Biotransformation of ethyl 2-(2′-nitrophenoxy)acetate to benzohydroxamic acid (D-DIBOA) by Escherichia coli

Article abstract of DOI:10.1016/j.procbio.2010.09.011

Benzohydroxamic acids, such as DIBOA, exhibit interesting biological properties (herbicidal, fungicidal and bactericidal). Recently, the synthesis of DIBOA has been simplified to only two steps. This paper explores the possibility of replacing the second stage in the chemical synthesis of D-DIBOA by a biotransformation using a strain of Escherichia coli and a strain of Serratia marcescens. Biotransformation experiments were carried out for both strains in the presence of different concentrations (0.25, 0.5 and 1 mg/mL) of the precursor (ethyl 2-(2′-nitrophenoxy)acetate) under aerobic and anaerobic conditions. Both strains tolerated the concentrations of precursor investigated here. Higher biotransformation yields were reached for E. coli under aerobic conditions. The UV/vis spectra and ¹H/¹³C spectroscopic data obtained from HPLC-DAD and NMR, respectively, for the compounds obtained in the biotransformation reaction confirmed the presence of D-DIBOA in cultures of E. coli. The maximum yields were obtained in experiments supplemented with 0.5 mg/mL of precursor and these were 20.14 ± 1.87% under aerobic conditions and 8.17 ± 0.94% under anaerobic conditions.

Full text of DOI:10.1016/j.procbio.2010.09.011

Process Biochemistry 46 (2011) 358–364
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
ꢀ
Biotransformation of ethyl 2-(2 -nitrophenoxy)acetate to benzohydroxamic acid
(
D-DIBOA) by Escherichia coli
a
a,∗
b
a
b
a
A. Valle , G. Cabrera , J.M.G. Molinillo , J.M. Gómez , F.A. Macías , D. Cantero
a
Department of Chemical Engineering and Food Technology, University of Cádiz, Avda. República Saharaui s/n, 11510 Puerto Real, Cádiz, Spain
Department of Organic Chemistry, University of Cádiz, Avda. República Saharaui s/n, 11510 Puerto Real, Cádiz, Spain
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2010
Received in revised form
Benzohydroxamic acids, such as DIBOA, exhibit interesting biological properties (herbicidal, fungicidal
and bactericidal). Recently, the synthesis of DIBOA has been simplified to only two steps. This paper
explores the possibility of replacing the second stage in the chemical synthesis of D-DIBOA by a bio-
transformation using a strain of Escherichia coli and a strain of Serratia marcescens. Biotransformation
1
5 September 2010
Accepted 15 September 2010
experiments were carried out for both strains in the presence of different concentrations (0.25, 0.5 and
ꢀ
1
mg/mL) of the precursor (ethyl 2-(2 -nitrophenoxy)acetate) under aerobic and anaerobic conditions. Both
Keywords:
Biotransformation
Escherichia coli
Serratia marcescens
Benzohydroxamic acid
Natural product
strains tolerated the concentrations of precursor investigated here. Higher biotransformation yields were
1
13
reached for E. coli under aerobic conditions. The UV/vis spectra and H/ C spectroscopic data obtained
from HPLC-DAD and NMR, respectively, for the compounds obtained in the biotransformation reaction
confirmed the presence of D-DIBOA in cultures of E. coli. The maximum yields were obtained in experi-
ments supplemented with 0.5 mg/mL of precursor and these were 20.14 ± 1.87% under aerobic conditions
and 8.17 ± 0.94% under anaerobic conditions.
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
The second step involves reduction of the nitro group followed by a
cyclization involving intramolecular addition/removal of the ethyl
chain from the ester. This step is heterogeneously catalysed by a
Pd/C catalyst and the conditions are critical due to the exothermic
character of the reaction and the release of hydrogen [6].
The isolation, structural elucidation, and synthesis of natu-
ral products constitute the main source of new molecules with
biological activity [1]. Compounds with a (2H)-1,4-benzoxazin-
3
(4H)-one skeleton (benzohydroxamic acids) have attracted the
attention of phytochemistry researchers since 2,4-dihydroxy-(2H)-
,4-benzoxazin-3(4H)-one (known as D-DIBOA, see Fig. 1) and 2,4-
One of the alternatives in the total chemical synthesis is the
replacement of some stage of the synthesis by a process involving
microbial action. This type of process is called biotransformation
and can be defined as the modification of a chemical compound
by a living organism. There are a wide range of products of sec-
ondary origin that can be metabolized and processed by enzymes
or microbial cells. These products are interesting in terms of stere-
oselectivity, regiospecificity and conversion into derivatives, but
also for the advantages related to the reaction conditions in which
they occur. Such products are currently applied in the production
of drugs and other substances of industrial interest.
