Appl Biochem Biotechnol (2014) 172:2769–2785 DOI 10.1007/s12010-014-0723-7
A Study of the Effects of Aeration and Agitation on the Properties and Production of Xanthan Gum from Crude Glycerin Derived from Biodiesel Using the Response Surface Methodology Denilson de Jesus Assis & Líllian Vasconcelos Brandão & Larissa Alves de Sousa Costa & Tamiris Vilas Boas Figueiredo & Luciane Santos Sousa & Francine Ferreira Padilha & Janice Izabel Druzian
Received: 30 September 2013 / Accepted: 2 January 2014 / Published online: 17 January 2014 # Springer Science+Business Media New York 2014
Abstract The effects of aeration and agitation on the properties and production of xanthan gum from crude glycerin biodiesel (CGB) by Xanthomonas campestris mangiferaeindicae 2103 were investigated and optimized using a response surface methodology. The xanthan gum was produced from CGB in a bioreactor at 28 °C for 120 h. Optimization procedures indicated that 0.97 vvm at 497.76 rpm resulted in a xanthan gum production of 5.59 g L−1 and 1.05 vvm at 484.75 rpm maximized the biomass to 3.26 g L−1. Moreover, the combination of 1.05 vvm at 499.40 rpm maximized the viscosity of xanthan at 0.5 % (m/v), 25 °C, and 25 s−1 (255.40 mPa s). The other responses did not generate predictive models. Low agitation contributed to the increase of xanthan gum production, biomass, viscosity, molecular mass, and the pyruvic acid concentration. Increases in the agitation contributed to the formation of xanthan gum with high mannose concentration. Decreases in the aeration contributed to the xanthan gum production and the formation of biopolymer with high mannose and glucose concentrations. Increases in aeration contributed to increased biomass, viscosity, and formation of xanthan gum with greater resistance to thermal degradation. Overall, aeration and agitation of CGB fermentation significantly influenced the production of xanthan gum and its properties.
D. de Jesus Assis (*) : L. V. Brandão : L. A. de Sousa Costa Department of Chemical Engineering, Polytechnic School, Federal University of Bahia, Aristides Novis Street, n° 2, Second Floor, Federação, Salvador, Bahia 40210-630, Brazil e-mail: [email protected] T. V. B. Figueiredo : L. S. Sousa : J. I. Druzian Department of Bromatological Analysis, College of Pharmacy, Federal University of Bahia, Barão of Geremoabo Street, s/n, Ondina, Salvador, Bahia 40171-970, Brazil F. F. Padilha Technology and Research Institute, Tiradentes University, Murilo Dantas Street, n° 300, Aracajú, Sergipe 49040-020, Brazil
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Keywords Xanthan gum . Crude glycerin . Aeration . Agitation . Properties
Introduction Xanthan gum is an extracellular biopolymer produced by various gram-negative bacteria of the genus Xanthomonas [1]. Due to its structure, xanthan gum exhibits pseudoplastic properties: high viscosity and solubility, enhanced stability over a wide range of temperature and pH values, and compatibility with many salts and other polysaccharides. This biopolymer is a favorable thickener and stabilizer of emulsions and suspensions used in the food industry, pharmaceutical formulations, paper milling, ceramic glazes, and agricultural products [2, 3]. Recently, the global xanthan gum market has been progressively increasing at an annual rate of 5–10 % [4]. The main chain of xanthan gum is composed of D-glucose (β-1,4) units that form a cellulose backbone with trisaccharide side chains composed of mannose (β-1,4) and glucuronic acid (β-1,2); mannose is attached to alternate glucose residues in the backbone by α-1,3 linkages [2, 5]. The composition structure, molecular mass, and viscosity depend on several factors, such as the composition of the culture medium, carbon and nitrogen sources, mineral salts, trace elements, type of Xanthomonas strains, and fermentation conditions (time, pH, temperature, oxygen concentration, and agitation rate) [6, 7]. Large-scale batch production processes of xanthan often use glucose as a substrate. Such processes are highly productive [8], but the increasing market price and demand suggest that glucose may no longer be an economically feasible raw material. Because the use of glucose or sucrose significantly impacts the production costs of this polysaccharide [3], other sources have also been tested as raw materials, such as raw cassava starch [9], date palm juice [4], coconut juice, unmodified starch [2], potato residue [10], apple residue [11], acid whey [12], and cassava serum [13]. Glycerin is a major by-product of the biodiesel manufacturing process. Generally, approximately 4.53 kg of crude glycerin is created for every 45.3 kg of biodiesel produced [14]. The worldwide biodiesel market is estimated to reach 37 billion gallons by 2016, which implies that approximately 4 billion gallons of crude glycerol will be produced [15]. Thus, new uses for crude glycerin are urgently needed. The glycerin derived from biodiesel production is impure, and the significant cost of purification prevents its use in the food and pharmaceutical industries [16]. A promising alternative use of this by-product is the microbial conversion of crude glycerin into valueadded products through biotechnological processes, such as polyhydroxybutyrate polymers [17], clavulanic acid [18], recombinant human erythropoietin [17], citric acid [19], and xanthan gum [20]. The response surface methodology (RSM) technique can optimize complex process by facilitating the arrangement and interpretation of experiments more easily compared to other traditional methods [21]. This study reports the effects of aeration and agitation speed on the production and properties of the xanthan gum production by Xanthomonas campestris from crude glycerin derived from biodiesel (CGB) during batch fermentation. Accordingly, this research aimed to optimize the selection process variables to maximize the dependent variables using response surface methodology. Specifically, the individual and simultaneous effects of the aeration and speed agitations were investigated on the production, apparent viscosity, composition, molecular mass, and initial temperature degradation of the polymers.
