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Biosynthesis, structure and antioxidant activities of xanthan gum from Xanthomonas campestris with additional furfural
Accepted Manuscript Title: Biosynthesis, structure and antioxidant activities of xanthan gum from Xanthomonas campestris with additional furfural Authors: Yan Kang, Panyu Li, Xiaotong Zeng, Xi Chen, Yi Xie, Yu Zeng, Yongkui Zhang, Tonghui Xie PII: DOI: Reference:
S0144-8617(19)30402-3 https://doi.org/10.1016/j.carbpol.2019.04.018 CARP 14792
To appear in: Received date: Revised date: Accepted date:
5 January 2019 15 March 2019 3 April 2019
Please cite this article as: Kang Y, Li P, Zeng X, Chen X, Xie Y, Zeng Y, Zhang Y, Xie T, Biosynthesis, structure and antioxidant activities of xanthan gum from Xanthomonas campestris with additional furfural, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biosynthesis, structure and antioxidant activities of xanthan gum from Xanthomonas campestris with additional furfural
Yan Kanga, Panyu Lia, Xiaotong Zenga, Xi Chena, Yi Xiea, Yu Zenga, Yongkui
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Zhanga,*, Tonghui Xiea,*
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Department of Pharmaceutical & Biological Engineering, School of Chemical
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Engineering, Sichuan University, Chengdu, Sichuan 610065, China
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Tonghui Xie, Yongkui Zhang
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Corresponding author
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Tel.: +86 28 85408255/Fax: +86 28 85403397
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Authors
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E-mail: [email protected] (T. Xie), [email protected] (Y. Zhang)
Yan Kang, [email protected] Panyu Li, [email protected]
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Xiaotong Zeng, [email protected] Xi Chen, [email protected] Yi Xie, [email protected] Yu Zeng, [email protected]
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Graphical abstract
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Highlights
The effect of additional furfural on xanthan gum production was explored.
Low concentration of furfural promoted cell growth and kept steady xanthan
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yield.
Furfural altered acetyl, pyruvate and glucuronic acid contents of xanthan
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products.
1 g/L of furfural provided product a desirable hydroxyl radical scavenging effect.
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Abstract: Lignocellulosic-like materials are potentially low-cost fermentation substrates, but their pretreatment brings about by-products. This work investigated the effects of furfural on xanthan gum (XG) production, and product quality was evaluated by
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structure, viscosity and antioxidant capacities. Xanthomonas campestris maintained steady polysaccharide yield (above 13 g·L–1) with enhanced cell growth at low
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furfural concentrations (below 3.2 g·L–1). The products were verified as XG by FT-IR, XRD, NMR and monosaccharide analysis. Moreover, they were found to have
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reduced acetyl, rising pyruvate and up-to-down glucuronic acid groups as increasing
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furfural concentration. Furthermore, XG product with 1 g·L–1 furfural addition
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showed the best hydroxyl scavenging effects, though reducing powers presented no
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variation. It was demonstrated that furfural, the common hydrolysis by-product, was
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not necessarily an inhibitor for fermentation, and an appropriate amount of furfural
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was beneficial to XG production with steady yield and good quality.
Keywords: Furfural; Xanthan gum; Xanthomonas campestris; antioxidant activity;
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polysaccharide structure
1.
Introduction Xanthan gum (XG) is a water-soluble heteropolysaccharide, formed by repeated
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pentasaccharide units consisting of glucose, mannose, and glucuronic acid (Garcíaochoa, Santos, Casas, & Gómez, 2000; Munish, Ashok, & Kuldeep, 2012). XG is widely applied in food, pharmaceutical, cosmetic, oil and textile industries because of its superior properties, such as excellent solubility, high viscosity at low
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concentrations, compatibility, stability, etc. (Jang, Zhang, Bo, & Choi, 2015; Song, Kim, & Chang, 2006; Verhoeven, Vervaet, & Remon, 2006). Especially, antioxidant
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activity of XG has attracted increasing attention, since health care products with antioxidation are popular in the last few decades (Gawlik, 2012).
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XG industry faces a challenge of high production cost. For example, traditional
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fermentation carbon sources (mainly glucose and sucrose) are expensive (Li et al.,
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2017). Low-cost substrates like lignocellulose are attractive for the potential to supply
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mixed sugars. Some biomass materials have been successfully adopted to XG
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fermentation, including tapioca pulp (Gunasekar, Reshma, Treesa, Gowdhaman, & Ponnusami, 2014), sugar cane broth (Faria et al., 2011), rice bran (Demirci, Arici, &
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Gumus, 2012), kitchen waste (Li et al., 2017) and so on. In order to solve the problem
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that microbe cannot effectively utilize organic macromolecules, it is necessary to hydrolyze biomass materials before fermentation. Among alternative pretreatment methods, acid hydrolysis is most commonly used on account of the advantages of
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high efficiency and low price, although its by-products furan derivatives are regarded as microbial metabolism inhibitors (Lin et al., 2015). In this context, it is important to investigate the effect of probable inhibitor on XG production. Furfural, the by-product released from Maillard reaction, is noticed in acid-
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converted dehydration of lignocellulosic biomass (Navarro, 1994). Its usual concentrations were found to be 1.5–3 g·L–1 during acidic hydrolysis of tapioca pulp (Gunasekar et al., 2014), wheat straw (Olofsson, Rudolf, & Lidén, 2008), oil palm empty (Rahman, Choudhury, & Ahmad, 2006) and other biomass waste (Cuevas,
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Quero, Hodaifa, López, & Sánchez, 2014). When utilizing tapioca pulp and kitchen waste in XG production, it failed to obtain expected xanthan yield as increasing
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hydrolysate nutrient (Gunasekar et al., 2014; Li et al., 2016). According to the
observed inhibiting effect of furfural on ethanol and hydrogen productions (Lin et al.,
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2015; Navarro, 1994), it was inferred that the detected furfural in culture was the
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limiting factor to XG production. However, the stimulation for acetone-butanol-
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ethanol fermentation by furfural was also found as a promoter (Zhang, Han, & Ezeji,
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2012). Besides, current studies about the influence of furfural on fermentation paid
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close attention to production output (Akobi, Hafez, & Nakhla, 2016; Zhang et al., 2012), and neglected biological activities of products. However, XG serves as a
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potential antioxidant and the information of its antioxidant activities is important.
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Thus, the characterization of additional furfural on XG production and product quality needs to be systematically studied for the utilization of renewable biomass. The biological activities of polysaccharides are highly associated with their
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structure, such as monosaccharide composition and substitutions (Chen et al., 2014; Lo, Cheng, Chiu, Tsay, & Jen, 2011; Wang, Zhang, Li, Hou, & Zeng, 2004). For instance, pyruvate acid contents could reflect linkage to radical scavenging activity of xanthan oligosaccharides (Xiong et al., 2013). Moreover, polyglucuronic-oxidized
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xanthan and O-acetylated algal polysaccharide showed improvements on hydroxyl radical scavenging activities (Delattre et al., 2015; Wang, Zhang, Yao, Zhao, & Qi, 2013). Figuring out whether and how furfural affects XG substitutions is helpful to understand antioxidant activity change of XG products.
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To address the above issues, the effect of furfural on XG production was studied. Cell growth, product yield, substrate consumption and furfural transformation were
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analyzed in a series of furfural systems. Spectral characteristics and Rheological property of products were evaluated. In addition, total carbohydrate content,
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monosaccharide composition and substitutions (acetyl, pyruvate and glucuronic acid
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groups) contents were determined. Furthermore, antioxidant capacities (hydroxyl
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2. Materials and methods
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radical scavenging and reducing power) of XG products were investigated.
2.1. Strain and inoculum preparation
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The wild-type strain Xanthomonas campestris LRELP-1 was from the Lab of
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Resource and Environmental Microorganism in Sichuan University, Chengdu, China. Yeast peptone (YP) substrate was used as inoculum medium, and seed preparation method was the same as the previous report (Li et al., 2016).
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2.2. XG production The fermentation medium was composed of sucrose 20 g·L–1, peptone 2.4 g·L–1,
K2HPO4 2 g·L–1, MgSO4·7H2O 0.12 g·L–1, CaCO3 3 g·L–1 and citric acid 0.4 g·L–1. Sterile-filtered furfural were added to the culture medium in the final concentrations
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(1, 2, 3, 4 and 5 g·L–1), without furfural as control. Seed broth was inoculated at 10% (v/v). Unless otherwise noted, sterilization was performed by autoclaving at 115°C for 30 min. All the fermentation experiments were carried out in 250 mL Erlenmeyer flasks on a rotary incubator shaker running at 180 rpm and 30°C for 72 h. The
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products from the control and experimental groups were labelled as XG-control, XG1 (1 g·L–1 furfural), XG-2 (2 g·L–1 furfural), XG-3 (3 g·L–1 furfural) and XG-4 (4
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g·L–1 furfural), respectively. Besides, blank media with furfural (same concentrations as experimental groups) and without cells were prepared simultaneously as negative
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controls, in order to exclude the interference of furfural volatilization.
