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
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bioreactor operation via the production of xanthan gum, an industrially relevant biopolymer, by
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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 En-
gineering, Laboratory Instruction, Hands-On Learning/Manipulatives, Biotechnology, Industrial
Chemistry, Laboratory Equipment/Apparatus, Laboratory Management, Polymerization

X anthan gum, obtained by fermentation, is the most commer-

cially produced industrial gum with an annual worldwide
production of 30,000 tons, which corresponds to a market of
subject and helped the students understand the different phe-
nomena taking place were given before the laboratory.

$408 million.1 Because of its commercial and industrial impor-

tance, xanthan gum was selected as a model biopolymer to design
a laboratory experience for undergraduate students of biotech-
nology. Xanthan gum is an extracellular hetero-polysaccharide,
which is produced by the aerobic fermentation of the Xantho-
monas campestris bacteria. Xanthan is composed of pentasacchar-
ide 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
’ EDUCATIONAL OBJECTIVES
The main educational objectives of this experiment were (i)
collection of the experimental data, their mathematical treat-
ment, 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 fermen-
tation 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
’ 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. cam-
pestris (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.
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 fermenta-
tion medium was adjusted to 7.0 after sterilization.
Published: June 02, 2011

Copyright r 2011 American Chemical Society and

Division of Chemical Education, Inc. 1175 dx.doi.org/10.1021/ed100681b | J. Chem. Educ. 2011, 88, 1175–1177
Journal of Chemical Education LABORATORY EXPERIMENT

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 bior-
eactor was operated at 150 rpm and 30 °C, and the pH was auto-
matically controlled at 7.0 by adding sterile solutions of either
NaOH (1 N) or HCl (1 N). Students monitored the fermenta-
tion 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 solu-
tion 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 re-
peated 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
1176
Figure 2. Reducing sugars and biomass obtained along the fermenta-
tion 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 pos-
sible 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.
dx.doi.org/10.1021/ed100681b |J. Chem. Educ. 2011, 88, 1175–1177
Journal of Chemical Education LABORATORY EXPERIMENT

’ ASSOCIATED CONTENT

bS Supporting Information
Student instructions; instructions for preparing Luria
Bertani medium and inoculum; and instructions for the deter-
mination of reducing sugars. This material is available via the
Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION
Corresponding Author
*E-mail: srodriguez@ceit.es.

’ 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).

1177 dx.doi.org/10.1021/ed100681b |J. Chem. Educ. 2011, 88, 1175–1177