ORI GI NAL RESEARCH PAPER

Streptomyces coelicolor increases the production

of undecylprodigiosin when interacted with Bacillus subtilis
Khalid Jaber Kadhum Luti

Ferda Mavituna
Received: 8 July 2010 / Accepted: 27 August 2010 / Published online: 29 September 2010
Springer Science+Business Media B.V. 2010
Abstract
Purpose of work Undecylprodigiosin has antimi-
crobial, immunosuppressive and anticancer proper-
ties. Pure cultures of Streptomyces coelicolor produce
only low concentrations of undecylprodigiosin. We
have therefore sought to increase this by interacting
the cells with a bacterium.
When interacted with live or heat-killed cells of
Bacillus subtilis, Streptomyces coelicolor A3 (2),
increased undecylprodigiosin production in shake-
asks and in a 2 l bioreactor. An inoculum of 2.5%
(v/v) B. subtilis containing 10
8
cells/ml added at the
beginning of the S. coelicolor culture gave the best
results for undecylprodigiosin production. In the
shake-asks, the increase was 175211% and in the
bioreactor, 256% in comparison to the pure cultures
of S. coelicolor.
Keywords Streptomyces coelicolor
Undecylprodigiosin Bacillus subtilis
Interspecies interaction Antibiotics
Introduction
Microbial populations in natural settings normally
consist of complex mixtures of different species.
Some of these species interact with each other which
may result in the production of some important
bioactive compounds including antibiotics (Oh et al.
2005). Although the function of antibiotics for the
producing organism is still not completely under-
stood, competition hypothesis suggests that antibiotics
give the producing organism an advantage over any
possible competitors in a nutritionally poor environ-
ment such as soil (Maplestone et al. 1992). Yet, in
industrial bioprocesses and laboratory research, the
common procedure is to use pure cultures which
masks the real biosynthetic potential of the organism
under investigation. Inter-species interactions on the
other hand, can induce the unexpressed biosynthetic
pathways for new compounds as well as improvement
needed in the productivity of the antibiotic-producing
strains (Petti 2009; Scherlach and Hertweck 2009). In
this context, a new antibiotic, pestalone, was produced
by a marine fungus Pestalotia sp. when challenged
with some marine bacteria in a mixed fermentation
(Cueto et al. 2001). Induction of the production of the
blue pigment, pyocyanin, by Pseudomonas aerogin-
osa was observed in a co-culture with Enterobacter
sp. (Angell et al. 2009). In addition, interaction with
some actinomycetes triggered the biosynthesis of
secondary metabolites in Aspergillus nidulans
(Schroeckh et al. 2009).
K. J. K. Luti F. Mavituna (&)
School of Chemical Engineering and Analytical Science,
The Mill, The University of Manchester, Oxford Road,
Manchester M13 9PL, UK
e-mail: ferda.mavituna@manchester.ac.uk
1 3
Biotechnol Lett (2011) 33:113118
DOI 10.1007/s10529-010-0401-y
Streptomyces coelicolor A3 (2) produces two
pigmented antibiotics, undecylprodigiosin (red) and
actinorhodin (blue) (Naeimpoor and Mavituna 2000).
In addition to its antimicrobial activities, undecyl-
prodigiosin has immunosuppressive and anticancer
properties (Williamson et al. 2006). The selective
apoptotic effect of undecylprodigiosin on breast
carcinoma cells was found, and consequently, it
was suggested to be useful as a novel anticancer
drug for the treatment of breast cancer (Ho et al.
2007).
Streptomyces coelicolor is a gram-positive, soil-
dwelling organism and, since its habitat is nutrition-
ally poor, it has biosynthetic pathways which infer
survival capabilities in the presence of competition
from other soil organisms such as Bacillus subtilis.
For this reason, the present study was designed to
investigate the effect of the presence of a second
gram-positive soil bacterium in the fermentation
medium on the production of undecylprodigiosin
by S. coelicolor. We describe for the rst time,
the increase in undecylprodigiosin production by
S. coelicolor when interacted with live or dead (heat-
killed) cells of B. subtilis in shake ask and
bioreactor cultures.
Materials and methods
Microorganisms and media composition
A chemically dened liquid medium described by
Hobbs et al. (1989) was used for both Streptomyces
coelicolor A3 (2) MT1110 pure culture and elicited
cultures. Mannitol/soya our (MS) agar (Choi et al.
2004) was used for maintenance and spore stocks of
S. coelicolor as well as solid co-cultured experiments.
Nutrient agar (NA) and broth were used for the main-
tenance and cultivation, respectively, of Bacillus
subtilis ATCC 6633.
Preparation of inocula
In all the experiments, vegetative inocula of
S. coelicolor were used and prepared as described
(Elibol et al. 1995). In all the experiments, the
number of spores that initiated the vegetative inoc-
ulum was kept to approximately 1.2 9 10
9
spores/ml.
This inoculum was shaken at 200 rpm for 2 days
at 28C and then used as vegetative inocula for
subsequent pure and interacted culture experiments.
