[Mmim]DMP and enzymatic

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Cite This: J. Agric. Food Chem. 2018, 66, 11709−11717

Pretreatment with γ‑Valerolactone/[Mmim]DMP and Enzymatic Hydrolysis on Corncob and Its Application in Immobilized Butyric Acid Fermentation Wenxiu Zheng,† Xujie Liu,‡ Liying Zhu,§ He Huang,† Tianfu Wang,∥ and Ling Jiang*,⊥ †

College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, PR China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 210009, PR China § College of Chemical and Molecular Engineering, Nanjing Tech University, Nanjing 210009, PR China ∥ Laboratory of Environmental Science and Technology, The Xinjiang Technical Institute of Physics & Chemistry, Key Laboratory of Functional Materials and Devices for Special Environments, Chinese Academy of Sciences, Urumqi 830011, PR China ⊥ College of Food Science and Light Industry, Nanjing Tech University, Nanjing 210009, PR China

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‡

S Supporting Information *

ABSTRACT: Corncob is a widely available raw material with high carbohydrate and low lignin content. To improve corncob conversion to the fermentable sugars, a novel method encompassing pretreatment using the γ-valerolactone (GVL)/1-methyl-3methylimidazolium dimethylphosphite ([Mmim]DMP) system integrated with cellulase hydrolysis was developed and optimized. It is confirmed that lignin was extracted efficiently after combined pretreatment and that the subsequent enzymatic saccharification efficiency could be significantly enhanced, resulting in the yield of 94.9% glucose from cellulose and 53.3% xylose from xylan, respectively. Furthermore, the above fermentable sugars were used as carbon source for Clostridium tyrobutyricum immobilized in macroporous Ca-alginate-lignin beads with the extracted lignin as the active ingredient to evaluate the fermentability of butyric acid. The results showed that high butyrate productivity of 0.47 g/L/h and yield of 0.45 g/g were obtained after 10 repeated batches of fermentation, demonstrating an effective process for the production of butyric acid from abundant corncob waste-biomass. KEYWORDS: γ-valerolactone, [Mmim]DMP, enzymatic hydrolysis, full component utilization, butyric acid fermentation

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and cellulase activity as reported by Li et al.5 However, it may be wasteful to utilize only a single component since ILs are still too expensive for many commercial applications. Recently, the biomass-derived solvent γ-valerolactone (GVL) was used to facilitate the mild pretreatment of lignocellulosic biomass.6−8 Thus, we hypothesized that GVL can be used as a cosolvent to assist [Mmim]DMP by overcoming the mass transfer limitation, allowing mild yet efficient pretreatment of corncobs to extract lignin as much as possible and obtain cellulose-rich material for subsequent enzymatic hydrolysis. Butyric acid, as a bulk chemical, is one of the most widely used short chain fatty acids (SCFA). Current industrial production of butyric acid is exclusively by chemical synthesis. However, bioproduction of butyric acid from renewable biomass is an inevitable trend as there are increasing demands for biobased butyric acid by food and pharmaceutical manufacturers who generally prefer food additives or pharmaceutical products produced biologically. Clostridium tyrobutyricum is a representative strain of the clostridia traditionally used for butyric acid fermentation which enable stable production of butyric acid in high purity and yield from

INTRODUCTION Corn (Zea mays) is one of the most widely grown grain crops in China, with a production of about 220 million tons in 2016 (China’s National Bureau of Statistics, 2018), 20 percent of which is corncob, one of the agricultural wastes. Corncob is a practical lignocellulosic biomass feedstock due to its low lignin and high carbohydrate content, which is an ideal source for the production of biofuels and high value-added chemicals.1 Consequently, it has been developed and utilized by the biorefinery industry to effectively prepare xylose, xylooligomers, and biological fermentation products such as xylitol, ethanol, butanediol, and furfural.2 However, before it can be used in biological fermentation processes, the raw materials must be pretreated, which deserves special consideration because the high crystallinity of cellulose and the presence of lignin will minimize enzyme access to the polymeric fibers and result in poor yields of fermentable sugars.3 Ionic liquids (ILs) are increasingly being regarded as the most promising new solvents for the pretreatment of lignocellulose, which possess a wide range of favorable physicochemical properties, such as low volatility, high thermal stability, nonflammability, unique solubilization characteristics, and low impact on the environment and human health.4 1-Methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) is a proven environmentally friendly solvent for the pretreatment of corncobs due to its biocompatibility with both lignocellulose solubility © 2018 American Chemical Society

Received: Revised: Accepted: Published: 11709

August 14, 2018 September 26, 2018 October 8, 2018 October 8, 2018 DOI: 10.1021/acs.jafc.8b04323 J. Agric. Food Chem. 2018, 66, 11709−11717

Article

Journal of Agricultural and Food Chemistry different substrates, including glucose, xylose,9 cane molasses,10 jerusalem artichoke,11 sweet sorghum juice,12 the hydrolysate of sugar cane bagasse,13 oilseed rape straw,14 wheat straw,15 and similar agricultural wastes.16 Recently, butyric acid fermentation based on various lignocellulosic substrates seems to have become the dominant trend. Therefore, the objective of this study was to achieve the whole-component utilization of corncob by means of chemical pretreatment combined with enzymatic methods and develop an efficient process for butyric acid production using immobilized C. tyrobutyricum. To our best knowledge, this is the first attempt to utilize GVL in conjunction with [Mmim]DMP to extract lignin from natural corncob efficiently, followed by enzymatic hydrolysis to obtain fermentable sugars. Finally, cultivations were performed in a fermentation tank with hydrolysates of the pretreated corncob via C. tyrobutyricum immobilized in the macroporous Ca-alginate-lignin (MCAL) beads, demonstrating the efficient production of butyric acid.

