text: https://doi.org/10.1016/j.bjm.2015.11.006
  Márcia de Mello Luvielmoa *
  Caroline Dellinghausen Borgesb
  Daniela de Oliveira Toyamac
  Claire Tondo Vendruscolod
  Adilma Regina Pippa Scamparinic
  aSchool of Chemistry and Food, Federal University of Rio Grande, RS, Brazil
  bCenter of Chemical, Pharmaceutical and Food Sciences, Universidade Federal de Pelotas, RS, Brazil
  cDepartment of Food Sciences, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, SP, Brazil
  dCenter of Technological Development, Biopolymeres, Universidade Federal de Pelotas, Pelotas, RS, Brazil
  Keywords Xanthomonas campestris; Xanthan production; Alkali stress; Transmission microscopy; Scanning electron microscopy
  Xanthan gum is a polysaccharide produced by bacterium species Xanthomonas campestris.1Xanthomonas cells are single straight rods, Gram-negative, 0.4–0.7 µm wide and 0.7–1.8 µm long.2 Several biological functions, attributed to the exopolysaccharide produced by these pathogenic bacteria, include protection against adverse environmental conditions such as drying, temperature oscillations, radiation, certain chemical products, and adhesion.3
  The production and commercialization of xanthan gum as thickener and stabilizer has progressively increased at an annual rate of 5–10%,4 due to its better physicochemical properties as compared to other available polysaccharides. The most significant properties of xanthan gum include its high viscosity at low concentrations and stability for a wide range of temperatures and pH (even in the presence of salts).5,6
  Both microorganism growth and xanthan production are influenced by several factors that include the type of bioreactor, operation mode (batch or continuous), medium composition, cultivation conditions (temperature, pH, stirrer speed, air flow rate), fermentation time, strain of the microorganisms and post-fermentation conditions (heat treatment, recovery, purification). Due to the wide application of xanthan gum and its worldwide market, several studies have been performed to optimize xanthan gum production.4,7–13 However, the effect of this polysaccharide on the ultrastructure of cells and behavior of gum at different production stages have not yet been addressed properly, and thus the present study is relevant and may provide greater understanding on this process.
  Peter et al.14 observed accumulation of xanthan around the cells cultured on agar using transmission electron microscopy, while Contreras et al.15 have reported plant-bacterial cell interactions during pathogenesis.
  Following media were used for the culture of the bacterium: standard solid and liquid YM medium,16 medium I7 and nutrient medium.17
  An aliquot of 50 mL of standard YM medium was placed in three 250 mL-Erlenmeyer flasks with vented caps. Ten standard scoops (platinum 3.0 mm diameter-scoop) of culture grown in standard solid YM for 48 h at 28 °C were added to each flask. The flasks were then incubated on an orbital shaker (New Brunswick Scientific Co. G27) at 28 °C and stirred at 250 rpm for 24 h. Cell suspensions with at least 2.6 × 109 CFU mL−118 were used as fermentation inoculum.
  The rheological behavior of the samples was evaluated by a HAAK rheometer RS 150, using coaxial cylinder type sensor DG 41. The viscosity of the samples was determined at 25 °C. For that, 1.0% (w/v) xanthan solutions were prepared using distilled water, the mixture was stirred for 2 h, and then heated at 60 °C for 20 min.19 The rheological analysis was performed in triplicate, using shear rate from 0.01 s−1 to 60 s−1, with 5100 mm slot for 300 s.
  Gum samples were first fixed overnight in a solution containing 0.1 M sodium cacodylate buffer pH 7.4, 25% glutaraldehyde and tannic acid, and then post-fixed in osmium tetroxide. Pellets were washed thrice with sodium cacodylate buffer pH 7.4, between the first and second fixation procedure, and at the end of the second fixation. The samples were then subjected to a dehydration series in 30%, 50%, 70%, and 90% (v/v) ethanol solutions for about 20 min each. Critical point drying of the samples was accomplished via Balzers CPD 030. The samples were fixed to metal stubs and gold coated with a sputter coater (Balzers CD 050 Sputter 5), followed by measurements at 15 kV in a scanning electron microscope JSM 5800 LV JEOL.20,21
  The samples were collected at their appropriate time point according to fermentation pathway, and stored at 4 °C. Then they were centrifuged at 12,200 × g for 5 min at room temperature and re-suspended in ultra-pure water, stirred, and centrifuged again under the same conditions. The procedure was repeated thrice. The pellets were fixed overnight in a solution containing 0.1 M sodium cacodylate buffer pH 7.4, 25% glutaraldehyde, and tannic acid, and then post-fixed in osmium tetroxide for 2 h. The pellets were washed thrice with sodium cacodylate buffer between the first and second fixation procedures and at the end of the second fixation. The samples were then subjected to a dehydration series in ethanol solutions, absolute ethanol, a mixture of propylene oxide and absolute ethanol (1:1), and propylene oxide alone, for approximately 20 min each, with the exception of propylene oxide. The resin and propylene oxide (1:1) remained on the rotator apparatus for 2 h. The oxide was removed and blended with the pure resin. The Eppendorf tubes remained inside the rotator overnight. After inclusion, polymerization was carried out at 60 °C for 48 h. The material was sectioned using ultramicrotome Leica Ultra Cut, and observed under a Zeiss LEO 906 Transmission Electron Microscope, at a magnification between 6000 and 27800 ×.20,22,23
  Low production rates were observed when pH was not controlled during processing. Papagianni et al.11 evaluated xanthan production by X. campestris ATCC 1395 under non-controlled pH conditions at different stirring speeds. The highest yield obtained by them was approximately 6.5 g L−1 at 600 rpm, which is lower than that obtained in the current study. In contrast, Borges et al.24 studied the xanthan production by Xanthomonas arboricola pv. pruni 106 under non-controlled pH conditions, and found slightly higher production rate of 10.2 g L−1 at 400 rpm.
