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Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16
Accepted Manuscript Title: Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16 Authors: Jing Li, Haiyang Xu, Xiangnan Chen, Linxiang Xu, Rui Cheng, Jianfa Zhang, Shiming Wang PII: DOI: Reference:
S0141-8130(16)32561-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.030 BIOMAC 7710
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
22-11-2016 25-5-2017 6-6-2017
Please cite this article as: Jing Li, Haiyang Xu, Xiangnan Chen, Linxiang Xu, Rui Cheng, Jianfa Zhang, Shiming Wang, Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.030 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.
Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16
Jing Li1,¶, Haiyang Xu1,¶, Xiangnan Chen1, Linxiang Xu1, Rui Cheng1, Jianfa Zhang1, Shiming Wang1,*
1 Center for Molecular Metabolism, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing 210094, China ¶ These authors contribute equally. * Corresponding author, Email: [email protected] (SW)
Highlights: 1. A newly isolated strain was identified as Paenibacillus edaphicus NUST16. 2. Molecular structure of the polysaccharide, POS16, was determined. 3. POS16 shows strong saline-tolerance.
Abstract Microbial exopolysaccharides (EPSs) have been commercially used for decades owing to their distinct rheological properties. New EPSs with special traits are being explored continuously. The strain Paenibacillus edaphicus NUST16 was isolated in this study. NUST16 produces 12.5 g/L EPS after 72 h cultivation in shaker flask. Moreover, the purified EPS, POS16, has the molecular weight of 1.2×107 Da and consists of five types of glycosides, namely, D-Glc, D-Man, L-Fuc, D-GlcA, and D-Gal. Methylation, NMR, and FT-IR results indicate that three glucosides and three mannosides make up the main chain of POS16 via 1→3 linkage. The Gal(1→3)Fuc(1→3)Fuc(1→ and GlcA(1→ branches link to the C6 positions of two mannosides, respectively. The other mannoside is partially derivatized by the acetyl group at C2 position. Rheological analysis shows that POS16 has typical shear-thinning pattern which fits the Hershel-Bulkley model well. More importantly, POS16 shows ideal saline tolerance as the apparent viscosity keeps high in solutions containing 100
g/L salts, such as NaCl, KCl, MgCl2, and CaCl2. Furthermore, POS16 behaves more viscous than xanthan gum and sodium carboxymethylcellulose in divalent ion solutions. The results suggest that POS16 is a potential rheological modulator that can be used in operations where high concentrations of salts exist. Keywords: Paenibacillus edaphicus; polysaccharide; structure; rheology
1. Introduction For centuries people have explored the utility of biopolymers. Among them, the microbial extracellular products, especially the polysaccharides, draw enormous attention for decades owing to the superior properties they have. Many microbes, such as bacteria, fungi, and actinomycetes, excrete exopolysaccharides (EPSs) during their lifecycles as protectants against the adverse environmental stresses or as nutrition for later use. The metabolism and production for EPSs have been extensively studied and the overviews can be found elsewhere [1-4]. Among a series of important properties, the rheological modulation ability of EPSs is of great prominence that makes the industrial processes, such as thickening, stabilizing, suspending, emulsifying, texturizing, gelling, and so on, easy to achieve. Nowadays, EPSs have been successfully applied to the commercial fields, for example, food, beverage, cosmetics, pharmaceuticals, construction, and oil drilling. At present, several bacteria EPSs that exhibit distinct properties are industrialized, for example, xanthan gum from Xanthomonas campestris [5], curdlan from Alcaligenes faecalis var. myxogenes [6], gellan from Sphingomonas elodea [7], and welan from Alcaligenes sp. [8]. As EPSs are indispensable to human activity, discovering novel EPSs with highlighted properties deserve the attention of researchers. For one reason, the dosage of EPSs in food or beverages is strictly administrated and the total cost is related with the addition of EPSs. The other fact is that the existence of high concentration of metal ions often affects the function of EPSs. Therefore, a more effective thickening polysaccharide will lower the additive dosage in food or beverage, which in turn is a potential cost-cutting strategy [9]. Except for modifying the existed EPSs [10, 11], exploring new microbes with EPS producing ability offers an alternative but effective way. A great number of microbes are reported to produce EPSs. Among them, Paenibacillus strains produce EPSs that possess
specific properties, for example, speeding up the sand aggregation in the desert [12], promoting the plant growth and fertilizing the soil [13-15], flocculating inorganic and organic suspended solids, and absorbing heavy metals in wastewater [16-18]. However, to date few studies have discussed the rheological aspects of these EPSs. Therefore, further efforts are still necessary to discover new EPSs or to explore new properties of EPSs in the reservoir of Paenibacillus strains. In this study we isolated a new strain from genus Paenibacillus that produced a novel exopolysaccharide with distinct rheological properties. The chemical structure was first speculated by means of the nuclear magnetic resonance (NMR), methylation, and Fourier transformed infrared (FT-IR) analysis. Then the rheological aspects of the EPS were examined including the effect of pH, temperature, and salts. The aim of this work is to examine the traits of a newly found exopolysaccharide and to explore the potential use of this biopolymer.
2. Materials and methods 2.1 Purification and identification of Paenibacillus edaphicus NUST16 The coastal soil from Yancheng, Jiangsu province, China was sampled and used in this study to screen the desired strain. One liter screening medium contains NaH2PO4 1 g, peptone 3 g, CaCl2 0.07 g, MgCl2 0.2 g, FeCl2·7H2O 0.0125 g, MnSO4 0.003 g, ZnCl2 0.0075 g, sucrose 20 g and agar 9 g. The pH of the medium is adjusted to 8.5 before autoclaved at 121°C for 20 min. Strain cultivation in this study was maintained at 30°C. All the chemicals were analytical grade and were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). The strain was identified after purification. In brief, 2 mL broth was centrifuged at 10000 g × 10 min to recover the pellet. After being washed with deionized water twice, the strain pellet was lysed to obtain the genomic DNA with the DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd., China) according to the manual. Then the partial 16S rDNA sequence was
amplified
by
PCR
method
(primer
Fwd:
CATGCAAGTCGARCG;
Rev:
GGTGTGACGGGCGGT. S1000 Thermal Cycler, Bio-Rad Laboratories, Inc., USA). The PCR product was sequenced by Shanghai Sunny Biotechnology Co., Ltd. Afterwards, the 16S