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the multidimensional space, represent them with original variables, and then guide the actual production operations.18 On the basis of this principle, we imported the data of the orthogonal table in the software, then the objective function was calculated by 100 iterations to generate the fitting curve, and after that the objective function contour was generated. According to the inverse mapping algorithm, we predicted the optimal experimental operating point. According to the literature we have surveyed,19−21 the pretreatment operational approaches of lignocellulose dissolved by ionic liquids are similar, and most investigators do not adjust the pH of pretreatment reaction. Thus, we used the same methods and considered that the lignin extraction yields may not be affected by different pH conditions when corncob pretreated by [Mmim]DMP and γ-valerolactone is used. The lignin extraction yields may depend on the properties of the solvent and cosolvent or catalyst. Corncob was pretreated as described by Qing et al.,19 with minor modifications as follows: 0.3 g of corncob was added to the specified volume of a [Mmim]DMP and GVL mixture in a 50 mL three-necked round-bottomed flask which was heated in a silicone oil bath under a nitrogen atmosphere with magnetic stirring at 1000 rpm for a preset time period. After a certain reaction time, the reaction mixture was diluted with the same volume of deionized water which was used as an antisolvent to precipitate the pretreated corncob powder. The resulting solid residue and filtrate were separated by vacuum filtration. The solid residue was washed repeatedly with deionized water to remove possible [Mmim]DMP residues and dried at 65 °C for 10 h, after which its lignin content was determined to calculate the lignin extraction yield. The filtrates after each pretreatment containing [Mmim]DMP and GVL were slowly acidified with 4 M HCl until the pH was adjusted to 2.0 and stored in a refrigerator at 4 °C for 48 h for complete regeneration of lignin. The lignin precipitate was washed with deionized water three times and dried for further use. All assays were done in triplicate and run under parallel conditions. The lignin extraction yield was calculated as eq 1:

MATERIALS AND METHODS

Materials. Corncob was purchased in the suburbs of Qingdao, China. It was composed of 43.61% cellulose, 35.23% hemicellulose, 15.84% lignin, and 5.32% other components. C. tyrobutyricum CCTCC W428 was from our own laboratory stock and was deposited in the China Center for Type Culture Collection, Wuhan under the identification code CCTCC M201690.17 The major chemicals used in this study were [Mmim]DMP (≥99%, Shanghai Cheng Jie Chemical Co. Ltd., China), GVL (98%, Sigma-Aldrich, USA), cellulase (3 × 105 U/g enzyme activity, extracted from Aspergillus niger, Ningxia Heshibi Biotechnology Co. Ltd., China), sodium alginate (Sinopharm Chemical Reagent Co. Ltd., China), calcium carbonate (Sinopharm Chemical Reagent Co. Ltd., China), and calcium chloride (Sinopharm Chemical Reagent Co. Ltd., China). All other chemicals were of analytical or reagent grade and directly used as purchased without further purification. Pretreatment of Corncob. Five hundred grams of corncob were milled into 80−120 mesh, washed three times with deionized water, and dried at 65 °C for 10 h in a vacuum oven. The dried corncob was cooled down and preserved in a valve bag for the pretreatment process. In order to extract lignin in the corncob as much as possible and obtain cellulose-enriched material, the volume of [Mmim]DMP, concentration of GVL, reaction temperature, and reaction time were optimized. As shown in UD table U9*(94) (Table 1), a four-factor

m zyz ji zz × 100 Y % = jjj1 − j z m 0{ k

where m is the lignin weight in pretreated corncob, and m0 is the lignin weight in raw corncob. The filtrates after each pretreatment containing [Mmim]DMP and GVL were slowly acidified with 4 M HCl until the pH was adjusted to 2.0 and stored in a refrigerator at 4 °C for 48 h for complete regeneration of lignin. The pH may affect the lignin regeneration amount, and we chose pH 2.0 to precipitate lignin, which was according to the methods of Fu et al.22 The lignin precipitate was washed with deionized water three times and dried for further use. All assays were done in triplicate and run under parallel conditions. Enzymatic Hydrolysis. The pretreated corncob obtained under the optimal pretreatment conditions predicted as described above was used as the substrate for enzymatic conversion to fermentable sugars. Enzymatic hydrolysis reactions were performed in 25 mL conical flasks with a total reaction volume of 5 mL in a thermostatic water bath oscillator at 150 rpm for the indicated time. On the basis of the preliminary experimental results, a five-factor (e.g., enzymatic concentration, substrate concentration, reaction temperature, pH, and reaction time) four-level orthogonal experimental design was performed to optimize the enzymatic hydrolysis conditions (Table S1). After incubation, the solution was boiled for 5 min to quench the enzymatic reaction and centrifugated at 10,000g for 5 min prior to analysis. Glucose and xylose in the supernatant were analyzed by high performance liquid chromatography (HPLC). The experiments were carried out in triplicate. Culture Conditions of C. tyrobutyricum and Preparation of MCAL Beads. The nutrient broth for C. tyrobutyricum contained 30 g/L glucose, 5 g/L yeast extract (OXOID, UK), 5 g/L tryptone (OXOID, UK), 6 g/L NaCl, 3 g/L (NH4)2SO4, 1.5 g/L K2HPO4, 0.6 g/L MgSO4·7H2O, 0.03 g/L FeSO4·7H2O, 0.3 g/L L-cysteine hydrochloride (Shanghai Shenggong Biological Engineering Co. Ltd., China), and 0.1% resazurin (Sigma, USA) at pH 6.0.10 The

