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Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii
Accepted Manuscript Title: Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii Author: Seyyed Vahid Niknezhad Mohammad Ali Asadollahi Akram Zamani Davoud Biria PII: DOI: Reference:
S0141-8130(15)30068-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.065 BIOMAC 5479
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
11-8-2015 28-9-2015 19-10-2015
Please cite this article as: S.V. Niknezhad, M.A. Asadollahi, A. Zamani, D. Biria, Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.10.065 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.
Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii
Department of Biotechnology, Faculty of Advanced Sciences and Technologies,
University of Isfahan, Isfahan 81746-73441, Iran
Swedish center for resource recovery, University of Borås, 50190 Borås, Sweden
*Corresponding author:
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E-mail address: [email protected]
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Seyyed Vahid Niknezhad1, Mohammad Ali Asadollahi1,*, Akram Zamani2, Davoud Biria1
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Abstract Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was
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investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2
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g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose
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and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three
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consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested
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that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells
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immobilized in CA beads for xanthan production.
Keywords: Xanthan gum, Immobilization, Xanthomonas
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1. Introduction Natural polymers including polysaccharides have attracted renewed attention in recent years due to their prominent properties such as biocompatibility, biodegradability, non-
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toxicity, and renewability [1-3]. Xanthan gum is a natural high molecular weight heteropolysaccharide which is mainly produced by the Gram-negative bacteria of the genus
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Xanthomonas. Xanthan gum has found numerous applications in many industries such as
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food, cosmetics, pharmaceutical, and oil recovery because of its unique rheological properties. For instance, xanthan gum has high water solubility (at both low and high
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temperatures), high viscosity even at low concentrations, excellent stability over a wide range of pH and temperatures, compatibility with most metallic salts, and high stability in
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aqueous solutions [4-6]. Xanthan is the most widely used industrial natural gum with a
10% [6,7].
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worldwide production of 30,000 tonnes/year which is increasing at an annual rate of 5-
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Most of the previous research on microbial xanthan production has focused on the type of carbon source [5-12], nutritional requirements of microorganisms [13-15], and optimization of operating conditions [4,16-18]. Using immobilized cells for production of microbial products offers several advantages over the conventional free cell systems including higher cell density, higher metabolic activity, possibility of cell recovery and reuse, lower downstream costs associated with cell separation, and improved cell resistance to toxic and inhibitory compounds. Although immobilized cell systems have been widely employed for the production of a broad range of biological products, there are only few reports [19,20] on using immobilized cells for
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xanthan production. Therefore, the aim of this study was to use immobilized Xanthomonas cells for microbial xanthan production. Entrapment of living cells in the beads of calcium alginate (CA) is one of the widely used
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methods for immobilization of living cells [21]. However, CA beads suffer from poor mechanical properties [22]. Enhancement of mechanical properties of calcium alginate
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beads by various methods such as adding fillers [23], using Cu2+ [24] and Ba2+ [25] instead
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of Ca2+ as gel inducing agents, and using a combination of alginate-chitosan [26] has been reported. Combining CA with polyvinyl alcohol (PVA), boric acid, and Ca(NO3)2 has
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improved the mechanical properties of the beads [22,27]. Presence of CA is expected to enhance the surface properties and hinder agglomeration of beads, while PVA improves
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mechanical properties of the beads.
In this study, xanthan gum production using immobilized cells of X. campestris and X.
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pelargonii in CA and CA-PVA-boric acid (CA-PVA) beads was examined. Glucose and
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hydrolyzed starch were used as carbon sources. 2. Materials and methods
2.1. Substrates and enzymes
Pure glucose monohydrate (Merck, Darmstadt, Germany) and a commercially available pure wheat flour starch (Shahdine Aran, Isfahan, Iran) were used as carbon sources in this study. The CHNS/O elemental analysis (CHNS-932, LECO Co., St. Joseph, MI) proved the purity of starch (98%). There was no indication of nitrogen and sulfur in starch and the weight percentages of carbon and hydrogen were 42.7% and 6.7%, respectively. αAmylase (Liquizyme, Novozymes A/S, Copenhagen, Denmark) and Glucoamylase
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(Dextrozyme GA, Novozymes A/S, Copenhagen, Denmark) were employed for enzymatic hydrolyzed of wheat flour. 2.2. Enzymatic hydrolysis of starch
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Granular starch was milled in a kitchen mill to obtain granules between 0.2 and 0.4 mm particle size (wheat flour starch). pH of a 1:5 starch-water suspension (200 g/L) was
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adjusted to 5.5 and α-amylase was added to this mixture to start liquefaction step. The
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enzyme loading was 1 µl/g starch. The liquefaction step was performed at 90 ˚C and 120 rpm for 2 h. Then, the mixture was cooled down to 65 ˚C and pH was adjusted to 4.5.
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Saccharification step was initiated by addition of 1 µl glucoamylase/g starch and continued at 65 ˚C for 20 h [12,28]. The mixture was centrifuged and supernatant (starch
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hydrolyzate) was separated and stored at 4 ˚C until use. Concentration of reducing sugars
190 g/L.
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in a mixture containing 200 g/L granular starch, as measured by DNS method [29], was
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2.3. Microorganisms and cultivation conditions X. campestris PTCC1473 and X. pelargonii PTCC1474 were purchased from Persian Type Culture Collection (PTCC) and used in this study. The microorganisms were cultivated on GYC plates containing (g/L): glucose 20, calcium carbonate 20, yeast extract 10, and agar 17 at pH 7 for 24 h at 30 ˚C [9,30].