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Materials and Methods Microorganism and Inoculum Preparation The Tropical Collection Culture of the Biological Institute (Campinas, São Paulo, Brazil) supplied the X. campestris mangiferaeindicae 2103 strain isolated from Brazil and used in this study. The strain was grown and maintained in yeast extract malt agar (YMA) with the following composition (in grams per liter): 10.0 glucose, 3.0 yeast extract, 3.0 malt extract, 5.0 peptone, and 15.0 agar. During the preparation, the media pH was adjusted to 7.0 and then was sterilized (121 °C/20 min). After growing for 48 h at 28 °C, the culture was maintained at 4 °C. To minimize losses in productivity and fluctuations in the results during subsequent cultivations due to intra-population variability, the cell cultures were preserved in glycerol (20 % v/v) at −80 °C. Each experiment was started by reviving one glycerol vial in vegetative culture [22]. Inoculum cultures were prepared in yeast malt (YM) broth containing (w/v) 1.0 % glucose, 0.5 % peptone, 0.3 % yeast extract, and 0.3 % malt extract [3]. The medium pH was adjusted to 7.0 before autoclaving (121 °C/15 min). X. campestris mangiferaeindicae 2103 cells were incubated in 250-mL Erlenmeyer flasks containing 50 mL of YM broth at 28±2 °C for 24 h. The flasks were placed in an orbital shaker (Tecnal mod. TE-424, São Paulo, Brazil) at 180 rpm. Cell growth was monitored spectrophotometrically (PerkinElmer model Lambda 20) by measuring the optical density at 620 nm after 48 h of incubation until the cell concentration reached 1011 UFC mL−1. Crude Glycerin Biodiesel Composition Crude glycerin biodiesel (CGB) was supplied by the Biodiesel Pilot Plant of the State University of Santa Cruz (Ilhéus, Bahia, Brazil). The following analyses were carried out on the crude glycerin in triplicate: acidity (pH), volatile content at 105 °C, protein (Kjeldahl method), ash [23], and total lipid content [24]. The carbohydrate content (glycerol) was calculated by difference [100−(ash+protein+volatiles+lipid) percentages]. Xanthan Gum Production Medium and Cultivation Conditions To enhance the process variables, all fermentations were conducted in a 4.5-L bioreactor (Tecnal Mod. TecBio, Piracicaba, São Paulo, Brazil) containing 3.0 L of production medium consisting of 2.0 % (v/v) CGB, 0.01 % (w/v) (NH2)2CO, 0.1 % (w/v) KH2PO4, and 0.1 % (v/v) antifoam. The medium’s pH was adjusted to 7.0, and the medium was then sterilized. The fermentation medium was inoculated (20 % v/v) at 28±2 °C, and the fermentation was allowed to proceed for 120 h. Cell growth was estimated by measuring the absorbance of the cell suspensions at 620 nm in a PerkinElmer model Lambda 35 spectrophotometer (Norwalk, USA). Biomass Concentration The cells were collected after centrifugation at 18,800×g for 30 min (HITACHI, CR-GIII). After the supernatant was discarded, the biomass was washed with 0.85 % NaCl solution and re-centrifuged. This process was repeated twice. Finally, the cells were dried in an oven for 24 h and weighed.