rDNA sequence was deposited to NCBI GenBank database under accession number KX962167. The 99% similarity to Paenibacillus edaphicus was calculated on the basis of the results of sequence alignment from NCBI database. This strain was named as P. edaphicus NUST16. Thereafter, the phylogenetic tree was constructed as previously described [19]. NUST16 colonies was pictured by a digital camera, and the cells stained by methylene blue (methylene blue 30 mg, 95% (v/v) ethanol 3 mL, 0.01% (m/v) potassium hydroxide 10 mL) were microscopically examined (Eclipse 800, Nikon Corp., Tokyo, Japan). 2.2 Fermentation kinetics of P. edaphicus NUST16 The cultivation medium consists of 2% sucrose, 0.1% NaH2PO4, 0.3% KNO3, 0.007% CaCl2, 0.02% MgCl2, 0.00125% FeSO4·7H2O, 0.0003% MnSO4, 0.00075% ZnCl2, pH8.5. One colony of P. edaphicus NUST16 was inoculated into a 100 mL flask containing 20 mL autoclaved medium and was cultivated at 30°C for 24 h to proliferate the strain. Afterwards, 5 mL broth was transferred into a 500 mL flask containing 100 mL newly prepared medium and incubated at 30°C for 108 h at 250 rpm. Utilization of sucrose was monitored in the time-course manner. In general, 1 mL broth was heated to 60°C for 10 min and then diluted up to 30 folds with double-distilled water. After being centrifuged at 5000 g × 30 min, the supernatant was hydrolyzed by 1 M HCl at 100°C for 1.5 min and then neutralized by 1.0 M NaOH to pH7. After that, the reducing sugar in the hydrolyzate was quantified by 3,5-dinitrosalicylic acid (DNS) method. Absorbance at 600 nm was adopted to determine the strain biomass in the broth. The concentration of exopolysaccharide (EPS) was obtained by measuring the dry weight. In brief, the broth was heated to 60°C for 10 min at first. Then the broth was diluted with two volumes of deionized water and centrifuged at 5000 g × 30 min to recover the supernatant. Next, three-fold volume of ethanol was added into the supernatant to precipitate the EPS. After being washed twice with 75% (v/v) ethanol, the EPS was dried for weighing. All experiments were carried out in triplicates. 2.3 Purification and characterization of the EPS (POS16) 2.3.1 Purification procedures The gross EPS was purified by precipitation and chromatographic method. In brief, 5 g/L EPS solution was mixed with one volume of phenol/chloroform solution (1:1, v/v) to
flocculate the impurities. This procedure was repeated six times. Then the aqueous phase containing EPS was dried and further purified by anion exchange chromatography (AEC) method. The AEC system equipped with a Toyopearl SuperQ-650M column (16 mm×300 mm, Tosoh Corp., Japan) was adopted. An amount of 100 mg EPS sample was loaded on the top of the column and eluted by the eluent at flow rate of 0.8 mL/min at 25°C. A gradient elution protocol was adopted, i.e. 0-20 min, 25 mM PBS (pH7.5); 20-120 min, 25 mM PBS (pH7.5) with sodium chloride which linearly increased from 0 to 1.2 M. Only one peak was detected during the process. This peak was manually collected for further analysis. Afterwards, the size exclusion column (Toyopearl HW-65F, 16 mm×300 mm, Tosoh Corp., Japan) was incorporated to remove the salts by taking deionized water was eluent. The purified sample was named as POS16. After being lyophilized (Virtis Benchtop 2K, Virtis Corp., USA), POS16 was stored at 4°C for later use. 2.3.2 Chemical reactions The concentration of purified POS16 was quantified on the basis of the phenol-sulfuric acid reaction [20] with modification. Briefly, 40 μL aliquot of the POS16 solution was blended with 40 μL 9% (v/v) phenol aqueous and 200 μL sulfuric acid, and then kept at 100°C for 5 min. Absorbance of the reacted liquid was monitored at 490 nm. 2.3.3 Determination of molecular weight (Mw) of POS16 The HPLC system (Waters Inc., USA) equipped with a W410 RI detector and a TSKgel G5000PWXL column (7.8 mm×300 mm, Tosoh Corp., Japan) was used. The system temperature was 30°C and deionized water was used as eluent. Twenty milliliters POS16 solution was injected into the column and eluted at the flow rate of 0.6 mL/min for analysis. The Mw of POS16 was calibrated using Dextran Standard Kit (21 kDa-2990 kDa, American Polymer Standards Corp., OH, USA). 2.3.4 Fourier-transform infrared (FT-IR) spectroscopy analysis Before inspection, POS16 was blended with potassium bromide powder, ground and pressed into a 1 mm pellet. Then FT-IR spectrum of POS16 was scanned within the wave number range of 4000–500 cm-1 on a Nicolet Nexus 470 spectrometer (USA) and the results were automatically recorded for further analysis.
2.3.5 Monosaccharide composition of POS16 The pre-column derivatization method was introduced to quantify the composition of monosaccharides in POS16 [21]. Briefly, POS16 was completely hydrolyzed by 1 M trifluoroactic acid (TFA) at 105°C for 10 h at first. Then, 150 μL hydrolyzate was blended with 150 μL, 0.3 M sodium hydroxide and 200 μL, 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP, in methanol) and then kept at 70°C for 30 min. The excessive PMP was removed by chloroform extraction. Afterwards, the aqueous phase was used for HPLC analysis. The Waters HPLC system (Waters Inc., USA) equipped with a Zorbax SB-Aq column (4.6 mm×150 mm, Agilent Technologies, Inc., CA, USA) was adopted to quantify the PMP-monosaccharides (at 248 nm) using acetonitrile/monopotassium phosphate aqueous solution (17.7:82.3 (v/v), 50 mM KH2PO4, pH5.6) as eluent. A series of monosaccharides were used as standards. As the uronic acids are prone to break down during acid hydrolysis [22], the glucuronate and galacturonate were firstly hydrolyzed as POS16 and then used as standards. D-fructose was identified by thin-layer chromatography. At first, 1 mL POS16
hydrolyzate was spotted on the TLC Silica gel 60 F254 plate (Merck KGaA, Darmstadt, Germany). Then the sample was developed by n-butanol solution (n-butanol: pyrindine: water: ammonia= 8:4:1.5:0.025) and colored by spraying of orcinol solution (900 mg orcinol, 25 mL dH2O, 375 mL ethanol, 50 mL sulfuric acid) at 105°C for 4 min. 2.3.6 Nuclear magnetic resonance (NMR) analysis Before use, POS16 was depolymerized by 1 M TFA at 100°C for 2 h to lower the viscosity. The hydrolyzate was neutralized by 1 M NaOH to pH7 and then fractionated by a Toyopearl HW-50F column (16 mm × 30 mm, Tosoh Corp., Japan). Low molecular weight (approx. 20000 Da) fraction was manually collected for later use. Afterwards, the low Mw fraction was lyophilized and re-dissolved into D2O to the final concentration of 60 g/L with sodium-3-trimethylsilylpropionate (Cambridge Isotope Laboratories, Inc., MA, USA) as internal standard. 1H and
13
C NMR spectra were recorded at 25°C by a Bruker DRX-500
spectrometer (Bruker GmbH, Rheinstetten, Germany). Chemical shifts were automatically recorded and expressed in ppm. 2.3.7 Methylation analysis
Methylation of POS16 was performed as previously described [23]. In brief, dry POS16 powder was dissolved in DMSO to the final concentration of 2.5 g/L. Moderate heating (50°C) and sonication were used to speed up this process. Then the fine powder of sodium hydroxide and methyl iodide were added sequentially into the solution. The absence of peak at 3300-3500 cm-1 by FT-IR indicates the completion of methylation. Then the methylated sample was depolymerized by TFA hydrolysis at 120°C for 4 h. Next, acetylation reaction was carried out using acetic anhydride-pyridine (1:1, v/v) at 100°C for 2 h. The alditol acetates were analyzed by gas chromatography-mass spectrometry method (Thermo Scientific™ ISQ™ LT, Thermo Fisher Scientific Inc., USA). 2.3.8 Rheological analysis To characterize the rheological properties of POS16, a series of analyses were carried out using the Physica MCR101 rheometer (Anton Paar Germany GmbH, Ostfildern, Germany) equipped with a plate geometry sensor (PP-50, 1.000 mm gap). Shear rate ranging from 0.01 s-1 to 1000 s-1 was generated to obtain the flow curve of apparent viscosity. The system temperature was 25°C for all the tests. The Hershel-Bulkley model (Eq. 1) was adopted to fit the results. 𝜏 = 𝜏0 + 𝐾 × 𝛾 𝑛 (Eq. 1) In Eq. 1, τ is the shear stress (Pa), τ0 is the yield stress (Pa) and, K, γ and n are consistency parameter, shear rate (s-1), and flow index, respectively. For temperature sweep analysis, 5 g/L POS16 solution was used. The shear rate was maintained at 1.0 s-1 during the process. The melting curve was calculated on the basis of the derivative of apparent viscosity (η) versus temperature (T). The peak value was determined as melting point (Tm). Xanthan gum and sodium carboxymethylcellulose (CMC.Na) were used as counterparts for saline tolerant analysis. Four mineral salts, namely sodium chloride, potassium chloride, magnesium chloride, and calcium chloride, were adopted. The experiments were performed at 25°C and the concentration of all samples was 5 g/L.