Four loops of cells were transferred from YPC plates to 100 ml of YPD solutions containing (g/L): glucose 20, peptone 20, and yeast extract 10. The cells were incubated at 28 ˚C and 180 rpm for 24 h and the grown cells were used as inoculum for production of xanthan. Solutions of 2 g/L KH2PO4, 0.2 g/L MgSO4, 2 g/L NH4NO3, 2 g/L citric acid, 0.006 g/L H3BO3, 0.006 g/L ZnCl2, 0.0024 g/L FeCl3, and 0.02 g/L CaCl2 were
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supplemented with 20 g/L glucose or starch hydrolyzate [12]. The pH of the solutions was adjusted to 6.6 before autoclaving at 121 ˚C for 20 min. Glucose and starch hydrolyzates were separately autoclaved and added to the culture medium. The sterile solutions were
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inoculated by adding 5% (v/v) of the inoculum for free cell culture and cultivations were performed at 28 ˚C and 180 rpm for 60 h in cotton plugged Erlenmeyer flasks [11,18].
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2.4. Cell immobilization
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Immobilization of cells in CA and CA-PVA beads is illustrated in Fig. 1. For cell immobilization in CA beads, 35 ml of sodium alginate (SA) solutions at concentrations of
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4, 4.5 and 5% (w/v) were prepared (solution A) [22].
For cell immobilization in CA-PVA, 35 ml of 2% (w/v) of PVA (average MW 77,000 to
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79,000) mixture in water was heated at 60 °C to completely dissolve PVA. The PVA solution was then cooled down to 35 °C and mixed with SA at concentrations of 4 and
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4.5% (w/v) and tween 80 as surfactant at concentration of 0.2% (v/v) (solution B) [31].
Fig 1. Cell Immobilization using (A) Calcium alginate, (B) Calcium alginate - PVA.
Five milliliters of microbial culture medium containing about 108 CFU of either X. campestris or X. pelargnii was added to each of the sterilized solutions of A and B and mixed thoroughly. Solutions A and B were extruded via a peristaltic pump through a thin needle into 200 ml of 2% (w/v) CaC12.2H2O and 200 ml of saturated boric acid and 2% (w/v) CaC12.2H2O, respectively [19]. The bead size and shape were controlled by adjusting the distance between needle tip and the surface of the liquid.
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The beads were gently stirred for 24 h to complete the bead formation [19,32], and then washed with distilled water to remove any excess boric acid, SA, and PVA. The bead diameters were measured using a vernier caliper.
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2.5. Estimation of immobilization efficiency For estimating immobilization efficiency, beads were separated from 200 ml of salt
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solutions. Fifty microliters of bead-free solutions were cultivated on YPD plates and
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incubated at 28 ˚C for 24 h. The numbers of colonies were reported as CFU/ml. Thus the number of alive cells that were not entrapped into the beads was estimated.
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2.6. Xanthan production using immobilized cells
The beads were transferred into 100 ml of xanthan production medium and fermentation
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was performed for 60 h. Beads were separated from culture medium and rinsed with
medium to start a new cycle.
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sterilized distilled water. The beads were subsequently transferred into a new culture
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2.7. Recovery and purification of xanthan gum To measure the amount of xanthan produced, 1.5 ml samples were taken at different time intervals and cells were separated by centrifuging the sample for 25 min at 10,000 g. Xanthan crude solution, i.e. cell free supernatant, was mixed with 3 ml solution of 0.1% CaCl2, KCl and NaCl in isopropanol. Precipitated xanthan was collected by centrifugation (30 min, 10,000 g) [9]. This was then dried at 50 ˚C for 48 h and weighed. 2.8. FT-IR spectra of xanthan gum
FT-IR spectra of the dried xanthan gum powders were recorded (FTIR, JASCO, FT/IR6300, Japan) to compare the functional groups of the synthesized xanthan gum with a standard pure xanthan sample (Sigma-Aldrich, USA). The dry sample powder was mixed
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with KBr (spectroscopic grade) and pressed into pellets under reduced pressure. The FTIR spectra were obtained by scanning between 4000 and 400 cm-1 [30]. 2.9. Preparation of samples for scanning electronic microscopy
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Samples of the beads were taken once the cultivation was over and examined by scanning electron microscopy (SEM). The samples were gently washed with o.1 N sulphuric acid
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and fixed with 2.5% glutaraldehyde solution for 20 min. Dehydration of samples was
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performed by ethanol at concentration gradients of 20%, 40%, 60%, 80% to 100% for 15 min. Samples were placed on specimen tube with a silver paint, gold-coated, and tested by
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SEM under a Philips Xl30 model [33].