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2.3. Fermentation analysis
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2.3.1. Determination of cell growth and xanthan yield
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Optical density (OD660) was determined to monitor cell growth using a UV-Vis
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spectrophotometer (UV-1800, Mapada, China). XG yield was measured by dry weight estimation. Culture supernatant was mixed with triple volume of 95% ethanol. The
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precipitated XG was collected by centrifugation at 4000 rpm for 15 min. Then the
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precipitate was dissolved in water and re-precipitated in order to eliminate impurities. Finally, the product was dried in a vacuum oven at 50°C.
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2.3.2. Determination of sucrose, furfural and furfuryl alcohol The amounts of sucrose, furfural and furfuryl alcohol were analyzed by high-
performance liquid chromatography (HPLC). Residual sucrose was detected by a HPLC system (Alltech, America) equipped with an evaporative light scattering detector (ELSD) and a Prevail Carbohydrate ES column (5 μm, 250 mm × 4.6 mm,
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Grace Davison Discovery Sciences). The gradient elution mobile phase was water mixed with acetonitrile at the ratios ranged from 0.3:0.7 to 0.5:0.5. The concentrations of surplus furfural and generated furfuryl alcohol were measured by the HPLC with an ultraviolet detector and a C-18 column (5 μm, 250 mm × 4.6 mm, Grace Davison
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Discovery Sciences), using an eluent of acetonitrile and water (15:85) at a flow rate of 1.0 mL min–1. The detection absorption wavelengths were 280 nm and 215 nm for
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furfural and furfuryl alcohol, respectively.
The utilization rate and the conversion rate of sucrose were calculated as Eqs. (1)
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Utilization rate (%) = (S initial – S residual)/S initial × 100
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and (2), respectively.
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Conversion rate (%) = P/(S initial − S residual) × 100
(1) (2)
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where S initial and S residual are initial and residual concentrations of sucrose, g·L–1; P is
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XG yield, g·L–1. 2.3.3. SEM analysis of microbe
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After centrifugation and wash, cells were suspended in fixated solution
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containing 2.5% (v/v) glutaraldehyde, and then dehydrated in a series of gradient ethanol-water solutions (55–100%, v/v). Finally, the fixed cells were dried at room temperature overnight and observed by a scanning electron microscopy (SSX-550,
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Shimadzu, Japan). 2.4. Characterization of products 2.4.1. FT-IR, XRD, 1H NMR and 13C NMR analysis The Fourier transform infrared (FT-IR) spectra were obtained using Nicolet 6700
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FT-IR spectrophotometer (Thermo Scientific, America). All the xanthan products were pressed into KBr pellets and scanned from 400 to 4000 cm−1 with 64 scans/sample with a resolution of 4 waves·cm−1. The X-ray diffractometry (XRD) patterns were performed by a Rigaku D/max-TTR III X-ray diffractometer using Cu
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Kα-radiation generated at 40 kV and 40 mA in the differential angle range (2θ) of 1080°.The proton nuclear magnetic resonance (1H NMR) spectra were determined by a
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Bruker advance 600 MHz spectrometer after dissolving about 20 mg XG in 0.6 mL
deuterated water (D2O). High resolution solid state 13C NMR spectra were obtained
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by cross-polarisation magic angle spinning nuclear magnetic resonance (CPMAS-
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NMR) (advance III 500MHz, Bruker).
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2.4.2. Determination of total carbohydrate content and monosaccharide
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composition
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The contents of total carbohydrates were detected by phenol-sulfuric acid (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Monosaccharide composition was
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determined by gas chromatography (GC) (6890A, Agilent, America). XG was totally
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hydrolyzed with trifluoroacetic acid, and the hydrolysis products were acetylated using acetic anhydride before subjected to GC analysis (Luo et al., 2010).
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2.4.3. Quantitative analysis of pyruvate, acetyl and glucuronic acid contents The contents of pyruvate, acetyl and glucuronic acid groups were analyzed by
chemical methods. The content of pyruvate group attached to XG was measured by 2,4-dinitrophenylhydrazone method (Rong, Lin, & Zhang, 2012). The content of acetyl group was measured by hydroxamic acid method (Mccomb, Mccready, &
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Chem, 1957). The determination of glucuronic acid group was operated using metahydroxydiphenyl assay (Filisetti-Cozzi & Carpita, 1991). 2.4.4. Rheological analysis XG products were dissolved in 0.1 mol·L–1 NaCl (0.4% w/v) and homogenized
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using magnetic stirring overnight at 25°C. then, viscosity was measured with a rotational rheometer (LVDV-1, Jing Tian, China). Meanwhile, USP-grade commercial
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XG (purchased from Aladdin) was analyzed to make a comparison. 2.4.5. Antioxidant activity assay
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Hydroxyl radicals scavenging activities were conducted based on the Fenton
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reaction of Fe2+/H2O2 (Leung, Venus, Zeng, & Tsopmo, 2018). Aqueous solutions
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(0.5-2.0 g·L–1) of XG were incubated with 1 mL of phosphate buffer (0.75 mmol·L–1,
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pH 7.4), 1 mL of 1, 10-phenanthroline (0.75 mmol·L–1), 1 mL of FeSO4 (0.75
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mmol·L–1) and 1 mL of 2% H2O2 for 30 min at 37°C, and then were detected at 510 nm. The scavenging activity was calculated as follow:
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Scavenging ability (%) = (A sample –A control 1)/(A control 2 – A control 1) × 100
(3)
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where A sample is the absorbance in the presence of XG, A control 1 is the absorbance in the absence of XG, and A control 2 is the absorbance in the absence of XG and H2O2. Reducing power was quantified by the reduction of ferric ion to ferrous ion (Fan,
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Li, Deng, & Ai, 2012). 0.5 mL of XG solution and 0.5 of mL potassium ferricyanide (1.0%) were orderly added into 0.5 of mL phosphate buffer (0.2 mol·L–1, pH 6.6). The mixture was placed at 50°C for 20 min, and then was added with 0.5 mL of trichloroacetic acid (10%). After centrifugation at 4°C for 10 min, 0.2 of mL ferric
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chloride (0.1%) was added into 1 mL of supernatant, and the solution was kept at room temperature for 10 min before measuring at 700 nm.
3. Results and discussion
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3.1. Fermentation analysis 3.1.1. Furfural conversion
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As described in Fig. 1a, furfural was consumed completely by X. campestris
LRELP-1 when initial concentrations below 4.3 g·L–1, and transformed to less toxic
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furfuryl alcohol (Zhang et al., 2012). The accumulation amount of furfuryl alcohol at
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the end of fermentation was linearly dependent on the additive amount of furfural, and
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the highest concentration of furfuryl alcohol was 3.62 g·L–1 when 4.3 g·L–1 furfural
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was added. Further increasing the dosage of furfural to 5.4 g·L–1, there was 1.71 g·L–1
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furfural left, and only 2.36 g·L–1 furfuryl alcohol was generated, indicating that the bioconversion capacity of X. campestris reduced. Besides, the interference of furfural
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volatilization during processes could be ignored, since the remaining amount of
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furfural after volatilization (negative control) was nearly equal to the output of furfuryl alcohol when low initial furfural concentrations or the sum of furfural remained and furfuryl alcohol generated when high initial furfural concentration
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(p>0.05, t-test). The results suggested that X. campestris had an excellent ability to tolerate furfural and convert it to furfuryl alcohol within a limit of about 4.3 g·L–1 furfural.
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3.1.2. Cell growth and XG accumulation Furfural affected cell growth of X. campestris (Fig. 1b). Furfural exhibited a stimulation effect on growth at low concentrations. The addition of 1.0–3.2 g·L–1 furfural enhanced cell density, and the highest biomass was increased by 13.3%
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compared to the control (without furfural). The facilitation was contrary to the inhibitory effect conjectured in applications of tapioca pulp and kitchen waste for XG
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production (Gunasekar et al., 2014; Li et al., 2016). The similar promotion phenomenon on microbial cell growth was also observed on Clostridium
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acetobutylicum when furfural concentration was less than 3 g·L–1 (Zhang et al., 2012).
