Improving Whole-Cell Biocatalysis by Addition of ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Improving Whole-Cell Biocatalysis by Addition of Deep Eutectic Solvents and Natural Deep Eutectic Solvents Tao-Xiang Yang,† Li-Qing Zhao,‡ Juan Wang,§ Guo-Li Song,§ Hai-Min Liu,† Hui Cheng,† and Zhen Yang*,§ †

College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology, ‡College of Chemistry and Environmental Engineering, and §College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen 518060, Guangdong, China S Supporting Information *

ABSTRACT: Being considered greener alternatives to ionic liquids (IL), deep eutectic solvents (DES) and natural deep eutectic solvents (NADES) have currently attracted broad interests from academics and industry. In this study, the transformation of isoeugenol to vanillin catalyzed by Lysinibacillus f usiformis CGMCC1347 cells was taken as the model reaction to examine the impacts of 24 DESs and 21 NADESs as cosolvents on whole-cell biocatalysis. Both types of cosolvents showed the ability of improving the production yields up to 142% and 132% of the ones obtained in (NA)DES-free aqueous systems, respectively. The data obtained by confocal laser scanning microscopy and flow cytometry tests and measurements of OD260 and OD280 agreed well with the bioconversion data, suggesting that addition of these cosolvents may be beneficial to whole-cell biocatalysis in enhancing the cellular membrane permeability. Interaction of DES with bacterial cell wall was discussed. The cells immobilized in PVA-alginate beads granted an augmented production yield in the presence of DES and NADES, up to 181% of the one obtained in a pure water system, and their catalytic activity was well maintained after being used for at least 13 cycles. KEYWORDS: Deep eutectic solvents (DES), Natural deep eutectic solvents (NADES), Isoeugenol, Vanillin, Whole-cell biocatalysis, Immobilized cells

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INTRODUCTION Biocatalysis can be performed by both whole cells and isolated enzymes. The extraordinary catalytic power, the high specificity, and the mild conditions under which the biotransformations can be carried out make this approach preferable to chemical reactions. As compared to the use of isolated enzymes, a major advantage of using whole cell catalysts is that cells provide a natural environment for the enzymes, allowing cofactor regeneration and also preventing enzymes from denaturation and inactivation that may occur under harsh conditions, including unconventional (i.e., nonaqueous) reaction media.1 Without the requirement for enzyme purification and cofactor addition, whole cell catalysts also represent the cheapest form of catalyst formulation. As a result, the combination of whole cell catalysts and unconventional reaction media is highly appealing, especially for hydrophobic substrates.2 Being considered a potentially greener alternative to conventional organic solvents and the more advanced ionic liquids (ILs),3 deep eutectic solvents (DESs) have currently attracted widespread academic and industrial interests, granting some attractive IL-like advantages such as low melting point, low volatility, high thermal stability, excellent designability, and high solubility for various substances.4,5 A DES can be prepared © 2017 American Chemical Society

by thermal mixing an ammonium salt (such as choline chloride) with a hydrogen-bond donor (HBD, such as urea and glycerol) at a specified stoichiometric ratio or by freeze-drying this mixture. Its formation is believed to arise from the interaction between the HBD and the salt anion through hydrogen bonding, thus forming an extensive H-bond network throughout the solvent. More recently, a new type of DESs, natural deep eutectic solvents (NADESs), has emerged with an enormous potential for applications due to their nontoxicity and even higher sustainability and environmental friendliness.6 NADESs are biobased solvents, composed of two or more compounds that are generally plant-based primary metabolites, i.e., organic acids, sugars, alcohols, amines, and amino acids. The greenness and sustainability of DESs and NADESs lie in the fact that the components involved in the solvent are inexpensive, biodegradable, nontoxic, and widely available in nature. It is all of these unique properties and advantages that have made (NA)DESs highly promising and attractive as a new Received: January 26, 2017 Revised: March 22, 2017 Published: April 4, 2017 5713

DOI: 10.1021/acssuschemeng.7b00285 ACS Sustainable Chem. Eng. 2017, 5, 5713−5722

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ACS Sustainable Chemistry & Engineering