Inoculum of B. subtilis was prepared as follows:
a loopful of B. subtilis from an overnight grown
culture on NA was inoculated into a 250 ml Erlen-
meyer ask containing 50 ml nutrient broth and
incubated at 37C for 24 h. The culture was centri-
fuged at 10,0009g for 10 min and the cells then
washed and re-suspended in equal volume of sterile
normal saline. A haemocytometer was used to adjust
the number of B. subtilis cells to be approximately
10
8
cells/ml by adding sterile normal saline if
necessary. If dead B. subtilis cells were required,
the B. subtilis cells suspension in normal saline was
placed in boiling water for 30 min.
Cultivation in shake asks and bioreactor
Pure cultures of S. coelicolor were prepared accord-
ing to Elibol et al. (1995). In interacted cultures, live
or heat-killed cell suspension of B. subtilis at 2.5%
(v/v) level was added to the asks already inoculated
with S. coelicolor. Flasks were shaken at 200 rpm at
30C for 7 days. All the experiments were accompa-
nied with a pure culture which was referred to as the
control, and each run conducted either in triplicate or
duplicate and the results represented the arithmetic
average.
Some of the experiments were carried out in a 2 l
bioreactor constructed of jacketed Pyrex glass vessel
with a working volume of 1.8 l. Medium (excluding
glucose, phosphate and trace elements) were added
to the vessel and then autoclaved at 121C for 45 min.
Glucose, trace elements and phosphate were sterilized
separately and then pumped into the bioreactor asep-
tically. The bioreactor was operated at 30C with
aeration at 2 l/min and agitation at 200 rpm. The pHof
the medium was initially adjusted to 7.2 and then
allowed to follow its natural course. Foam control was
achieved by using antifoam Struktol J647.
Analyses
Undecylprodigiosin concentration was determined
using a colorimetric method (Kang et al. 1998). The
growth of S. coelicolor in pure and interacted cultures
was measured as the dry weight of cell material
according to the method described before which
separates the two species (Rasool and Wimpenny
114 Biotechnol Lett (2011) 33:113118
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1982). An Analox glucose analysis machine was used
to determine the glucose concentration.
Results and discussion
Streptomyces coelicolor interaction with
B. subtilis on solid medium
A preliminary investigation for the effect of the
interaction with B. subtilis on the antibiotic produc-
tion by S. coelicolor was carried out by growing the
two bacteria on the same MS agar smeared adja-
cently. A signicant stimulatory effect on the
production of red-pigmented undecylprodigiosin by
S. coelicolor was seen at the interface of B. subtilis
and S. coelicolor biolms as the dark border shown
in Fig. 1 within 3 days. This production of undecyl-
prodigiosin was not seen on those sections of the
S. coelicolor biolm not in direct contact with
B. subtilis. Furthermore, in the pure cultures of
S. coelicolor on MS agar, there was no production of
undecylprodigiosin even after 10 days. This obser-
vation suggested that the production of undecylpro-
digiosin correlated to some interaction between the
two bacteria. In order to exploit this interaction, live
and heat-killed cells of B. subtilis were tested as
possible inducers in both shake-asks and bioreactor
cultures of S. coelicolor.
Streptomyces coelicolor interaction with
B. subtilis in liquid medium
Bacillus subtilis did not grow in the dened medium
designed for S. coelicolor. However, when live cells
of B. subtilis were added to the S. coelicolor shake-
ask cultures, production of undecylprodigiosin
increased. Furthermore, an earlier onset of undecyl-
prodigiosin production in the growth phase was
observed. As illustrated in Fig. 2, undecylprodigiosin
on the rst in the interacted culture was 0.28 mg/l
which increased to 2.83 mg/l after 5 days of incuba-
tion. The maximum concentration of this antibiotic in
the control culture was only 1.02 mg/l.
Similar to the effect of live cells of B. subtilis,
heat-killed cells also stimulated undecylprodigiosin
production by S. coelicolor. As can be seen in Fig. 3,
production of undecylprodigiosin was higher in the
cultures stimulated with heat-killed cells of B. subtilis
Fig. 1 Growth of S. coelicolor and B. subtilis on MS agar and
production of undecylprodigiosin at the co-culture interface
Fig. 2 Time course of undecylprodigiosin production by
S. coelicolor in both pure (dotted line with lled triangle)
and interacted culture (continuous line with lled square) with
live cells of B. subtilis
Fig. 3 Time course of undecylprodigiosin production by
S. coelicolor in both pure (dotted line with lled triangle)
and interacted culture (continuous line with lled square) with
heat-killed cells of B. subtilis
Biotechnol Lett (2011) 33:113118 115
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compared with the control. At the end of the rst day
of incubation, the concentration of undecylprodigio-
sin in the interacted culture was 0.46 mg/l compared
with 0.07 mg/l in the control. Undecylprodigiosin
reached a maximum of 2.79 mg/l on day 7 in the
interacted culture whereas the maximum undecylpro-
digiosin concentration in the control culture was
attained on day 5 but was only 0.89 mg/l. In both
control and interacted cultures, pH values were
approximately the same and ranged from the initial
pH 7.2 to pH 8.5 at the end of incubation.
