LABORATORY EXPERIMENT pubs.acs.org/jchemeduc
Production of a Biopolymer at Reactor Scale: A Laboratory Experience Rukan Genc-† and Susana Rodríguez-Couto*,‡,§ †
Department of Chemical Engineering, Rovira i Virgili University, Av. Països Catalans 26, 43007 Tarragona, Spain CEIT, Unit of Environmental Engineering. Paseo Manuel de Lardizabal 15, 20018 San Sebastian, Spain § IKERBASQUE, Basque Foundation for Science. Alameda de Urquijo 36, 48011, Bilbao Spain ‡
bS Supporting Information ABSTRACT: Undergraduate students of biotechnology became familiar with several aspects of bioreactor operation via the production of xanthan gum, an industrially relevant biopolymer, by Xanthomonas campestris bacteria. The xanthan gum was extracted from the fermentation broth and the yield coefficient and productivity were calculated. KEYWORDS: Graduate Education/Research, Upper-Division Undergraduate, Chemical Engineering, Laboratory Instruction, Hands-On Learning/Manipulatives, Biotechnology, Industrial Chemistry, Laboratory Equipment/Apparatus, Laboratory Management, Polymerization
X
anthan gum, obtained by fermentation, is the most commercially produced industrial gum with an annual worldwide production of 30,000 tons, which corresponds to a market of $408 million.1 Because of its commercial and industrial importance, xanthan gum was selected as a model biopolymer to design a laboratory experience for undergraduate students of biotechnology. Xanthan gum is an extracellular hetero-polysaccharide, which is produced by the aerobic fermentation of the Xanthomonas campestris bacteria. Xanthan is composed of pentasaccharide repeating units containing D-glucose, D-mannose, D-glucoronic acid (at a ratio 2:2:1), acetal-linked pyruvic acid, and D-acetyl groups.2 Owing to its excellent rheological properties, xanthan gum is used in many applications, mainly in food industry as thickening, suspending, and stabilizing agent.3
subject and helped the students understand the different phenomena taking place were given before the laboratory.
’ EXPERIMENT The results presented are the experimental data from the 2007 2008 course. The 10 students worked in teams of five guided by the instructors. Before the first laboratory class, the instructors prepared the inoculum. The microorganism X. campestris (ATCC 33913) was used.4 The inoculum was prepared as follows: X. campestris was grown in a medium containing tryptone (tryptic digest of casein) (1% w/v), yeast extract (0.5% w/v), NaCl (0.5% w/v), and glucose (1% w/v) (Luria Bertani medium plus glucose, LBG medium).5 Single colonies of X. campestris were transferred to 250 mL Erlenmeyer flasks containing 100 mL of LBG medium. The flasks were plugged with cotton stoppers, which permit a passive aeration, and incubated on an orbital shaker at 25 °C and 200 rpm for 72 h.
’ EDUCATIONAL OBJECTIVES The main educational objectives of this experiment were (i) collection of the experimental data, their mathematical treatment, and construction and comprehension of different graphics; (ii) use of analytical techniques; (iii) bioreactor setup; and (iv) downstream process: recovery of xanthan gum from the fermentation broth. To test whether the objectives were fulfilled, the students were asked to give a report of the research performed in the laboratory. This report was a mini-research project and had to include all the results with their respective calculations, graphical representations, and comments. Lectures that introduced the Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
First Laboratory Class (3 h): Medium Preparation and Sterilization
Students prepared the fermentation medium (LBG medium) and autoclaved it in a 2.5 L Minifors bioreactor (Infors, Switzerland) (working volume of 2 L). The pH of the fermentation medium was adjusted to 7.0 after sterilization. Published: June 02, 2011 1175
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Figure 1. Calibration curve for reducing sugar determination.