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Xanthan Gum Recovery and Purification The xanthan gum product was recovered from the supernatants of centrifugation by precipitation with 98 % ethanol at a 3:1 ratio (v/v). The precipitated xanthan gum was collected and dried in an oven (Tecnal TE 394/2, São Paulo, Brazil) at 30±2 °C for 72 h. The production was expressed as grams per liter of fermentation broth. The aqueous xanthan gum solutions were dialyzed (cutoff 12,000 Da) against purified water at 100 rpm and 25 °C for 72 h in an orbital shaker (Tecnal TE-424, São Paulo, Brazil). The solutions were lyophilized (LIOBRAS L101) for 48 h and then stored in hermetical flasks. Apparent Viscosity of the Xanthan Gum Solution To study the rheological characteristics of the gums, 0.5 % (m/v) xanthan gum solutions were prepared using distilled water, stirred for 5 min to complete dissolution, and then maintained at room temperature for 12 h before testing. The apparent viscosities were measured in a concentric cylinder rheometer (Haake Rheotest mod 2.1, Medingen, Germany) coupled with a wash bath for temperature control and shear rate of 25 to 1,000 s−1. The rheological data were fitted to the Ostwald–de Waele model: μ ¼ Kγ ðn−1Þ
ð1Þ
where μ is the apparent viscosity, K is the consistency index, γ is the shear rate, and n is the flow behavior index. Experimental Methodology The individual and interactive effects of aeration (X1) (0.5–1.5 vvm) and agitation (X2) (300– 700 rpm) on xanthan gum production (Y1), biomass (Y2), apparent viscosity (Y3), glucose content (Y4), mannose content (Y5), glucuronic acid content (Y6), pyruvic acid content (Y7), average molecular mass (Y8), and maximum thermal degradation temperature (Y9) were determined using response surface methodology [25]. The procedure performed to determine the ideal agitation and aeration conditions to optimize polymer production and properties followed a full factorial experimental design of type 22, with three central points and four axial at a distance α=±1.412, totaling 11 experiments. The levels (in coded values) were −1, 0, and +1, where 0 corresponded to the central point. The coded values were calculated according to the following equation: Coded value ¼
actual value−ðhigh level þ low levelÞ=2 ðhigh level−low levelÞ=2
ð2Þ
The responses of the dependent variables were analyzed using the Statistica software for Windows version 7, and the significance level was 5 %. The levels chosen were based on preliminary tests conducted by our research group. Mathematical models were fitted to the experimental points and from the analysis of variance (ANOVA) model. The predictive power of the regression was tested using the Fisher (F) test and coefficients (R2) [25]. Fit testing and model predicting were set at a 5 % significance level (P value ≤0.05). To find the values that maximize or minimize each estimated response, the stationary point of the surface was calculated by canonical analysis [25] to determine optimal combination of aeration and agitation speed. Furthermore, the nature of the surface was evaluated based on the
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signals from the root characteristics of the quadratic equations (λ1e λ2) from the surface of the second-order model. Xanthan Gum Average Molecular Mass The average molecular mass of xanthan gum was estimated by size-exclusion chromatography (GPC HPLC systems (PerkinElmer Series 200; Shelton, USA)) with Shodex OHpak SB 803, 804, 805, and 806 columns in series (Kawasaki-ku, Japan) using 0.5 % (w/v) NaNO3 as the eluant at a flow rate of 1 mL min−1. A Refractive Index (RI) PerkinElmer Series 200 (Shelton, USA) was used as the detector. The column was calibrated with dextran patterns (102,000; 207,200; 431,800; 655,200; 759,400; 1,360,000; 2,025,000; 2,800,000; 3,450,000; and 5,900,000 Da) (American Polymer Patterns, USA). An 80-μL aliquot (0.3 % m/v) of dextran pattern and xanthan gum aqueous solutions was injected in triplicate. The molecular mass of xanthan gum was calculated using the calibration curve log molecular mass (dextran patterns molecular mass)×retention time (RT) and compared with the molecular mass of Sigma xanthan gum (reference). Xanthan Gum Chemical Composition The glucose, mannose, glucuronic acid, and pyruvate contents in xanthan gum synthesized by X. campestris mangiferaeindicae 2103 were quantified. The samples (10.0 mg) were initially hydrated with purified water (0.5 mL) for 12 h, hydrolyzed with 1 M trifluoroacetic acid (TFA) at 0.5 mL for 10 h at 100 °C, subsequently dried with nitrogen gas, lyophilized to completely remove any TFA residue, and dissolved with 1 mL chromatographic grade water. To determine the sugar concentrations, the hydrolyzed polymer solutions were injected into the HPLC-IR (PerkinElmer 200 series) using a Polypore Ca pre-column (30 mm×10 mm× 4.6 mm) followed by a Polypore Ca column (220 mm×4.6 mm×10 mm), both of which were placed in an oven at 80 °C. The mobile phase used was chromatographic grade water flowing at 0.1 mL min−1. The injection volume was 5 μL. The sugars were identified by comparing the RT between the glucose and mannose peaks from the patterns with the hydrolyzed xanthan gum sample. The peaks were quantified using external aqueous solutions of