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The facilitation might be ascribed to more supply of nicotinamide adenine
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dinucleotide (phosphate) (NAD(P)H), an indispensable cofactor involved in
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biological detoxification of furfural (Ujor, Agu, Gopalan, & Ezeji, 2014). NAD(P)H
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also plays an important role in cell metabolism. The increasing amount of NAD(P)+ during the detox process might enhance tricarboxylic acid cycle and part of
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glycolysis, thereby improving cell growth (Letisse, Chevallereau, Simon, & Lindley,
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2002). Nevertheless, when further increasing furfural concentration to 4.3 g·L–1, cell density decreased by 13.3% compared to the control. As reported by Almeida, Bertilsson, Gorwa-Grauslund, Gorsich, and Lidén (2009), hexokinase and
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glyceraldehyde-3-phosphate dehydrogenase, two critical enzymes in glycolysis, are vulnerable to furfural. When the adverse effect overwhelmed the stimulation of NAD(P)+, inhibitory effect was observed. When exposed to 5.4 g·L–1 furfural, X. campestris cells could barely grow. From the SEM images (Fig. 2), these cells
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collapsed, while the intact cells presented long rods and smooth edges as observed in the control group. It was demonstrated that cells would suffer serious damage in the circumstance of high furfural concentration. Therefore, suitable furfural concentration
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was beneficial to cell growth but excessive furfural was lethal to X. campestris.
The effect of furfural on XG accumulation is also exhibited in Fig. 1b. XG yield
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maintained over 13 g·L–1 when below 3 g·L–1 furfural, but significantly declined
beyond 3 g·L–1 furfural (p<0.05, t-test). Even no xanthan product was collected when
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5.4 g·L–1 furfural was added. In terms of XG yield per unit cells (expressed as g
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XG/OD660), the value continuously descended along with increasing furfural
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concentration. Thus, furfural ought to be a limiting factor to XG accumulation. The
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inhibition might be due to the undesirable interference of furan to hexokinase and
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glyceraldehyde-3-phosphate dehydrogenase, which were crucial for xanthan biosynthesis (Letisse et al., 2002). Owing to improved cell growth, there was a steady
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output of XG below 3 g·L–1 furfural.
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3.1.3. Sugar consumption
The consumption of sucrose is illustrated in Fig. 3. Under various levels of
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furfural, residual sucrose contents ranged from 0.47 to 17.65 g·L–1. The least residual sucrose and the highest utilization rate of 97.6% occurred at the addition of 2.2 g·L–1 furfural. Accordingly, the maximum cell density was obtained in the condition (Fig. 1b), as active cell proliferation consumed these carbon nutrients. Moreover, the
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utilized carbon source was converted to XG product, and the conversion rate kept above 70% when furfural concentration below 4 g·L–1. The conversion rates were superior compared to literature reports (Table 1). Further, the conversion rate decreased to 58.6% at 4.3 g·L–1 furfural, accompanied with the highest level of
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transformational furfuryl alcohol (Fig. 1a). It was inferred that biological detoxification of furfural also consumed carbon source. The speculation was
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supported by detected sucrose utilization at 5.4 g·L–1 furfural. Although neither cell
growth nor XG product was observed, 9% of carbon consumption might be used for
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detoxification of furfural. Similar phenomenon was also found in ethanol
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fermentation (Navarro, 1994). In conclusion, carbon source was consumed for cell
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growth, polysaccharide accumulation, and furfural detoxification.
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3.2. Structure and rheological characterization of XG products 3.2.1. Spectral characteristics Fig. 4a exhibits the FT-IR spectra of XG products. The strong broad absorption peak observed at 3405-3423 cm-1 was owing to the stretching frequency of O–H
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group. The C–H stretching absorption peak was located at 2924-2926 cm-1. The absorption peaks at 1727-1729 cm-1 and 1609-1619 cm-1 could be attributed to the
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stretching of carbonyl group. The vibration peak around 1064 cm-1 was due to acetyl group. Deflection angle of C–H was found at 1404-1414 cm-1. The presence of
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absorption peak around 1249 cm-1 could be attributed to C–O–C group and the peak
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around 604 cm-1 was due to C–H bending vibration. Above all, the FT-IR peaks of XG
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produced under different furfural concentrations appeared at the same positions,
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which also matched with the previous report (Li et al., 2016).
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The XRD patterns of biosynthetic XG are described in Fig. 4b. Similar broad diffraction peaks were observed in all products from diverse furfural concentrations.
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The results suggested that these XG products were amorphous, in accordance with Li
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et al. (2016).
The 1H NMR results are shown in Fig. 4c. The peaks appearing at around 1.3
ppm and 2.0 ppm corresponded to acetate and pyruvate groups, respectively.
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Glucuronic acid of hexose or pentose located at 3.5 ppm. Hydroxyl group of xanthan was found around 4.0 ppm (Gunasekar et al., 2014). The evident peaks at 4.7 ppm were due to D2O. The solid state 13C CPMAS NMR results are listed in Fig. 4d. Although the
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peaks were broad like Abbaszadeh et al. (2015), the discernible peaks around 78 ppm and 116 ppm were attributable to C1 and the rest carbon of XG components, respectively. The signal around 28 ppm was from methyl of acetyl and pyruvate, while the signal at 180 ppm was due to carbonyl (Horton, Mols, Walaszek, & Wernau,
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1985). Combining with these FT-IR, XRD and NMR results, the fermentation products
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in this study could be confirmed as XG. Additional furfural into the culture had no
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impact on the primary structure of XG products.
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3.2.2. Polysaccharide structure
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The total carbohydrate content and monosaccharide composition of XG products from furfural-contained media are shown in Table 2. The total carbohydrate contents of
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all products were around 80%, and predominant monosaccharides were glucose and
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mannose. Moreover, the molar ratios of glucose, mannose and glucuronic acid were close to the theoretic value of 2.0:2.0:1.0 (Faria et al., 2011; Wang, Wu, Zhu, & Zhan,
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2017). Combined with Garcíaochoa et al. (2000), the molecular structural formula of
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XG was concluded as Fig 5a.
Glucuronic acid group is one of main components of XG pentasaccharide units.
As exhibited in Fig. 5b, the maximum content of glucuronic acid (14.55%) was from XG-1, and furfural concentration below 2.2 g·L–1 presented advantage on glucuronic acid content. Glucuronic acid group was closely related to uridine diphosphate 16
(UDP)-glucose dehydrogenase, the NAD(P)+/--relied enzyme of catalyzing the oxidation of UDP-glucose to UDP-glucuronic acid (Blanch, Legaz, & Vicente, 2008; Campbell, Sala, Van, & Tanner, 1997). The bioconversion of furfural to furfuryl alcohol could stimulate more NAD(P)+/- as discussed above, thereby leading to the
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improvement of glucuronic acid contents of XG-1 and XG-2. However, furfural over 2.2 g·L–1 decreased the number of glucuronic acid group, and the downward trend
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might be resulted from lowered activity of UDP-glucose dehydrogenase.
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The amounts of acetyl and pyruvate groups in XG are variable according to
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culture conditions (Faria et al., 2011). Acetyl group is the internal substituent of
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mannose while pyruvate group located at the terminal of mannose. As shown in Fig.
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5b, the acetyl contents gradually decreased from 4.41% to 2.54% with the increase of
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furfural concentration from 0 g·L–1 to 4.3 g·L–1. On the other side, the pyruvate contents first went up and then a little dropped beyond 3.2 g·L–1 furfural. The
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maximum pyruvate content of 6.43% was observed from XG-3, and the value of XG-
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4 decreased to 5.84% which was still 28.92% higher than XG-control. Increased pyruvate contents and decreased acetyl contents of XG could be attributed to pyruvate retention in glycolysis. Pyruvate dehydrogenase links pyruvate and acetyl-CoA in
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central carbon metabolism, but it is sensitive to furfural in vitro (Akobi et al., 2016; Modig & Liden GTaherzadeh, 2002). Under appropriate furfural environment, lowered pyruvate dehydrogenase activity brought about more pyruvate groups in XG. Additionally, slight decrease of XG-4 might be caused by the profound suppression of
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furfural to glycolysis as discussed in cell growth. Furthermore, acetyl group is related to intramolecular association (Fitzpatrick, Meadows, Ratcliffe, & Williams, 2013), and pyruvate-rich XG tends to disorder its own ordered form (Li & Feke, 2015). Thus, decreased acetyl group and increased pyruvate group could make XG more
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flexible and easier to associate, which could offer XG products a wider application (Izawa et al., 2014). It indicated that XG products from furfural-contained systems
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might be popular. 3.2.3. Rheological analysis
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Rheological parameters of XG may be influenced by fermentation processes
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(Faria et al., 2011). As seen from Fig. 6, the viscosities of XG products from furfural-
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contained environments exhibited a similar change trend to commercial XG. All XG
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solutions showed decreased apparent viscosity along with increased shear rate, and
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the pseudoplastic behavior is typical of polymers with high molecular weight (Miranda et al., 2018). Moreover, the products with furfural had slightly lower
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viscosities than the control without furfural. The variation might be caused by the
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different contents of acetate and pyruvate groups, since the substituents can strongly influence rheological properties of XG (Li & Feke, 2015; Tako & Nakamura, 1984). In general, rheological property of XG products from furfural-contained systems was
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a little weaker than that of commercial XG but still higher than those from some other substrates, such as glycerol (Wang et al., 2017).