3. Results and discussion 3.1 Identification and fermentation kinetics of Paenibacillus edaphicus NUST16
A bacterial strain was isolated from the costal soil in Yancheng, China. The 1241-bp 16S rDNA sequence shows more than 99% similarity to the Paenibacillus edaphicus strains on the basis of the nucleotide alignment (Fig. 1A). This unique sequence was deposited to NCBI GenBank as KX962167. This strain was named as Paenibacillus edaphicus NUST16. After 2 days cultivation, NUST16 formed mucoid and translucent colonies. The sizes of free cells in fresh medium were 0.2-0.4 μm in width and 0.4-1.0 μm in length (Supporting Fig. S1). Physiological analysis showed that NUST16 was Gram-positive and the catalase reaction was positive. When using glucose as the sole carbon source, NUST16 produced detectable acids. The optimal temperature and pH for growth were 30°C and 8.5, respectively. NUST16 grew well in medium containing 10 g/L NaCl whereas the growth was inhibited by high concentration of salts (100 g/L NaCl). The batch fermentation kinetics of NUST16 was determined using sucrose as carbon source. As illustrated in figure 1B, the biomass reached plateau after 12 h cultivation and the pH dropped rapidly to 6.3. The maximal specific growth rate was 0.56 h-1 at the logarithmic growth phase. Besides, the yield of exopolysaccharide (EPS) was 12.5±0.5 g/L after 72 h fermentation, which corresponds to the YP/S of 0.66 g/g and EPS productivity of 0.17 g/L/h. Moreover, the non-coupled relation between cell growth and EPS production was observed in NUST16. P. edaphicus strain was formerly classified as Bacillus edaphicus [24]. It has been used as a plant growth-promoting bacterium owing to its potassium-mobilizing ability from illite [25]. In addition, the strain P. edaphicus NBT shows lead-adsorption capability [26]. However, to date information on extracellular products of P. edaphicus strains has not been fully addressed. Our results suggest that P. edaphicus NUST16 produced desirable amount of EPS which is a new source for the microbial biopolymers. (Position for Fig. 1) 3.2 Structural characterization of POS16 from P. edaphicus NUST16 A series of purification procedures were introduced before structure analysis. Proteins in purified EPS of NUST16 (POS16) were scarcely detected, as the curve of second-order derivative spectrometry was highly close to zero around 280 nm (Supporting Fig. S2). The results imply that POS16 has small possibility to be a proteoglycan. In addition, only one
peak was detected during chromatographic analysis (Supporting Fig. S3), which suggests that P. edaphicus NUST16 produces one type of polysaccharide. The weight-average molecular weight of POS16 was 1.2×107 Da, which was extrapolated from the retention time of standards (Fig. 2A). Monosaccharide analysis showed that POS16 was comprised of D-mannose, D-glucuronic acid, D-glucose, D-galactose, and L-fucose with the molar ratio of 3.11:0.85:3.00:0.90:1.89. In addition, the ketose, D-fructose,
was not detected by the thin-layer chromatography and by the methylation-GC/MS analysis. To the best of our knowledge, POS16 shows different composition of monosaccharides from other biopolymers. The content of L-fucose in POS16 is higher than many others except for FucoPol, a fucose-rich bacterial EPS, which contains 32-36 mol% L-fucose in its molecule. However, other glycosides in FucoPol differ from these in POS16 [27] and D-mannoside is absent in FucoPol [28]. Besides, no similarities are found between POS16
and other polysaccharides produced by Peanibacillus strains [29-32]. (Position for Fig. 2) The NMR technique shows paramount importance in glycans’ structural analysis [33, 34], and the methylation-GC/MS analysis gives useful information on glycosidic linkage [23]. For this reason, these methods were introduced to determine the structure of POS16. Figure 3 shows the 1H and 13C NMR spectra. As shown in Fig. 3A, the chemical shifts, δ 1.21 and 1.29 ppm, are typical values of protons in methyl groups that correspond to the C6 moieties of two L-fucosides in POS16. In addition, the chemical shifts in
C spectrum, δ
13
15.27 and 15.40 ppm, give further evidence for the existence of two L-fucosides (Fig. 3B, α and β). The weak signals, δ 2.16 ppm in 1H spectrum and δ 20.36 ppm in 13C spectrum, imply the existence of acetyl group which can be further determined in methylation analysis. The molar ratio of the acetyl group to POS16 repeating unit was 0.52, which agrees well with the intensity of NMR signals (Table 1). Ten anomeric carbon signals are strong (Fig. 3B, 1-10). They are in good correlation with the proton signals (Fig. 3A, a-f3) and in line with the analysis results of monosaccharide composition (Fig. 2C). Additionally, the strong signal, δ 175.74 ppm, confirms the existence of glucuronate (Fig. 3B, γ). As the chemical shifts from δ 59.71 ppm to 61.39 ppm account for seven C6 signals (Fig. 3B), another conclusion can be drawn is that the number of glycosidic
linkage from C6 position in the repeat unit of POS16 is two. Taking reference to the confirmed information [35], ten glycosides were designated with most possibility as follows (from a to f3); α-GlcA (98.31, 5.50), α-Man (99.96, 5.15), α-Man (101.23, 5.15), α-Fuc (100.46, 4.92), α-Fuc (100.64, 4.92), β-Gal (102.65, 4.86), β-Man (103.35, 4.60), β-Glc (101.50, 4.50), β-Glc (101.79, 4.50), and β-Glc (102.26, 4.50). (Position for Fig. 3) Linkage information was obtained from methylation results (Table 1). Molar ratio of each glycoside agreed well with the results from monosaccharide analysis. Three glucosides were linked via 1,3-linkage. Two mannosides were linked via 1,3-linkage while the C6 positions were both branch points. The third mannoside had a C2 position to be derivatized by acetyl group (molar ratio 0.52:1.0). Additional experiment of partial acidic hydrolysis using Sun et al.’s method [36] proved that the glucuronide, fucoside and galactoside were most probably in the branched-chain, as the content of these glycosides in POS16 residues was remarkably
decreased
after
hydrolysis
(data
not
shown).