All preparation steps including washing, fixation, and dehydration, were performed with
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minimum physical disturbance of the sample content. 3. Results
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3.1. Cell immobilization in CA beads
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Different concentrations of SA were used for immobilization of X. campestris and X. pelargonii cells. The concentration of SA and the distance between needle tip and the liquid surface influenced the shape and size of the beads. Various needle tip to liquid surface distances were examined. At distances below 7 cm, the beads did not have a primary structure whereas bead deformation was observed at distances over 8 cm. It was noted that the solutions containing 4 and 4.5% (w/v) of SA resulted in beads with tails which disturbed the uniform spherical structure of the beads (data not shown). Therefore, 5% (w/v) of SA and 7-8 cm distance from needle tip to the liquid surface were chosen as optimum conditions for the CA bead formation and cell immobilization. At these
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conditions uniform spherical beads with the approximate diameter of 2.5 mm were obtained. 3.2. Cell immobilization in CA-PVA beads
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A mixture containing 1% (w/w) PVA, 4% (w/w) SA, and 0.2% (v/v) tween 80 was used for cell immobilization and again different needle tip to liquid surface distances were
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examined. However, none of the beads had spherical shape. By increasing PVA
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concentration to 2% (w/w), spherical beads with a tail were observed (Fig. 2A and B). When concentration of SA was increased to 4.5% (w/w), at needle tip to liquid surface
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distances of 6-7 cm, uniform spherical beads with an average size of 2.6 mm were obtained
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(Fig. 2C).
Fig. 2. Influence of immobilization conditions on the structure of CA- PVA beads
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observed at 24 h: (A) 1% (w/w) PVA, 4% (w/w) SA, and 0.2% (v/v) tween 80, (B) 1%
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(w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80, (C) 2% (w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80.
3.3. Estimation of cell immobiliztion efficiency Beads were maintained in CaCl2 solution (for CA beads) and in 200 ml saturated CaCl2boric acid solution (for CA-PVA beads) for 24 h to complete the crosslinking process. Then, beads were removed by filtration and 50 µl of the filtrate was cultured on YPD plates. Low number of colonies (about 150) on YPD plates after 24 h suggested high efficiency of cell immobilization.
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3.4. Xanthan production by immobilized cells in CA beads X. campestris cells immobilized in CA beads were used for xanthan production with glucose and hydrolyzed starch as carbon sources. For the recovery of xanthan gum, three
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different salts (NaCl, KCl, and CaCl2) each at concentration of 1 g/L and ratio of 2:1 to the supernatant were dissolved in isopropanol. Separation of xanthan gum after 52 h using
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CaCl2 led to precipitation of 3.5 g/L xanthan in the culture medium containing glucose
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while 2.8 and 3.3 g/L xanthan was precipitated for KCl and NaCl as precipitating agents, respectively. In the culture medium containing hydrolyzed starch as carbon source 3.3, 2.7,
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and 3.1 g/L of xanthan was precipitated using CaCl2, KCl, and NaCl, respectively (Fig. 3). Therefore, CaCl2 was chosen as the precipitating agent for the precipitation of xanthan
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gum.
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Fig. 3. Xanthan concentrations obtained by cells of X. campestris immobilized in CA
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beads in culture media containing glucose or hydrolyzed starch after 52 h. Xanthan gum was precipitated by NaCl, CaCl2 or KI.
3.5. Xanthan production by immobilized cells in CA-PVA complex Xanthan concentrations using immobilized cells of X. campestris and X. pelargonii in CAPVA beads were followed in the culture media containing glucose and hydrolyzed starch from the beginning until xanthan concentrations reached a plateau (Fig. 4). Xanthan titers of 8.2 and 9.2 g/L were obtained for X. campestris after 48 h using glucose and hydrolyzed starch as carbon sources, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively.
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Fig. 4. Profiles of xanthan production using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose or hydrolyzed starch.
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After each cycle, beads were separated and rinsed with sterile distilled water. Washed beads were added to the fresh medium and a new cycle was started. This was continued for
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three cycles. Although lower xanthan titers were obtained for the next cycles, higher
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xanthan concentrations were observed compared to those with CA beads (Fig. 5).
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Fig. 5. Xanthan concentrations in the first, second, and third batch cycles using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose (g)
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or hydrolyzed starch (s)
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Xanthan titers were also followed in the culture media inoculated with free cells. After 52
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h xanthan concentrations reached a plateau. Xanthan concentrations were 7 and 6.5 g/L for X. campestris and 6.7 and 6.3 g/L for X. pelarggonii cells grown in culture media containing 2% (w/v) glucose and hydrolyzed starch, respectively (Fig. 6).
Fig. 6. Profiles of xanthan production by free cells of X. campestris and X. pelargonii grown on glucose (g) or hydrolyzed starch (s) 3.6. FT-IR spectra of xanthan gum
The FTIR spectra of synthesized xanthan gum for identifying functional groups were obtained. FTIR spectra of xanthan indicated the presence of hydroxyl (at 3426 cm-1), carbonyl (at 1617 cm-1), and acetal (at 1032 cm-1) groups. Xanthan produced using both
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strains had slightly higher proportions of acetal groups (1028, 1031 cm-1 for X. campestris and X. pelargonii, respectively) than the standard xanthan gum (1032 cm-1). More acetal groups in xanthan gum result in a higher solubility of xanthan which is favored for
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industrial applications [15,30]. The spectra of functional groups are summarized in Table
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1.
3.7. Image of optimized immobilized cells by SEM
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Table 1. The spectra of functional groups
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The surface and central structures of the beads containing immobilized cells of X. campestis were identified with SEM photographs. Fig. 7 shows the SEM images of the CA
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and CA-PVA beads. It was found that each bead was filled with microbial cells. There was a clear difference between the surface part (Fig. 7A) and the center part (Fig. 7B) of the
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CA beads since the surface part of the CA beads was more compact than that of the center
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part. A highly macroporous structure was found on the surfaces of the CA-PVA bead (Fig. 7C) which favors diffusion of substrates and metabolites and thus could explain higher xanthan gum titers using the cells immobilized in the CA-PVA beads. Fig. 7D shows the central part of the CA-PVA beads and indicates suitable porosity for xanthan gum production.