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3.3. Antioxidant activities of XG products Xanthan possesses protective effect against hydroxyl radical (•OH) which is the most harmful reactive oxygen species (ROS) to biological tissues (Wu et al., 2013). Fig. 7a depicts the hydroxyl radical scavenging effects of XG products from furfural-
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contained systems. As shown, all XG had the ability for scavenging hydroxyl radical, and the scavenging effect depended on the concentration. XG-1 exhibited the
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strongest scavenging ability, while XG-4 had the lowest scavenging ability.
Spasojević et al. (2009) suggested that monosaccharide levels played an important
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role in scavenging abilities of hydroxyl radical, but the abilities of main components
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of XG (glucose and mannose) were much alike. Besides, polyglucuronic acid sodium
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salt, pyruvate acid and O–acetylation of xanthan were found to be beneficial to the
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property in vitro (Delattre et al., 2015; Wang et al., 2013; Xiong et al., 2013). On the
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basis, the varied contents of pyruvate, acetyl and glucuronic acid groups might be responsible for the difference of hydroxyl radical scavenging abilities. Among the
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three groups, glucuronic acids were the biggest contributor, for the hydroxyl radical
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scavenging ability displayed a similar changing trend with the content of glucuronic acids (Fig. 5b). Overall, 1 g·L–1 furfural addition enhanced the hydroxyl radical
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scavenging ability of XG product.
Reducing power evaluates the ability of donating electrons, and is one of the significant indicators of potential antioxidant activities (Xiong et al., 2013). From Fig. 7b, all XG products exhibited reducing powers, and showed no distinction (p>0.05, t-
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test). Reductones are deemed to be related to reducing property, which functions by donating hydrogen atoms (Gordon, 1990). Nevertheless, main groups of XG do not belong to reductones, and pyruvate acid group had been demonstrated to have no connection with reducing power (Delattre et al., 2015). In addition, reducing power
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could be influenced by polysaccharide monomers (Lo et al., 2011), but the monosaccharide compositions of XG products were similar. Thereby, the addition of
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furfural exerted no impacts on reducing powers of XG products.
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4. Conclusion
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The effects of furfural on XG production were systematically evaluated. During
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XG fermentation, X. campestris LRELP-1 could convert furfural to furfuryl alcohol.
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Although furfural inhibited XG accumulation, below 3.2 g·L–1 of furfural showed
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stimulation on cell growth and had stable XG yields above 13 g·L–1. Moreover, the addition of furfural reduced acetyl contents but rose pyruvate contents, and glucuronic
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acid contents increased first and then decreased with increasing furfural
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concentration. Accordingly, XG-1 showed the highest hydroxyl radical scavenging effect. The results provided guidance to the application of lignocellulosic-like raw
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materials in polysaccharide fermentation.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant number 21506132) and the Basic Application Program of Department of
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Science and Technology of Sichuan Province (Grant No. 2015JY0241).
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Mccomb, E. A., Mccready, R. M., & Chem, A. (1957). Determination of ccetyl in pectin and in acetylated carbohydrate polymers. Analytical Chemistry, 29(5), 819-821. Miranda, A. L., Costa, S. S., Assis, D. D. J., Andrade, B. B., Souza, C. O. D., Guimarães, A. G., & Druzian, J. I. (2018). Investigation of cellular fatty acid composition of Xanthomonas spp. as chemical markers of productivity and quality of xanthan gum. Carbohydrate Polymers, 192, S0144861718303424. Modig, T., & Liden GTaherzadeh, M. J. (2002). Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochemical Journal, 363(3), 769-776. Munish, A., Ashok, K., & Kuldeep, S. (2012). Synthesis, characterization and in vitro release behavior of
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carboxymethyl xanthan. International Journal of Biological Macromolecules, 51(5), 1086-1090. Navarro, A. R. (1994). Effects of furfural on ethanol fermentation by Saccharomyces cerevisiae : Mathematical models. Current Microbiology, 29(2), 87-90.
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for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. Journal of Biotechnology, 134(1–2), 112-120.
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Rong, C., Lin, L., & Zhang, Y. (2012). Hydrogen peroxide (H2O2 ) supply significantly improves xanthan Microbiology & Biotechnology, 39(5), 799-803.
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gum production mediated by Xanthomonas campestris in vitro. Journal of Industrial Song, K. W., Kim, Y. S., & Chang, G. S. (2006). Rheology of concentrated xanthan gum solutions: Steady
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Tako, M., & Nakamura, S. (1984). Rheological properties of deacetylated xanthan in aqueous media. Journal of the Agricultural Chemical Society of Japan, 48(12), 2987-2993. Ujor, V., Agu, C. V., Gopalan, V., & Ezeji, T. C. (2014). Glycerol supplementation of the growth medium
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enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation. Applied Microbiology and Biotechnology, 98(14), 6511-6521. Verhoeven, E., Vervaet, C., & Remon, J. P. (2006). Xanthan gum to tailor drug release of sustained-release
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ethylcellulose mini-matrices prepared via hot-melt extrusion: in vitro and in vivo evaluation. European Journal of Pharmaceutics and Biopharmaceutics, 63(3), 320-330.
Wang, X., Zhang, Z., Yao, Q., Zhao, M., & Qi, H. (2013). Phosphorylation of low-molecular-weight polysaccharide from Enteromorpha linza with antioxidant activity. Carbohydrate Polymers, 96(2), 371-375.
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Wang, Y., Zhang, L., Li, Y., Hou, X., & Zeng, F. (2004). Correlation of structure to antitumor activities of five derivatives of a β-glucan from Poria cocos sclerotium. Carbohydrate Research, 339(15), 2567-2574.
Wang, Z., Wu, J., Zhu, L., & Zhan, X. (2016). Activation of glycerol metabolism in Xanthomonas campestris by adaptive evolution to produce a high-transparency and low-viscosity xanthan gum from glycerol. Bioresource Technology, 211, 390-397. Wang, Z., Wu, J., Zhu, L., & Zhan, X. (2017). Characterization of xanthan gum produced from glycerol by a mutant strain Xanthomonas campestris CCTCC M2015714. Carbohydrate Polymers, 157, 521-
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526. Wu, S. J., Wu, J. H., Xia, L. Z., Chu, C., Liu, D., & Gong, M. (2013). Preparation of xanthan-derived oligosaccharides and their hydroxyl radical scavenging activity. Carbohydrate Polymers, 92(2), 1612-1614. Xiong, X., Li, M., Xie, J., Jin, Q., Xue, B., & Sun, T. (2013). Antioxidant activity of xanthan oligosaccharides prepared by different degradation methods. Carbohydrate Polymers, 92(2), 1166-1171. Zhang, Y., Han, B., & Ezeji, T. C. (2012). Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation. New Biotechnology,
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29(3), 345-351.
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Fig. 1. Concentrations of residual furfural and generated furfuryl alcohol (a), cell
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growth, product yield and product content per unit cells (g XG/OD660) (b) under
various furfural concentrations. Error bars indicate the standard deviation of the mean
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(n=3).
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Fig. 2. SEM images of damaged cells in 5.4 g·L–1 furfural (a) and intact cells in
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control (b).
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Fig. 3. Sucrose consumption and utilization\conversion rate of sucrose under various
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furfural concentrations. Error bars indicate the standard deviation of the mean (n=3).
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Fig. 4. FT-IR spectra (a), XRD patterns (b), 1H NMR spectra (c) and 13C NMR spectra
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(d) of XG products from various furfural concentrations.
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Fig. 5. Molecular structural formula of XG (a), and glucuronic acid, acetyl and
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pyruvate contents of XG products from furfural-contained media (b). Error bars
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indicate the standard deviation of the mean (n=3).
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Fig. 6. Viscosity curves of XG products from various furfural concentrations and
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commercial XG.
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Fig. 7. Hydroxyl radical scavenging effects (a) and reducing powers (b) of XG
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products from various furfural additions.