More
likely
two
6-deoxy-galactosides (L-fucosides) were linked with the terminal galactoside to form one branch in C6 of the mannoside. Glucuronide was linked to C6 of another mannoside. (Position for Table 1) The FT-IR spectrum of POS16 showed the typical polysaccharide profile (Fig. 4) [31, 37, 38]. The broaden peak at 3313.11 cm-1 indicated the stretching vibration of O-H at association status. The peaks at 2912.95, 1402.00, and 1362.94 cm-1 implied the existence of methyl groups of L-fucose. The strong peak at 1595.81 cm-1 gave evidence of carboxyl group. 913.18, 886.13, and 791.15 cm-1 suggested that both β and α conformation existed in POS16 molecules. As a result, a possible structure of POS16 was given in terms of the above listed information (Fig. 3C). Three glucosides and three mannosides consecutively constituted the backbone of POS16. The Gal(1→3)Fuc(1→3)Fuc(1→ and GlcA(1→ branches were linked to the C6 of two mannosides, respectively. The other mannoside was partially derivatized by the acetyl group at C2 position. (Position for Fig. 4) 3.3 Rheological characterization of POS16
The most fascinating aspect of biopolymers is their rheological modulatory property that enlarges the commodity lists in many industries and facilitates the pharmaceutical application in medical field. Not surprisingly, POS16 showed satisfactory rheological properties. As shown in figure 5, POS16 behave evident shear-thinning patterns that fit the Hershel-Bulkley model well [39]. The apparent viscosity (η) dropped down when speeding up the shear rate. When the concentration of POS16 increased, the solution became more viscous. Moreover, higher value of viscosity was observed near neutral conditions (pH6-9) than in acidic or alkaline conditions (Fig. 5B). A similar phenomenon was reported in xanthan gum solution at low pH. The reason was partially attributed to the alteration of charge density of the xanthan molecules [40]. The glucuronides in the nonionic form suppress the electrostatic repulsion, which consequently allows a more compact conformation and reduces the viscosity. In alkaline solutions the decrease of viscosity was subjected to the alteration of charge density and change of the hydrodynamic volume of the polymer molecules [28, 41]. Meanwhile, it is reported that the hydrogen bonds are weakened by hydroxyl ions [42]. Besides, ζ-potentials gave further information on POS16 stability which in turn affects the solution viscosity (Supporting Table S1). Owing to its macromolecular weight, POS16 possesses some colloid properties. In acidic conditions, the magnitude of charge of POS16 reduced because the glucuronides tended to be nonionic form, which generated a smallζ-potential (-21.8 mV, 1 g/L POS16 at pH4). ζ-Potentials increased in higher pH solutions. In neutral condition, the value -70.7 mV was detected (1 g/L POS16 at pH7) whereas -54.6 mV was obtained in alkaline condition (pH11). Due to the reduction of electrostatic repulsion, macromolecules are prone to aggregate or to form compact conformation at low ζ-potentials. Therefore, POS16 showed high viscosity in neutral condition where the ζ-potential had the highest value. In addition, temperature sweep curves gave information on the interaction between POS16 molecules (Fig. 6). When the solution was heated, the viscosity of POS16 decreased with temperature. Remarkable attenuation was observed when the temperature was over 50°C. This phenomenon was due to the thermo motion beyond the interactive forces between molecules. However, the melting points of POS16 in acidic and alkaline conditions were lower than these in neutral solutions (more than 1°C). The results indicate that the intermolecular interaction at high temperature was strong in neutral condition (Fig. 5B).
Despite the variation of melting points, POS16 showed promising viscosity at moderate temperature. (Position for Fig. 5) (Position for Fig. 6) It is common in practice that various metal ions exist during unit operation of biopolymers. However, in some occasions, high content metal ions affect the behavior of biopolymers, which in turn impacts the quality of final products. Interestingly, POS16 showed remarkable saline-tolerance as the apparent viscosity kept the high values in different ion solutions where 100 g/L salts existed (Fig. 7). Taking xanthan gum and sodium carboxymethylcellulose (CMC.Na) as counterparts, POS16 showed ideal results in solutions containing high concentration of salts (Fig. 8). The viscosity of POS16 was comparable with xanthan gum whereas it was much higher than CMC.Na in the whole shear rate range. When monovalent ions (Na+ and K+) were added, the viscosity of all polymers decreased and POS16 was slightly lower than it of xanthan gum. Interestingly, different results were obtained when divalent ions (Mg2+ and Ca2+) were added. As xanthan gum and CMC.Na were partially soluble in 100 g/L CaCl2 solution, POS16 showed superior advantage to both counterparts. The parameters of Hershel-Bulkley model were listed in supporting Table S2, which gave the quantified comparison between these polymers. These results suggest that POS16 is suitable for applying to operations where high concentrations of divalent salts exist. In general, POS16 shows potential use in future as a rheological modulator. Despite these results, further investigation should be performed, including the tests of viscoelastic dynamics, freezing-thawing, and stability properties. (Position for Fig. 7) (Position for Fig. 8) 4. Conclusion Exploring new microbial species and discovering new polysaccharides with novel traits are essential not only for research concerns but also for daily application. In this work, we reported the structure and rheological properties of an exopolysaccharide, POS16, produced by a new strain, Paenibacillus edaphicus NUST16. POS16 has a considerably large molecular weight of 1.2×107 Da. The repeating unit of POS16 is comprised of five types of glycosides,
namely
D-Glc,
D-Man,
L-Fuc,
D-GlcA,
and
D-Gal
with the
molar
ratio
of
3.00:3.11:1.89:0.85:0.90. As a potential rheological additive, POS16 shows typical shear-thinning pattern which fits the Hershel-Bulkley model well. The ideal saline-tolerance in solutions containing high concentration of mono- or divalent metal ions makes POS16 a suitable candidate in the processes where suspending, thickening, texturizing, or mixing is involved.
Acknowledgements This work is supported by the research grants from the National Natural Science Foundation of China (No. 31500418), the Natural Science Foundation of Jiangsu Province (Grants No. BK20150773), and the Fundamental Research Funds for the Central Universities (No. 30915011101, 30920130121013). The authors would like to thank these organizations for financial supports.
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Figure Captions Fig. 1 Phylogenetic tree (A) and fermentation kinetics (B) of Paenibacillus edaphicus NUST16 (A, phylogenetic tree was constructed on the basis of the 16S rDNA sequences from NCBI database. Figure B shows one of three batch fermentation results that performed in the 500 mL shaker flask.)
Fig. 2 Weight-average molecular weight and monosaccharide composition of POS16 (A, weight-average molecular weight was analyzed using the Tskgel G5000PWXL column. Calibration curve of dextran standards is listed in the upper left corner. Composition of monosaccharides was analyzed using the ZorBax SB-Aq column. B, monosaccharide standards derivatized by 1-phenyl-3-methyl-5-pyrazolone. C, POS16 hydrolyzate.)
Fig. 3 1D NMR spectra and the structure of POS16 (A1, full range 1H spectrum of POS16.
A2, partial 1H spectrum. B1, full range 13C spectrum. B2, partial 13C spectrum. C, structure of POS16. a-f3 in A2 and 1-10 in B1 indicate the signals of anomeric protons and carbons. α and β in A1 and B2 show the signals of C6 methyl groups in fucosides.)
Fig. 4 FT-IR spectrum of POS16
Fig. 5 Effect of concentration and pH on the shear-thinning patterns of POS16 solutions (The experiments were carried out at 25°C. A, viscosity curves of different POS16 solutions at pH7. B, the effect of pH on the viscosity of 10 g/L POS16 solution.)
Fig. 6 temperature sweep curves of POS16 solution at different pH (The shear rate was fixed at 1 s-1 and the concentration was 5 g/L. A, temperature sweep curves. B, melting curves were based on the first order derivative of viscosity versus temperature.)
Fig. 7 Apparent viscosity of POS16 in different salt solutions (The shear rate and temperature were maintained at 0.5 s-1 and 25°C, respectively. The final concentration of POS16 was 5 g/L.)
Fig. 8 Effect of salts on the viscosity of POS16, xanthan gum, and sodium carboxymethylcellulose (The experiments were carried out at 25°C. The final concentration of all polymers was 5 g/L. The final concentration of all salts was 100 g/L. Xanthan gum and sodium carboxymethylcellulose (CMC.Na) were partially soluble in 100 g/L CaCl 2 solution. Therefore, the supernatants of these samples were used for analysis.)
Table 1 Methylation results of POS16 Methylated sugar a
Type of linkage
Retention time b
Molar ratio
2,3,4-Me3-GlcAp
terminal GlcAp
1.000
0.42
2,3,4,6-Me4-Galp
terminal Galp
1.043
0.78
2,4-Me2-Fucp
1,3-linked Fucp
1.145
1.82
2,4,6-Me3-Glcp
1,3-linked Glcp
1.151
3.00
2,4,6-Me3-Manp
1,3-linked Manp
1.175
0.46
4,6-Me2-Manp
1,2,3-linked Manp
1.184
0.52
2,4-Me2-Manp
1,3,6-linked Manp
1.218
2.14
4-Me-Glcp
1,2,3,6-linked Glcp
1.241
trace
a
2,3,4,6-Me4-Galp is the abbreviation of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol,
etc. b
Retention time is relative to the time of 2,3,4-Me3-GlcAp.