Table 1. Factors and Levels of Orthogonal Experimental Design level

reaction time (h)

volume of [Mmim]DMP (mL)

concentration of GVL (wt %)

reaction temperature (°C)

1 2 3

1.0 2.0 3.0

5 10 15

10 12 14

90 110 130

(1)

three-level experimental design with 9 single experiments was conducted to obtain the optimum pretreatment conditions. After this set of experiments, we used intelligent visualization software (version 1.0, China) developed by the Wuhan University of Technology Process Systems Engineering Institute to further analyze and process the experimental results and calculate the predicted outputs. The basic principle of the visualization optimization method is to map the sample data of the multidimensional space to a twodimensional plane through a neural network and automatically generate contours of the objective function on the plane. From this, the best point or optimum operation direction can be intuitively determined. Then, the inverse mapping algorithm is used to inverse map the optimal points and optimization areas defined in the plane to 11710

DOI: 10.1021/acs.jafc.8b04323 J. Agric. Food Chem. 2018, 66, 11709−11717

Article

Journal of Agricultural and Food Chemistry

system (Shimadzu, Japan) equipped with a Sepax HP-Amino column (4.6 mm × 250 mm × 5 μm; Sepax, USA) and a refractive index detector. The column temperature was held at 30 °C, and acetonitrile−water (77:23) was used as the mobile phase at a flow rate of 0.6 mL/min.18 The measurements were carried out in triplicate. FTIR Analysis. The native and [Mmim]DMP/GVL-treated corncob was analyzed by Fourier transform infrared spectrometry on a VERTEX 33 instrument (Bruker, Germany). The method was the same as that published by Auxenfans et al.25 Determination of Bead Size. The average diameters of 100 dried beads were measured by an optical microscopic method using an optical microscope (Olympus). The ocular micrometer was previously calibrated by a stage micrometer.26 SEM Analysis. An SU8010 scanning electron microscope (SEM; Hitachi, Japan) was used to obtain images of native and [Mmim]DMP/GVL-treated corncob samples to record the changes of their surface morphological features. The macroporous structure within the blank beads and the images of C. tyrobutyricum immobilized in MCAL beads were also recorded by SEM. In order to fix the cells firmly, the beads were incubated with 2.0% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 3 h. Finally, increasing ethanol concentrations of 30, 50, 70, 85, 95, and 100% (v/ v) were used to dehydrate the fixed beads, after which they were freeze-dried in an FD-1A-50 vacuum freeze-dryer (Shanghai Bilon Instruments Co. Ltd., China) for 24 h.27 After the dried beads were fractured in half in liquid nitrogen, the samples were inspected by SEM. Analysis of Biomass and Butyric Acid Yields. Cell density was analyzed by measuring the optical density of the cell suspension at a wavelength of 600 nm (OD600) using an Ultrospec 3300 pro spectrophotometer (Amersham Bioscience). Quantitative analysis of butyric acid was performed by gas chromatography on an Agilent 6820 GE instrument (Agilent Technologies, USA) equipped with a AT·SE-54 column (30 m × 0.32 mm × 0.5 μm; Agilent, USA), according to a published method.10