Fig. 7. SEM images of X. campestris cells immobilized in CA or CA-PVA beads. (A) surface of the CA bead, (B) center of the CA bead. (C) surface of the CA-PVA bead, (D) center of the CA-PVA bead.
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4. Discussion Microbial xanthan production was investigated using immobilized cells of X. campestris and X. pelargonii in CA and CA-PVA beads. Glucose and hydrolyzed starch were used as
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carbon sources. Spherical beads with an average diameter of 2.5 mm were formed at SA concentration of 5% (w/v). A needle tip to surface liquid of 7-8 cm resulted in spherical
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bead shape. These beads could be separated easily from culture medium and exhibit large
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surface area for mass transfer [32].
A mixture containing isopropanol and either of CaCl2, NaCl, or KCl salts was used for
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xanthan separation. The highest precipitation yield was obtained with 1 g/L of CaCl2 in isopropanol which resulted in 25% and 6% higher recovery efficiency compared to those
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for KCl and NaCl, respectively (Fig. 3). This could be explained by higher valency of calcium compared to the two other salts resulting in lower solubility of xanthan gum [7].
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The CA beads provided adequate mass transfer properties, protected the cells from the
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influence of external conditions, decreased the sensitivity of the cells and enzymes to the external conditions, which increased the stability and bioconversion activity of cells [32]. However, they were broken after the first cycle which made them inappropriate for repeated batch fermentation. Addition of PVA as an inexpensive and non-toxic polymer to CA enhanced the mechanical stability of the beads [19,34]. Consistent with these results, Wu et al. [22] used Pseudomonas bacteria immobilized in PVA-alginate beads for phenol degradation in a continuous fluidized bed bioreactor. They did not observe any noticeable degradation of the beads even after two weeks in a continuous operation. Since xanthan gum is a large molecule, a combination of SA and PVA was used to enhance mass transfer properties by allowing proper pore size distribution and quite
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mechanical stability. To improve oxygen transfer into the beads and prevent bead floatation, tween 80 at concentration of 0.2% (v/v) was used [31,35]. When glucose was used as carbon source, immobilized cells of X. campestris and X. pelargonii in CA-PVA
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beads in the first cycle resulted in respectively 17% and 19% higher xanthan titers than those observed for free cells due to higher oxygen and culture medium mass transfer. A
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similar trend was obtained when hydrolyzed starch was used as carbon source. In this case,
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40% and 28% increase in xanthan titers were obtained for X. campestris and X. pelargonii, respectively. The results of this study suggested that immobilized cells are advantageous
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over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan
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production. The Xanthomonas immobilized cells in CA-PVA beads were successfully used
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[31] S.H. Song, S.S. Choi, K. Park, Y.J. Yoo, Novel hybrid immobilization of
Ac ce pt e
Technol. 37 (2005) 567-573.
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microorganisms and its applications to biological denitrification, Enzyme Microb.
[32] J. Sun, J. Liu, Y. Liu, Z. Li, J. Nan, Optimization of entrapping conditions of nitrifying bacteria and selection of entrapping agent, Procedia Environ. Sci. 8 (2011) 166172.
[33] W. Yujian, Y. Xiaojuan, L. Hongyu, T. Wei, Immobilization of Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate, Polym. Degrad. Stab. 91 (2006) 2408-2414.
[34] L.U. Yan, M.E.I. Lehe, Production of indigo by immobilization of E. coli BL21 (DE3) cells in calcium-alginate gel capsules, Chin. J. Chem. Eng. 15 (2007) 387-390.
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[35] E. Galindo, G. Salcedo, Detergents improve xanthan yield and polimer quality in
cr
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cultures of Xanthomonas campestris, Enzyme Microb. Technol. 19 (1996) 145-149.
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Figure captions:
Fig 1. Cell Immobilization using (A) Calcium alginate, (B) Calcium alginate - PVA.
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Fig. 2. Influence of immobilization conditions on the structure of CA- PVA beads observed at 24 h: (A) 1% (w/w) PVA, 4% (w/w) SA, and 0.2% (v/v) tween 80, (B) 1%
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(w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80, (C) 2% (w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80.
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Fig. 3. Xanthan concentrations obtained by cells of X. campestris immobilized in CA
Ac ce pt e
beads in culture media containing glucose or hydrolyzed starch after 52 h. Xanthan gum was precipitated by NaCl, CaCl2 or KI.
Fig. 4. Profiles of xanthan production using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose or hydrolyzed starch. Fig. 5. Xanthan concentrations in the first, second, and third batch cycles using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose (g) or hydrolyzed starch (s)
Fig. 6. Profiles of xanthan production by free cells of X. campestris and X. pelargonii grown on glucose (g) or hydrolyzed starch (s)
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Fig. 7. SEM images of X. campestris cells immobilized in CA or CA-PVA beads. (A) surface of the CA bead, (B) center of the CA bead. (C) surface of the CA-PVA bead, (D)
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center of the CA-PVA bead.