glucose and mannose as standards (0.10 to 1.10 mg mL−1) to obtain standard curves. To determine the concentration of uronic acid, hydrolyzed polymer solutions were injected into the HPLC adapted with an ultraviolet (UV) detector operating at a wavelength of 195 nm (PerkinElmer 200 series) using a Polypore H pre-column (4.6 mm×30 mm×10 mm) followed by a Polypore H column (220 mm×4.6 mm×10 mm). The columns were placed in an oven at 50 °C. The mobile phase used was aqueous H2SO4, pH 1.9 at a flow rate of 0.4 mL min−1. The injection volume was 10 μL. The samples were identified by comparing the retention time between the peaks of the patterns of glucuronic acid (2.5 to 100.0 mg L−1) and pyruvic acid (2.0 to 82.0 mg L−1) with the samples of hydrolyzed xanthan gum. The peaks were quantified using pattern curves generated by external aqueous solution standards. Thermal Analysis of Xanthan Gum (TG/DTG) Non-isothermal experiments were carried out on a PerkinElmer thermogravimetric apparatus using masses of sample of approximately 7.0 mg, a maximum temperature of 1,000 °C, and a heating rate of 10 °C min−1 under a dynamic atmosphere of nitrogen with a flow rate of
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20 mL min−1. The thermogravimetric (TG) curves and their derivatives (DTG) were used to determine mass loss (in percent), temperature ranges (in degree Celsius), and the maximum temperature of thermal degradation, respectively. Both were calculated with the Pyris software Manager.
Results and Discussion Alternative Substrate Composition The CGB was composed of 53.50±0.01 % volatiles, 3.40±0.01 % ash, 6.70±0.02 % total lipids, 2.71±0.03 % crude protein, and 33.69±0.02 % glycerol, including minerals, organic nitrogen, and total lipids from biodiesel processing. The C and N sources supplied as nutrients for bacterial growth are known to affect xanthan gum production, depending on their composition and relative amounts. The C/N ratio in CGB is approximately 15:1. The glycerin chemical composition found here satisfies the non-limiting nitrogen concentration condition required for rapid cell growth, while excess carbon and low nitrogen concentration are essential for the production of xanthan gum with suitable rheological properties [26]. The nutrient source influences the pathway by which polymers are synthesized [27]. Glycerol and lipids as free fatty acids (soaps) are two major components in crude glycerin that contains a variety of elements such as calcium (3–15 ppm), magnesium (1–2 ppm), phosphorous (8–13 ppm), and sulfur (22–26 ppm), independent of the feedstock source (canola, rapeseed, and soybean) [28]. Thus, a fermentative medium richer in nutrients and micronutrients, and adaptation by the bacterium to an alternative medium may contribute to an increase in xanthan gum production. The composition of the CGB indicated that xanthan gum could possibly be produced from this waste with minimal supplementation. Some authors [12, 13] produced xanthan gum from alternative media only by supplementing the fermentation with urea (0.01 % w/v) and K2HPO4 (0.1 % w/v), resulting in lower production costs. To produce higher amounts of xanthan gum, cell growth and polysaccharide biosynthesis must be regulated throughout the process. The effect of fermentation time on xanthan gum production was evaluated at different CGB concentrations (1.0, 2.0, 4.0, and 6.0 % m/v) at 1.0 vvm and 400 rpm. The experiment using 2.0 % of CGB showed the maximum accumulation of xanthan gum (2.34 g L−1) after 120 h of fermentation, whereas at 1.0 % CGB, only 0.84 g L−1 of xanthan gum was accumulated. Experiments using 4 and 6.0 % of CGB resulted in significant foaming and subsequent loss of culture medium. Thus, we used 2.0 % GRB and a fermentation time of 120 h. These conditions are consistent with various studies on the nutritional requirements for fermentation using Xanthomonas, aiming at the sustainability of the process regarding cost-effectiveness of the production [3, 11, 29]. Effect of Aeration and Agitation on Xanthan Production (Y1), Biomass (Y2), and Apparent Viscosity (Y3) The responses to 11 tests of the crude glycerin fermentation as a function of independent variables (X1=aeration, X2=agitation) are presented in Table 1. The coefficients of multiple determinations (R2) give the percentage variations in the response explained by our regression model. Thus, we can explain Y1 and Y2 variations in 96.85 % and Y3 variations in 97.06 % (Table 2). The P value
A Study of the Effects of Aeration and Agitation on the Properties and Production of Xanthan Gum from Crude Glycerin Derived from Biodiesel Using the Response Surface Methodology Denilson de Jesus Assis & Líllian Vasconcelos Brandão & Larissa Alves de Sousa Costa & Tamiris Vilas Boas Figueiredo & Luciane Santos Sousa & Francine Ferreira Padilha & Janice Izabel Druzian
Received: 30 September 2013 / Accepted: 2 January 2014 / Published online: 17 January 2014 # Springer Science+Business Media New York 2014