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Table 1 Comparison of the conversion rate of carbon source to XG Carbon source
XG yield
Conversion rate of
source
concentration (g·L–1)
(g·L–1)
carbon source (%)
Sucrose
40
23.2
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Reference
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Carbon
(Wang, Wu, Zhu, &
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Zhan, 2016)
40
11.0
27.5
(Wang et al., 2016)
Sucrose
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15.8
63.2
(Faria, Vieira,
Sucrose
20
34
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50
14.0
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Sucrose
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Glycerol
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Resende, França, & Cardoso, 2009)
84 (in fermentor)
(Rong et al., 2012)
75.2
This work
Table 2 Total sugar content and monosaccharide composition of XG products under various furfural concentrations Molar ratio of glucose: mannose:
content (%)
glucuronic acid
XG-control
80.2±0.4
2:1.96:0.92
XG-1
78.9±1.2
2:1.87:0.94
XG-2
79.5±0.6
XG-3
79.1±3.4
XG-4
77.9±0.4
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Total carbohydrate
2:1.86:0.91
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2:1.88:0.82 2:1.72:0.77
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XG product
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S0144-8617(19)30402-3 https://doi.org/10.1016/j.carbpol.2019.04.018 CARP 14792
To appear in: Received date: Revised date: Accepted date:
5 January 2019 15 March 2019 3 April 2019
Please cite this article as: Kang Y, Li P, Zeng X, Chen X, Xie Y, Zeng Y, Zhang Y, Xie T, Biosynthesis, structure and antioxidant activities of xanthan gum from Xanthomonas campestris with additional furfural, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biosynthesis, structure and antioxidant activities of xanthan gum from Xanthomonas campestris with additional furfural
Yan Kanga, Panyu Lia, Xiaotong Zenga, Xi Chena, Yi Xiea, Yu Zenga, Yongkui
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Zhanga,*, Tonghui Xiea,*
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Department of Pharmaceutical & Biological Engineering, School of Chemical
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Engineering, Sichuan University, Chengdu, Sichuan 610065, China
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Tonghui Xie, Yongkui Zhang
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Corresponding author
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Tel.: +86 28 85408255/Fax: +86 28 85403397
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Authors
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E-mail: [email protected] (T. Xie), [email protected] (Y. Zhang)
Yan Kang, [email protected] Panyu Li, [email protected]
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Xiaotong Zeng, [email protected] Xi Chen, [email protected] Yi Xie, [email protected] Yu Zeng, [email protected]
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Graphical abstract
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Highlights
The effect of additional furfural on xanthan gum production was explored.
Low concentration of furfural promoted cell growth and kept steady xanthan
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yield.
Furfural altered acetyl, pyruvate and glucuronic acid contents of xanthan
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products.
1 g/L of furfural provided product a desirable hydroxyl radical scavenging effect.
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Abstract: Lignocellulosic-like materials are potentially low-cost fermentation substrates, but their pretreatment brings about by-products. This work investigated the effects of furfural on xanthan gum (XG) production, and product quality was evaluated by
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structure, viscosity and antioxidant capacities. Xanthomonas campestris maintained steady polysaccharide yield (above 13 g·L–1) with enhanced cell growth at low
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furfural concentrations (below 3.2 g·L–1). The products were verified as XG by FT-IR, XRD, NMR and monosaccharide analysis. Moreover, they were found to have
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reduced acetyl, rising pyruvate and up-to-down glucuronic acid groups as increasing
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furfural concentration. Furthermore, XG product with 1 g·L–1 furfural addition
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showed the best hydroxyl scavenging effects, though reducing powers presented no
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variation. It was demonstrated that furfural, the common hydrolysis by-product, was
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not necessarily an inhibitor for fermentation, and an appropriate amount of furfural
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was beneficial to XG production with steady yield and good quality.
Keywords: Furfural; Xanthan gum; Xanthomonas campestris; antioxidant activity;
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polysaccharide structure
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Introduction Xanthan gum (XG) is a water-soluble heteropolysaccharide, formed by repeated
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pentasaccharide units consisting of glucose, mannose, and glucuronic acid (Garcíaochoa, Santos, Casas, & Gómez, 2000; Munish, Ashok, & Kuldeep, 2012). XG is widely applied in food, pharmaceutical, cosmetic, oil and textile industries because of its superior properties, such as excellent solubility, high viscosity at low
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concentrations, compatibility, stability, etc. (Jang, Zhang, Bo, & Choi, 2015; Song, Kim, & Chang, 2006; Verhoeven, Vervaet, & Remon, 2006). Especially, antioxidant
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activity of XG has attracted increasing attention, since health care products with antioxidation are popular in the last few decades (Gawlik, 2012).
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XG industry faces a challenge of high production cost. For example, traditional
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fermentation carbon sources (mainly glucose and sucrose) are expensive (Li et al.,
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2017). Low-cost substrates like lignocellulose are attractive for the potential to supply
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mixed sugars. Some biomass materials have been successfully adopted to XG
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fermentation, including tapioca pulp (Gunasekar, Reshma, Treesa, Gowdhaman, & Ponnusami, 2014), sugar cane broth (Faria et al., 2011), rice bran (Demirci, Arici, &
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Gumus, 2012), kitchen waste (Li et al., 2017) and so on. In order to solve the problem
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that microbe cannot effectively utilize organic macromolecules, it is necessary to hydrolyze biomass materials before fermentation. Among alternative pretreatment methods, acid hydrolysis is most commonly used on account of the advantages of
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high efficiency and low price, although its by-products furan derivatives are regarded as microbial metabolism inhibitors (Lin et al., 2015). In this context, it is important to investigate the effect of probable inhibitor on XG production. Furfural, the by-product released from Maillard reaction, is noticed in acid-
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converted dehydration of lignocellulosic biomass (Navarro, 1994). Its usual concentrations were found to be 1.5–3 g·L–1 during acidic hydrolysis of tapioca pulp (Gunasekar et al., 2014), wheat straw (Olofsson, Rudolf, & Lidén, 2008), oil palm empty (Rahman, Choudhury, & Ahmad, 2006) and other biomass waste (Cuevas,
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Quero, Hodaifa, López, & Sánchez, 2014). When utilizing tapioca pulp and kitchen waste in XG production, it failed to obtain expected xanthan yield as increasing
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hydrolysate nutrient (Gunasekar et al., 2014; Li et al., 2016). According to the
observed inhibiting effect of furfural on ethanol and hydrogen productions (Lin et al.,
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2015; Navarro, 1994), it was inferred that the detected furfural in culture was the
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limiting factor to XG production. However, the stimulation for acetone-butanol-
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ethanol fermentation by furfural was also found as a promoter (Zhang, Han, & Ezeji,
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2012). Besides, current studies about the influence of furfural on fermentation paid
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close attention to production output (Akobi, Hafez, & Nakhla, 2016; Zhang et al., 2012), and neglected biological activities of products. However, XG serves as a
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potential antioxidant and the information of its antioxidant activities is important.
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Thus, the characterization of additional furfural on XG production and product quality needs to be systematically studied for the utilization of renewable biomass. The biological activities of polysaccharides are highly associated with their
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structure, such as monosaccharide composition and substitutions (Chen et al., 2014; Lo, Cheng, Chiu, Tsay, & Jen, 2011; Wang, Zhang, Li, Hou, & Zeng, 2004). For instance, pyruvate acid contents could reflect linkage to radical scavenging activity of xanthan oligosaccharides (Xiong et al., 2013). Moreover, polyglucuronic-oxidized
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xanthan and O-acetylated algal polysaccharide showed improvements on hydroxyl radical scavenging activities (Delattre et al., 2015; Wang, Zhang, Yao, Zhao, & Qi, 2013). Figuring out whether and how furfural affects XG substitutions is helpful to understand antioxidant activity change of XG products.
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To address the above issues, the effect of furfural on XG production was studied. Cell growth, product yield, substrate consumption and furfural transformation were
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analyzed in a series of furfural systems. Spectral characteristics and Rheological property of products were evaluated. In addition, total carbohydrate content,
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monosaccharide composition and substitutions (acetyl, pyruvate and glucuronic acid
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groups) contents were determined. Furthermore, antioxidant capacities (hydroxyl
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2. Materials and methods
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radical scavenging and reducing power) of XG products were investigated.
2.1. Strain and inoculum preparation
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The wild-type strain Xanthomonas campestris LRELP-1 was from the Lab of
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Resource and Environmental Microorganism in Sichuan University, Chengdu, China. Yeast peptone (YP) substrate was used as inoculum medium, and seed preparation method was the same as the previous report (Li et al., 2016).