broth was boiled to remove oxygen. The carbon and nitrogen sources were sterilized separately by autoclaving at 115 °C for 30 min. C. tyrobutyricum was cultivated at 37 °C for 48 h after inoculation of the broth with 5% bacterial solution. After three generations of activation, the third-generation seed broth was used as the mother liquid for further cultivation. The same cultivation process was used when the cells reached an exponential growth phase and then were centrifuged at 3000g for 10 min to collect the cell pellet, which was washed and resuspended in 1 mL of 0.1 M phosphate buffer (pH 7.4). Because of the extracted lignin recovered from acid precipitation after each pretreatment (0.3 g of pretreated corncob each) was very little, under the optimal pretreatment condition, only 16.84 mg of regenerated lignin was obtained. On the basis of mass balance calculations, the mass of extracted lignin was about 44.20 mg, which may be explained by the fact that partial lignin dissolved steadily in the solvent. Above all, when these small amounts of lignin were washed with deionized water, there were some lost during flushing; thus, we collected all the pretreatment solvent to precipitate lignin and make the final amount of lignin enough to prepare the MCAL beads and immobilize C. tyrobutyricum as described previously by Zhang et al.23 The amount of cells loaded was tested for optimal enzyme activity and macroscopic molecule performance by comparing the glucose consumption yield. Before immobilization, all the chemicals and solutions were sterilized at 121 °C for 20 min. After that, 20 mL of a 20 mg/mL sodium alginate aqueous solution containing five different concentrations (1 g/L, 5 g/L, 10 g/L, 15 g/L, and 20 g/L of wet thallus), 5 mg/mL lignin, and 50 mg/mL CaCO3 as a porogenic diluent was dropped from a 10 mL syringe into 100 mL of a magnetically stirred 11 mg/mL CaCl2 aqueous solution to prepare the spherical beads. The rounded beads with approximate diameters were collected and incubated at 4 °C for 4 h. After incubation, 0.5 M HCl was used to wash the beads three times until no gas bubbles were visible, followed by extra washing with phosphate buffer. 10% w/v beads, immobilized by C. tyrobutyricum separately prepared by different conditions, were incubated at 37 °C for 48 h in a 100 mL anaerobic bottle with 50 mL of broth, and the initial glucose concentration and glucose concentration after 48 h were monitored. Equivalent spherical beads without cells as a blank control were prepared using the same procedure. In order to investigate the fermentability of the enzymatic hydrolysates obtained from the pretreated corncob, butyric acid fermentation was performed by cultivating C. tyrobutyricum as free cells and immobilized in MCAL beads. After preprocessing and saccharification, the hydrolysates were filtered and collected for subsequent fermentation. The fermentation was conducted in a 5 L stirred-tank fermenter (B. Braun Biotech International, Germany) with 2 L of nutrient broth comprising 50 g/L carbon source (glucose, xylose, and enzymatic hydrolysate, respectively) and inorganic salts the same as growth medium except for L-cysteine hydrochloride and resazurin at pH 6.0. Before inoculation, the fermentation device was sterilized at 115 °C for 30 min, followed by repeated sterilization after cooling overnight. Anaerobic conditions were maintained by initially sparging with pure N2. To start the fermentation, either free cells or MCAL beads with an optimal cell load (5% inoculation amount) were introduced into the fermenter, which was operated at 37 °C, 150 rpm for 48 h, and the pH controlled at 6.0 by adding 6 M NaOH and 3 M H2SO4 via an online sensing and dosing system. The fermentation was conducted both in single and repeated batch modes. In repeated batch mode, the immobilized cells were allowed to grow continuously batch after batch, while the fermentation broth was replaced with fresh sterile carbon source to start a new batch whenever the concentration of sugars fell to zero. Samples comprising 10 mL for the measurement of substrate, biomass, and product concentrations were taken at regular intervals. Analysis of the Composition of Solids and Sugars. The composition of corncob and the solid residue obtained from pretreated corncob was determined according to the standard procedure published by the National Renewable Energy Laboratory (NREL).24 The monomeric sugar contents of the enzymatic hydrolysate supernatants were measured on an LC-20A HPLC

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RESULTS AND DISCUSSION Optimization of the Lignin Extraction Yield. Pretreatment and component separation are key for the high value utilization of lignocellulose. The main lignocellulosic pretreatment methods have concentrated on the removal of lignin and reduction of cellulose crystallinity. The ionic liquid [Mmim]DMP can be used as an efficient solvent for the pretreatment of lignocellulose due to its excellent lignocellulose solubility.19 Moreover, it was recently reported by several research groups that ionic liquids not only efficiently pretreat lignocellulose and enhance the subsequent enzymatic hydrolysis of polysaccharides but also have lower toxicity and thermal stability, and are almost nonvolatile and recyclable, which make them environmentally friendly.28 However, ionic liquids have high viscosity, which can slow down reactions due to mass-transfer problems. Consequently, many researchers added different kinds of cosolvents or catalysts such as H2O, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), ethanolamine, and mineral acids to help reduce the ionic liquids’ viscosity and overcome mass transfer resistance.19,29,30 In this study, we chose GVL as the cosolvent to assist [Mmim]DMP in the extraction of corncob lignin. On the basis of research by Boissou et al., the addition of GVL not only helps fluidify ionic liquids but also increases their cellulose dissolution ability.31 Inspired by these studies, we investigated the pretreatment and fractionation of corncob using GVL in combination with [Mmim]DMP. To optimize the pretreatment conditions, an L9 (34) orthogonal experimental design was performed (Table 1), 11711


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[Mmim]DMP to solubilize lignocellulose, resulting in the lower solubility of corncob.31 In order to further predict the optimal lignin extraction conditions, we took advantage of intelligent visualization software which can use dimensionreducing mapping to analyze the experimental results. On the basis of the contour of the objective function, we can predict the optimization direction and obtain the optimal operating point. Figure 1 shows the mapping diagram of the extraction yield. As shown in the figure, sample data in multidimensional space were mapped and reduced to a two-dimensional plane using the visualization method. The nine empty green points in the mapping diagram represent nine groups of experiments, while the solid black line represents the contours of each group’s extraction yield.32 According to the inversion mapping algorithm, the optimal point took points 6 and 8 as references and used a step size of 2 through extrapolation in the direction of the arrow, which led to the predicted optimal conditions comprising a reaction temperature of 90 °C, 4 h reaction time, 5 mL of [Mmim]DMP, and 16 wt % GVL, corresponding to a predicted yield of 93.6%. When these conditions were used experimentally for practical verification, the yield of lignin extraction was 93.0%, which indicated that the actual result was consistent with the result predicted by the visual optimization software. It is reported that the addition of inorganic acids, especially HCl, to ionic liquid pretreatment can promote the conversion of cellulose and hemicellulose to monomeric sugars and further increase the lignin extraction rate.19 However, the improvement was not as obvious as expected previously based on our results (data not shown). Thus, based on the above experimental data, GVL as cosolvent combined with [Mmim]DMP as solvent is well-suited to pretreat lignocellulose. Characterization of the Corncob Biomass. In order to investigate the structural changes between natural and [Mmim][DMP]/GVL pretreated corncobs, scanning electron microscopy (SEM) analysis was employed. SEM has been proved useful to monitor the altered structure of lignocellulosic biomass pretreated with various ionic liquids. It was apparent from the micrographs that the natural corncob Figure 2a had a smooth surface and compact structure with a complete form, while the structure of the pretreated corncob Figure 2b was

and the results are presented in Table 2. Note that the amount of biomass in each pretreated condition was set to 0.3 g. Table 2. Design and Results of the Orthogonal Pretreatment Experimenta

sample

reaction time (h)

volume of [Mmim] DMP (mL)

concentration of GVL (wt %)

reaction temperature (°C)

lignin extraction yield (%)