Abstract The effects of aeration and agitation on the properties and production of xanthan gum from crude glycerin biodiesel (CGB) by Xanthomonas campestris mangiferaeindicae 2103 were investigated and optimized using a response surface methodology. The xanthan gum was produced from CGB in a bioreactor at 28 °C for 120 h. Optimization procedures indicated that 0.97 vvm at 497.76 rpm resulted in a xanthan gum production of 5.59 g L−1 and 1.05 vvm at 484.75 rpm maximized the biomass to 3.26 g L−1. Moreover, the combination of 1.05 vvm at 499.40 rpm maximized the viscosity of xanthan at 0.5 % (m/v), 25 °C, and 25 s−1 (255.40 mPa s). The other responses did not generate predictive models. Low agitation contributed to the increase of xanthan gum production, biomass, viscosity, molecular mass, and the pyruvic acid concentration. Increases in the agitation contributed to the formation of xanthan gum with high mannose concentration. Decreases in the aeration contributed to the xanthan gum production and the formation of biopolymer with high mannose and glucose concentrations. Increases in aeration contributed to increased biomass, viscosity, and formation of xanthan gum with greater resistance to thermal degradation. Overall, aeration and agitation of CGB fermentation significantly influenced the production of xanthan gum and its properties.
D. de Jesus Assis (*) : L. V. Brandão : L. A. de Sousa Costa Department of Chemical Engineering, Polytechnic School, Federal University of Bahia, Aristides Novis Street, n° 2, Second Floor, Federação, Salvador, Bahia 40210-630, Brazil e-mail: [email protected] T. V. B. Figueiredo : L. S. Sousa : J. I. Druzian Department of Bromatological Analysis, College of Pharmacy, Federal University of Bahia, Barão of Geremoabo Street, s/n, Ondina, Salvador, Bahia 40171-970, Brazil F. F. Padilha Technology and Research Institute, Tiradentes University, Murilo Dantas Street, n° 300, Aracajú, Sergipe 49040-020, Brazil
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Keywords Xanthan gum . Crude glycerin . Aeration . Agitation . Properties
Introduction Xanthan gum is an extracellular biopolymer produced by various gram-negative bacteria of the genus Xanthomonas [1]. Due to its structure, xanthan gum exhibits pseudoplastic properties: high viscosity and solubility, enhanced stability over a wide range of temperature and pH values, and compatibility with many salts and other polysaccharides. This biopolymer is a favorable thickener and stabilizer of emulsions and suspensions used in the food industry, pharmaceutical formulations, paper milling, ceramic glazes, and agricultural products [2, 3]. Recently, the global xanthan gum market has been progressively increasing at an annual rate of 5–10 % [4]. The main chain of xanthan gum is composed of D-glucose (β-1,4) units that form a cellulose backbone with trisaccharide side chains composed of mannose (β-1,4) and glucuronic acid (β-1,2); mannose is attached to alternate glucose residues in the backbone by α-1,3 linkages [2, 5]. The composition structure, molecular mass, and viscosity depend on several factors, such as the composition of the culture medium, carbon and nitrogen sources, mineral salts, trace elements, type of Xanthomonas strains, and fermentation conditions (time, pH, temperature, oxygen concentration, and agitation rate) [6, 7]. Large-scale batch production processes of xanthan often use glucose as a substrate. Such processes are highly productive [8], but the increasing market price and demand suggest that glucose may no longer be an economically feasible raw material. Because the use of glucose or sucrose significantly impacts the production costs of this polysaccharide [3], other sources have also been tested as raw materials, such as raw cassava starch [9], date palm juice [4], coconut juice, unmodified starch [2], potato residue [10], apple residue [11], acid whey [12], and cassava serum [13]. Glycerin is a major by-product of the biodiesel manufacturing process. Generally, approximately 4.53 kg of crude glycerin is created for every 45.3 kg of biodiesel produced [14]. The worldwide biodiesel market is estimated to reach 37 billion gallons by 2016, which implies that approximately 4 billion gallons of crude glycerol will be produced [15]. Thus, new uses for crude glycerin are urgently needed. The glycerin derived from biodiesel production is impure, and the significant cost of purification prevents its use in the food and pharmaceutical industries [16]. A promising alternative use of this by-product is the microbial conversion of crude glycerin into valueadded products through biotechnological processes, such as polyhydroxybutyrate polymers [17], clavulanic acid [18], recombinant human erythropoietin [17], citric acid [19], and xanthan gum [20]. The response surface methodology (RSM) technique can optimize complex process by facilitating the arrangement and interpretation of experiments more easily compared to other traditional methods [21]. This study reports the effects of aeration and agitation speed on the production and properties of the xanthan gum production by Xanthomonas campestris from crude glycerin derived from biodiesel (CGB) during batch fermentation. Accordingly, this research aimed to optimize the selection process variables to maximize the dependent variables using response surface methodology. Specifically, the individual and simultaneous effects of the aeration and speed agitations were investigated on the production, apparent viscosity, composition, molecular mass, and initial temperature degradation of the polymers.