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2.2. XG production The fermentation medium was composed of sucrose 20 g·L–1, peptone 2.4 g·L–1,
K2HPO4 2 g·L–1, MgSO4·7H2O 0.12 g·L–1, CaCO3 3 g·L–1 and citric acid 0.4 g·L–1. Sterile-filtered furfural were added to the culture medium in the final concentrations
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(1, 2, 3, 4 and 5 g·L–1), without furfural as control. Seed broth was inoculated at 10% (v/v). Unless otherwise noted, sterilization was performed by autoclaving at 115°C for 30 min. All the fermentation experiments were carried out in 250 mL Erlenmeyer flasks on a rotary incubator shaker running at 180 rpm and 30°C for 72 h. The
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products from the control and experimental groups were labelled as XG-control, XG1 (1 g·L–1 furfural), XG-2 (2 g·L–1 furfural), XG-3 (3 g·L–1 furfural) and XG-4 (4
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g·L–1 furfural), respectively. Besides, blank media with furfural (same concentrations as experimental groups) and without cells were prepared simultaneously as negative
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controls, in order to exclude the interference of furfural volatilization.
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2.3. Fermentation analysis
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2.3.1. Determination of cell growth and xanthan yield
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Optical density (OD660) was determined to monitor cell growth using a UV-Vis
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spectrophotometer (UV-1800, Mapada, China). XG yield was measured by dry weight estimation. Culture supernatant was mixed with triple volume of 95% ethanol. The
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precipitated XG was collected by centrifugation at 4000 rpm for 15 min. Then the
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precipitate was dissolved in water and re-precipitated in order to eliminate impurities. Finally, the product was dried in a vacuum oven at 50°C.
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2.3.2. Determination of sucrose, furfural and furfuryl alcohol The amounts of sucrose, furfural and furfuryl alcohol were analyzed by high-
performance liquid chromatography (HPLC). Residual sucrose was detected by a HPLC system (Alltech, America) equipped with an evaporative light scattering detector (ELSD) and a Prevail Carbohydrate ES column (5 μm, 250 mm × 4.6 mm,
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Grace Davison Discovery Sciences). The gradient elution mobile phase was water mixed with acetonitrile at the ratios ranged from 0.3:0.7 to 0.5:0.5. The concentrations of surplus furfural and generated furfuryl alcohol were measured by the HPLC with an ultraviolet detector and a C-18 column (5 μm, 250 mm × 4.6 mm, Grace Davison
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Discovery Sciences), using an eluent of acetonitrile and water (15:85) at a flow rate of 1.0 mL min–1. The detection absorption wavelengths were 280 nm and 215 nm for
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furfural and furfuryl alcohol, respectively.
The utilization rate and the conversion rate of sucrose were calculated as Eqs. (1)
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Utilization rate (%) = (S initial – S residual)/S initial × 100
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and (2), respectively.
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Conversion rate (%) = P/(S initial − S residual) × 100
(1) (2)
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where S initial and S residual are initial and residual concentrations of sucrose, g·L–1; P is
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XG yield, g·L–1. 2.3.3. SEM analysis of microbe
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After centrifugation and wash, cells were suspended in fixated solution
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containing 2.5% (v/v) glutaraldehyde, and then dehydrated in a series of gradient ethanol-water solutions (55–100%, v/v). Finally, the fixed cells were dried at room temperature overnight and observed by a scanning electron microscopy (SSX-550,
A
Shimadzu, Japan). 2.4. Characterization of products 2.4.1. FT-IR, XRD, 1H NMR and 13C NMR analysis The Fourier transform infrared (FT-IR) spectra were obtained using Nicolet 6700
8
FT-IR spectrophotometer (Thermo Scientific, America). All the xanthan products were pressed into KBr pellets and scanned from 400 to 4000 cm−1 with 64 scans/sample with a resolution of 4 waves·cm−1. The X-ray diffractometry (XRD) patterns were performed by a Rigaku D/max-TTR III X-ray diffractometer using Cu
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Kα-radiation generated at 40 kV and 40 mA in the differential angle range (2θ) of 1080°.The proton nuclear magnetic resonance (1H NMR) spectra were determined by a
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Bruker advance 600 MHz spectrometer after dissolving about 20 mg XG in 0.6 mL
deuterated water (D2O). High resolution solid state 13C NMR spectra were obtained
U
by cross-polarisation magic angle spinning nuclear magnetic resonance (CPMAS-
N
NMR) (advance III 500MHz, Bruker).
A
2.4.2. Determination of total carbohydrate content and monosaccharide
M
composition
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The contents of total carbohydrates were detected by phenol-sulfuric acid (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Monosaccharide composition was
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determined by gas chromatography (GC) (6890A, Agilent, America). XG was totally
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hydrolyzed with trifluoroacetic acid, and the hydrolysis products were acetylated using acetic anhydride before subjected to GC analysis (Luo et al., 2010).
A
2.4.3. Quantitative analysis of pyruvate, acetyl and glucuronic acid contents The contents of pyruvate, acetyl and glucuronic acid groups were analyzed by
chemical methods. The content of pyruvate group attached to XG was measured by 2,4-dinitrophenylhydrazone method (Rong, Lin, & Zhang, 2012). The content of acetyl group was measured by hydroxamic acid method (Mccomb, Mccready, &
9
Chem, 1957). The determination of glucuronic acid group was operated using metahydroxydiphenyl assay (Filisetti-Cozzi & Carpita, 1991). 2.4.4. Rheological analysis XG products were dissolved in 0.1 mol·L–1 NaCl (0.4% w/v) and homogenized
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using magnetic stirring overnight at 25°C. then, viscosity was measured with a rotational rheometer (LVDV-1, Jing Tian, China). Meanwhile, USP-grade commercial
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XG (purchased from Aladdin) was analyzed to make a comparison. 2.4.5. Antioxidant activity assay
U
Hydroxyl radicals scavenging activities were conducted based on the Fenton
N
reaction of Fe2+/H2O2 (Leung, Venus, Zeng, & Tsopmo, 2018). Aqueous solutions
A
(0.5-2.0 g·L–1) of XG were incubated with 1 mL of phosphate buffer (0.75 mmol·L–1,
M
pH 7.4), 1 mL of 1, 10-phenanthroline (0.75 mmol·L–1), 1 mL of FeSO4 (0.75
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mmol·L–1) and 1 mL of 2% H2O2 for 30 min at 37°C, and then were detected at 510 nm. The scavenging activity was calculated as follow:
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Scavenging ability (%) = (A sample –A control 1)/(A control 2 – A control 1) × 100
(3)
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where A sample is the absorbance in the presence of XG, A control 1 is the absorbance in the absence of XG, and A control 2 is the absorbance in the absence of XG and H2O2. Reducing power was quantified by the reduction of ferric ion to ferrous ion (Fan,
A
Li, Deng, & Ai, 2012). 0.5 mL of XG solution and 0.5 of mL potassium ferricyanide (1.0%) were orderly added into 0.5 of mL phosphate buffer (0.2 mol·L–1, pH 6.6). The mixture was placed at 50°C for 20 min, and then was added with 0.5 mL of trichloroacetic acid (10%). After centrifugation at 4°C for 10 min, 0.2 of mL ferric
10
chloride (0.1%) was added into 1 mL of supernatant, and the solution was kept at room temperature for 10 min before measuring at 700 nm.
3. Results and discussion
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3.1. Fermentation analysis 3.1.1. Furfural conversion
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As described in Fig. 1a, furfural was consumed completely by X. campestris
LRELP-1 when initial concentrations below 4.3 g·L–1, and transformed to less toxic
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furfuryl alcohol (Zhang et al., 2012). The accumulation amount of furfuryl alcohol at
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the end of fermentation was linearly dependent on the additive amount of furfural, and
A
the highest concentration of furfuryl alcohol was 3.62 g·L–1 when 4.3 g·L–1 furfural
M
was added. Further increasing the dosage of furfural to 5.4 g·L–1, there was 1.71 g·L–1
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furfural left, and only 2.36 g·L–1 furfuryl alcohol was generated, indicating that the bioconversion capacity of X. campestris reduced. Besides, the interference of furfural
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volatilization during processes could be ignored, since the remaining amount of
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furfural after volatilization (negative control) was nearly equal to the output of furfuryl alcohol when low initial furfural concentrations or the sum of furfural remained and furfuryl alcohol generated when high initial furfural concentration
A
(p>0.05, t-test). The results suggested that X. campestris had an excellent ability to tolerate furfural and convert it to furfuryl alcohol within a limit of about 4.3 g·L–1 furfural.
11
3.1.2. Cell growth and XG accumulation Furfural affected cell growth of X. campestris (Fig. 1b). Furfural exhibited a stimulation effect on growth at low concentrations. The addition of 1.0–3.2 g·L–1 furfural enhanced cell density, and the highest biomass was increased by 13.3%
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compared to the control (without furfural). The facilitation was contrary to the inhibitory effect conjectured in applications of tapioca pulp and kitchen waste for XG
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production (Gunasekar et al., 2014; Li et al., 2016). The similar promotion phenomenon on microbial cell growth was also observed on Clostridium
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acetobutylicum when furfural concentration was less than 3 g·L–1 (Zhang et al., 2012).