1 2 3 4 5 6 7 8 9 K1 K2 K3 k1 k2 k3 R

1.0 1.0 1.0 2.0 2.0 2.0 3.0 3.0 3.0 161.1 174.6 207.2 53.70 58.20 69.07 15.37

5 10 15 5 10 15 5 10 15 180.8 191.3 170.8 60.27 63.77 56.93 6.83

10 12 14 14 10 12 12 14 10 196.7 154.8 191.4 64.73 66.00 50.23 15.77

90 110 130 110 130 90 130 90 110 194.2 198 150.7 65.57 51.60 63.80 13.97

63.3 54.1 43.7 67.3 56.8 50.5 50.2 80.4 76.6

a

K, sum of yields at each level; k, average of K at each level; R, range, the difference between the maximal k and minimal k. The value is expressed as the mean (n = 3).

According to the range analysis of the orthogonal experimental design, the strength of the effects of individual factors on the lignin extraction yield was in the following order: concentration of GVL > reaction time > reaction temperature > volume of [Mmim]DMP. In addition, based on these data we found that a longer time, higher concentration of GVL, and a lower temperature were beneficial for the extraction of lignin, while the lignin extraction yield did not significantly change as the volume of [Mmim]DMP was increased. However, it was worth noticing that the concentration of GVL was not the higher the better in that more GVL may inhibit the ability of

Figure 1. Contours on the mapping plane for the lignin extraction yield. The solid line represents contours, small circles and numbers represent sample data, and arrows represent the direction of data optimization. 11712


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Figure 2. SEM micrographs of the (a) natural corncob and (b) pretreated corncob with [Mmim][DMP]/GVL.

intensity of the 1250 cm−1 band assigned to ether bonds was slightly lower in the prtreated than in the natural corncob, which also confirmed the dissolution of ether linkages between lignin and carbohydrates. The peaks in other regions were almost unchanged, indicating that the basic functional groups in natural corncobs were largely unaffected during the process of lignin removal. Enzymatic-Hydrolysis of Pretreated Corncobs. Previous studies have shown that enzymatic hydrolysis is always applied after ionic liquid pretreatment of lignocellulose to completely deal with residues because of its mild reaction conditions and specificity. As such, the corncob biomass obtained under optimal ionic liquid pretreatment conditions was taken for enzymatic hydrolysis experiments with the selected cellulose. Details of the experimental parameter design of enzymatic hydrolysis are displayed in Table S2. It was found that, from the orthogonal experimental results, the optimal conditions of the enzymatic hydrolysis of pretreated corncobs was obtained with 45 FPU/g cellulase, 50 g/L substrate, 50 °C, pH 5.0, and 72 h reaction time, in which the yield of glucose, one of the main hydrolytic products, could be as high as 93.2%. Because of the tight crystalline cellulose structure and higher contents of hemicelluloses and lignin, it always takes a relatively long time (for example, 72 h) with enzymatic hydrolysis to obtain a higher conversion ratio from lignocellulosic biomass. However, the enzymatic hydrolysis time could be shortened effectively in the case of either increasing the amount of enzyme or using the enzyme mixtures (e.g., cellulase, hemicellulase, and pectinase).37,38 Taking the total yield of glucose and xylose as the index, the effect of enzymatic hydrolysis was further investigated in terms of corncob biomass digestibility obtained for each sample pretreated via ionic liquid in the previous treatment process. Note that under the optimal enzymatic hydrolysis conditions, the glucose and xylose yield of pretreated corncobs varied significantly based on the different ionic liquid pretreatment process, and the concentration of xylose was relatively smaller than that of glucose. The pretreated corncob samples prepared under optimal conditions exhibited the highest digestibility with a glucose and xylose yield of 94.9% and 53.3%, respectively (Figure S1), which, in turn, confirmed that the predicted best pretreatment condition was indeed from the most degradable sample in the following enzymatic hydrolysis process. This result indicated that the corncob pretreated with [Mmim][DMP] and GVL was enriched in cellulosic material because a large proportion of lignin was extracted and removed. The glucose and xylose in the hydrolysates can be

disrupted and broken, so that many fractures appeared, and the whole structure became disordered and loosened. This observation can be explained if the corncob’s cell walls were partly dissolved and lignin was partly extracted by [Mmim][DMP]/GVL, which would be beneficial for cellulases to access the external and internal surface area of cellulose, in turn increasing the efficiency of enzymatic saccharification.30 To investigate the chemical modifications after pretreatment, the natural and pretreated corncobs were investigated using Fourier-transform infrared spectroscopy (FTIR) analysis. According to the literature,33,34 Figure 3 clearly illustrates the