Optimising the amount and timing of B. subtilis
addition
In the light of the effect of both live and heat-killed
cells of B. subtilis on the production of undecylpro-
digiosin, which were approximately similar, heat-
killed cells were chosen for further experiments. The
best level of heat-killed B. subtilis cells was 2.5%
(v/v) (approximately 10
8
cells/ml) for the S. coeli-
color culture (Fig. 4). The best time for the addition
of B. subtilis cells to achieve maximum enhancement
of undecylprodigiosin production was at the begin-
ning (Fig. 5).
Bioreactor experiments
We studied the stimulation of undecylprodigiosin
production with the heat-killed B. subtilis in a 2 l
bioreactor. Production of undecylprodigiosin was
again stimulated and the production started on day
1 and reached its maximum of 1.48 mg/l on day 5.
Undecylprodigiosin in the control appeared on day 2
and reached only 0.41 mg/l on day 4 (Fig. 6).
Undecylprodigiosin is normally produced during
the late growth phase; however, earlier production
can occur as a result of some environmental and
Fig. 4 Production of undecylprodigiosin by S. coelicolor in
cultures interacted with different amounts of heat-killed cells
of B. subtilis (10
8
cells/ml) at zero time compared with the
control
Fig. 5 Production of undecylprodigiosin by S. coelicolor in
cultures interacted with 2.5% (v/v) heat-killed cells of
B. subtilis (10
8
cells/ml) at different cultivation times (zero,
rst, second, third and the fourth day of the incubation)
compared with the control
Fig. 6 Production of undecylprodigiosin by S. coelicolor in
2 l bioreactor in both pure culture (dotted line with lled
triangle) and that interacted with heat-killed cells of B. subtilis
(continuous line with lled square)
Fig. 7 Growth of S. coelicolor and glucose consumption in
both pure and that interacted with B. subtilis using chemically
dened synthetic medium. Growth in pure culture (dotted line
with lled triangle), growth in mixed culture (continuous line
with lled square), glucose consumption in pure culture (dotted
line with empty triangle), and glucose consumption in mixed
culture (continuous line with empty square)
116 Biotechnol Lett (2011) 33:113118
1 3
physiological inuences such as nutritional stress,
signalling molecules or low growth rates (Sevcikova
and Kormanec 2004; Bibb 1996). In our experiments,
although S. coelicolor showed an increase in the
production of undecylprodigiosin earlier in the
growth phase compared with the control, this could
not have been due to nutritional stress since
B. subtilis cells were dead and there was plenty of
glucose in the medium during undecylprodigiosin
production. In addition, growth of S. coelicolor was
also stimulated (Fig. 7). In both cultures, glucose
consumption was concomitant with growth and
reected the differences in growth trends. pH was
similar in both cultures ranging from 7.2 to 8.5.
B. subtilis dead cells may have lysed during
S. coelicolor cultivation and hence contributed some
important growth and undecylprodigiosin stimulants
or compounds that may act as precursors for the
product. In order to test the possibility of elicitation
by the B. subtilis cell wall, we added 2.5% (v/v)
0.010.5 mg peptidoglycan/l (SigmaAldrich) to the
control culture at zero time. There was no effect.
N-Acetylglucosamine is a component of the peptido-
glycan and there are some reports that it may trigger
antibiotic production (Rigali et al. 2008). When we
added 2.5% (v/v) of 0.010.5 mg N-acetylglucosa-
mine/l (SigmaAldrich), again, there was no stimu-
lation of undecylprodigiosin production.
Production of antifungal tolytoxin by Scytonema
osellatum was stimulated using fungal cell homoge-
nates of Penicillium notatum and Cylindrocladium
spathiphylli (Patterson and Bolis 1997). Other exam-
ples of elicitation in the literature focus on using
some chemical compounds such as acid hydrolysed
alginate oligosaccharides and enzyme-hydrolysed
pectin oligosaccharides (Asilonu et al. 2000; Murphy
et al. 2008).
In conclusion, when live or heat-killed cells of
B. subtilis were added to S. coelicolor cultures,
production of undecylprodigiosin was increased as
much as 256% compared to the pure cultures.
Furthermore, the onset of undecylprodigiosin produc-
tion was earlier when there was such stimulation. Our
work supports the use of interspecies interactions in
order to enhance the production of secondary metab-
olites at bioreactor scale as a preliminary step for
industrial scale applications. The fact that dead
cells are just as effective for such enhancement
strengthens the case for industrial use. We are currently
investigating the mechanism of this interaction
between these two bacteria.
Acknowledgments We would like to acknowledge the
nancial support from The Ministry of Higher Education and
Scientic Research of Iraq for Khalid Jaber Kadhum Luti. We
would also like to thank Vandana Bhatt for the preliminary
tests on microbial interactions.
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