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Materials and Methods Microorganism and Inoculum Preparation The Tropical Collection Culture of the Biological Institute (Campinas, São Paulo, Brazil) supplied the X. campestris mangiferaeindicae 2103 strain isolated from Brazil and used in this study. The strain was grown and maintained in yeast extract malt agar (YMA) with the following composition (in grams per liter): 10.0 glucose, 3.0 yeast extract, 3.0 malt extract, 5.0 peptone, and 15.0 agar. During the preparation, the media pH was adjusted to 7.0 and then was sterilized (121 °C/20 min). After growing for 48 h at 28 °C, the culture was maintained at 4 °C. To minimize losses in productivity and fluctuations in the results during subsequent cultivations due to intra-population variability, the cell cultures were preserved in glycerol (20 % v/v) at −80 °C. Each experiment was started by reviving one glycerol vial in vegetative culture [22]. Inoculum cultures were prepared in yeast malt (YM) broth containing (w/v) 1.0 % glucose, 0.5 % peptone, 0.3 % yeast extract, and 0.3 % malt extract [3]. The medium pH was adjusted to 7.0 before autoclaving (121 °C/15 min). X. campestris mangiferaeindicae 2103 cells were incubated in 250-mL Erlenmeyer flasks containing 50 mL of YM broth at 28±2 °C for 24 h. The flasks were placed in an orbital shaker (Tecnal mod. TE-424, São Paulo, Brazil) at 180 rpm. Cell growth was monitored spectrophotometrically (PerkinElmer model Lambda 20) by measuring the optical density at 620 nm after 48 h of incubation until the cell concentration reached 1011 UFC mL−1. Crude Glycerin Biodiesel Composition Crude glycerin biodiesel (CGB) was supplied by the Biodiesel Pilot Plant of the State University of Santa Cruz (Ilhéus, Bahia, Brazil). The following analyses were carried out on the crude glycerin in triplicate: acidity (pH), volatile content at 105 °C, protein (Kjeldahl method), ash [23], and total lipid content [24]. The carbohydrate content (glycerol) was calculated by difference [100−(ash+protein+volatiles+lipid) percentages]. Xanthan Gum Production Medium and Cultivation Conditions To enhance the process variables, all fermentations were conducted in a 4.5-L bioreactor (Tecnal Mod. TecBio, Piracicaba, São Paulo, Brazil) containing 3.0 L of production medium consisting of 2.0 % (v/v) CGB, 0.01 % (w/v) (NH2)2CO, 0.1 % (w/v) KH2PO4, and 0.1 % (v/v) antifoam. The medium’s pH was adjusted to 7.0, and the medium was then sterilized. The fermentation medium was inoculated (20 % v/v) at 28±2 °C, and the fermentation was allowed to proceed for 120 h. Cell growth was estimated by measuring the absorbance of the cell suspensions at 620 nm in a PerkinElmer model Lambda 35 spectrophotometer (Norwalk, USA). Biomass Concentration The cells were collected after centrifugation at 18,800×g for 30 min (HITACHI, CR-GIII). After the supernatant was discarded, the biomass was washed with 0.85 % NaCl solution and re-centrifuged. This process was repeated twice. Finally, the cells were dried in an oven for 24 h and weighed.