S0141-8130(16)32561-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.030 BIOMAC 7710
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
22-11-2016 25-5-2017 6-6-2017
Please cite this article as: Jing Li, Haiyang Xu, Xiangnan Chen, Linxiang Xu, Rui Cheng, Jianfa Zhang, Shiming Wang, Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.030 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.
Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16
Jing Li1,¶, Haiyang Xu1,¶, Xiangnan Chen1, Linxiang Xu1, Rui Cheng1, Jianfa Zhang1, Shiming Wang1,*
1 Center for Molecular Metabolism, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing 210094, China ¶ These authors contribute equally. * Corresponding author, Email: [email protected] (SW)
Highlights: 1. A newly isolated strain was identified as Paenibacillus edaphicus NUST16. 2. Molecular structure of the polysaccharide, POS16, was determined. 3. POS16 shows strong saline-tolerance.
Abstract Microbial exopolysaccharides (EPSs) have been commercially used for decades owing to their distinct rheological properties. New EPSs with special traits are being explored continuously. The strain Paenibacillus edaphicus NUST16 was isolated in this study. NUST16 produces 12.5 g/L EPS after 72 h cultivation in shaker flask. Moreover, the purified EPS, POS16, has the molecular weight of 1.2×107 Da and consists of five types of glycosides, namely, D-Glc, D-Man, L-Fuc, D-GlcA, and D-Gal. Methylation, NMR, and FT-IR results indicate that three glucosides and three mannosides make up the main chain of POS16 via 1→3 linkage. The Gal(1→3)Fuc(1→3)Fuc(1→ and GlcA(1→ branches link to the C6 positions of two mannosides, respectively. The other mannoside is partially derivatized by the acetyl group at C2 position. Rheological analysis shows that POS16 has typical shear-thinning pattern which fits the Hershel-Bulkley model well. More importantly, POS16 shows ideal saline tolerance as the apparent viscosity keeps high in solutions containing 100
g/L salts, such as NaCl, KCl, MgCl2, and CaCl2. Furthermore, POS16 behaves more viscous than xanthan gum and sodium carboxymethylcellulose in divalent ion solutions. The results suggest that POS16 is a potential rheological modulator that can be used in operations where high concentrations of salts exist. Keywords: Paenibacillus edaphicus; polysaccharide; structure; rheology
1. Introduction For centuries people have explored the utility of biopolymers. Among them, the microbial extracellular products, especially the polysaccharides, draw enormous attention for decades owing to the superior properties they have. Many microbes, such as bacteria, fungi, and actinomycetes, excrete exopolysaccharides (EPSs) during their lifecycles as protectants against the adverse environmental stresses or as nutrition for later use. The metabolism and production for EPSs have been extensively studied and the overviews can be found elsewhere [1-4]. Among a series of important properties, the rheological modulation ability of EPSs is of great prominence that makes the industrial processes, such as thickening, stabilizing, suspending, emulsifying, texturizing, gelling, and so on, easy to achieve. Nowadays, EPSs have been successfully applied to the commercial fields, for example, food, beverage, cosmetics, pharmaceuticals, construction, and oil drilling. At present, several bacteria EPSs that exhibit distinct properties are industrialized, for example, xanthan gum from Xanthomonas campestris [5], curdlan from Alcaligenes faecalis var. myxogenes [6], gellan from Sphingomonas elodea [7], and welan from Alcaligenes sp. [8]. As EPSs are indispensable to human activity, discovering novel EPSs with highlighted properties deserve the attention of researchers. For one reason, the dosage of EPSs in food or beverages is strictly administrated and the total cost is related with the addition of EPSs. The other fact is that the existence of high concentration of metal ions often affects the function of EPSs. Therefore, a more effective thickening polysaccharide will lower the additive dosage in food or beverage, which in turn is a potential cost-cutting strategy [9]. Except for modifying the existed EPSs [10, 11], exploring new microbes with EPS producing ability offers an alternative but effective way. A great number of microbes are reported to produce EPSs. Among them, Paenibacillus strains produce EPSs that possess
specific properties, for example, speeding up the sand aggregation in the desert [12], promoting the plant growth and fertilizing the soil [13-15], flocculating inorganic and organic suspended solids, and absorbing heavy metals in wastewater [16-18]. However, to date few studies have discussed the rheological aspects of these EPSs. Therefore, further efforts are still necessary to discover new EPSs or to explore new properties of EPSs in the reservoir of Paenibacillus strains. In this study we isolated a new strain from genus Paenibacillus that produced a novel exopolysaccharide with distinct rheological properties. The chemical structure was first speculated by means of the nuclear magnetic resonance (NMR), methylation, and Fourier transformed infrared (FT-IR) analysis. Then the rheological aspects of the EPS were examined including the effect of pH, temperature, and salts. The aim of this work is to examine the traits of a newly found exopolysaccharide and to explore the potential use of this biopolymer.
2. Materials and methods 2.1 Purification and identification of Paenibacillus edaphicus NUST16 The coastal soil from Yancheng, Jiangsu province, China was sampled and used in this study to screen the desired strain. One liter screening medium contains NaH2PO4 1 g, peptone 3 g, CaCl2 0.07 g, MgCl2 0.2 g, FeCl2·7H2O 0.0125 g, MnSO4 0.003 g, ZnCl2 0.0075 g, sucrose 20 g and agar 9 g. The pH of the medium is adjusted to 8.5 before autoclaved at 121°C for 20 min. Strain cultivation in this study was maintained at 30°C. All the chemicals were analytical grade and were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). The strain was identified after purification. In brief, 2 mL broth was centrifuged at 10000 g × 10 min to recover the pellet. After being washed with deionized water twice, the strain pellet was lysed to obtain the genomic DNA with the DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd., China) according to the manual. Then the partial 16S rDNA sequence was
amplified
by
PCR
method
(primer
Fwd:
CATGCAAGTCGARCG;
Rev:
GGTGTGACGGGCGGT. S1000 Thermal Cycler, Bio-Rad Laboratories, Inc., USA). The PCR product was sequenced by Shanghai Sunny Biotechnology Co., Ltd. Afterwards, the 16S