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Table 1. The spectra of functional groups Hydroxyl
Carbonyl
Carboxyl
Acetal
Standard Xanthan gum
3426
1617
1412
1032
3402
1613
1432
1028
Xanthan gum synthesized with X. pelargonii
3408
1616
1408
1031
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Xanthan gum synthesized with X. campestris
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Functional group/ Wave number (cm-1)
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Fig. 1
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A- 4, 4.5, 5% (w/v) sodium alginate
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5 ml suspension of bacteria
B- 4, 4.5% (w/v) sodium alginate with 2% (w/v) PVA + 0.2% (v/v) tween
A- Mix and drop into solution of saturated boric acid + 2% (w/v) CaCl2
A- Mix and drop into solution of 2% (w/v) CaCl2
A and B Allow hardening in solution for 12 and 24 h
A- 2.5 mm diameter calcium alginate immobilized beads
A and B- Rinse with distilled water
B- 2.6 mm diameter calcium alginate-PVA immobilized beads
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S0141-8130(15)30068-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.065 BIOMAC 5479
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
11-8-2015 28-9-2015 19-10-2015
Please cite this article as: S.V. Niknezhad, M.A. Asadollahi, A. Zamani, D. Biria, Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.10.065 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.
Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii
Department of Biotechnology, Faculty of Advanced Sciences and Technologies,
University of Isfahan, Isfahan 81746-73441, Iran
Swedish center for resource recovery, University of Borås, 50190 Borås, Sweden
*Corresponding author:
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E-mail address: [email protected]
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Seyyed Vahid Niknezhad1, Mohammad Ali Asadollahi1,*, Akram Zamani2, Davoud Biria1
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Abstract Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was
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investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2
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g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose
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and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three
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consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested
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that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells
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immobilized in CA beads for xanthan production.
Keywords: Xanthan gum, Immobilization, Xanthomonas
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1. Introduction Natural polymers including polysaccharides have attracted renewed attention in recent years due to their prominent properties such as biocompatibility, biodegradability, non-
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toxicity, and renewability [1-3]. Xanthan gum is a natural high molecular weight heteropolysaccharide which is mainly produced by the Gram-negative bacteria of the genus
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Xanthomonas. Xanthan gum has found numerous applications in many industries such as
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food, cosmetics, pharmaceutical, and oil recovery because of its unique rheological properties. For instance, xanthan gum has high water solubility (at both low and high
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temperatures), high viscosity even at low concentrations, excellent stability over a wide range of pH and temperatures, compatibility with most metallic salts, and high stability in
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aqueous solutions [4-6]. Xanthan is the most widely used industrial natural gum with a
10% [6,7].
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worldwide production of 30,000 tonnes/year which is increasing at an annual rate of 5-
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Most of the previous research on microbial xanthan production has focused on the type of carbon source [5-12], nutritional requirements of microorganisms [13-15], and optimization of operating conditions [4,16-18]. Using immobilized cells for production of microbial products offers several advantages over the conventional free cell systems including higher cell density, higher metabolic activity, possibility of cell recovery and reuse, lower downstream costs associated with cell separation, and improved cell resistance to toxic and inhibitory compounds. Although immobilized cell systems have been widely employed for the production of a broad range of biological products, there are only few reports [19,20] on using immobilized cells for
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xanthan production. Therefore, the aim of this study was to use immobilized Xanthomonas cells for microbial xanthan production. Entrapment of living cells in the beads of calcium alginate (CA) is one of the widely used
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methods for immobilization of living cells [21]. However, CA beads suffer from poor mechanical properties [22]. Enhancement of mechanical properties of calcium alginate
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beads by various methods such as adding fillers [23], using Cu2+ [24] and Ba2+ [25] instead
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of Ca2+ as gel inducing agents, and using a combination of alginate-chitosan [26] has been reported. Combining CA with polyvinyl alcohol (PVA), boric acid, and Ca(NO3)2 has
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improved the mechanical properties of the beads [22,27]. Presence of CA is expected to enhance the surface properties and hinder agglomeration of beads, while PVA improves
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mechanical properties of the beads.
In this study, xanthan gum production using immobilized cells of X. campestris and X.
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pelargonii in CA and CA-PVA-boric acid (CA-PVA) beads was examined. Glucose and
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hydrolyzed starch were used as carbon sources. 2. Materials and methods
2.1. Substrates and enzymes
Pure glucose monohydrate (Merck, Darmstadt, Germany) and a commercially available pure wheat flour starch (Shahdine Aran, Isfahan, Iran) were used as carbon sources in this study. The CHNS/O elemental analysis (CHNS-932, LECO Co., St. Joseph, MI) proved the purity of starch (98%). There was no indication of nitrogen and sulfur in starch and the weight percentages of carbon and hydrogen were 42.7% and 6.7%, respectively. αAmylase (Liquizyme, Novozymes A/S, Copenhagen, Denmark) and Glucoamylase
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(Dextrozyme GA, Novozymes A/S, Copenhagen, Denmark) were employed for enzymatic hydrolyzed of wheat flour. 2.2. Enzymatic hydrolysis of starch
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Granular starch was milled in a kitchen mill to obtain granules between 0.2 and 0.4 mm particle size (wheat flour starch). pH of a 1:5 starch-water suspension (200 g/L) was
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adjusted to 5.5 and α-amylase was added to this mixture to start liquefaction step. The
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enzyme loading was 1 µl/g starch. The liquefaction step was performed at 90 ˚C and 120 rpm for 2 h. Then, the mixture was cooled down to 65 ˚C and pH was adjusted to 4.5.
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Saccharification step was initiated by addition of 1 µl glucoamylase/g starch and continued at 65 ˚C for 20 h [12,28]. The mixture was centrifuged and supernatant (starch
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hydrolyzate) was separated and stored at 4 ˚C until use. Concentration of reducing sugars
190 g/L.