1
dihydroxy-7-methoxy-(2H)-1,4-benzoxacin-3(4H)-one (known as
D-DIMBOA, see Fig. 1) were isolated from plants belonging to the
Poaceae family [2,3]. These compounds exhibit interesting bio-
logical profiles that include phytotoxic, antimicrobial, antifeedant,
antifungal, and insecticidal properties [4,5]. A number of synthe-
ses of benzohydroxamic acids have been reported in the literature
[
6–9].
Recently, the synthesis of D-DIBOA has been simplified to only
two steps (Fig. 2). The first step involves functionalization of the
phenol by nucleophilic substitution to introduce the appropriate
side chain (from ethyl bromoacetate) under mild conditions using
The hypothesis suggested in this study is that it is possible
to reduce the nitro group of ethyl 2-(2 -nitrophenoxy)acetate by a
ꢀ
biotransformation that replaces the second stage of the synthe-
sis described above. The biological reduction of nitro groups in
nitrophenol as the starting material. The reaction product is ethyl
ꢀ
ꢀ
2
-(2 -nitrophenoxy)acetate and this is obtained in yields up to 99%.
a molecule analogous to ethyl 2-(2 -nitrophenoxy)acetate, namely
trinitrotoluene (TNT), has been documented in several reports. The
reductive transformation of TNT has been documented for many
bacterial genera, e.g. Serratia [11], Escherichia [12], Desulfovibrio
∗ Corresponding author at: Departamento de Ingeniería Química y Tecnología de
Alimentos, Avda. República Saharahui s/n, 11510 Puerto Real, Cádiz, Spain.
Tel.: +34 956 016554; fax: +34 956 016411.
[
13], Clostridium [14,15], and Pseudomonas [16]. Several of these
publications indicate that transformation of the nitro group, in
aerobic or anaerobic conditions, to an amine occurs through the
E-mail address: gema.cabrera@uca.es (G. Cabrera).
1
359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.09.011

A. Valle et al. / Process Biochemistry 46 (2011) 358–364
359
O
OH
O
H CO
O
OH
O
samples for aerobic and anaerobic conditions, respectively, were withdrawn at 0,
3
9
and 24 h. The samples were harvested by centrifugation for 10 min at 10,509 × g
and the supernatants were filtered through 0.22 ␮m nylon filters prior to analysis
by high-performance liquid chromatography (HPLC).
N
N
Two kinds of control experiments were performed: a biotic control, i.e. a bacte-
rial culture without addition of precursor, and several abiotic controls, i.e. culture
medium supplemented with the same concentrations of precursor tested with-
out inoculum. The controls were kept under the same conditions of agitation and
temperature as for the related experiments.
OH
OH
DIBOA
DIMBOA
Fig. 1. Natural benzoxazinones. 2,4-dihydroxy-(2H)-1,4-benzoxazin-3-(4H)-one
DIBOA) and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3-(4H)-one (DIM-
A biotransformation experiment was subsequently carried out in order to con-
firm the identities of the reaction products. In this process, E. coli was cultivated in
the presence of 0.75 mg/mL of precursor. The culture was monitored by Thin Layer
Chromatography (TLC) during 48 h. After this time, the bacterial culture was cen-
trifuged for 10 min at 10,509 × g. The supernatant was withdrawn and the pellet
was resuspended in distilled water and centrifuged again. The total supernatant
from both centrifugations (free of cells) was extracted ten times with ethyl acetate
(EtOAc) at room temperature. The solvent was removed from the organic phase on
(
BOA) [10].
formation of the corresponding hydroxylamine [12,15–17]. This
kind of biotransformation is quite similar to the second stage of the
process outlined in Fig. 2. This fact suggested that these microor-
ganisms could be capable of carrying out this step.