rDNA sequence was deposited to NCBI GenBank database under accession number KX962167. The 99% similarity to Paenibacillus edaphicus was calculated on the basis of the results of sequence alignment from NCBI database. This strain was named as P. edaphicus NUST16. Thereafter, the phylogenetic tree was constructed as previously described [19]. NUST16 colonies was pictured by a digital camera, and the cells stained by methylene blue (methylene blue 30 mg, 95% (v/v) ethanol 3 mL, 0.01% (m/v) potassium hydroxide 10 mL) were microscopically examined (Eclipse 800, Nikon Corp., Tokyo, Japan). 2.2 Fermentation kinetics of P. edaphicus NUST16 The cultivation medium consists of 2% sucrose, 0.1% NaH2PO4, 0.3% KNO3, 0.007% CaCl2, 0.02% MgCl2, 0.00125% FeSO4·7H2O, 0.0003% MnSO4, 0.00075% ZnCl2, pH8.5. One colony of P. edaphicus NUST16 was inoculated into a 100 mL flask containing 20 mL autoclaved medium and was cultivated at 30°C for 24 h to proliferate the strain. Afterwards, 5 mL broth was transferred into a 500 mL flask containing 100 mL newly prepared medium and incubated at 30°C for 108 h at 250 rpm. Utilization of sucrose was monitored in the time-course manner. In general, 1 mL broth was heated to 60°C for 10 min and then diluted up to 30 folds with double-distilled water. After being centrifuged at 5000 g × 30 min, the supernatant was hydrolyzed by 1 M HCl at 100°C for 1.5 min and then neutralized by 1.0 M NaOH to pH7. After that, the reducing sugar in the hydrolyzate was quantified by 3,5-dinitrosalicylic acid (DNS) method. Absorbance at 600 nm was adopted to determine the strain biomass in the broth. The concentration of exopolysaccharide (EPS) was obtained by measuring the dry weight. In brief, the broth was heated to 60°C for 10 min at first. Then the broth was diluted with two volumes of deionized water and centrifuged at 5000 g × 30 min to recover the supernatant. Next, three-fold volume of ethanol was added into the supernatant to precipitate the EPS. After being washed twice with 75% (v/v) ethanol, the EPS was dried for weighing. All experiments were carried out in triplicates. 2.3 Purification and characterization of the EPS (POS16) 2.3.1 Purification procedures The gross EPS was purified by precipitation and chromatographic method. In brief, 5 g/L EPS solution was mixed with one volume of phenol/chloroform solution (1:1, v/v) to
flocculate the impurities. This procedure was repeated six times. Then the aqueous phase containing EPS was dried and further purified by anion exchange chromatography (AEC) method. The AEC system equipped with a Toyopearl SuperQ-650M column (16 mm×300 mm, Tosoh Corp., Japan) was adopted. An amount of 100 mg EPS sample was loaded on the top of the column and eluted by the eluent at flow rate of 0.8 mL/min at 25°C. A gradient elution protocol was adopted, i.e. 0-20 min, 25 mM PBS (pH7.5); 20-120 min, 25 mM PBS (pH7.5) with sodium chloride which linearly increased from 0 to 1.2 M. Only one peak was detected during the process. This peak was manually collected for further analysis. Afterwards, the size exclusion column (Toyopearl HW-65F, 16 mm×300 mm, Tosoh Corp., Japan) was incorporated to remove the salts by taking deionized water was eluent. The purified sample was named as POS16. After being lyophilized (Virtis Benchtop 2K, Virtis Corp., USA), POS16 was stored at 4°C for later use. 2.3.2 Chemical reactions The concentration of purified POS16 was quantified on the basis of the phenol-sulfuric acid reaction [20] with modification. Briefly, 40 μL aliquot of the POS16 solution was blended with 40 μL 9% (v/v) phenol aqueous and 200 μL sulfuric acid, and then kept at 100°C for 5 min. Absorbance of the reacted liquid was monitored at 490 nm. 2.3.3 Determination of molecular weight (Mw) of POS16 The HPLC system (Waters Inc., USA) equipped with a W410 RI detector and a TSKgel G5000PWXL column (7.8 mm×300 mm, Tosoh Corp., Japan) was used. The system temperature was 30°C and deionized water was used as eluent. Twenty milliliters POS16 solution was injected into the column and eluted at the flow rate of 0.6 mL/min for analysis. The Mw of POS16 was calibrated using Dextran Standard Kit (21 kDa-2990 kDa, American Polymer Standards Corp., OH, USA). 2.3.4 Fourier-transform infrared (FT-IR) spectroscopy analysis Before inspection, POS16 was blended with potassium bromide powder, ground and pressed into a 1 mm pellet. Then FT-IR spectrum of POS16 was scanned within the wave number range of 4000–500 cm-1 on a Nicolet Nexus 470 spectrometer (USA) and the results were automatically recorded for further analysis.
2.3.5 Monosaccharide composition of POS16 The pre-column derivatization method was introduced to quantify the composition of monosaccharides in POS16 [21]. Briefly, POS16 was completely hydrolyzed by 1 M trifluoroactic acid (TFA) at 105°C for 10 h at first. Then, 150 μL hydrolyzate was blended with 150 μL, 0.3 M sodium hydroxide and 200 μL, 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP, in methanol) and then kept at 70°C for 30 min. The excessive PMP was removed by chloroform extraction. Afterwards, the aqueous phase was used for HPLC analysis. The Waters HPLC system (Waters Inc., USA) equipped with a Zorbax SB-Aq column (4.6 mm×150 mm, Agilent Technologies, Inc., CA, USA) was adopted to quantify the PMP-monosaccharides (at 248 nm) using acetonitrile/monopotassium phosphate aqueous solution (17.7:82.3 (v/v), 50 mM KH2PO4, pH5.6) as eluent. A series of monosaccharides were used as standards. As the uronic acids are prone to break down during acid hydrolysis [22], the glucuronate and galacturonate were firstly hydrolyzed as POS16 and then used as standards. D-fructose was identified by thin-layer chromatography. At first, 1 mL POS16
hydrolyzate was spotted on the TLC Silica gel 60 F254 plate (Merck KGaA, Darmstadt, Germany). Then the sample was developed by n-butanol solution (n-butanol: pyrindine: water: ammonia= 8:4:1.5:0.025) and colored by spraying of orcinol solution (900 mg orcinol, 25 mL dH2O, 375 mL ethanol, 50 mL sulfuric acid) at 105°C for 4 min. 2.3.6 Nuclear magnetic resonance (NMR) analysis Before use, POS16 was depolymerized by 1 M TFA at 100°C for 2 h to lower the viscosity. The hydrolyzate was neutralized by 1 M NaOH to pH7 and then fractionated by a Toyopearl HW-50F column (16 mm × 30 mm, Tosoh Corp., Japan). Low molecular weight (approx. 20000 Da) fraction was manually collected for later use. Afterwards, the low Mw fraction was lyophilized and re-dissolved into D2O to the final concentration of 60 g/L with sodium-3-trimethylsilylpropionate (Cambridge Isotope Laboratories, Inc., MA, USA) as internal standard. 1H and
13
C NMR spectra were recorded at 25°C by a Bruker DRX-500
spectrometer (Bruker GmbH, Rheinstetten, Germany). Chemical shifts were automatically recorded and expressed in ppm. 2.3.7 Methylation analysis
Methylation of POS16 was performed as previously described [23]. In brief, dry POS16 powder was dissolved in DMSO to the final concentration of 2.5 g/L. Moderate heating (50°C) and sonication were used to speed up this process. Then the fine powder of sodium hydroxide and methyl iodide were added sequentially into the solution. The absence of peak at 3300-3500 cm-1 by FT-IR indicates the completion of methylation. Then the methylated sample was depolymerized by TFA hydrolysis at 120°C for 4 h. Next, acetylation reaction was carried out using acetic anhydride-pyridine (1:1, v/v) at 100°C for 2 h. The alditol acetates were analyzed by gas chromatography-mass spectrometry method (Thermo Scientific™ ISQ™ LT, Thermo Fisher Scientific Inc., USA). 2.3.8 Rheological analysis To characterize the rheological properties of POS16, a series of analyses were carried out using the Physica MCR101 rheometer (Anton Paar Germany GmbH, Ostfildern, Germany) equipped with a plate geometry sensor (PP-50, 1.000 mm gap). Shear rate ranging from 0.01 s-1 to 1000 s-1 was generated to obtain the flow curve of apparent viscosity. The system temperature was 25°C for all the tests. The Hershel-Bulkley model (Eq. 1) was adopted to fit the results. 𝜏 = 𝜏0 + 𝐾 × 𝛾 𝑛 (Eq. 1) In Eq. 1, τ is the shear stress (Pa), τ0 is the yield stress (Pa) and, K, γ and n are consistency parameter, shear rate (s-1), and flow index, respectively. For temperature sweep analysis, 5 g/L POS16 solution was used. The shear rate was maintained at 1.0 s-1 during the process. The melting curve was calculated on the basis of the derivative of apparent viscosity (η) versus temperature (T). The peak value was determined as melting point (Tm). Xanthan gum and sodium carboxymethylcellulose (CMC.Na) were used as counterparts for saline tolerant analysis. Four mineral salts, namely sodium chloride, potassium chloride, magnesium chloride, and calcium chloride, were adopted. The experiments were performed at 25°C and the concentration of all samples was 5 g/L.