Second Laboratory Class (3 h): Setting Up the Bioreactor
After reconstruction of the reactor, students used two flasks of X. campestris (provided by the instructors) as inoculum, which was added to the student-prepared culture medium. The bioreactor was operated at 150 rpm and 30 °C, and the pH was automatically controlled at 7.0 by adding sterile solutions of either NaOH (1 N) or HCl (1 N). Students monitored the fermentation by taking samples every hour for reducing sugar and biomass determination. The DNS (3,5-dinitrosalicylic acid) method6 was chosen to measure the reducing sugar concentration (details are in the Supporting Information). The calibration curve was calculated with a glucose standard solution of 1 g/L. The DNS solution was prepared by dissolving 10.6 g of DNS and 19.8 g of NaOH in 1416 mL of distilled water and then 306 g of Rochelle salts (KNa tartrate; C4H4KNaO6 3 4H2O), 7.6 mL of phenol (melt at 50 °C), and 8.3 g of sodium metabisulfite were added. The prepared solution was stored at room temperature in the dark. To determine reducing sugar concentration, 3 mL of DNS solution was added to the reaction tubes (10 mL test tubes) containing the samples (diluted 1:10 in water). After agitation, the tubes were boiled for 5 min followed by quick cooling under tap water. Absorbance was, then, measured at 640 nm using a spectrophotometer (Ultrospec 2100, Amersham Biosciences) and absorbance versus corresponding glucose concentrations were plotted to get the linear fitting to obtain the slope (Figure 1). Biomass determination was performed by dry cell-weight estimations. Following the sample centrifugation (2500g for 10 min), the supernatant was discarded and cells were collected and washed with NaCl (0.9% w/v) and recentrifuged. This was repeated twice. Finally, the cells were dried in an oven for 24 h and weighted. Third Laboratory Class (3 h): Continuing with the Fermentation
Students monitored the fermentation and took a sample for reducing sugar and biomass determination before starting with the downstream process. Reducing sugars and biomass were plotted as a function of time as shown in Figure 2. Fourth Laboratory Class (3 h): Xanthan Gum Recovery
Students stopped the fermentation and determined the xanthan gum produced. To recover the xanthan, the cells were first removed either by filtration or centrifugation. Then, the xanthan gum was precipitated using isopropyl alcohol (the ratio
Figure 2. Reducing sugars and biomass obtained along the fermentation process.
was 1 vol of xanthan sample and 3 vol of isopropyl alcohol). After the precipitation process, the product was filtered and dried at 40 °C overnight. Fifth Laboratory Class (3 h): Ending the Experiment
The produced xanthan gum was weighted by students, and yield coefficient YP/S and the productivity were calculated.7 The following values were obtained: 0.72 ( 0.05 g/g and 0.128 ( 0.008 g/(L h), respectively. Finally, sterilization and cleaning of the bioreactor and other tools were done again by students under the guidance of the authors.
’ HAZARDS 3,5-Dinitrosalycilic acid (DNS) and sodium metabisulfite may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Phenol is very hazardous and causes irritation to skin, eyes, and respiratory tract and is a possible carcinogen. Sodium hydroxide is caustic; it causes burns to any area of contact. The students were required to wear a laboratory coat and safety goggles at all times and latex gloves when handling hazardous solutions and compounds. Also, the students were asked to read the material safety data sheets (MSDS) of the hazardous chemicals used. ’ CONCLUSION This experience introduced a number of areas to the students: sugars and biomass determination, biopolymer production, product recovery, and bioreactor operation. 1176
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’ ASSOCIATED CONTENT
bS
Supporting Information Student instructions; instructions for preparing Luria Bertani medium and inoculum; and instructions for the determination of reducing sugars. This material is available via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was developed with the financial support of the Department of Chemical Engineering, Rovira i Virgili University (Tarragona, Spain). The authors gratefully acknowledge J.M. Borras and P. Obon for their technical assistant in preparing the experiments. The authors would also thank the students C. Gomez, S. Samino, E. Guiu, M. Guerrero, E. Valverde, J. Bori, J. Teichenne, M. Parrilla, D. Fernandez, and C. Ruíz from the 3rd course on Biotechnology at the Rovira i Virgili University (Tarragona, Spain) for their companion and participation. ’ REFERENCES (1) Kalogiannis, S.; Iakovidou, G.; Liakopoulou-Kyriakides, M.; Kyriakidis, D. A.; Skaracis, G. N. Process Biochem. 2003, 39, 249–256. (2) Baird, J. K. Encyclopedia of Polymer Science and Engineering; Wiley: New York, 1989; pp 901 918. (3) Katzbauer, B. Polym. Degrad. Stab. 1998, 59, 81–84. (4) Xanthomonas campestris was purchased from the CECT (Spanish Type Culture Collection, University of Valencia, Spain). (5) Tryptone and yeast extract were supplied by Scharlau Chemie (Barcelona, Spain) and the rest of the reagents by Sigma Aldrich (St. Louis, MO). (6) Ghose, T. K. Pure Appl. Chem. 1987, 59, 257–268. (7) YP/S is calculated as the ratio between the formed product (g) and the consumed substrate (g). The productivity is calculated as the formed product (g) per unit of volume (L) and unit of time (h).
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