The reduction of the nitro groups via hydroxylamines can be
attributed to nitroreductases [18,19]. In the Enterobacteriaceae
family, in addition to the species Escherichia coli, several enzymes
with nitroreductase activity have been characterized in Enterobac-
ter cloacae [20], particularly a xenobiotic reductase that reduces
nitro groups on the ring to give the hydroxylamine derivative of
TNT [21].
◦
a rotary evaporator under reduced pressure at 35 C. The resulting crude extract
was chromatographed on silica gel columns, using hexane/EtOAc with increasing
polarity (from 1:4 to 1:1 (v/v)).
2
.4. Analytical methods
2.4.1. Spectrophotometry
Bacterial growth experiments were monitored by following the increase in
turbidity of the culture at 600 nm using a UV spectrophotometer (Model HITACHI U-
2001). The bacterial population was represented by counting colony-forming units
The aim of the work described here was to explore the possibility
of replacing the second step in the chemical synthesis of D-DIBOA
by a biotransformation using a strain of E. coli and a strain of Serratia
marcescens.
per mL (CFU/mL); this measure was correlated with turbidity by the McFarland scale
[23]. All cultures were carried out in duplicate; the values presented are the mean
of two experiments.
2
.4.2. High-performance liquid chromatography (HPLC)
Reverse-phase HPLC analysis was performed on a Merck HITACHI HPLC system
2
. Materials and methods
equipped with an L-7100 LaChrom quaternary gradient pump, an L-7200 LaChrom
autoinjector and an L-7455 LaChrom diode array detector (DAD). Data were collected
and processed using a Merck HITACHI D-7000 HPLC data system. Instrumental con-
2.1. Microorganisms and chemicals
®
ditions were Phenomenex Gemini C18 (4.6 mm × 250 mm) reversed-phase column
The strain E. coli JM109 (ATCC 53323)/pGEM4Z (Promega) was obtained from
◦
at 25 C. Mobile phases were as follows: water: 1% AcOH (A) and MeOH:1% AcOH (B)
ATCC. S. marcescens strain N2 belongs to the microbial collection of the Metal
Biotechnology Laboratory, Biology Faculty, Havana University. This strain was iso-
lated from a lateritic deposit from Moa, Holguin (Cuba) [22].
at a flow rate of 1 mL/min. The injection volume was 15 ␮L. The following gradient
was used for separation: at 0 min, 0% B; 7 min, 30% B; 30 min, 40% B; 35 min, 100%
B.
ꢀ
The starting compound used to study the biotransformation was ethyl 2-(2 -
Chromatogram analysis was carried out using HPLC System Manager software
Version 4.1 with the diode array detector set at 253 nm by comparing the retention
times (RT) and ultraviolet/visible spectra with those of the chemical standards.
The fractions eluted from the silica gel column were further purified by HPLC
on semipreparative silica gel (Lichrospher Si-60, 10 ␮m) columns using a mixture
of hexane/EtOAc (6:4, v/v).
nitrophenoxy)acetate, which is henceforth referred to as the precursor.
Chemical standards of the precursor and D-DIBOA were synthesized from 2-
nitrophenol as described by Macías et al. [6]. The reagents were purchased from
Sigma–Aldrich and the culture media from Panreac, FLUKA and Cultimed. All organic
solvents were HPLC grade.
2
.2. Media
2
.4.3. Nuclear magnetic resonance (NMR)
1H and 13C NMR spectra were run on Varian INOVA-400 spectrometers. Chemical
The inoculums required for experimentation were obtained by seeding bacteria
1
shifts are given in parts per million (ppm) with respect to the residual H signal of
deuterated chloroform (CDCl ) (ı 7.25) and the C signal of chloroform (CHCl ) (ı
3 3
7
from conservation strains in Luria-Bertani broth (LB). The inoculum/medium ratio
was 1:10 (v/v) and the bacteria were grown overnight at 30 C in Erlenmeyer flasks.
These pre-inoculums were used to inoculate (10%, v/v) test systems for both aerobic
and anaerobic conditions.
1
3
◦
7.00).