(both in dimethyl sulfoxide) was purchased from ThermoFisher Scientific. Preparation of DES and NADES. Twenty-four DESs were prepared by mixing 2 cholinium salts (ChCl and ChAc) and 4 HBDs (A, U, G, and EG) at a molar ratio of 1:2, 1:1, or 2:1 respectively.27,28 21 NADESs were prepared by mixing the two components listed in Table 1 at a specified molar ratio, with addition of water until a final content of 20% (w/w). The mixture was mixed at about 80 °C for 1−2 h on a hot plate until a colorless clear liquid was formed, which was then kept in a sealed bottle at room temperature. The components of DESs and NADESs used in this study are listed in Scheme 2. Cultivation of Lysinibacillus f usiformis CGMCC1347 Cells. The strain of Lysinibacillus f usiformis CGMCC1347 was isolated from soil and is kept in the China General Microbiological Culture Collection Center (CGMCC). The cells were cultured in a growth medium consisting of 10.0 g/L peptone, 5.2 g/L KH2PO4, 14.0 g/L K2HPO4·3H2O, 1.0 g/L MgSO4·7H2O (pH 7.0) at 37 °C and 220 rpm for 24 h. Immobilization of Lysinibacillus f usiformis CGMCC1347 Cells. Cells were immobilized in PVA-alginate beads following the procedures described in ref 29 with some modifications. A PVAalginate solution was prepared by mixing 4.0 g of poly(vinyl alcohol) (PVA, average MW 74,800−79,200) in 20.0 mL water and 0.75 g sodium alginate in 5.0 mL water thoroughly until both solutes were completely dissolved. The centrifuged cells (5.0 g wet weight) were then added and mixed gently by addition of water to reach a final volume of 50 mL. This PVA-alginate cell suspension was then extruded through a syringe into a 100 mL solution consisting of saturated boric acid and 4.0% (w/v) CaCl2. The resulting beads were kept gently stirred in this solution for 4 h to complete the solidification and then rinsed three times with distilled water to remove any excess boric acid, before being kept in distilled water at 4 °C for future use. Determination of Isoeugenol Solubility. Isoeugenol (0.05 g) was added to 2.0 mL phosphate buffer (50 mM, pH 7.0) containing an (NA)DES (20%, v/v) in an Eppendorf tube, which was then shaken in an incubator/shaker at 30 °C and 200 rpm for 5 h. After centrifugation, the supernatant was obtained for HPLC analysis to determine the concentration of isoeugenol as its solubility. General Procedure for Biocatalytic Conversion of Isoeugenol to Vanillin. After rinsed with phosphate buffer (pH 7.0, 50 mM), followed by centrifugation, cells obtained from 10 mL of the fermentation broth were resuspended in 10.0 mL phosphate buffer containing a certain amount of a DES or NADES. 0.25 g isoeugenol was added to start the reaction. The reaction flask was placed in a shaking incubator with agitation of 200 rpm at 30 °C for 72 h and then stopped by addition of 10 mL of 95% ethanol. After centrifugation, 100 μL of the supernatant was taken and diluted with 900 μL of 95% ethanol before being injected for HPLC analysis. For reactions with immobilized cells, 5.0 g of the PVA-alginate beads was added to 10 mL of distilled water containing 0.25 g isoeugenol to start the reaction. The reaction conditions were the same as above. After 72 h, the immobilized cells were recovered by filtration and washed twice with distilled water before being placed in the same substrate solution as above for the next round of reaction. HPLC Analysis. A 10 μL sample obtained above was subjected to HPLC analysis with an Agilent 1260 HPLC equipped with a UV detector and a 150 × 4.6 mm, 5 μm inertsil ODS-SP column (GL Sciences Inc. Japan). A solvent mixture of acetic acid (0.1%, v/v) and methanol was employed as the mobile phase with gradient elution: The volumetric ratio of methanol to acetic acid solution was increased from 20% to 60% within the initial 5 min, keeping constant at 60% for the later 10 min, and then decreased from 60% to 20% during the forthcoming 5 min. The absorbance at 270 nm was followed within 20 min. The retention times for isoeugenol and vanillin were 6.645 and 11.859 min, respectively. Linear standard curves for them two were obtained, with a regression coefficient of 0.99912 and 0.99989, respectively. The yields reported were the amounts of vanillin produced (g/L), determined by HPLC analysis. All of the reactions were carried out at least in triplicate, subject to less than ±10% error for each data point.