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Xanthan Gum Recovery and Purification The xanthan gum product was recovered from the supernatants of centrifugation by precipitation with 98 % ethanol at a 3:1 ratio (v/v). The precipitated xanthan gum was collected and dried in an oven (Tecnal TE 394/2, São Paulo, Brazil) at 30±2 °C for 72 h. The production was expressed as grams per liter of fermentation broth. The aqueous xanthan gum solutions were dialyzed (cutoff 12,000 Da) against purified water at 100 rpm and 25 °C for 72 h in an orbital shaker (Tecnal TE-424, São Paulo, Brazil). The solutions were lyophilized (LIOBRAS L101) for 48 h and then stored in hermetical flasks. Apparent Viscosity of the Xanthan Gum Solution To study the rheological characteristics of the gums, 0.5 % (m/v) xanthan gum solutions were prepared using distilled water, stirred for 5 min to complete dissolution, and then maintained at room temperature for 12 h before testing. The apparent viscosities were measured in a concentric cylinder rheometer (Haake Rheotest mod 2.1, Medingen, Germany) coupled with a wash bath for temperature control and shear rate of 25 to 1,000 s−1. The rheological data were fitted to the Ostwald–de Waele model: μ ¼ Kγ ðn−1Þ
ð1Þ
where μ is the apparent viscosity, K is the consistency index, γ is the shear rate, and n is the flow behavior index. Experimental Methodology The individual and interactive effects of aeration (X1) (0.5–1.5 vvm) and agitation (X2) (300– 700 rpm) on xanthan gum production (Y1), biomass (Y2), apparent viscosity (Y3), glucose content (Y4), mannose content (Y5), glucuronic acid content (Y6), pyruvic acid content (Y7), average molecular mass (Y8), and maximum thermal degradation temperature (Y9) were determined using response surface methodology [25]. The procedure performed to determine the ideal agitation and aeration conditions to optimize polymer production and properties followed a full factorial experimental design of type 22, with three central points and four axial at a distance α=±1.412, totaling 11 experiments. The levels (in coded values) were −1, 0, and +1, where 0 corresponded to the central point. The coded values were calculated according to the following equation: Coded value ¼
actual value−ðhigh level þ low levelÞ=2 ðhigh level−low levelÞ=2
ð2Þ
The responses of the dependent variables were analyzed using the Statistica software for Windows version 7, and the significance level was 5 %. The levels chosen were based on preliminary tests conducted by our research group. Mathematical models were fitted to the experimental points and from the analysis of variance (ANOVA) model. The predictive power of the regression was tested using the Fisher (F) test and coefficients (R2) [25]. Fit testing and model predicting were set at a 5 % significance level (P value ≤0.05). To find the values that maximize or minimize each estimated response, the stationary point of the surface was calculated by canonical analysis [25] to determine optimal combination of aeration and agitation speed. Furthermore, the nature of the surface was evaluated based on the
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signals from the root characteristics of the quadratic equations (λ1e λ2) from the surface of the second-order model. Xanthan Gum Average Molecular Mass The average molecular mass of xanthan gum was estimated by size-exclusion chromatography (GPC HPLC systems (PerkinElmer Series 200; Shelton, USA)) with Shodex OHpak SB 803, 804, 805, and 806 columns in series (Kawasaki-ku, Japan) using 0.5 % (w/v) NaNO3 as the eluant at a flow rate of 1 mL min−1. A Refractive Index (RI) PerkinElmer Series 200 (Shelton, USA) was used as the detector. The column was calibrated with dextran patterns (102,000; 207,200; 431,800; 655,200; 759,400; 1,360,000; 2,025,000; 2,800,000; 3,450,000; and 5,900,000 Da) (American Polymer Patterns, USA). An 80-μL aliquot (0.3 % m/v) of dextran pattern and xanthan gum aqueous solutions was injected in triplicate. The molecular mass of xanthan gum was calculated using the calibration curve log molecular mass (dextran patterns molecular mass)×retention time (RT) and compared with the molecular mass of Sigma xanthan gum (reference). Xanthan Gum Chemical Composition The glucose, mannose, glucuronic acid, and pyruvate contents in xanthan gum synthesized by X. campestris mangiferaeindicae 2103 were quantified. The samples (10.0 mg) were initially hydrated with purified water (0.5 mL) for 12 h, hydrolyzed with 1 M trifluoroacetic acid (TFA) at 0.5 mL for 10 h at 100 °C, subsequently dried with nitrogen gas, lyophilized to completely remove any TFA residue, and dissolved with 1 mL chromatographic grade water. To determine the sugar concentrations, the hydrolyzed polymer solutions were injected into the HPLC-IR (PerkinElmer 200 series) using a Polypore Ca pre-column (30 mm×10 mm× 4.6 mm) followed by a