In aerobic experiments LB broth was inoculated (100 mL final volume) in Erlen-
meyer flasks that were kept in an orbital incubator at 150 rpm and 30 C. For
2.4.4. Thin layer chromatography (TLC)
◦
TLC was carried out on Alugram Sil G/UV254 plates (Merck) with a layer thick-
ness of 0.25 mm with fluorescent indicator. Chromatography plates were developed
by treatment with oleum prepared with a solution of sulphuric acid, water and acetic
anaerobic cultures LB broth was inoculated (50 mL final volume) in sealed vials and
◦
incubation was carried out at 30 C under static conditions.
◦
acid (1:4:20) (v/v) followed by heating at 150 C. The sample spots were observed
2.3. Biotransformation experiments
using a UV lamp at 254 or 360 nm.
Strains used in this study were selected for their ability to reduce nitro groups
2.5. Biotransformation yield
of TNT as this indicates that they may be capable of performing the reduction of the
nitro group in the precursor.
For quantitative analysis, stock solutions of each individual chemical standard
were prepared by dissolving accurate amounts in LB broth with 1% MeOH (v/v).
Working D-DIBOA standard solutions of 0.25, 0.125, 0.0625, 0.0313 mg/mL were
obtained by dilution of the 0.5 mg/mL stock. These solutions were used to generate
the external standard response calibration curves for subsequent measurements
of D-DIBOA concentrations from bacteria culture samples. Hydroxamic acids could
The transformation of the precursor was initiated by adding 1 mL of stock pre-
cursor solutions diluted in methanol (MeOH) for the aerobic culture, or 0.5 mL for the
anaerobic culture, to achieve concentrations of 0.25, 0.5 and 1.0 mg of precursor/mL.
The inoculation was performed from cultures of strains E. coli and S. marcescens in
the exponential phase of growth (10%, v/v). During the incubation, 1 mL and 0.5 mL
ꢀ
Fig. 2. Benzohydroxamic acid synthesis: (A) first step: synthesis of ethyl 2-(2 -nitrophenoxy)acetate (2) (the precursor) using nitrophenol (1) as starting material and (B)
second step: reduction of the precursor nitro group to obtain D-DIBOA (3) [6].

3
60
A. Valle et al. / Process Biochemistry 46 (2011) 358–364
aerobic
Fig. 3. Bacteria growth curves. Cultures in LB medium supplemented with increasing concentrations of precursor. (A) Nic2 and (B) E. coli in aerobic conditions; (C) Nic2 and
D) E. coli in anaerobic conditions. (—᭹—) 0 mg/mL control precursor; (· · ·ꢀ· · ·) 0.25 mg/mL precursor; (—ꢁ—) 0.5 mg/mL precursor and (- -♦- -) 1.0 mg/mL precursor.
(
be good chelator of iron, then the quantification of D-DIBOA could be affected if
iron complex is formed. For this reason, we determined the influence of the iron
The different behaviours observed are due to the different
natures of the two strains, the different culture conditions tested
(
III) concentration present in the medium to D-DIBOA through the examination of
(aerobic/anaerobic) and the possible inhibitory action exerted
UV/vis spectra and the peaks areas of the chromatograms (data not shown). Finally,
we can conclude that the iron content in LB broth does not affect the quantification
of D-DIBOA in the culture conditions.
by the products that appear in the medium due to microbial
metabolism.
Data for biotransformation yields are presented as percentage of biotransfor-
mation, which was calculated thorough the molar relation of D-DIBOA at 24 h and
the initial quantity of precursor supplemented in each experiment.
3.2. Identification of compounds by HPLC
The samples were monitored at 253 nm and their compo-
nent/s identified by diode array detector. Three chromatographic
peaks were observed in chromatograms of samples from the cul-
tivation of E. coli and S. marcescens at 9 and 24 h: compound I
(Fig. 4(A)) and compound II (Fig 4(B)) appear at RT of 20.41 and
3
. Results
3.1. Growth curves and response to precursor
2
0.74 ± 0.04 min, respectively, and compound III (Fig. 4(A) and (B))
Tracking the growth of S. marcescens and E. coli in CFU/mL for
at 24.69 ± 0.04 min.
4
8 h allowed us to establish the tolerance of these strains to the
Compounds I and II were identified as D-DIBOA as they eluted
very close to the reference standard (RT 20.43 min).
precursor. Growth was observed in both cultures under aerobic
and anaerobic conditions and this was affected by increasing the
concentration of precursor. Concentrations above 1 mg/mL were
tested but these proved to be inhibitory for cell growth (data not
shown), hence biotransformation experiments were conducted at
concentrations below this level.