type of nonaqueous solvents/cosolvents for a lot of applications including biocatalysis.5,7,8 While compatibility of DESs with isolated enzymes has been demonstrated already for enzymes of different classes,5,9 very few reports have been given regarding the use of DESs as solvents/cosolvents for whole-cell catalysts, including Baker’s yeast,10,11 Arthrobacter simplex,12 recombinant E. coli,13 Acetobacter sp. CCTCC M209061, 14,15 and Acetobacter pasteurianus G1M1.158.16 No research has been reported on carrying out whole cell catalysis in NADES systems. While three studies have demonstrated that DESs are effective in altering the enantioselectivity of the whole-cell catalysts,10,11,13 the conversions obtained by these catalysts in DES or DES/ H2O systems were always lower than those obtained in aqueous solution. In this study, bioconversion of isoeugenol to vanillin catalyzed by Lysinibacillus f usiformis CGMCC1347 cells was taken as the model reaction (Scheme 1). Vanillin (4-hydroxy-3methoxybenzaldehyde) is one of the most commonly used flavors in the world and has been applied extensively in the food, beverage, perfumery, pharmaceutical, and medical industries.17 As the annual world market demand of vanillin could not be met by natural extraction and chemical synthesis, biotechnological approaches by virtue of whole cell catalysts have become much attractive to make production of biovanillin economically viable. Microbial transformation of isoeugenol to vanillin has been explored by the use of a yeast strain (Candida galli strain PGO6)18 and a series of bacterial ones such as Bacillus subtilis,19,20 Bacillus pumilus S-1,21 Pseudomonas chlororaphis,22 Pseudomonas putida,23,24 Psychrobacter sp. strain CSW4,25 and Lysinibacillus f usiformis CGMCC1347.26 Only one group has reported the work of immobilizing the cells in calcium alginate gels for the transformation.26 The major goals of this current study were: (1) to assess the influence of DES and NADES on the bioconversion by screening 24 DESs and 21 NADESs; (2) to examine the impacts of DES as cosolvent on cellular viability and membrane permeability and, in turn, to elucidate the influences induced by (NA)DES on the bioconversion; and (3) to improve the recyclability of the whole-cell catalyst by immobilization of the cells in poly(vinyl alcohol) (PVA)-alginate beads.

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EXPERIMENTAL SECTION

Materials. Vanillin (99.5%) and isoeugenol (cis + trans, 98+%) were purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. Choline acetate (ChAc) (all 99%) was obtained from ShangHai Cheng Jie Chemical Co., Ltd. Choline chloride (ChCl), acetamide (A), urea (U), glycerol (G), ethylene glycol (EG), and all other reagents used were of analytical grade from local manufacturers. The LIVE/DEAD BacLight Bacterial Viability Kit (#L7012) containing solutions of 3.34 mM SYTO9 and 20 mM propidium iodide (PI)

Scheme 1. Production of Vanillin from Isoeugenol with Lysinibacillus f usiformis CGMCC1347 Cells As the Catalyst

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Table 1. List of NADESs Used in This Study (All Containing 20% w/w H2O) and the Yields Obtained in the Reaction System Containing 20% (v/v) of These NADESs Relative to the One Obtained in the NADES-Free Solution component 2 (C2) no.

component 1 (C1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

type

C1:C2 molar ratio

NADES abbreviation

relative yield (%)

organic acids

lactic acid malonic acid maleic acid malic acid citric acid oxalic acid tartaric acid

1:1 1:1 1:1 1:1 1:1 1:1 1:1

ChCl/LA (1:1) ChCl/MA (1:1) ChCl/MaleicA (1:1) ChCl/MalicA (1:1) ChCl/CA (1:1) ChCl/OA (1:1) ChCl/TA (1:1)

98.86 94.99 95.01 94.09 58.05 31.29 103.73

alcohols

ethylene glycol propylene glycol glycerol xylitol sorbitol

1:1 1:1 1:1 5:2 5:2

ChCl/EG (1:1) ChCl/PG (1:1) ChCl/G (1:1) ChCl/Xylitol (5:2) ChCl/Sor (5:2)

110.65 104.31 117.14 98.89 125.64

sugars

xylose glucose fructose mannose galactose sucrose maltose lactose raffinose

ChCl/Xyl (5:2) ChCl/Glc (5:2) ChCl/Fru (5:2) ChCl/Man (5:2) ChCl/Gal (5:2) ChCl/Suc (4:1) ChCl/Mal (4:1) ChCl/Lac (4:1) ChCl/Raf (11:2)