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The facilitation might be ascribed to more supply of nicotinamide adenine
A
dinucleotide (phosphate) (NAD(P)H), an indispensable cofactor involved in
M
biological detoxification of furfural (Ujor, Agu, Gopalan, & Ezeji, 2014). NAD(P)H
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also plays an important role in cell metabolism. The increasing amount of NAD(P)+ during the detox process might enhance tricarboxylic acid cycle and part of
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glycolysis, thereby improving cell growth (Letisse, Chevallereau, Simon, & Lindley,
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2002). Nevertheless, when further increasing furfural concentration to 4.3 g·L–1, cell density decreased by 13.3% compared to the control. As reported by Almeida, Bertilsson, Gorwa-Grauslund, Gorsich, and Lidén (2009), hexokinase and
A
glyceraldehyde-3-phosphate dehydrogenase, two critical enzymes in glycolysis, are vulnerable to furfural. When the adverse effect overwhelmed the stimulation of NAD(P)+, inhibitory effect was observed. When exposed to 5.4 g·L–1 furfural, X. campestris cells could barely grow. From the SEM images (Fig. 2), these cells
12
collapsed, while the intact cells presented long rods and smooth edges as observed in the control group. It was demonstrated that cells would suffer serious damage in the circumstance of high furfural concentration. Therefore, suitable furfural concentration
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was beneficial to cell growth but excessive furfural was lethal to X. campestris.
The effect of furfural on XG accumulation is also exhibited in Fig. 1b. XG yield
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maintained over 13 g·L–1 when below 3 g·L–1 furfural, but significantly declined
beyond 3 g·L–1 furfural (p<0.05, t-test). Even no xanthan product was collected when
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5.4 g·L–1 furfural was added. In terms of XG yield per unit cells (expressed as g
N
XG/OD660), the value continuously descended along with increasing furfural
A
concentration. Thus, furfural ought to be a limiting factor to XG accumulation. The
M
inhibition might be due to the undesirable interference of furan to hexokinase and
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glyceraldehyde-3-phosphate dehydrogenase, which were crucial for xanthan biosynthesis (Letisse et al., 2002). Owing to improved cell growth, there was a steady
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output of XG below 3 g·L–1 furfural.
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3.1.3. Sugar consumption
The consumption of sucrose is illustrated in Fig. 3. Under various levels of
A
furfural, residual sucrose contents ranged from 0.47 to 17.65 g·L–1. The least residual sucrose and the highest utilization rate of 97.6% occurred at the addition of 2.2 g·L–1 furfural. Accordingly, the maximum cell density was obtained in the condition (Fig. 1b), as active cell proliferation consumed these carbon nutrients. Moreover, the
13
utilized carbon source was converted to XG product, and the conversion rate kept above 70% when furfural concentration below 4 g·L–1. The conversion rates were superior compared to literature reports (Table 1). Further, the conversion rate decreased to 58.6% at 4.3 g·L–1 furfural, accompanied with the highest level of
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transformational furfuryl alcohol (Fig. 1a). It was inferred that biological detoxification of furfural also consumed carbon source. The speculation was
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supported by detected sucrose utilization at 5.4 g·L–1 furfural. Although neither cell
growth nor XG product was observed, 9% of carbon consumption might be used for
U
detoxification of furfural. Similar phenomenon was also found in ethanol
N
fermentation (Navarro, 1994). In conclusion, carbon source was consumed for cell
A
CC E
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ED
M
A
growth, polysaccharide accumulation, and furfural detoxification.
14
3.2. Structure and rheological characterization of XG products 3.2.1. Spectral characteristics Fig. 4a exhibits the FT-IR spectra of XG products. The strong broad absorption peak observed at 3405-3423 cm-1 was owing to the stretching frequency of O–H
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group. The C–H stretching absorption peak was located at 2924-2926 cm-1. The absorption peaks at 1727-1729 cm-1 and 1609-1619 cm-1 could be attributed to the
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stretching of carbonyl group. The vibration peak around 1064 cm-1 was due to acetyl group. Deflection angle of C–H was found at 1404-1414 cm-1. The presence of
U
absorption peak around 1249 cm-1 could be attributed to C–O–C group and the peak
N
around 604 cm-1 was due to C–H bending vibration. Above all, the FT-IR peaks of XG
A
produced under different furfural concentrations appeared at the same positions,
M
which also matched with the previous report (Li et al., 2016).
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The XRD patterns of biosynthetic XG are described in Fig. 4b. Similar broad diffraction peaks were observed in all products from diverse furfural concentrations.
PT
The results suggested that these XG products were amorphous, in accordance with Li
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et al. (2016).
The 1H NMR results are shown in Fig. 4c. The peaks appearing at around 1.3
ppm and 2.0 ppm corresponded to acetate and pyruvate groups, respectively.
A
Glucuronic acid of hexose or pentose located at 3.5 ppm. Hydroxyl group of xanthan was found around 4.0 ppm (Gunasekar et al., 2014). The evident peaks at 4.7 ppm were due to D2O. The solid state 13C CPMAS NMR results are listed in Fig. 4d. Although the
15
peaks were broad like Abbaszadeh et al. (2015), the discernible peaks around 78 ppm and 116 ppm were attributable to C1 and the rest carbon of XG components, respectively. The signal around 28 ppm was from methyl of acetyl and pyruvate, while the signal at 180 ppm was due to carbonyl (Horton, Mols, Walaszek, & Wernau,
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1985). Combining with these FT-IR, XRD and NMR results, the fermentation products
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in this study could be confirmed as XG. Additional furfural into the culture had no
N
U
impact on the primary structure of XG products.
A
3.2.2. Polysaccharide structure
M
The total carbohydrate content and monosaccharide composition of XG products from furfural-contained media are shown in Table 2. The total carbohydrate contents of
ED
all products were around 80%, and predominant monosaccharides were glucose and
PT
mannose. Moreover, the molar ratios of glucose, mannose and glucuronic acid were close to the theoretic value of 2.0:2.0:1.0 (Faria et al., 2011; Wang, Wu, Zhu, & Zhan,
CC E
2017). Combined with Garcíaochoa et al. (2000), the molecular structural formula of
A
XG was concluded as Fig 5a.
Glucuronic acid group is one of main components of XG pentasaccharide units.
As exhibited in Fig. 5b, the maximum content of glucuronic acid (14.55%) was from XG-1, and furfural concentration below 2.2 g·L–1 presented advantage on glucuronic acid content. Glucuronic acid group was closely related to uridine diphosphate 16
(UDP)-glucose dehydrogenase, the NAD(P)+/--relied enzyme of catalyzing the oxidation of UDP-glucose to UDP-glucuronic acid (Blanch, Legaz, & Vicente, 2008; Campbell, Sala, Van, & Tanner, 1997). The bioconversion of furfural to furfuryl alcohol could stimulate more NAD(P)+/- as discussed above, thereby leading to the
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improvement of glucuronic acid contents of XG-1 and XG-2. However, furfural over 2.2 g·L–1 decreased the number of glucuronic acid group, and the downward trend
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might be resulted from lowered activity of UDP-glucose dehydrogenase.
U
The amounts of acetyl and pyruvate groups in XG are variable according to
N
culture conditions (Faria et al., 2011). Acetyl group is the internal substituent of
A
mannose while pyruvate group located at the terminal of mannose. As shown in Fig.
M
5b, the acetyl contents gradually decreased from 4.41% to 2.54% with the increase of
ED
furfural concentration from 0 g·L–1 to 4.3 g·L–1. On the other side, the pyruvate contents first went up and then a little dropped beyond 3.2 g·L–1 furfural. The
PT
maximum pyruvate content of 6.43% was observed from XG-3, and the value of XG-
CC E
4 decreased to 5.84% which was still 28.92% higher than XG-control. Increased pyruvate contents and decreased acetyl contents of XG could be attributed to pyruvate retention in glycolysis. Pyruvate dehydrogenase links pyruvate and acetyl-CoA in
A
central carbon metabolism, but it is sensitive to furfural in vitro (Akobi et al., 2016; Modig & Liden GTaherzadeh, 2002). Under appropriate furfural environment, lowered pyruvate dehydrogenase activity brought about more pyruvate groups in XG. Additionally, slight decrease of XG-4 might be caused by the profound suppression of
17
furfural to glycolysis as discussed in cell growth. Furthermore, acetyl group is related to intramolecular association (Fitzpatrick, Meadows, Ratcliffe, & Williams, 2013), and pyruvate-rich XG tends to disorder its own ordered form (Li & Feke, 2015). Thus, decreased acetyl group and increased pyruvate group could make XG more
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flexible and easier to associate, which could offer XG products a wider application (Izawa et al., 2014). It indicated that XG products from furfural-contained systems
SC R
might be popular. 3.2.3. Rheological analysis
U
Rheological parameters of XG may be influenced by fermentation processes
N
(Faria et al., 2011). As seen from Fig. 6, the viscosities of XG products from furfural-
A
contained environments exhibited a similar change trend to commercial XG. All XG
M
solutions showed decreased apparent viscosity along with increased shear rate, and
ED
the pseudoplastic behavior is typical of polymers with high molecular weight (Miranda et al., 2018). Moreover, the products with furfural had slightly lower
PT
viscosities than the control without furfural. The variation might be caused by the
CC E
different contents of acetate and pyruvate groups, since the substituents can strongly influence rheological properties of XG (Li & Feke, 2015; Tako & Nakamura, 1984). In general, rheological property of XG products from furfural-contained systems was
A
a little weaker than that of commercial XG but still higher than those from some other substrates, such as glycerol (Wang et al., 2017).