In general, it appears that the aerobic growth of both strains is
similar, with the cell population showing maximum decreases as
the concentration of precursor in the medium was increased. How-
ever, the absolute CFU/mL values achieved are significantly higher
for S. marcescens than for E. coli (Fig. 3(A) and (B)). Under anaerobic
conditions the behaviour of the two strains was very different, with
S. marcescens practically reaching a stationary phase at 10 h while
E. coli peaks are reached at the end of the experiment. In any case,
the absolute values for both strains are 10 times smaller than under
aerobic conditions.
In addition, the UV/vis spectra of compounds I and II showed a
high correlation to that of the D-DIBOA reference under both sets of
conditions, with a correlation coefficient of 0.9999 for compound I
and 0.9342 for compound II (Fig. 5(A) and (B), respectively).
The presence of compounds I and II was not evidenced in the
chromatograms corresponding to biotic and abiotic controls, thus
demonstrating that production of these compounds is the result of
the transformation of the precursor by bacteria.
Compound III eluted more slowly than the precursor
(24.51 ± 0.16 min) and the UV/vis spectrum had a high corre-
lation coefficient with the precursor (0.9962) (Fig. 5(C)). This
compound was detected in all of the analyzed samples from both
cultures and also the abiotic controls, in both cases with the
precursor, but it was not present in the biotic control. These find-
ings seem to indicate that this compound is formed by chemical

A. Valle et al. / Process Biochemistry 46 (2011) 358–364
361
0
0
0
0
0
.5
.4
.3
.2
.1
0
3.3. Analysis of metabolites in culture supernatants
(
A)
Since the experiments with E. coli showed better biotrans-
formation yields of the precursor, it was decided to analyze the
metabolites in order to identify and characterize the compounds
obtained in these experiments. To this end, a liquid–liquid extrac-
tion was carried out on the culture supernatant with ethyl acetate.
Precursor
D-DIBOA
0
5
10
15
20
25
30
35
Retention Time (min)
0
0
0
0
0
.5
.4
.3
.2
.1
0
(B)
Compound III
Compound I
0
5
10
15
20
25
30
35
Retention Time (min)
0
0
0
0
0
.5
.4
.3
.2
.1
0
(
C)
Compound III
Compound II
0
5
10
15
20
25
30
35
Retention Time (min)
Fig. 4. E. coli and S. marcescens chromatograms. (A) Chromatographic peak areas for
reference compounds, D-DIBOA and precursor (B) chromatographic peak areas for
compounds in E. coli culture (C) chromatographic peak areas for compounds in S.
marcescens culture.
degradation of the precursor. However, the structure of compound
III remains unknown.
The graphs shown in Fig. 6 represent the chromatographic peak
areas obtained for the precursor and compounds I and II under
the different conditions tested for E. coli and S. marcescens cul-
tures. It can be seen that the precursor concentration decreases
significantly at 9 h (Fig. 6(A)) and reaches levels below the limit of
detection at 24 h – except for S. marcescens cultures under anaerobic
conditions, where the concentration is almost constant between 9
and 24 h (Fig. 6(C)). In the case of E. coli, the decrease in the con-
centration of precursor during the experiment can be correlated
with the increase in the concentration of compound I, as can be
seen by the peak areas for this compound in the chromatograms
recorded at the two measurement times (Fig. 6(B) and (D)). How-
ever, this relationship is not observed in the case of compound II. In
this case the disappearance of the precursor cannot be correlated
with the appearance of compound II. Moreover, it is evident that S.
marcescens under anaerobic conditions is not able to transform the
precursor, which suggests some inhibitory effect for this compound
on the metabolic activity of this strain in the absence of oxygen.
Fig. 5. UV–vis spectra of compounds present in the biotransformation experiments.
Comparison of normalized UV–vis spectra of: (A) (—) D-DIBOA analytical standard
and (– –) compound I from E. coli culture sample identified as D-DIBOA with a 0.9999
correlation coefficient. (B) (—) D-DIBOA analytical standard and (– –) compound II
from Nic2 culture sample identified as D-DIBOA with a 0.9342 correlation coeffi-
cient. (C) (– –) compound III and (—) precursor analytical standard with a 0.9962
correlation coefficient.