129.45 126.70 85.02 108.86 129.91 114.08 121.21 131.72 130.73

choline chloride (ChCl)

name

5:2 5:2 5:2 5:2 5:2 4:1 4:1 4:1 11:2

Confocal Laser Scanning Microscopy (CLSM) and Flow Cytometry (FCM) Tests. Cells were incubated in a solution containing 1.6 mL of 0.85% (w/v) NaCl solution and 0.4 mL of a specified DES at 30 °C for 72 h. After being washed and appropriately diluted in 0.85% (w/v) NaCl solution, 300 μL of the cell suspension was taken and stained with 1 μL of the premixed dye solution (a 2:1 mixture of the SYTO9 and PI stains provided by the LIVE/DEAD BacLight Bacterial Viability Kit) for 15 min at room temperature in the dark, before being subjected to CLSM tests by using the Zeiss LSM 710 confocal laser scanning microscope with 100× magnification and FCM tests using the BD FACSCalibur flow cytometer (BD Biosciences, USA). For CLSM tests, the excitation/emission wavelengths were 488/525 nm for the SYTO9 stain and 488/678 nm for the PI stain, and images were acquired and analyzed by using the Zeiss ZEN software. For FCM tests, the sample was excited at 488 nm, and the emission wavelengths were 515−545 nm for the SYTO9 stain and >650 nm for the PI stain, and data were analyzed by using the Flowing Software, a free flow cytometry data analysis software (http://www. uskonaskel.fi/flowingsoftware/). Measurements of OD260 and OD280. Cells were treated with a specified DES as above. After centrifugation, the supernatant was taken for measurement of absorbance at 260 and 280 nm with Shimadzu UV-1800 UV−vis spectrophotometer.

higher than those obtained when ChCl-based DESs were present. A bell-shaped relationship was observed between the conversion and the DES content in the reaction system, with an optimum obtained when the volume of DES added reached 20% (v/v) (Figure S1). The later decline in the production yield when more DES was added might be partially related to the high viscosity of the DES, which may interfere with the mass transfer in the reaction system. Another reason may be the entrapment of the substrate (due to the presence of the hydroxy and methoxy groups) within the DES through Hbonding, thus minimizing its availability for the reaction. This idea was initially proposed by Bubalo et al.30 when they carried out a lipase-catalyzed esterification reaction of acetic anhydride with 1-butanol in various DES solutions (e.g., ChCl/EG (1:2)), and the H-bonding between 1-butanol (a cosubstrate) and the hydroxyl groups on both choline and ethylene glycol has been confirmed by using nuclear overhauser enhancement spectroscopy (NOESY). Upon addition of DES up to 20% (v/v), the pH of the phosphate buffer (50 mM, pH 7.0) remained fairly constant at 7.0, while the solubility of isoeugenol was enhanced which is 2.0−2.3 times the one obtained in DES-free solution, suggesting that the DESs used in this study had no effect on changing the buffer pH and that their accelerating power may be related to an augmentation in the substrate solubility. In fact, one of the most attractive features of (NA)DESs is their excellent solubilization power.5 They are capable of dissolving a wide range of poorly water-soluble chemical compounds.7 However, when plotting the production yield against the solubility of isoeugenol in DES solution (Figure S2), one can see a slight decrease in the yields accompanied by an increase in the isoeugenol solubility. It is worth noting that the isoeugenol solubility in the DES-free solution was 0.90 g/L. Therefore,

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RESULTS AND DISCUSSION Effect of DES on the Bioconversion. The screening test with addition of 1% (v/v) DES into the reaction system (Figure 1) has shown that all the 24 DESs tested (except ChCl/U (2:1)) triggered a higher conversion (up to 142% of the one obtained in the DES-free system), with a maximum obtained by addition of ChAc/U (1:1) and ChAc/EG (1:1). This manifests that DESs possess an accelerating power in the biocatalytic conversion of isoeugenol to vanillin. The production yields did not seem to have any obvious correlation with either the HBD or the salt/HBD molar ratio. But comparatively, the yields obtained in the presence of ChAc-based DES were slightly 5715