Figure 3. FTIR spectra of (a) natural and (b) pretreated corncobs with [Mmim][DMP]/GVL under optimized conditions.

typical absorbance bands expected for a lignocellulosic polymer, with similar spectral profiles and relative intensities of the signals, indicating a structure analogous to lignocellulose. There were two broad adsorption peaks around 3380 and 2910 cm−1, corresponding to the stretching vibrations of −OH and −CH groups, respectively.5 In addition, a strong absorption between 1150 and 1000 cm−1 was obvious, which was attributed to the C−O−C pyranose ring skeletal vibration mainly present in cellulose and hemicelluloses, and sparse in lignin.35 An obvious peak at 1730 cm−1 almost disappeared in the spectrum of the [Mmim]DMP/GVL pretreated corncob compared with the spectrum of the natural corncob, which suggested that the pretreatment effectively broke the ester linkages between carbohydrates and lignin.36 Furthermore, the 11713


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Figure 4. Optical image (a) and SEM micrographs (b−e) of both external and interior structures of MCAL beads. (a) Surface profile of beads; (b) cross-sectioned microstructure of an empty bead; (c) planar view of the cross-section; (d) view of plenty of immobilized C. tyrobutyricum; (e) enlarged view of a single immobilized C. tyrobutyricum.

into alginate beads and using these MCAL beads to immobilize the C. tyrobutyricum responsible for the production of butyrate. The morphology of the obtained MCAL beads is shown in Figure 4a, with the average diameter ranging from 3.92 ± 0.28 to 4.2 ± 0.15 mm. The particle size of the MCAL was a little bit larger than the uncoated beads without lignin, which coincided with the results of other studies.23 It was noticed that the introduction of lignin contents in the formulations could lead to a higher porosity, which was in accordance with the conclusion drawn by Rudaz.45 SEM micrographs clearly revealed the morphological characteristics of the interior structure of MCAL beads contained with the immobilized bacteria. As can be seen in Figure 4b, the cross-sectioned microstructure of the beads resembled a honeycomb with the diameters of macropores in them to be 10−50 μm. The inner walls of the interior cavities were rough (Figure 4c), so that the C. tyrobutyricum was able to adhere to the walls as much as possible (Figure 4d). Moreover, Figure 4e clearly showed the embedding phenomenon, with bacteria engulfed by the carrier material. The macropores and interior cavities that developed due to the addition of CaCO3 in the MCAL beads provide more space for the growth of cells. With these, an intensified transport of substrate and nutrients to the interior of the MCAL beads can be expected, which will thus facilitate the growth of C. tyrobutyricum. Lignin is expected to reduce the hydrophilicity of alginate and hence provide more suitable environments for cells to adhere and grow.43 In fact, as

further used as fermentation carbon sources, which were evaluated using C. tyrobutyricum immobilized in MCAL beads to produce butyric acid. Characterization of Immobilized C. tyrobutyricum. The immobilized-cell culture is more effective in shielding environmental perturbations such as pH and metabolic toxic byproducts compared to the suspension culture. Our group conducted multiple studies on butyric acid fermentation in a fibrous bed bioreactor with immobilized C. tyrobutyricum with good results.9,10,39 Herein, we made an attempt to use alginate to immobilize C. tyrobutyricum in beads. Alginate, composed of β-1,4-linked D-mannuronic acid and α-1,4-linked L-guluronic acid, is one of the abundant natural polymers with good properties such as low cost and lack of toxicity to microorganisms.40 It has a unique property of instantaneously undergoing ionotropic gelation to form bead-shaped hydrogels in aqueous solution when contacted with multivalent cations (e.g., Ca2+, etc.), which has been extensively employed to encapsulate enzymes and living cells used for the treatment of metal ions and to fabricate alginate-based drug delivery carriers.41,42 However, two major problems with this matrix are the high hydrophilic nature of the alginate chains that lead to hampered cell adhesion43 and the frangibility of pure calcium alginate so that it cannot be used alone.44 With the appreciation of the porous feature of lignin, we arrived at the idea of incorporating lignin, the byproduct of corncob material, 11714


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Table 3. Comparison of Cell Growth and Acid Production with Different Sugar Sources between Free-Cell and Immobilized Fermentationsa cell growth fermentation model free cell

immobilized cell

sugar sources

special growth rate (h−1) ± ± ± ±

glucose xylose hydrolysates glucose

0.19 0.07 0.14 0.17

0.05 0.01 0.06 0.03

xylose hydrolysates

0.06 ± 0.01 0.13 ± 0.05

acid production

biomass yield (g/g) 0.16 0.13 0.14 0.15

± ± ± ±

butyric acid conc. (g/L)

0.02 0.04 0.05 0.04

0.12 ± 0.03 0.13 ± 0.05

22.8 22.2 22.2 27.6

± ± ± ±

3.23 2.31 4.23 3.87

24.0 ± 2.48 26.4 ± 2.55

butyric acid yield (g/g) 0.46 0.44 0.44 0.55

± ± ± ±

0.03 0.02 0.03 0.04

0.48 ± 0.02 0.53 ± 0.04

acetic acid conc. (g/L) 3.6 5.4 4.8 1.2

± ± ± ±

0.43 0.32 0.37 0.21

2.4 ± 0.79 1.8 ± 0.27

acetic acid yield (g/g)

