Biotechnology Letters 22: 407–411, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

407

Direct biosynthesis of ascorbic acid from glucose by Xanthomonas

campestris through induced free-radicals

Y. Manjula Rao & G. K. Sureshkumar∗

Biochemical Engineering Group, Department of Chemical Engineering, Indian Institute of Technology, Bombay,
Powai, Mumbai 400 076, India
∗ Author for correspondence (Fax: +91 (22) 578 3480; E-mail: gksuresh@che.iitb.ernet.in)

Received 22 November 1999; Revisions requested 14 December 1999; Revisions received 12 January 2000; Accepted 12 January 2000

Key words: ascorbic acid, free-radicals, oxidative stress, Xanthomonas campestris

Abstract
Ascorbic acid (20.4 g l−1 in 50 h) was synthesized directly from glucose by Xanthomonas campestris as an adaptive
response to induced free-radicals through HOCl treatment. Identity of ascorbic acid was confirmed through IR and
NMR spectroscopy.

Introduction direct synthesis of ascorbic acid from glucose (the

Ascorbic acid is an antioxidant and has many ap-
plications in medicine, food, photography and the
metallurgical industries (Jaffe 1984). Ascorbic acid is
produced industrially through a multi-step process of
which only one step, namely, the conversion of (+)-
sorbitol to (−)-sorbose, is mediated by microorgan-
isms (Jaffe 1984). The direct biosynthesis of ascorbic
acid from a carbohydrate source by higher organisms,
such as yeast (Delic et al. 1989), plant and animal
(liver) cells (Smirnoff & Wheeler 1999) are known,
but such processes have not been used for industrial
production, probably due to low yields (1.5 g l−1 , even
with yeast). However, the direct synthesis of ascorbic
acid from a carbohydrate source by bacteria has not
yet been reported. At most, only a few intermediate
steps in the multi-step ascorbic acid production from
a carbohydrate source are mediated by bacteria (Delic
et al. 1989).
Although free-radicals damage cells (Cacciuttolo
et al. 1993) when present in high concentrations, they
are suspected to be important modulators of mam-
malian cell growth (Burdon 1994), product synthesis
by plant cells (Jeong et al. 1999) and in our re-
lated work we have shown that free-radicals mediate
secondary metabolite (xanthan gum) production by
the bacterium Xanthomonas campestris. In this work,
carbohydrate source), by Xanthomonas campestris,
through a free-radical inducing treatment is reported.
Materials and methods
Xanthomonas campestris (MTCC (Microbial Type
Culture Collection)) 2286, Institute of Microbial Tech-
nology (IMTECH), India) was grown in 40 g glu-
cose l−1 , 3 g yeast extract l−1 , 5 g K2 HPO4 l−1 , 0.6 g
MgSO4 ·7H2 O l−1 and 0.4 g urea l−1 (Sriram et al.
1998).
Free-radical induction procedure
Free-radicals were induced in Xanthomonas campestris
through HOCl treatment (Sriram et al. 1998) (Fig-
ure 1). Cells in the stationary phase were treated
with HOCl in three stages. The concentrations of
HOCl used at the beginning of each stage were: 1st
stage: 0.66 mmol (g cell)−1 , 2nd stage: 4.65 mmol
(g cell)−1 , and 3rd stage: 20 mmol (g cell)−1 . Each
stage consisted of HOCl exposure followed by 40 min
of incubation in dark, at 30 ◦ C, with gentle shaking.
After the third incubation, the free chlorine left in
the flasks was quenched by using sterile sodium thio-
sulfate. Culturable bacteria were assayed by plating
408
Fig. 1. Schematic of the free-radical induction and measurement
procedure.
on LB agar and colonies were counted after 10 h of
incubation.
Free-radical estimations
The procedure given by Katsuwon & Anderson
(1989) was used to obtain the cytoplasmic fraction of
cells for free-radical and cellular antioxidant analyses.
To measure oxygen-centered radicals formed, a spin
trapping technique (Schellhorn et al. 1987) was em-
ployed. Briefly, the cells at 0.13 g l−1 were taken and
the cytoplasmic fraction was obtained in the presence
of 100 mM 5,5-dimethyl-pyrroline-N-oxide (DMPO)
(Sigma). Losses were minimized by employing cold
conditions throughout the separation process (Janzen
1984). The free-radical levels were estimated using
electron spin resonance spectroscopy (Varian). The
free-radical concentration is proportional to the area
under the absorption curve (Borg 1976). The area
under the absorption curve was obtained by double
integration of the obtained derivative spectrum, using
computer programs. The free radical concentrations
were obtained by comparison with concentrations of
the known standard (Hayaishi & Asada 1977).
Cellular antioxidant estimations
Intracellular activities of superoxide dismutase, cata-
lase and glutathione were estimated using the proce-
dures given by Nandi & Chatterjee (1988), Beer &
Sizer (1951), and Ellman (1959), respectively.
Spectroscopic analysis
The procedure given by Rosenberg (1945) for ascor-
bic acid extraction from citrus fruit juice was used,
in principle, to extract ascorbic acid from the super-
natant of Xanthomonas campestris. Infra-red analy-
sis (Nicolet) and proton nuclear magnetic resonance
analysis (Varian) of both the sample and standard were
performed.
Results and discussion
Free-radical concentrations
From analysis of the hyperfine split patterns in the
derivative Electron Spin Resonance (ESR) spectra
(Borg 1976, Rosen & Rauckman 1984), the free-
radicals were identified to be either superoxide- (3
splits) or hydroxyl- (4 splits) free radicals. The free-
radical concentrations were measured as mentioned in
Materials and methods. During the stationary-phase
(50 h) (Table 1), 0.45 mmol (g cell)−1 hydroxyl free
radicals were obtained in the cytoplasm of HOCl-
treated cells, but 0.08 mmol (g cell)−1 superoxide free
radicals were obtained in the cytoplasm of wild-type
cells. Hydroxyl free radicals are known to be much
more reactive than superoxide free radicals (Fridovich
1976).
Cellular antioxidant activities
Cellular antioxidants such as superoxide dismutase,
catalase and glutathione counter the damaging effects
of free radicals. Induced oxidative stress conditions in
the bacterium Xanthomonas campestris, through ex-
tracellular HOCl treatment, led to an increase in such
cellular antioxidants (Table 1). Superoxide dismutase
(SOD) activity increased from 6.25 U (mg protein)−1
to 108.8 U (mg protein)−1 in the stationary phase, as
an adaptive response. Also, catalase, the antioxidant
enzyme which eliminates the metabolic H2 O2 formed
from superoxide and hydroxyl free radicals, was ex-
pressed in the stationary phase at a concentration of
204.36 U (mg protein)−1, whereas in the wild-type
 409