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ACS Sustainable Chemistry & Engineering Scheme 2. Components of DESs (a−f) and NADESs (a, e−y) Used in This Study

these data reinforced our previous comment31,32 that a high substrate solubility is beneficial in maximizing the substrate concentration available for the reaction, but too high a substrate solubility may also be disadvantageous in stabilizing the substrate ground state so as to enhance the activation energy, thereby lowering the reaction rate. Effect of NADES on the Bioconversion. The 21 NADESs were also screened for their impact on the bioconversion of isoeugenol to vanillin. As shown in Table 1, more than half of the NADESs tested (20%, v/v) exhibited the ability of improving the production yield, and ChCl/Lac (4:1) and

ChCl/Raf (11:2) were the two that gave the highest conversions (respectively 132% and 131% relative to the yield obtained in the NADES-free solution). Besides, the yields seem to be sensitive to the choice of the second component selected for the preparation of the NADES, varying in the order of organic acids < alcohols < sugars. A look at the pH change upon addition of NADES to the phosphate buffer (pH 7.0) reveals that NADESs with organic acids as the component 2 triggered an abrupt pH drop in the solution, and ChCl/OA (1:1) was the one that dropped pH the most (down to pH 0.82). This indicates that the organic acids 5716


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Figure 1. Bioconversion of isoeugenol to vanillin catalyzed by Lysinibacillus f usiformis CGMCC1347 cells in aqueous solution containing 1% (v/v) DES. The relative yield (%) refers to the percentage of the yield obtained in the DES-containing solution relative to the one obtained in the DES-free solution.

similar as for DES above (see Figure S2), confirming again the depressing effect of substrate solubility. Cell Viability and Its Impact on the Bioconversion. The results reported above clearly show us that the catalytic activity of the Lysinibacillus f usiformis CGMCC1347 cells in converting isoeugenol to vanillin can be affected by addition of DESs and NADESs. It is reasonable to assume that one major impact of these additives on the bioconversion might be through their interaction with the cells, which may affect the integrity and hence permeability of the cellular membranes. Cellular membrane integrity is commonly taken as a criterion to distinguish whether the cells are damaged or not. In this study, we took DES as the affecting agents to treat the Lysinibacillus f usiformis CGMCC1347 cells, in order to assess

involved in the NADES had a significant impact on lowering the buffer pH and thus may explain why ChCl/OA (1:1) yielded the poorest conversion, while all the NADESs with organic acids as the component 2 triggered lower yields as compared to those with alcohols and sugars involved (Table 1). Zong’s group14,16 has observed no ketone reduction in the presence of ChCl/OA (1:2) and ChCl/MA (1:2), when catalyzed by Acetobacter sp. CCTCC M209061 and Acetobacter pasteurianus G1M1.158 cells, respectively. Poor yields were also obtained in the baker’s yeast-mediated asymmetric reduction of ethyl 3-oxobutanoate in the aqueous solution of the two DESs: ChCl/OA (1:1) and ChCl/MA (1:1).11 The detrimental impact of these organic acid-containing DESs on the biotransformation is understandable when taking into account that the environmental pH significantly influences the activity and selectivity of the enzymes involved in the reaction, the coenzyme regeneration inside the cells,11 and the bacterial growth by altering the cellular proliferation and metabolic properties.33 As the optimal pH for the Lysinibacillus f usiformis CGMCC1347 cells in bioconversion of isoeugenol to vanillin was 7.0,34 it is not surprising that a pH as low as 0.82 would be disastrous to the reaction. The biocompatibility tests on two Gram-positive and two Gram-negative bacteria carried out by Zhao et al.33 have manifested that among the 20 DESs they tested, the 6 organic acid-based DESs dramatically inhibited the bacterial growth, while all other DESs containing amines, alcohols, and sugars did not. The DESs and NADESs containing organic acids have been reported to present serious cytotoxicity and phytotoxicity,8,35,36 while the viability of the baker’s yeast was also found to be tremendously low when the cells were exposed to the two organic acid-based DESs (ChCl/ OA and ChCl/MA), relative to the DESs containing other HBDs.11 Like the situation with DES, addition of all the 21 NADESs in the buffer solution can also induce an enhancement in the solubility of isoeugenol that is 1.9−2.7 times the one in the NADES-free solution. A plot of the production yield against the isoeugenol solubility in the NADES solutions shows the trend

Figure 2. CLSM images of Lysinibacillus f usiformis CGMCC1347 cells after being treated for 72 h at 30 °C in aqueous solution containing 20% (v/v) and 50% (v/v) of ChAc/EG (1:1) and ChCl/G (1:1), respectively. 5717