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in a mixture containing 200 g/L granular starch, as measured by DNS method [29], was
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2.3. Microorganisms and cultivation conditions X. campestris PTCC1473 and X. pelargonii PTCC1474 were purchased from Persian Type Culture Collection (PTCC) and used in this study. The microorganisms were cultivated on GYC plates containing (g/L): glucose 20, calcium carbonate 20, yeast extract 10, and agar 17 at pH 7 for 24 h at 30 ˚C [9,30].
Four loops of cells were transferred from YPC plates to 100 ml of YPD solutions containing (g/L): glucose 20, peptone 20, and yeast extract 10. The cells were incubated at 28 ˚C and 180 rpm for 24 h and the grown cells were used as inoculum for production of xanthan. Solutions of 2 g/L KH2PO4, 0.2 g/L MgSO4, 2 g/L NH4NO3, 2 g/L citric acid, 0.006 g/L H3BO3, 0.006 g/L ZnCl2, 0.0024 g/L FeCl3, and 0.02 g/L CaCl2 were
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supplemented with 20 g/L glucose or starch hydrolyzate [12]. The pH of the solutions was adjusted to 6.6 before autoclaving at 121 ˚C for 20 min. Glucose and starch hydrolyzates were separately autoclaved and added to the culture medium. The sterile solutions were
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inoculated by adding 5% (v/v) of the inoculum for free cell culture and cultivations were performed at 28 ˚C and 180 rpm for 60 h in cotton plugged Erlenmeyer flasks [11,18].
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2.4. Cell immobilization
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Immobilization of cells in CA and CA-PVA beads is illustrated in Fig. 1. For cell immobilization in CA beads, 35 ml of sodium alginate (SA) solutions at concentrations of
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4, 4.5 and 5% (w/v) were prepared (solution A) [22].
For cell immobilization in CA-PVA, 35 ml of 2% (w/v) of PVA (average MW 77,000 to
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79,000) mixture in water was heated at 60 °C to completely dissolve PVA. The PVA solution was then cooled down to 35 °C and mixed with SA at concentrations of 4 and
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4.5% (w/v) and tween 80 as surfactant at concentration of 0.2% (v/v) (solution B) [31].
Fig 1. Cell Immobilization using (A) Calcium alginate, (B) Calcium alginate - PVA.
Five milliliters of microbial culture medium containing about 108 CFU of either X. campestris or X. pelargnii was added to each of the sterilized solutions of A and B and mixed thoroughly. Solutions A and B were extruded via a peristaltic pump through a thin needle into 200 ml of 2% (w/v) CaC12.2H2O and 200 ml of saturated boric acid and 2% (w/v) CaC12.2H2O, respectively [19]. The bead size and shape were controlled by adjusting the distance between needle tip and the surface of the liquid.
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The beads were gently stirred for 24 h to complete the bead formation [19,32], and then washed with distilled water to remove any excess boric acid, SA, and PVA. The bead diameters were measured using a vernier caliper.
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2.5. Estimation of immobilization efficiency For estimating immobilization efficiency, beads were separated from 200 ml of salt
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solutions. Fifty microliters of bead-free solutions were cultivated on YPD plates and
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incubated at 28 ˚C for 24 h. The numbers of colonies were reported as CFU/ml. Thus the number of alive cells that were not entrapped into the beads was estimated.
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2.6. Xanthan production using immobilized cells
The beads were transferred into 100 ml of xanthan production medium and fermentation
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was performed for 60 h. Beads were separated from culture medium and rinsed with
medium to start a new cycle.
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sterilized distilled water. The beads were subsequently transferred into a new culture
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2.7. Recovery and purification of xanthan gum To measure the amount of xanthan produced, 1.5 ml samples were taken at different time intervals and cells were separated by centrifuging the sample for 25 min at 10,000 g. Xanthan crude solution, i.e. cell free supernatant, was mixed with 3 ml solution of 0.1% CaCl2, KCl and NaCl in isopropanol. Precipitated xanthan was collected by centrifugation (30 min, 10,000 g) [9]. This was then dried at 50 ˚C for 48 h and weighed. 2.8. FT-IR spectra of xanthan gum
FT-IR spectra of the dried xanthan gum powders were recorded (FTIR, JASCO, FT/IR6300, Japan) to compare the functional groups of the synthesized xanthan gum with a standard pure xanthan sample (Sigma-Aldrich, USA). The dry sample powder was mixed
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with KBr (spectroscopic grade) and pressed into pellets under reduced pressure. The FTIR spectra were obtained by scanning between 4000 and 400 cm-1 [30]. 2.9. Preparation of samples for scanning electronic microscopy
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Samples of the beads were taken once the cultivation was over and examined by scanning electron microscopy (SEM). The samples were gently washed with o.1 N sulphuric acid
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and fixed with 2.5% glutaraldehyde solution for 20 min. Dehydration of samples was
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performed by ethanol at concentration gradients of 20%, 40%, 60%, 80% to 100% for 15 min. Samples were placed on specimen tube with a silver paint, gold-coated, and tested by
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SEM under a Philips Xl30 model [33].