B/A ratio

± ± ± ±

0.01 0.01 0.01 0.01

6.3 4.1 4.6 23.0

0.05 ± 0.01 0.04 ± 0.01

10.0 14.7

0.07 0.11 0.10 0.02

a

The B/A ratio means the ratio of butyric acid to acetic acid based on yield. The total concentration of sugars used in the system was close to 50 g/ L.

that they produced a higher purity of butyric acid than free cells, as evidenced by the high butyric to acetic acid concentration (B/A) ratio and the decreased yield of acetic acid. The increased butyrate yield and less acetic acid may be partially attributed to reduce cell growth which would need less energy, so that more substrate could be metabolized to butyric acid.9 In addition, during the fermentation process, the absorbed butyric acid and acetic acid concentration were also increased with the concentration of butyric acid and acetic acid, but the amount of adsorption was less than 1% in the end, which therefore can be ignored (Figure S3). The adsorption capacity of MCAL beads can be contributed by the lignin encapsulated in beads, which has been proved by other research groups.23,47 In addition to the batch mode, we also carried out repeated-batch fermentations under the same culture conditions for the purpose of comparing and evaluating the long-term stability of butyric acid fermentation by immobilized cells. Figure 5 shows the results of the

reported by Zhang et al., the presence of lignin in MCA beads to immobilize Phanerochaete chrysosporium also led to a 28.7fold increase in the phenanthrene adsorption capacity compared with that by conventional MCA beads because of the high mass transfer rate.23 Furthermore, glucose was applied as carbon source to evaluate the growth of C. tyrobutyricum immobilized in MCAL beads. The maximum growth of C. tyrobutyricum was obtained in the case of immobilization in MCAL beads with a cell loading of 10 g/L when using the glucose consumption yield as an index of mass transfer performance (Figure S2). It might be a result of the competitive adhesion of cells at the higher cell loading, which will accelerate the consumption of nutrients, degrade the microenvironment, and thereby affect the metabolism activity of immobilized cells.46 In addition, it can be seen from Figure 4d that the cells uniformly distributed on the inner surface of the macropores; thus, the MCAL beads we prepared here were appropriate to immobilize C. tyrobutyricum responsible for the production of butyrate. Application of Pretreated Corncobs in Butyrate Fermentations. To investigate the performance of butyric acid fermentation using enzymatic hydrolysates of the pretreated corncob, free and immobilized-cell fermentations were conducted, alongside control fermentations with glucose and xylose as the carbon source. A change in pH would affect the performance of MCAL immobilized C. tyrobutyricum cells in butyric acid production. In our previous study, the effects of pH on butyric acid production in batch fermentation have been systematically researched.10 Thus, the results clearly indicated that the optimal pH on microorganism growth and butyric acid production was 6.0, which was used directly as a condition for the fermentation experiments here. Table 3 shows a comparison between free cells and immobilized cells in single batch fermentation for butyric acid production using different carbon sources. As shown in the table, both free and immobilized cells had good utilization rates of glucose, xylose, and pretreated corncob hydrolysate to produce butyric acid, although there were some minor differences between the yields of butyric acid when glucose was used as the carbon source since C. tyrobutyricum uses glucose first. In addition, the concentration and yield of butyric acid produced by immobilized cells was superior to that of free cells, although the growth rate of immobilized cells was somewhat lower with any of the three carbon sources, most likely because C. tyrobutyricum cells were spatially constrained in the MCAL beads, which limited their reproduction. Although the immobilized cells grew in a small space, it was noteworthy

Figure 5. Kinetics of repeated-batch fermentation of corncob hydrolysates by C. tyrobutyricum immobilized in MCAL beads.

fermentation kinetics of butyric acid production by immobilized cells of C. tyrobutyricum with corncob hydrolysate as the substrate. The kinetics of hydrolysate consumption, product formation, OD600, and byproduct formation were similar in all 10 batches (Figure 5), which suggested that this system was quite stable. When each new batch was initiated, the cells grew fast, and butyric acid was quickly produced. Even though the beads were fermented for 10 batches, immobilized C. 11715