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ACS Sustainable Chemistry & Engineering the membrane integrity of the treated cells by using CLSM and FCM tests, both offering powerful techniques to examine the membrane integrity of the cells, as dead cells exhibit red fluorescence (due to the staining with PI), while live cells look green (due to the staining with SYTO9). As can be seen from the CLSM micrographs (typical examples are shown in Figure 2), addition of DES did to some degree disrupt the membrane of the cells leading to their death, and the disruption became much more severe when the cells were treated with 50% DES relative to 20% DES. This was confirmed by performing an FCM test: A sigmoid relationship was obtained between the DES concentration in the solution and the proportion of the dead cells (Figure S3). Bubalo et al.11 have also observed a decrease in the viability of the baker’s yeast as a result of a high DES content in the medium and have explained this by the high osmotic pressure imposed on the yeast cells, leading to diffusion of water out of the cells. The FCM tests on the cells being treated with 20% (v/v) DES for 72 h and with 50% (v/v) DES for 24 h (Figure 3) have further supported the damaging effect of DES on the cellular membranes. In both situations, there seemed to be relatively more cells survived in the presence of the ChCl-based DESs as compared to those after being treated with the ChAc-based ones. This is in accordance with the bioconversion data showing a slightly higher production yield obtained in the reaction system containing ChAc-based DESs than when ChClbased DESs were added (Figure 1). In support of this, the plot shown in Figure 4a illustrates a slight decrease in the conversion along with an increase in the cell viability. Determination of the change in OD260 and OD280 after removal of the cells offers another indicator for the damage to the cell membranes, as an increase in both absorbances can be taken as a measure of the release of intracellular components (primarily nucleic acids and proteins) into the medium. After the cells were exposed to DES (20%, v/v)-containing aqueous solution for a period of time followed by their removal, both OD260 and OD280 of the remaining solution had a sharp increase during the initial 24 h of treatment and then gradually leveled off along with time (Figure S4), implying that the most damaging effect of DES occurred within the initial 24 h. However, a comparison of the absorbance data obtained at 24 h (Figure S5) indicates that relatively more intracellular components were released from the cells being treated with

Figure 4. Correlation of production yield with cell viability (a) and OD280 (b). The yields were obtained in the presence of 1% (v/v) DES after 72 h, the cell viability was determined by FCM after the cells were treated for 24 h in the presence of 50% (v/v) DES, while OD280 and OD260 were measured spectrophotometrically after the cells were exposed to 20% (v/v) DES for 24 h.

ChAc-based DES relative to those with ChCl-based ones, and a higher release of proteins (OD280) and nucleic acids (OD260) was accompanied by a slight increase in the bioconversion yield (Figure 4b), both in good support of the above FCM results. Therefore, the above experiments have demonstrated that treatment with DES can trigger the cellular membranes damaged and thus their permeability augmented, while also making the cells more active in promoting the bioconversion. By correlating the cell viability and bioconversion yield together, it is reasonable to postulate that the improvement in the production yields upon addition of (NA)DESs may derive from the enhanced cellular membrane permeability induced by these additives. The disrupted cellular membranes allow the substrate to get a better access to the enzyme responsible for the bioconversion by easily diffusing into the cells, while also facilitating the product to pass more easily out of the cells. Additionally, because the enzyme may leak out through the disrupted/broken cellular membranes, it may have a direct contact with the substrate outside the cells for the reaction to occur. In fact, the importance of the permeability issue in whole-cell biocatalysis has been stressed by Ni et al.37 [2006], who have testified that an enhancement in the permeability of the outer membrane (by mutations on its structure) of the bacterial cells could lead to a striking enhancement in the whole-cell catalytic efficiency. On the other hand, Wachtmeister and Rother2 have pointed out that the limitation in mass transfer, which is the prominent disadvantage of using whole-cell biocatalysts, can be improved by modifying the cell wall with surfactants, chelating agents, or organic solvents. Our study has thus offered another type of reagents for such modification.