Polypore Ca column (220 mm×4.6 mm×10 mm), both of which were placed in an oven at 80 °C. The mobile phase used was chromatographic grade water flowing at 0.1 mL min−1. The injection volume was 5 μL. The sugars were identified by comparing the RT between the glucose and mannose peaks from the patterns with the hydrolyzed xanthan gum sample. The peaks were quantified using external aqueous solutions of glucose and mannose as standards (0.10 to 1.10 mg mL−1) to obtain standard curves. To determine the concentration of uronic acid, hydrolyzed polymer solutions were injected into the HPLC adapted with an ultraviolet (UV) detector operating at a wavelength of 195 nm (PerkinElmer 200 series) using a Polypore H pre-column (4.6 mm×30 mm×10 mm) followed by a Polypore H column (220 mm×4.6 mm×10 mm). The columns were placed in an oven at 50 °C. The mobile phase used was aqueous H2SO4, pH 1.9 at a flow rate of 0.4 mL min−1. The injection volume was 10 μL. The samples were identified by comparing the retention time between the peaks of the patterns of glucuronic acid (2.5 to 100.0 mg L−1) and pyruvic acid (2.0 to 82.0 mg L−1) with the samples of hydrolyzed xanthan gum. The peaks were quantified using pattern curves generated by external aqueous solution standards. Thermal Analysis of Xanthan Gum (TG/DTG) Non-isothermal experiments were carried out on a PerkinElmer thermogravimetric apparatus using masses of sample of approximately 7.0 mg, a maximum temperature of 1,000 °C, and a heating rate of 10 °C min−1 under a dynamic atmosphere of nitrogen with a flow rate of
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20 mL min−1. The thermogravimetric (TG) curves and their derivatives (DTG) were used to determine mass loss (in percent), temperature ranges (in degree Celsius), and the maximum temperature of thermal degradation, respectively. Both were calculated with the Pyris software Manager.
Results and Discussion Alternative Substrate Composition The CGB was composed of 53.50±0.01 % volatiles, 3.40±0.01 % ash, 6.70±0.02 % total lipids, 2.71±0.03 % crude protein, and 33.69±0.02 % glycerol, including minerals, organic nitrogen, and total lipids from biodiesel processing. The C and N sources supplied as nutrients for bacterial growth are known to affect xanthan gum production, depending on their composition and relative amounts. The C/N ratio in CGB is approximately 15:1. The glycerin chemical composition found here satisfies the non-limiting nitrogen concentration condition required for rapid cell growth, while excess carbon and low nitrogen concentration are essential for the production of xanthan gum with suitable rheological properties [26]. The nutrient source influences the pathway by which polymers are synthesized [27]. Glycerol and lipids as free fatty acids (soaps) are two major components in crude glycerin that contains a variety of elements such as calcium (3–15 ppm), magnesium (1–2 ppm), phosphorous (8–13 ppm), and sulfur (22–26 ppm), independent of the feedstock source (canola, rapeseed, and soybean) [28]. Thus, a fermentative medium richer in nutrients and micronutrients, and adaptation by the bacterium to an alternative medium may contribute to an increase in xanthan gum production. The composition of the CGB indicated that xanthan gum could possibly be produced from this waste with minimal supplementation. Some authors [12, 13] produced xanthan gum from alternative media only by supplementing the fermentation with urea (0.01 % w/v) and K2HPO4 (0.1 % w/v), resulting in lower production costs. To produce higher amounts of xanthan gum, cell growth and polysaccharide biosynthesis must be regulated throughout the process. The effect of fermentation time on xanthan gum production was evaluated at different CGB concentrations (1.0, 2.0, 4.0, and 6.0 % m/v) at 1.0 vvm and 400 rpm. The experiment using 2.0 % of CGB showed the maximum accumulation of xanthan gum (2.34 g L−1) after 120 h of fermentation, whereas at 1.0 % CGB, only 0.84 g L−1 of xanthan gum was accumulated. Experiments using 4 and 6.0 % of CGB resulted in significant foaming and subsequent loss of culture medium. Thus, we used 2.0 % GRB and a fermentation time of 120 h. These conditions are consistent with various studies on the nutritional requirements for fermentation using Xanthomonas, aiming at the sustainability of the process regarding cost-effectiveness of the production [3, 11, 29]. Effect of Aeration and Agitation on Xanthan Production (Y1), Biomass (Y2), and Apparent Viscosity (Y3) The responses to 11 tests of the crude glycerin fermentation as a function of independent variables (X1=aeration, X2=agitation) are presented in Table 1. The coefficients of multiple determinations (R2) give the percentage variations in the response explained by our regression model. Thus, we can explain Y1 and Y2 variations in 96.85 % and Y3 variations in 97.06 % (Table 2). The P value