18
3.3. Antioxidant activities of XG products Xanthan possesses protective effect against hydroxyl radical (•OH) which is the most harmful reactive oxygen species (ROS) to biological tissues (Wu et al., 2013). Fig. 7a depicts the hydroxyl radical scavenging effects of XG products from furfural-
IP T
contained systems. As shown, all XG had the ability for scavenging hydroxyl radical, and the scavenging effect depended on the concentration. XG-1 exhibited the
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strongest scavenging ability, while XG-4 had the lowest scavenging ability.
Spasojević et al. (2009) suggested that monosaccharide levels played an important
U
role in scavenging abilities of hydroxyl radical, but the abilities of main components
N
of XG (glucose and mannose) were much alike. Besides, polyglucuronic acid sodium
A
salt, pyruvate acid and O–acetylation of xanthan were found to be beneficial to the
M
property in vitro (Delattre et al., 2015; Wang et al., 2013; Xiong et al., 2013). On the
ED
basis, the varied contents of pyruvate, acetyl and glucuronic acid groups might be responsible for the difference of hydroxyl radical scavenging abilities. Among the
PT
three groups, glucuronic acids were the biggest contributor, for the hydroxyl radical
CC E
scavenging ability displayed a similar changing trend with the content of glucuronic acids (Fig. 5b). Overall, 1 g·L–1 furfural addition enhanced the hydroxyl radical
A
scavenging ability of XG product.
Reducing power evaluates the ability of donating electrons, and is one of the significant indicators of potential antioxidant activities (Xiong et al., 2013). From Fig. 7b, all XG products exhibited reducing powers, and showed no distinction (p>0.05, t-
19
test). Reductones are deemed to be related to reducing property, which functions by donating hydrogen atoms (Gordon, 1990). Nevertheless, main groups of XG do not belong to reductones, and pyruvate acid group had been demonstrated to have no connection with reducing power (Delattre et al., 2015). In addition, reducing power
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could be influenced by polysaccharide monomers (Lo et al., 2011), but the monosaccharide compositions of XG products were similar. Thereby, the addition of
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furfural exerted no impacts on reducing powers of XG products.
U
4. Conclusion
N
The effects of furfural on XG production were systematically evaluated. During
A
XG fermentation, X. campestris LRELP-1 could convert furfural to furfuryl alcohol.
M
Although furfural inhibited XG accumulation, below 3.2 g·L–1 of furfural showed
ED
stimulation on cell growth and had stable XG yields above 13 g·L–1. Moreover, the addition of furfural reduced acetyl contents but rose pyruvate contents, and glucuronic
PT
acid contents increased first and then decreased with increasing furfural
CC E
concentration. Accordingly, XG-1 showed the highest hydroxyl radical scavenging effect. The results provided guidance to the application of lignocellulosic-like raw
A
materials in polysaccharide fermentation.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant number 21506132) and the Basic Application Program of Department of
20
Science and Technology of Sichuan Province (Grant No. 2015JY0241).
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Tako, M., & Nakamura, S. (1984). Rheological properties of deacetylated xanthan in aqueous media. Journal of the Agricultural Chemical Society of Japan, 48(12), 2987-2993. Ujor, V., Agu, C. V., Gopalan, V., & Ezeji, T. C. (2014). Glycerol supplementation of the growth medium
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enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation. Applied Microbiology and Biotechnology, 98(14), 6511-6521. Verhoeven, E., Vervaet, C., & Remon, J. P. (2006). Xanthan gum to tailor drug release of sustained-release
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ethylcellulose mini-matrices prepared via hot-melt extrusion: in vitro and in vivo evaluation. European Journal of Pharmaceutics and Biopharmaceutics, 63(3), 320-330.
Wang, X., Zhang, Z., Yao, Q., Zhao, M., & Qi, H. (2013). Phosphorylation of low-molecular-weight polysaccharide from Enteromorpha linza with antioxidant activity. Carbohydrate Polymers, 96(2), 371-375.
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Wang, Y., Zhang, L., Li, Y., Hou, X., & Zeng, F. (2004). Correlation of structure to antitumor activities of five derivatives of a β-glucan from Poria cocos sclerotium. Carbohydrate Research, 339(15), 2567-2574.
Wang, Z., Wu, J., Zhu, L., & Zhan, X. (2016). Activation of glycerol metabolism in Xanthomonas campestris by adaptive evolution to produce a high-transparency and low-viscosity xanthan gum from glycerol. Bioresource Technology, 211, 390-397. Wang, Z., Wu, J., Zhu, L., & Zhan, X. (2017). Characterization of xanthan gum produced from glycerol by a mutant strain Xanthomonas campestris CCTCC M2015714. Carbohydrate Polymers, 157, 521-
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526. Wu, S. J., Wu, J. H., Xia, L. Z., Chu, C., Liu, D., & Gong, M. (2013). Preparation of xanthan-derived oligosaccharides and their hydroxyl radical scavenging activity. Carbohydrate Polymers, 92(2), 1612-1614. Xiong, X., Li, M., Xie, J., Jin, Q., Xue, B., & Sun, T. (2013). Antioxidant activity of xanthan oligosaccharides prepared by different degradation methods. Carbohydrate Polymers, 92(2), 1166-1171. Zhang, Y., Han, B., & Ezeji, T. C. (2012). Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation. New Biotechnology,
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29(3), 345-351.
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Fig. 1. Concentrations of residual furfural and generated furfuryl alcohol (a), cell
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growth, product yield and product content per unit cells (g XG/OD660) (b) under
various furfural concentrations. Error bars indicate the standard deviation of the mean
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N
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(n=3).
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Fig. 2. SEM images of damaged cells in 5.4 g·L–1 furfural (a) and intact cells in
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control (b).
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Fig. 3. Sucrose consumption and utilization\conversion rate of sucrose under various
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N
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furfural concentrations. Error bars indicate the standard deviation of the mean (n=3).
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Fig. 4. FT-IR spectra (a), XRD patterns (b), 1H NMR spectra (c) and 13C NMR spectra
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(d) of XG products from various furfural concentrations.
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Fig. 5. Molecular structural formula of XG (a), and glucuronic acid, acetyl and
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pyruvate contents of XG products from furfural-contained media (b). Error bars
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U
indicate the standard deviation of the mean (n=3).
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Fig. 6. Viscosity curves of XG products from various furfural concentrations and
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N
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commercial XG.
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Fig. 7. Hydroxyl radical scavenging effects (a) and reducing powers (b) of XG
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products from various furfural additions.
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Table 1 Comparison of the conversion rate of carbon source to XG Carbon source
XG yield
Conversion rate of
source
concentration (g·L–1)
(g·L–1)
carbon source (%)
Sucrose
40
23.2
58
Reference
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Carbon
(Wang, Wu, Zhu, &
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Zhan, 2016)
40
11.0
27.5
(Wang et al., 2016)
Sucrose
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15.8
63.2
(Faria, Vieira,
Sucrose
20
34
M
50
14.0
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Sucrose
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N
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Glycerol
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Resende, França, & Cardoso, 2009)
84 (in fermentor)
(Rong et al., 2012)
75.2
This work
Table 2 Total sugar content and monosaccharide composition of XG products under various furfural concentrations Molar ratio of glucose: mannose:
content (%)
glucuronic acid
XG-control
80.2±0.4
2:1.96:0.92
XG-1
78.9±1.2
2:1.87:0.94
XG-2
79.5±0.6
XG-3
79.1±3.4
XG-4
77.9±0.4
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Total carbohydrate
2:1.86:0.91
U
2:1.88:0.82 2:1.72:0.77
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A
N
XG product
33