Figure 3. Viability of Lysinibacillus f usiformis CGMCC1347 cells after being treated for 72 h in the presence of 20% (v/v) DES and for 24 h in the presence of 50% (v/v) DES, respectively, as determined by FCM. 5718


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Figure 5. Immobilized cells (a) and their scanning electron micrographs before (b,c) and after (d,e) being recycled for 13 times.

present in the polysaccharide and peptide chains of the bacterial cell wall; and the hydroxyl groups rich in the sugar moieties of peptidoglycan and teichoic acids of the bacterial cell wall may compete with the HBD of the DES to H-bond with the anions either still involved in, or already dissociated from, the DES. As has been proposed in our recent study,39 the Hofmeister effect40 combined with the concept of matching water affinities41 provides a convincing elucidation regarding the interaction of cholinium salts and their DESs with cellular membranes from different living organisms. According to the “principle of matching water affinities” proposed by Collins,41 in aqueous solution oppositely charged ions tend to bind strongly with each other to form a compact ion-pair if they have equal affinities for water but will separate if their water affinities are very different. Because choline is a chaotropic cation for the two anions, acetate is kosmotropic, and chloride is relatively chaotropic, ChCl forms a stronger ion-pair than ChAc in aqueous solution. Therefore, as opposed to the ChCl-based DESs, the ChAc-based ones may have a higher tendency of releasing the acetate anions so that they can interact with the bacterial cell wall through H-bonding and electrostatic interactions as mentioned above. This can explain why as compared to the ChCl-based DESs, the ChAc-based ones exerted a more pronounced impact on the cellular membranes, as has been observed in this study. Hayyan et al.36 have also suggested that the propensity of DES/NADES species to permeate through cellular membranes follow the Hofmeister effect, which could provide a strong tool for prediction of the disruption to the cell walls caused by these mixtures. In fact, in our recent study assessing the DES biotoxicity,39 we have already observed that ChAc-based DESs were obviously more toxic to E. coli, a Gram-negative bacterium, than the ChCl-based ones, in favor of the results obtained in this study. Moreover, a series of choline42−44 or choline-like45 salts have been proven to exert antimicrobial activities, and some quaternary ammonium compounds (QACs) have already been widely used as antiseptics and disinfectants.46 Although the mechanism is not yet fully understood, it is believed to be related to the interaction of these salts with, or even penetration into, the membrane structure of microorganisms, leading to a subsequent loss of membrane integrity and their aggregation and accumulation on the cellular surface.47 Cornmell et al.48 have noticed the accumulation of quaternary ammonium and phosphonium salts in the membrane fraction of the E. coli cells.

It is worth noting that in their recent study on the ketone reduction catalyzed by Acetobacter pasteurianus G1M1.158 cells in DES/aqueous solutions, Xu et al.16 have reported that among the 7 DESs tested, ChCl/EG (1:2) gave the fastest initial reaction rate and highest production yield while being biocompatible (by sugar metabolic activity retention (MAR) test) and triggering the cells to maintain relatively high cell membrane integrity (by FCM) and to release relatively small amounts of intracellular nucleic acids and proteins (by OD260 and OD280 measurements). We have to admit that the impact of (NA)DES on whole-cell biocatalysis involves a complicated situation, which cannot be simply explained by a single interpretation. In addition to the cellular membrane permeability, other important factors may also count, including viscosity and polarity of the reaction medium, toxicity of the additives to the cells, and direct interactions of these additives with the cells, with the enzymes responsible for the reaction, and also with the substrates and/or the products. It is of great interest to further investigate the mechanisms behind. Understanding the Interaction of DES with Bacterial Cell Wall. The influence of DES on the cell membrane integrity can be understood when taking the bacterial cell wall structure into consideration. The bacterial cell wall differs from that of all other organisms by the presence of peptidoglycan that forms a mesh-like layer immediately outside the plasma membrane. Responsible for the rigidity and strength of the bacterial cell wall, peptidoglycan is a polysaccharide consisting of alternating N-acetylmuramic acid and N-acetylglucosamine residues in equal amounts, cross-linked by short peptides including amino acid residues such as Gly, Ala, Glu, and Lys. The cell wall of Gram-positive bacteria has also a major constituent, teichoic acids, which are copolymers of ribitol or glycerol phosphate and carbohydrates linked via phosphodiester bonds. They are covalently bound to peptidoglycan or anchored to the phospholipid bilayer plasma membrane. According to Gutierrez et al.,38 high hydration of a DES results in the rupture of the H-bonding network in the DES. Therefore, in its aqueous solution, the DES may be partially dissociated, releasing its cations (i.e., cholinium), anions (i.e., Cl− or Ac−), and H-bond donors (HBD) to interact with the cell wall, leading to cell wall distortion or disruption. For instance, the cholinium cation can interact with the negatively charged carboxylic and phosphate groups, while the chloride and acetate anions with the positively charged amino groups, all 5719