All preparation steps including washing, fixation, and dehydration, were performed with
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minimum physical disturbance of the sample content. 3. Results
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3.1. Cell immobilization in CA beads
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Different concentrations of SA were used for immobilization of X. campestris and X. pelargonii cells. The concentration of SA and the distance between needle tip and the liquid surface influenced the shape and size of the beads. Various needle tip to liquid surface distances were examined. At distances below 7 cm, the beads did not have a primary structure whereas bead deformation was observed at distances over 8 cm. It was noted that the solutions containing 4 and 4.5% (w/v) of SA resulted in beads with tails which disturbed the uniform spherical structure of the beads (data not shown). Therefore, 5% (w/v) of SA and 7-8 cm distance from needle tip to the liquid surface were chosen as optimum conditions for the CA bead formation and cell immobilization. At these
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conditions uniform spherical beads with the approximate diameter of 2.5 mm were obtained. 3.2. Cell immobilization in CA-PVA beads
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A mixture containing 1% (w/w) PVA, 4% (w/w) SA, and 0.2% (v/v) tween 80 was used for cell immobilization and again different needle tip to liquid surface distances were
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examined. However, none of the beads had spherical shape. By increasing PVA
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concentration to 2% (w/w), spherical beads with a tail were observed (Fig. 2A and B). When concentration of SA was increased to 4.5% (w/w), at needle tip to liquid surface
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distances of 6-7 cm, uniform spherical beads with an average size of 2.6 mm were obtained
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(Fig. 2C).
Fig. 2. Influence of immobilization conditions on the structure of CA- PVA beads
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observed at 24 h: (A) 1% (w/w) PVA, 4% (w/w) SA, and 0.2% (v/v) tween 80, (B) 1%
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(w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80, (C) 2% (w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80.
3.3. Estimation of cell immobiliztion efficiency Beads were maintained in CaCl2 solution (for CA beads) and in 200 ml saturated CaCl2boric acid solution (for CA-PVA beads) for 24 h to complete the crosslinking process. Then, beads were removed by filtration and 50 µl of the filtrate was cultured on YPD plates. Low number of colonies (about 150) on YPD plates after 24 h suggested high efficiency of cell immobilization.
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3.4. Xanthan production by immobilized cells in CA beads X. campestris cells immobilized in CA beads were used for xanthan production with glucose and hydrolyzed starch as carbon sources. For the recovery of xanthan gum, three
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different salts (NaCl, KCl, and CaCl2) each at concentration of 1 g/L and ratio of 2:1 to the supernatant were dissolved in isopropanol. Separation of xanthan gum after 52 h using
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CaCl2 led to precipitation of 3.5 g/L xanthan in the culture medium containing glucose
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while 2.8 and 3.3 g/L xanthan was precipitated for KCl and NaCl as precipitating agents, respectively. In the culture medium containing hydrolyzed starch as carbon source 3.3, 2.7,
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and 3.1 g/L of xanthan was precipitated using CaCl2, KCl, and NaCl, respectively (Fig. 3). Therefore, CaCl2 was chosen as the precipitating agent for the precipitation of xanthan
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gum.
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Fig. 3. Xanthan concentrations obtained by cells of X. campestris immobilized in CA
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beads in culture media containing glucose or hydrolyzed starch after 52 h. Xanthan gum was precipitated by NaCl, CaCl2 or KI.
3.5. Xanthan production by immobilized cells in CA-PVA complex Xanthan concentrations using immobilized cells of X. campestris and X. pelargonii in CAPVA beads were followed in the culture media containing glucose and hydrolyzed starch from the beginning until xanthan concentrations reached a plateau (Fig. 4). Xanthan titers of 8.2 and 9.2 g/L were obtained for X. campestris after 48 h using glucose and hydrolyzed starch as carbon sources, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively.
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Fig. 4. Profiles of xanthan production using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose or hydrolyzed starch.
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After each cycle, beads were separated and rinsed with sterile distilled water. Washed beads were added to the fresh medium and a new cycle was started. This was continued for
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three cycles. Although lower xanthan titers were obtained for the next cycles, higher
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xanthan concentrations were observed compared to those with CA beads (Fig. 5).
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Fig. 5. Xanthan concentrations in the first, second, and third batch cycles using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose (g)
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or hydrolyzed starch (s)
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Xanthan titers were also followed in the culture media inoculated with free cells. After 52
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h xanthan concentrations reached a plateau. Xanthan concentrations were 7 and 6.5 g/L for X. campestris and 6.7 and 6.3 g/L for X. pelarggonii cells grown in culture media containing 2% (w/v) glucose and hydrolyzed starch, respectively (Fig. 6).
Fig. 6. Profiles of xanthan production by free cells of X. campestris and X. pelargonii grown on glucose (g) or hydrolyzed starch (s) 3.6. FT-IR spectra of xanthan gum
The FTIR spectra of synthesized xanthan gum for identifying functional groups were obtained. FTIR spectra of xanthan indicated the presence of hydroxyl (at 3426 cm-1), carbonyl (at 1617 cm-1), and acetal (at 1032 cm-1) groups. Xanthan produced using both
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strains had slightly higher proportions of acetal groups (1028, 1031 cm-1 for X. campestris and X. pelargonii, respectively) than the standard xanthan gum (1032 cm-1). More acetal groups in xanthan gum result in a higher solubility of xanthan which is favored for
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industrial applications [15,30]. The spectra of functional groups are summarized in Table
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1.