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(2) Lei, C.; Zhang, J.; Xiao, L.; Bao, J. An alternative feedstock of corn meal for industrial fuel ethanol production: delignified corncob residue. Bioresour. Technol. 2014, 167, 555−559. (3) Gao, K.; Rehmann, L. ABE fermentation from enzymatic hydrolysate of NaOH-pretreated corncobs. Biomass Bioenergy 2014, 66, 110−115. (4) Wang, Y.; Radosevich, M.; Hayes, D.; Labbé, N. Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass. Biotechnol. Bioeng. 2011, 108, 1042−1048. (5) Li, Q.; Jiang, X.; He, Y.; Li, L.; Xian, M.; Yang, J. Evaluation of the biocompatibile ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite pretreatment of corn cob for improved saccharification. Appl. Microbiol. Biotechnol. 2010, 87, 117−126. (6) Luterbacher, J. S.; Rand, J. M.; Alonso, D. M.; Han, J.; Youngquist, J. T.; Maravelias, C. T.; et al. Nonenzymatic sugar production from biomass using biomass-derived γ-valerolactone. Science 2014, 343, 277−280. (7) Shuai, L.; Questell-Santiago, Y. M.; Luterbacher, J. S. A mild biomass pretreatment using γ-valerolactone for concentrated sugar production. Green Chem. 2016, 18, 937−943. (8) Wu, M.; Liu, J. K.; Yan, Z. Y.; Wang, B.; Zhang, X. M.; Xu, F.; Sun, R. C. Efficient recovery and structural characterization of lignin from cotton stalk based on a biorefinery process using a γvalerolactone/water system. RSC Adv. 2016, 6, 6196−6204. (9) Jiang, L.; Wang, J.; Liang, S.; Wang, X.; Cen, P.; Xu, Z. Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor. Appl. Biochem. Biotechnol. 2010, 160, 350−359. (10) Jiang, L.; Wang, J. F.; Liang, S. Z.; Wang, X. N.; Cen, P. L.; Xu, Z. N. Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses. Bioresour. Technol. 2009, 100, 3403−3409. (11) Huang, J.; Cai, J.; Wang, J.; Zhu, X.; Huang, L.; Yang, S. T.; Xu, Z. N. Efficient production of butyric acid from jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor. Bioresour. Technol. 2011, 102, 3923−3926. (12) Wang, L.; Ou, M. S.; Nieves, I.; Erickson, J. E.; Vermerris, W.; Ingram, L. O.; Shanmugam, K. T. Fermentation of sweet sorghum derived sugars to butyric acid at high titer and productivity by a moderate thermophile Clostridium thermobutyricum at 50°C. Bioresour. Technol. 2015, 198, 533−539. (13) Wei, D.; Liu, X.; Yang, S. T. Butyric acid production from sugarcane bagasse hydrolysate by Clostridium tyrobutyricum, immobilized in a fibrous-bed bioreactor. Bioresour. Technol. 2013, 129, 553− 560. (14) Huang, J.; Zhu, H.; Tang, W.; Wang, P.; Yang, S. T. Butyric acid production from oilseed rape straw by Clostridium tyrobutyricum, immobilized in a fibrous bed bioreactor. Process Biochem. 2016, 51, 1930−1934. (15) Baroi, G. N.; Gavala, H. N.; Westermann, P.; Skiadas, I. V. Fermentative production of butyric acid from wheat straw: economic evaluation. Ind. Crops Prod. 2017, 104, 68−80. (16) Suo, Y.; Fu, H.; Ren, M.; Yang, X.; Liao, Z.; Wang, J. Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing Class I heat shock protein GroESL. Bioresour. Technol. 2018, 250, 691−698. (17) Wu, Q.; Zhu, L.; Xu, Q.; Huang, H.; Jiang, L.; Yang, S. T. Tailoring the oxidative stress tolerance of Clostridium tyrobutyricum cctcc w428 by introducing trehalose biosynthetic capability. J. Agric. Food Chem. 2017, 65, 8892−8901. (18) Yang, X.; Zhu, L.; Jiang, L.; Xu, Q.; Xu, X.; Huang, H. Optimization of bioconversion process for trehalose production from enzymatic hydrolysis of kudzu root starch using a visualization method. Bioresour Bioprocess 2015, 2, 37. (19) Qing, Q.; Hu, R.; He, Y.; Zhang, Y.; Wang, L. Investigation of a novel acid-catalyzed ionic liquid pretreatment method to improve biomass enzymatic hydrolysis conversion. Appl. Microbiol. Biotechnol. 2014, 98, 5275−5286.

tyrobutyricum could still produce 22.5 g/L of butyric acid with the productivity of 0.47 g/L/h and yield of 0.45 g/g in the tenth repeated batch, which was not dramatically lower than that of the first batch (vs 26.4 g/L, 0.55 g/L/h, and 0.46 g/g, respectively). Moreover, the concentration of the byproduct acetic acid also dropped slightly after each batch. The results also showed that C. tyrobutyricum could efficiently use glucose and xylose at the same time, which was consistent with a previous study,9,48 whereas the likely trace amounts of degradation compounds present in corncob hydrolysate had no obvious effects on cell growth and butyric acid production. In conclusion, the ionic liquid [Mmim]DMP in conjunction with GVL was evaluated in the pretreatment of corncobs, which have the potential to become a novel pretreatment to improve the enzymatic hydrolysis of biomass. MCAL beads were used for the first time as a carrier to immobilize C. tyrobutyricum for butyric acid production from pretreated corncob hydrolysate. This novel approach was proved to be practicable and enabled us to obtain higher yields of purer butyric acid. This research is expected to improve the available lignocellulose pretreatment options, enabling the economical production of butyric acid on an industrial scale.

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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/acs.jafc.8b04323. Comparison of glucose and xylose yields; glucose consumption yield of MCAL beads; adsorption isotherms; factors and levels of orthogonal experimental design of enzymatic hydrolysis conditions; and design and results of the orthogonal enzymatic hydrolysis experiments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ling Jiang: 0000-0001-6625-5557 Author Contributions

W.Z. was the main author of this work who carried out the main experiments and wrote the manuscript. X.L. helped collect and analyze the data. Z.L. contributed reagents and materials. H.H. assisited W.Z. in revising the manuscript. L.J. and T.W. contributed to designing the experimental scheme and gave general guidance to the writing of the paper. Funding

This work was supported by the National Key R&D Program of China (2017YFC1600404), the National Science Foundation for Young Scholars of China (U1603112 and 21506101), the Natural Science Foundation of Jiangsu Province (BK20171461 and BK20180038), the Six Talent Peaks Project in Jiangsu Province (2015-JY-009), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTE1838). Notes

The authors declare no competing financial interest.

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REFERENCES

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