3. Results and discussion 3.1 Identification and fermentation kinetics of Paenibacillus edaphicus NUST16
A bacterial strain was isolated from the costal soil in Yancheng, China. The 1241-bp 16S rDNA sequence shows more than 99% similarity to the Paenibacillus edaphicus strains on the basis of the nucleotide alignment (Fig. 1A). This unique sequence was deposited to NCBI GenBank as KX962167. This strain was named as Paenibacillus edaphicus NUST16. After 2 days cultivation, NUST16 formed mucoid and translucent colonies. The sizes of free cells in fresh medium were 0.2-0.4 μm in width and 0.4-1.0 μm in length (Supporting Fig. S1). Physiological analysis showed that NUST16 was Gram-positive and the catalase reaction was positive. When using glucose as the sole carbon source, NUST16 produced detectable acids. The optimal temperature and pH for growth were 30°C and 8.5, respectively. NUST16 grew well in medium containing 10 g/L NaCl whereas the growth was inhibited by high concentration of salts (100 g/L NaCl). The batch fermentation kinetics of NUST16 was determined using sucrose as carbon source. As illustrated in figure 1B, the biomass reached plateau after 12 h cultivation and the pH dropped rapidly to 6.3. The maximal specific growth rate was 0.56 h-1 at the logarithmic growth phase. Besides, the yield of exopolysaccharide (EPS) was 12.5±0.5 g/L after 72 h fermentation, which corresponds to the YP/S of 0.66 g/g and EPS productivity of 0.17 g/L/h. Moreover, the non-coupled relation between cell growth and EPS production was observed in NUST16. P. edaphicus strain was formerly classified as Bacillus edaphicus [24]. It has been used as a plant growth-promoting bacterium owing to its potassium-mobilizing ability from illite [25]. In addition, the strain P. edaphicus NBT shows lead-adsorption capability [26]. However, to date information on extracellular products of P. edaphicus strains has not been fully addressed. Our results suggest that P. edaphicus NUST16 produced desirable amount of EPS which is a new source for the microbial biopolymers. (Position for Fig. 1) 3.2 Structural characterization of POS16 from P. edaphicus NUST16 A series of purification procedures were introduced before structure analysis. Proteins in purified EPS of NUST16 (POS16) were scarcely detected, as the curve of second-order derivative spectrometry was highly close to zero around 280 nm (Supporting Fig. S2). The results imply that POS16 has small possibility to be a proteoglycan. In addition, only one
peak was detected during chromatographic analysis (Supporting Fig. S3), which suggests that P. edaphicus NUST16 produces one type of polysaccharide. The weight-average molecular weight of POS16 was 1.2×107 Da, which was extrapolated from the retention time of standards (Fig. 2A). Monosaccharide analysis showed that POS16 was comprised of D-mannose, D-glucuronic acid, D-glucose, D-galactose, and L-fucose with the molar ratio of 3.11:0.85:3.00:0.90:1.89. In addition, the ketose, D-fructose,
was not detected by the thin-layer chromatography and by the methylation-GC/MS analysis. To the best of our knowledge, POS16 shows different composition of monosaccharides from other biopolymers. The content of L-fucose in POS16 is higher than many others except for FucoPol, a fucose-rich bacterial EPS, which contains 32-36 mol% L-fucose in its molecule. However, other glycosides in FucoPol differ from these in POS16 [27] and D-mannoside is absent in FucoPol [28]. Besides, no similarities are found between POS16
and other polysaccharides produced by Peanibacillus strains [29-32]. (Position for Fig. 2) The NMR technique shows paramount importance in glycans’ structural analysis [33, 34], and the methylation-GC/MS analysis gives useful information on glycosidic linkage [23]. For this reason, these methods were introduced to determine the structure of POS16. Figure 3 shows the 1H and 13C NMR spectra. As shown in Fig. 3A, the chemical shifts, δ 1.21 and 1.29 ppm, are typical values of protons in methyl groups that correspond to the C6 moieties of two L-fucosides in POS16. In addition, the chemical shifts in
C spectrum, δ
13
15.27 and 15.40 ppm, give further evidence for the existence of two L-fucosides (Fig. 3B, α and β). The weak signals, δ 2.16 ppm in 1H spectrum and δ 20.36 ppm in 13C spectrum, imply the existence of acetyl group which can be further determined in methylation analysis. The molar ratio of the acetyl group to POS16 repeating unit was 0.52, which agrees well with the intensity of NMR signals (Table 1). Ten anomeric carbon signals are strong (Fig. 3B, 1-10). They are in good correlation with the proton signals (Fig. 3A, a-f3) and in line with the analysis results of monosaccharide composition (Fig. 2C). Additionally, the strong signal, δ 175.74 ppm, confirms the existence of glucuronate (Fig. 3B, γ). As the chemical shifts from δ 59.71 ppm to 61.39 ppm account for seven C6 signals (Fig. 3B), another conclusion can be drawn is that the number of glycosidic
linkage from C6 position in the repeat unit of POS16 is two. Taking reference to the confirmed information [35], ten glycosides were designated with most possibility as follows (from a to f3); α-GlcA (98.31, 5.50), α-Man (99.96, 5.15), α-Man (101.23, 5.15), α-Fuc (100.46, 4.92), α-Fuc (100.64, 4.92), β-Gal (102.65, 4.86), β-Man (103.35, 4.60), β-Glc (101.50, 4.50), β-Glc (101.79, 4.50), and β-Glc (102.26, 4.50). (Position for Fig. 3) Linkage information was obtained from methylation results (Table 1). Molar ratio of each glycoside agreed well with the results from monosaccharide analysis. Three glucosides were linked via 1,3-linkage. Two mannosides were linked via 1,3-linkage while the C6 positions were both branch points. The third mannoside had a C2 position to be derivatized by acetyl group (molar ratio 0.52:1.0). Additional experiment of partial acidic hydrolysis using Sun et al.’s method [36] proved that the glucuronide, fucoside and galactoside were most probably in the branched-chain, as the content of these glycosides in POS16 residues was remarkably
decreased
after
hydrolysis
(data
not
shown).