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CCTCC M209061 cells to catalyze the asymmetric reduction of ketones: After 5 cycles of reaction (6 h each time), the cells retained 80.0% of its initial activity in the aqueous solution containing 5% (v/v) ChCl/U (1:2), while 50.4% relative activity remained in the DES-free buffer solution;14 in their more recent study,15 the reaction was carried out in an IL ([BMIm][PF6])/DES (ChCl/G)-buffer biphasic system, and the immobilized cell remained around 72% active after 9 cycles. Mao et al.12 have immobilized Arthrobacter simplex TCCC 11037 cells in calcium alginate, which was then used as catalyst for steroid 1-en-dehydrogenation. After five cycles, the substrate conversion decreased from 93% to 81% in the aqueous solution containing 6% ChCl/U (1:2), while from 72% to 53% in the DES-free solution.

Notoriously, as compared to Gram-negative bacteria with an outer membrane (rich in phospholipids and lipopolysaccharides) right on top of the peptidoglycan cell wall, the Grampositive ones are less protective against the attack of many biocides.46 As Lysinibacillus fusiformis CGMCC1347 cells are Gram-positive,49 our study provides another example to support the above finding: The Gram-positive cells are susceptive to treatment by DESs, in particular, the ChAcbased ones. Recyclability of Immobilized Lysinibacillus f usiformis CGMCC1347 cells. Figure 5a shows the immobilized cells, which are spherical beads with a diameter of 2−4 mm. They present a highly amorphous structure as can be seen in their scanning electron micrographs (Figure 5b,c), suggesting that the cells inside remain intact and are readily accessible for the reaction to proceed. The catalytic activity of the immobilized cells in the reaction systems containing 20% (v/v) DES or NADES has been assessed. Among the 8 DESs (with a salt/HBD molar ratio of 1:1) and 21 NADESs tested, ChCl/Gal (5:2) gave the highest production yield, which was 181.4% of the one obtained in the pure water system, followed by ChCl/PG (1:1) and ChCl/EG (1:1), yielding 151.7% and 145.0% of the one obtained in pure water, respectively. These results reiterate the activating power of DES and NADES. The immobilized cells also exhibited an excellent operational stability, as has been demonstrated in their recycle tests (Figure 6): Their catalytic activity was well maintained for at least 13 cycles (72 h reaction for each cycle), after which the reaction was terminated. The initial increase in the production during the first four cycles may be ascribed to the possibility that the cells were still continuously growing by taking the substrate isoeugenol as the nutrient. By comparing the scanning electron micrographs of the immobilized cells before and after the use of 13 cycles (Figure 5b−e), one can also see that the amorphous structure of the beads was as before, while the cells inserted inside still remained highly intact and healthy, further manifesting the excellent operational stability of these immobilized cells. For comparison, Zhao et al.26 have tried to immobilize the same cells by entrapment into calcium alginate gels. When catalyzing the same transformation (i.e., isoeugenol to vanillin) at 37 °C, the immobilized cells retained 76% and 19% of the original activity after 5 and 8 cycles (72 h for each cycle), respectively. In addition, Zong’s group has employed the similar immobilization method as ours for immobilizing Acetobacter sp.

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CONCLUSIONS Above we have assessed the impacts of 24 DESs and 21 NADESs as cosolvents on the transformation of isoeugenol to vanillin, catalyzed by Lysinibacillus f usiformis CGMCC1347 cells. All these cosolvents have shown the ability of promoting the bioconversion, which seems to be correlated with the enhanced cellular membrane permeability induced by these additives. Comparatively, the ChAc-based DESs were superior to the ChCl-based ones in improving the production yield, which is in accordance with their more pronounced influence on cellular membranes. The cells immobilized in PVA-alginate beads exerted not only good activity but also excellent operational stability. The combination of utilizing DES/ NADES as cosolvent and the PVA-alginate immobilized cells as the catalyst offers a promising design for whole-cell biocatalytic transformation processes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00285. Additional data on bioconversion yields, FCM tests, and OD260 and OD280 measurements in the presence of eight DESs were provided (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86 755 2653 4152. Fax: +86 755 2653 4277. ORCID

Zhen Yang: 0000-0003-4594-9271 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant no. 21276159). REFERENCES

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Figure 6. Operational stability of the immobilized cells. 5720


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