerant and alkali-tolerant cellulolytic enzymes and endo-b-1,4-D-glucanase, respectively [25,26]. To the best of our knowledge, the function of no CMCase from a Nocardiopsis actinomycete has been investigated in the presence of an ionic liquid. We demonstrated that strain SSC4 produces CMCase in the presence of 1.0% (v/v, 48.1 mmol/L) [EMIM]CF 3 SO 3 . CMCase activity was detected in the presence of 1.0% (v/ v, 48.1 mmol/L) [EMIM]CF 3 SO 3 and hydrolysed CMC at 37 C. Using CMCase, development of a bioconversion system of cellulose-rich materials to produce fermentable reducing sugars at close to room temperature is expected. We plan to purify and elucidate the properties of CMCase from strain SSC4.
To produce wood-based biofuels, pretreatment of wood with ionic liquids has advantages over other methods currently used for degradation of lignocellulose [11]. Ionic liquidsdue to their low volatility and low flammabilityare intriguing as potentially 'green' solvents posing little environmental hazard. However, ionic liquids often inhibit the enzymes used subsequently for biomass conversion. Therefore, the [EMIM]CF 3 SO 3 -tolerant CMCase is considered to be useful for development of a novel bioconversion system.  considered to be 'green' solvents for conversion of wood to industrially useful compounds. Further study needs to be done to purify and characterize CMCase against ionic liquids that are able to develop novel efficient systems for bioconversion of wood with these ionic liquids.

Disclosure statement
No potential conflict of interest was reported by the authors.