More
likely
two
6-deoxy-galactosides (L-fucosides) were linked with the terminal galactoside to form one branch in C6 of the mannoside. Glucuronide was linked to C6 of another mannoside. (Position for Table 1) The FT-IR spectrum of POS16 showed the typical polysaccharide profile (Fig. 4) [31, 37, 38]. The broaden peak at 3313.11 cm-1 indicated the stretching vibration of O-H at association status. The peaks at 2912.95, 1402.00, and 1362.94 cm-1 implied the existence of methyl groups of L-fucose. The strong peak at 1595.81 cm-1 gave evidence of carboxyl group. 913.18, 886.13, and 791.15 cm-1 suggested that both β and α conformation existed in POS16 molecules. As a result, a possible structure of POS16 was given in terms of the above listed information (Fig. 3C). Three glucosides and three mannosides consecutively constituted the backbone of POS16. The Gal(1→3)Fuc(1→3)Fuc(1→ and GlcA(1→ branches were linked to the C6 of two mannosides, respectively. The other mannoside was partially derivatized by the acetyl group at C2 position. (Position for Fig. 4) 3.3 Rheological characterization of POS16
The most fascinating aspect of biopolymers is their rheological modulatory property that enlarges the commodity lists in many industries and facilitates the pharmaceutical application in medical field. Not surprisingly, POS16 showed satisfactory rheological properties. As shown in figure 5, POS16 behave evident shear-thinning patterns that fit the Hershel-Bulkley model well [39]. The apparent viscosity (η) dropped down when speeding up the shear rate. When the concentration of POS16 increased, the solution became more viscous. Moreover, higher value of viscosity was observed near neutral conditions (pH6-9) than in acidic or alkaline conditions (Fig. 5B). A similar phenomenon was reported in xanthan gum solution at low pH. The reason was partially attributed to the alteration of charge density of the xanthan molecules [40]. The glucuronides in the nonionic form suppress the electrostatic repulsion, which consequently allows a more compact conformation and reduces the viscosity. In alkaline solutions the decrease of viscosity was subjected to the alteration of charge density and change of the hydrodynamic volume of the polymer molecules [28, 41]. Meanwhile, it is reported that the hydrogen bonds are weakened by hydroxyl ions [42]. Besides, ζ-potentials gave further information on POS16 stability which in turn affects the solution viscosity (Supporting Table S1). Owing to its macromolecular weight, POS16 possesses some colloid properties. In acidic conditions, the magnitude of charge of POS16 reduced because the glucuronides tended to be nonionic form, which generated a smallζ-potential (-21.8 mV, 1 g/L POS16 at pH4). ζ-Potentials increased in higher pH solutions. In neutral condition, the value -70.7 mV was detected (1 g/L POS16 at pH7) whereas -54.6 mV was obtained in alkaline condition (pH11). Due to the reduction of electrostatic repulsion, macromolecules are prone to aggregate or to form compact conformation at low ζ-potentials. Therefore, POS16 showed high viscosity in neutral condition where the ζ-potential had the highest value. In addition, temperature sweep curves gave information on the interaction between POS16 molecules (Fig. 6). When the solution was heated, the viscosity of POS16 decreased with temperature. Remarkable attenuation was observed when the temperature was over 50°C. This phenomenon was due to the thermo motion beyond the interactive forces between molecules. However, the melting points of POS16 in acidic and alkaline conditions were lower than these in neutral solutions (more than 1°C). The results indicate that the intermolecular interaction at high temperature was strong in neutral condition (Fig. 5B).
Despite the variation of melting points, POS16 showed promising viscosity at moderate temperature. (Position for Fig. 5) (Position for Fig. 6) It is common in practice that various metal ions exist during unit operation of biopolymers. However, in some occasions, high content metal ions affect the behavior of biopolymers, which in turn impacts the quality of final products. Interestingly, POS16 showed remarkable saline-tolerance as the apparent viscosity kept the high values in different ion solutions where 100 g/L salts existed (Fig. 7). Taking xanthan gum and sodium carboxymethylcellulose (CMC.Na) as counterparts, POS16 showed ideal results in solutions containing high concentration of salts (Fig. 8). The viscosity of POS16 was comparable with xanthan gum whereas it was much higher than CMC.Na in the whole shear rate range. When monovalent ions (Na+ and K+) were added, the viscosity of all polymers decreased and POS16 was slightly lower than it of xanthan gum. Interestingly, different results were obtained when divalent ions (Mg2+ and Ca2+) were added. As xanthan gum and CMC.Na were partially soluble in 100 g/L CaCl2 solution, POS16 showed superior advantage to both counterparts. The parameters of Hershel-Bulkley model were listed in supporting Table S2, which gave the quantified comparison between these polymers. These results suggest that POS16 is suitable for applying to operations where high concentrations of divalent salts exist. In general, POS16 shows potential use in future as a rheological modulator. Despite these results, further investigation should be performed, including the tests of viscoelastic dynamics, freezing-thawing, and stability properties. (Position for Fig. 7) (Position for Fig. 8) 4. Conclusion Exploring new microbial species and discovering new polysaccharides with novel traits are essential not only for research concerns but also for daily application. In this work, we reported the structure and rheological properties of an exopolysaccharide, POS16, produced by a new strain, Paenibacillus edaphicus NUST16. POS16 has a considerably large molecular weight of 1.2×107 Da. The repeating unit of POS16 is comprised of five types of glycosides,
namely
D-Glc,
D-Man,
L-Fuc,
D-GlcA,
and
D-Gal
with the
molar
ratio
of
3.00:3.11:1.89:0.85:0.90. As a potential rheological additive, POS16 shows typical shear-thinning pattern which fits the Hershel-Bulkley model well. The ideal saline-tolerance in solutions containing high concentration of mono- or divalent metal ions makes POS16 a suitable candidate in the processes where suspending, thickening, texturizing, or mixing is involved.
Acknowledgements This work is supported by the research grants from the National Natural Science Foundation of China (No. 31500418), the Natural Science Foundation of Jiangsu Province (Grants No. BK20150773), and the Fundamental Research Funds for the Central Universities (No. 30915011101, 30920130121013). The authors would like to thank these organizations for financial supports.
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Figure Captions Fig. 1 Phylogenetic tree (A) and fermentation kinetics (B) of Paenibacillus edaphicus NUST16 (A, phylogenetic tree was constructed on the basis of the 16S rDNA sequences from NCBI database. Figure B shows one of three batch fermentation results that performed in the 500 mL shaker flask.)
Fig. 2 Weight-average molecular weight and monosaccharide composition of POS16 (A, weight-average molecular weight was analyzed using the Tskgel G5000PWXL column. Calibration curve of dextran standards is listed in the upper left corner. Composition of monosaccharides was analyzed using the ZorBax SB-Aq column. B, monosaccharide standards derivatized by 1-phenyl-3-methyl-5-pyrazolone. C, POS16 hydrolyzate.)
Fig. 3 1D NMR spectra and the structure of POS16 (A1, full range 1H spectrum of POS16.
A2, partial 1H spectrum. B1, full range 13C spectrum. B2, partial 13C spectrum. C, structure of POS16. a-f3 in A2 and 1-10 in B1 indicate the signals of anomeric protons and carbons. α and β in A1 and B2 show the signals of C6 methyl groups in fucosides.)
Fig. 4 FT-IR spectrum of POS16
Fig. 5 Effect of concentration and pH on the shear-thinning patterns of POS16 solutions (The experiments were carried out at 25°C. A, viscosity curves of different POS16 solutions at pH7. B, the effect of pH on the viscosity of 10 g/L POS16 solution.)
Fig. 6 temperature sweep curves of POS16 solution at different pH (The shear rate was fixed at 1 s-1 and the concentration was 5 g/L. A, temperature sweep curves. B, melting curves were based on the first order derivative of viscosity versus temperature.)
Fig. 7 Apparent viscosity of POS16 in different salt solutions (The shear rate and temperature were maintained at 0.5 s-1 and 25°C, respectively. The final concentration of POS16 was 5 g/L.)
Fig. 8 Effect of salts on the viscosity of POS16, xanthan gum, and sodium carboxymethylcellulose (The experiments were carried out at 25°C. The final concentration of all polymers was 5 g/L. The final concentration of all salts was 100 g/L. Xanthan gum and sodium carboxymethylcellulose (CMC.Na) were partially soluble in 100 g/L CaCl 2 solution. Therefore, the supernatants of these samples were used for analysis.)
Table 1 Methylation results of POS16 Methylated sugar a
Type of linkage
Retention time b
Molar ratio
2,3,4-Me3-GlcAp
terminal GlcAp
1.000
0.42
2,3,4,6-Me4-Galp
terminal Galp
1.043
0.78
2,4-Me2-Fucp
1,3-linked Fucp
1.145
1.82
2,4,6-Me3-Glcp
1,3-linked Glcp
1.151
3.00
2,4,6-Me3-Manp
1,3-linked Manp
1.175
0.46
4,6-Me2-Manp
1,2,3-linked Manp
1.184
0.52
2,4-Me2-Manp
1,3,6-linked Manp
1.218
2.14
4-Me-Glcp
1,2,3,6-linked Glcp
1.241
trace
a
2,3,4,6-Me4-Galp is the abbreviation of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol,
etc. b
Retention time is relative to the time of 2,3,4-Me3-GlcAp.