3
62
A. Valle et al. / Process Biochemistry 46 (2011) 358–364
Fig. 6. Areas of precursor, compound I and compound II. Precursor areas (a.u.) in E. coli and Nic2 culture samples at: (A) 9 h and (C) 24 h. Compound I and II areas (a.u.)
identified as D-DIBOA in E. coli and Nic2 culture samples respectively at: (B) 9 h and (D) 24 h. Precursor concentration [mg/mL]: 0.25, 0.5 and 1 mg/mL precursor concentration
added at the start of the lag time. Culture bacteria conditions: experiments were carried out aerobically and anaerobically in both strains. Graph (A) represents the area
corresponding to abiotic control at 0 and 9 h to facilitate comparison with the rest of the experiments.
Table 1
The solvent was removed on a rotary evaporator to give 82.5 mg of
Biotransformation yield in E. coli. E.coli culture at 24 h in aerobic and anaerobic con-
extract (AcOEt-A). The extract (AcOEt-A) was separated into four
fractions with hexane/AcOEt (6:4) and methanol and the follow-
ditions. Precursor added to initial culture. The results are shown as mean ± Standard
Deviation (S.D.).
ing amounts were obtained: 1 (6.5 mg), 2 (6.3 mg), 3 (1.7 mg) and 4
Precursor concentration [mg/mL]
Biotransformation yield %
(
33.7 mg). These fractions were subsequently chromatographed by
reverse phase HPLC (C-18 column). In these fractions, the only prod-
uct identified as D-DIBOA on comparing the RT and UV/vis spectra
Aerobic
Anaerobic
0
0.5
1
.25
11.2 ± 0.8
20.1 ± 1.9
10.8 ± 2.2
9.8 ± 0.8
8.2 ± 0.9
8.2 ± 0.6
(
Fig. 4(A)) was detected in fraction 4 (33.7 mg). Furthermore, frac-
tion 4 contained impurities and was therefore purified further on a
silica gel column to give 8 mg of pure product.
The H NMR (Fig. 7(B)) and 13C NMR spectra of the pure product
1
in CDCl were identical to that of D-DIBOA (Fig. 7(A)) obtained using
4. Discussion
3
the procedure described by Macías et al. [6], which unequivocally
confirms the structure.
In tests with concentrations up to 0.5 mg/mL, the amount of
compound I obtained was proportional to the initial concentration
of precursor. At higher concentrations a further increase in bio-
transformation was not observed, a finding that can be attributed
to inhibitory effects. Aerobic conditions gave better results in the
biotransformation and this could be due in part to the fact that,
during these trials, agitation was used and this helps mass transfer,
The biotransformation yields obtained for E. coli are shown in
Table 1. The best results were obtained under aerobic conditions in
the experiment involving the addition of precursor at 0.5 mg/mL to
give a yield of 20.14%. Under anaerobic conditions the yields were
lower – between 8.17 and 9.19% for the three precursor concentra-
tions tested.

A. Valle et al. / Process Biochemistry 46 (2011) 358–364
363
insensitive (Type I) and an oxygen-sensitive (Type II) [26] type.
In our case, it is possible that type I is involved as the biotrans-
formation occurs equally well in both oxygenic and anoxygenic
conditions.
As far as biotransformation yields are concerned, the biological
process is still inefficient. These results highlight several variables
that could be changed in future studies to achieve better operating
performance: e.g. the culture medium composition, the concentra-
tion of precursor, the effect of iron concentration, the agitation rate
and the purification method.
In conclusion, these results confirm that the strain E. coli used
in this study is capable of biotransforming the precursor to D-
DIBOA, although the yield of this biotransformation is still low.
The results also suggest that the enzymes of E. coli nitroreduc-
tases, as described in the literature, may be present in the strains
used and may participate in the reduction of the nitro group of the
precursor. The objectives for future work are the identification of
the enzymes responsible for the biotransformation using molecular
biology techniques and the optimization of operational variables in
order to increase the production yields.
Acknowledgments
This work was supported by the Consejería de Innovación, Cien-
cia y Empresa de la Junta de Andalucía through the Project for
Excellence P06-01399 (2006) The authors wish to thank the Fac-
ulty of Biology, Universidad de la Habana (Cuba), who provided the
strain S. marcescens used in this work.
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