3.7. Image of optimized immobilized cells by SEM
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Table 1. The spectra of functional groups
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The surface and central structures of the beads containing immobilized cells of X. campestis were identified with SEM photographs. Fig. 7 shows the SEM images of the CA
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and CA-PVA beads. It was found that each bead was filled with microbial cells. There was a clear difference between the surface part (Fig. 7A) and the center part (Fig. 7B) of the
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CA beads since the surface part of the CA beads was more compact than that of the center
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part. A highly macroporous structure was found on the surfaces of the CA-PVA bead (Fig. 7C) which favors diffusion of substrates and metabolites and thus could explain higher xanthan gum titers using the cells immobilized in the CA-PVA beads. Fig. 7D shows the central part of the CA-PVA beads and indicates suitable porosity for xanthan gum production.
Fig. 7. SEM images of X. campestris cells immobilized in CA or CA-PVA beads. (A) surface of the CA bead, (B) center of the CA bead. (C) surface of the CA-PVA bead, (D) center of the CA-PVA bead.
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4. Discussion Microbial xanthan production was investigated using immobilized cells of X. campestris and X. pelargonii in CA and CA-PVA beads. Glucose and hydrolyzed starch were used as
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carbon sources. Spherical beads with an average diameter of 2.5 mm were formed at SA concentration of 5% (w/v). A needle tip to surface liquid of 7-8 cm resulted in spherical
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bead shape. These beads could be separated easily from culture medium and exhibit large
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surface area for mass transfer [32].
A mixture containing isopropanol and either of CaCl2, NaCl, or KCl salts was used for
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xanthan separation. The highest precipitation yield was obtained with 1 g/L of CaCl2 in isopropanol which resulted in 25% and 6% higher recovery efficiency compared to those
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for KCl and NaCl, respectively (Fig. 3). This could be explained by higher valency of calcium compared to the two other salts resulting in lower solubility of xanthan gum [7].
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The CA beads provided adequate mass transfer properties, protected the cells from the
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influence of external conditions, decreased the sensitivity of the cells and enzymes to the external conditions, which increased the stability and bioconversion activity of cells [32]. However, they were broken after the first cycle which made them inappropriate for repeated batch fermentation. Addition of PVA as an inexpensive and non-toxic polymer to CA enhanced the mechanical stability of the beads [19,34]. Consistent with these results, Wu et al. [22] used Pseudomonas bacteria immobilized in PVA-alginate beads for phenol degradation in a continuous fluidized bed bioreactor. They did not observe any noticeable degradation of the beads even after two weeks in a continuous operation. Since xanthan gum is a large molecule, a combination of SA and PVA was used to enhance mass transfer properties by allowing proper pore size distribution and quite
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mechanical stability. To improve oxygen transfer into the beads and prevent bead floatation, tween 80 at concentration of 0.2% (v/v) was used [31,35]. When glucose was used as carbon source, immobilized cells of X. campestris and X. pelargonii in CA-PVA
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beads in the first cycle resulted in respectively 17% and 19% higher xanthan titers than those observed for free cells due to higher oxygen and culture medium mass transfer. A
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similar trend was obtained when hydrolyzed starch was used as carbon source. In this case,
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40% and 28% increase in xanthan titers were obtained for X. campestris and X. pelargonii, respectively. The results of this study suggested that immobilized cells are advantageous
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over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan
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production. The Xanthomonas immobilized cells in CA-PVA beads were successfully used
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Figure captions:
Fig 1. Cell Immobilization using (A) Calcium alginate, (B) Calcium alginate - PVA.
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Fig. 2. Influence of immobilization conditions on the structure of CA- PVA beads observed at 24 h: (A) 1% (w/w) PVA, 4% (w/w) SA, and 0.2% (v/v) tween 80, (B) 1%
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(w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80, (C) 2% (w/w) PVA, 4.5% (w/w) SA, and 0.2% (v/v) tween 80.
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Fig. 3. Xanthan concentrations obtained by cells of X. campestris immobilized in CA
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beads in culture media containing glucose or hydrolyzed starch after 52 h. Xanthan gum was precipitated by NaCl, CaCl2 or KI.
Fig. 4. Profiles of xanthan production using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose or hydrolyzed starch. Fig. 5. Xanthan concentrations in the first, second, and third batch cycles using cells of X. campestris and X. pelargonii immobilized in CA-PVA complex and grown on glucose (g) or hydrolyzed starch (s)
Fig. 6. Profiles of xanthan production by free cells of X. campestris and X. pelargonii grown on glucose (g) or hydrolyzed starch (s)
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Fig. 7. SEM images of X. campestris cells immobilized in CA or CA-PVA beads. (A) surface of the CA bead, (B) center of the CA bead. (C) surface of the CA-PVA bead, (D)
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center of the CA-PVA bead.
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Table 1. The spectra of functional groups Hydroxyl
Carbonyl
Carboxyl
Acetal
Standard Xanthan gum
3426
1617
1412
1032
3402
1613
1432
1028
Xanthan gum synthesized with X. pelargonii
3408
1616
1408
1031
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Xanthan gum synthesized with X. campestris
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Functional group/ Wave number (cm-1)
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Fig. 1
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A- 4, 4.5, 5% (w/v) sodium alginate
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5 ml suspension of bacteria
B- 4, 4.5% (w/v) sodium alginate with 2% (w/v) PVA + 0.2% (v/v) tween
A- Mix and drop into solution of saturated boric acid + 2% (w/v) CaCl2
A- Mix and drop into solution of 2% (w/v) CaCl2
A and B Allow hardening in solution for 12 and 24 h
A- 2.5 mm diameter calcium alginate immobilized beads
A and B- Rinse with distilled water
B- 2.6 mm diameter calcium alginate-PVA immobilized beads
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
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