cells, it was not expressed in the stationary phase at

all. Also, there was a 2-fold increase in the intracellu-
lar glutathione level as an adaptive response to HOCl
treatment.

Ascorbic acid secretion as an adaptive response to

induced free-radicals

Ascorbic acid is used by plant and animal cells as

an antioxidant to protect themselves against oxidative
stresses (Smirnoff & Wheeler 1999). Therefore, we
suspected ascorbic acid to be formed under oxidative
stress conditions as employed in this study, in the
bacterium, Xanthomonas campestris also. Further, the
immediate precursor of ascorbic acid in industrial syn-
thesis, 2-ketogulonic acid, is known to be present in
other Xanthomonas species (Delic et al. 1989).
As an adaptive response to HOCl treatment, when
the treated cells were grown again in normal medium,
the cells were found to secrete ascorbic acid. Analysis
of the supernatant at 240 nm indicated the presence
of ascorbic acid, which was first suspected from the
unexpected observation of H2 O2 degradation in the
extracellular broth (medium without cells) of HOCl-
treated cells. Subsequent infra-red (IR) (Figure 2) and
nuclear magnetic resonance (NMR) (Figure 3) spec-
tral analyses confirmed the presence of ascorbic acid
in the extracellular broth of HOCl-treated cells. In
IR spectroscopy, there was a good correspondence
Fig. 2. Infra-red spectra of ascorbic acid (A) standard ascorbic acid
between spectra of the sample and reference, espe- (B) ascorbic acid purified from Xanthomonas campestris medium.
cially in the expected wave number range of 1885– They show an infra-red absorption at 1885–1820 cm−1 correspond-
1820 cm−1 corresponding to the range expected for ing to lactones.
lactones (Pouchert 1981). In NMR spectroscopy, the
chemical shift spectra showed excellent correspon-
type cells. However, the mechanism of conversion
dence between the sample and the reference. The
of 2-ketogulonic acid to ascorbic acid, i.e., whether
concentration of ascorbic acid found in the extracellu-
free-radicals make the lactonization possible through
lar broth (Table 1) was 20.36 g l−1 , which is 13.6-fold
generation of appropriate free-radicals, or, whether the
of that found in yeast cells which directly synthesize
genetic mechanisms induced in response to oxidative
ascorbic acid from glucose.
stress conditions also convert 2-ketogulonic acid to
Ascorbic acid is chemically produced from 2-
ascorbic acid, is unknown.
ketogulonic acid by lactonization (Rosenberg 1945).
Further, if the production of ascorbic acid is truly
As 2-ketogulonic acid is present in Xanthomonas,
free-radical mediated, then other free-radical generat-
Xanthomonas campestris probably converts 2-keto-
ing procedures must also lead to ascorbic acid produc-
gulonic acid into ascorbic acid under oxidative stress
tion. We investigated ascorbic acid production when
conditions. A higher level of cytoplasmic free-radicals
cells were grown with hydrogen peroxide as the oxy-
was obtained in HOCl-treated cells compared to that
gen source (Sriram et al. 1998), as well as when
in wild-type cells. Further, the more reactive hydroxyl
menadione treated cells were grown; menadione is a
free radicals were found in HOCl-treated cells com-
free-radical inducer. The response to peroxide addi-
pared to the superoxide free radicals in wild-type cells.
tion was a direct response to free-radical induction and
These clearly indicate that the HOCl-treated cells were
the response to menadione treatment was an adaptive
under higher oxidative stress conditions than wild-
410
Table 1. The various parameters measured in the stationary phase (50 h). The average values from
three different cultivations along with the deviations are given.

Parameter Wild-type cells HOCl-treated cells

Free-radical concentration 0.08 ± 0.009 mmol (g cell)−1
superoxide free radical
Superoxide dismutase 6.25 ± 0.6 U (mg protein)−1
Catalase None
Glutathione 4.9 ± 0.5 mg (g cell)−1
Ascorbic acid None
0.45 ± 0.03 mmol (g cell)−1
hydroxyl free radical
108.8 ± 1.2 U (mg protein)−1
204.36 ± 9.6 U (mg protein)−1
9.8 ± 0.6 mg (g cell)−1
20.36 ± 1.4 g l−1
(3.42 ± 0.24 g (g cell)−1 )

Acknowledgement

Research funding from the Department of Science and

Technology, India, is acknowledged.

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Fig. 3. Nuclear magnetic resonance (NMR) spectra of (A) stan-
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