  Different behaviors have been observed under non-controlled pH conditions, as the medium pH might either decrease from neutral to near pH 5 or even increase, depending on several factors including medium composition, strain used and processing conditions.11,24,25
  However, the present study demonstrates that the gum production tends to increase with alkali stress or pH control. Gupte and Kamat26 evaluated xanthan production by X. campestris ICA-125 at different pH values (4–9), and observed the highest yield at pH 6 with a production rate of 18.6 g L−1, which decreased and reached 3.1 g L−1 at pH 9. In contrast, Borges et al.24 found a slight reduction in xanthan production at pH 9 when compared to pH 7, with values of 16.05 and 16.9 g L−1, respectively. Liakopoulou-Kyriakides et al.27 investigated xanthan production by X. campestris XLM1521 and found an increase from pH 6 to 8, reaching 17.3 g L−1 at pH 8. These values were close to those obtained in the current study using alkali stress.
  As shown in Fig. 2, the 1% solutions prepared from xanthan gums obtained from the conventional fermentation alkali stress free (W/AS) and with alkali stress process for 1 h (AS1h), 24 h (AS24h), and 48 h (AS48h) exhibited a pseudoplastic fluid behavior, as also observed by other authors.6,28–31 The samples not subjected to alkali stress (W/AS) exhibited apparent viscosity of 317 mPa s at a shear rate of 10 s−1, while the samples subjected to AS for 1 h, 24 h, and 48 h yielded viscosity values of approximately 117 mPa s, under the same shear rate. It is worth noting that the quality of the gum obtained after the alkali stress was 64% lower as compared to the gum obtained without the alkali stress process while the time of alkali stress did not affect the quality of the gum obtained in the present study.
  Similar results were observed by Gupte and Kamat26 and Borges et al.,24 who also evaluated the viscosity of the gum produced at different pH values.
  As reported by Gupte and Kamat,26 the viscosity of the xanthan solution increased by 1% when produced at pH 4–7 (153 mPa s to 559 mPa s, respectively), with a decrease at pH 8 and pH 9 (280 mPa s to 124 mPa s, respectively).
  Borges et al.24 have shown that the gum produced under non-controlled pH conditions and at pH 5 (76.3 mPa s and 77.7 mPa s, respectively) had higher viscosity rates when compared to that produced at pH 7 and pH 9 (52.8 mPa s and 50.4 mPa s, respectively), at a shear rate of 19 s−1, using 0.5% solution. According to these authors, this phenomenon could be due to the lower molecular weight of the xanthan produced at higher pH values.
  Besides molecular weight, the concentration of xanthan might affect the viscosity when associated with salts. Zhong et al.32 demonstrated that at low xanthan concentration, addition of salt (Na+ and Ca2+) lowered the solution viscosity, while at higher xanthan concentration (5000 mg/L), the salt addition increased the viscosity.
  As depicted in Fig. 3A and B, the gum without alkali stress showed a polygonal structure, whereas the gum subjected to alkali stress had star-shaped structure. While comparing these results with those found by Borges et al.,24 it was assumed that no reduction in molecular weight occurred. It has also been suggested that the high sodium concentration due to the alkali might lead to the structure compaction due to the repulsion caused by the positively charged ions. The present results corroborate with those obtained by Carrington et al.33 on the effect of salts on the extension flow properties. In fact, high salt concentration may change xanthan molecule from an expanded to a compact conformation. The changes in the molecule hydrodynamic size can lead to a decrease in viscosity, as observed in the current study.
  However, opposite behavior was observed by Savi-Junior et al.,34 who found that the viscosity decreased at low salt concentration as the polymer coil became contracted, providing a smaller hydrodynamic volume.
  The production of xanthan gum around some cells in very low quantity was also observed. During the preparation of inoculum, an increase in cell concentration was observed, and only a small amount of gum was produced. The gum surrounding the cell prevented the transport of nutrients and extended the growth phase.35,36
  The extracellular polysaccharides may be classified into two types: (I) polymers physically attached to the microbial cell in a capsule form, and (II) polymers excreted by the microbial cells in the medium in the form of loose slime.37 The present results suggest a revision of such classification, since xanthan gum can be classified either as type (I) or type (II) depending on the medium conditions.
  Co-occurrence of acetic and pyruvic acid molecules with xanthan molecules results in anion-type of polysaccharides.5,37,38 It is believed that when this biopolymer is subjected to strong alkaline conditions, the charges are neutralized prior to the electrostatic repulsion. In the absence of repulsion, there might be some changes in the conformation of the molecule (Fig. 3B) and in the way the gum is deposited (Fig. 5C).
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