Journal of Basic Microbiology 2008, 48, 53 – 58 53

Short Communication
Gentamicin production by Micromonospora echinospora
(Me-22) in stirred tank reactor: effect of various parameters

Himabindu Meenavilli, Ravichandra Potumarthi and Annapurna Jetty

Indian Institute of Chemical Technology, Hyderabad, India

Effect of production medium components, initial starch and soyabean meal concentrations, for
the enhanced production of gentamicin by Micromonospora echinospora (Me-22) was studied
in a lab scale stirred tank reactor. Also effect of different aeration (0.5, 1, 2, and 4 vvm)
and agitation rates (100, 200, 300 and 400 rpm) in a stirred tank reactor was examined.
A maximum gentamicin concentration of 2.68 g l–1 was achieved in the medium having low
concentrations of initial starch (7.5 g l-1) and high concentrations of initial soyabean meal
(4 g l–1). Both aeration and agitation significantly affected gentamicin concentration,
productivity and biomass formation. The maximum gentamicin concentration of 4.12 g l–1 and
the highest yield of gentamicin on substrate 0.967 g g–1 were obtained at impeller speed of
200 rpm and aeration rate of 2 vvm. Under optimal culture conditions in STR the production of
gentamicin could be increased 3 fold when compared with shake flask.

Keywords: Gentamicin / Stirred tank reactor / Aeration / Agitation / Media optimization

Received: April 04, 2007; accepted: July 11, 2007

DOI 10.1002/jobm.200700116

Introduction* logical properties of the culture may also play crucial

Gentamicins are bactericidal aminoglycoside antibiotics
that inhibit protein synthesis by rapid binding to bacte-
rial ribosomes. In addition to its use as antibacterial
agent, the potential antiviral properties of some gen-
tamicin conjugates recently have been demonstrated
[1]. Nearly 150 aminoglycoside antibiotics have been
isolated from Micromonospora species, highlighting the
industrial potential of this genus. Studies of Micromono-
spora for gentamicin production in reactors have been
few when compared to other actinomycetes genera
such as Streptomyces and consequently the physiology of
the genus is poorly understood. Selection of appropri-
ate carbon, nitrogen and other nutrients is one of the
most critical stages in the development of an efficient
and economic bioprocess for gentamicin production [2].
Aeration in aerobic fermentations is extremely im-
portant since growth and production can be seriously
affected by the dissolved oxygen concentration. Rheo-
Correspondence: Dr. Annapurna Jetty, BEEC, Indian Institute of
Chemical Technology, Hyderabad-500 007, India
E-mail: annapurna@iict.res.in; annapurnajetty@gmail.com
Phone: +91-40-27160123 extn: 2663
Fax: +91-40-27193159
roles in fermentation. For example, the highly viscous
nature of Streptomyces liquid cultures can significantly
obstruct the oxygen transfer. Therefore, in order to
minimize the effect of the oxygen limitation problem, a
sufficient supply of oxygen is necessary for microorgan-
isms [3].
Among other factors having an impact on the operat-
ing conditions during fermentation in bioreactors is
agitation and mixing. Agitation is important for uni-
form mixing of the medium components within the
fermenter (dispersion of cells and nutrients) as well as
mass transfer phenomena (e.g., oxygen transfer rates)
[4].
In our previous communications we have optimized
the gentamicin production medium by Micromonospora
echinospora parent culture under shake flask conditions
[5]. We have conducted subsequent experiments to
produce mutant culture of M. echinospora by different
methods [6] and found that M. echinospora EtBr-22
(Me-22) was producing the maximum gentamicin con-
centration in shake flask experiments using production
medium as defined previously [5]. The purpose of the
present study was to optimize the initial concentration
of carbon and nitrogen sources to study kinetic aspects

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

54 H. Meenavilli et al.
of Me-22 fermentation in stirred tank reactor (STR).
Effects of aeration rate and agitation rate were also
studied in order to understand the variation in produc-
tion kinetics in STR.
Materials and methods
Me-22 obtained by mutagenic treatment with Ethidium
bromide [6] was used in the present study. Each 250 ml
Erlenmeyer flasks contained 50 ml inoculum medium
having composition (g l–1): beef extract, 3; glucose, 1;
soluble starch, 24; yeast extract, 5; CaCO3, 4; pH 7.6 and
inoculated with Me-22 (one slant for each flask) under
aseptic conditions. The inoculated flasks were kept on a
rotary shaker at 200 rpm at 27 ± 2 °C for 72 h.
Different experiments were carried out in a lab scale
1.5-litre bioreactor (B-Braun Biostat, Germany) with one
litre working volume, fixed with two-stage rushton
type impeller of 50 mm diameter for the production of
gentamicin by Me-22. In the initial phase, STR was op-
erated to study the effect of starch concentrations on
gentamicin production. Further the STR was operated
to optimize initial soybean meal concentration at two
levels of initial starch concentrations for the produc-
tion of gentamicin. After optimizing the starch and
soyabean meal levels further experiments were con-
ducted at three levels of airflow rates, 0.5 vvm, 1 vvm,
2 vvm and 4 vvm. Effect of agitation rate on gentamicin
production was also investigated by doing the experi-
ments in the levels of 100, 200, 300 and 400 rpm at
2 vvm of airflow rate. For all batches of STR operation
6% inoculum was used and cultivated for 7 days at
28 ± 2 °C. The fermentation medium contained (g l–1):
starch, 9; soyabean meal 3; K2HPO4 0.9; CaCO3 4; FeSO4
0.03 and CoCl2 0.001 was used in the present bioreactor
operation. Starch concentration was varied (2.5, 5, 7.5,
10 and 12.5 g l–1) to test the effect on gentamicin pro-
duction, biomass formation. Different concentrations
of soyabean meal (2, 3, 4 g l–1) were also tested at low
and high starch concentrations. Estimation of carbohy-
drates, biomass, DO and gentamicin production was
carried out at every 24 hr interval.
At specified intervals, production of gentamicin was
determined by an agar disc technique using Staphylo-
coccus aureus MTCC 737 as the assay organism [7]. After
cultivation, growth was measured as the weight of
cell mass obtained from a culture by vacuum filtra-
tion (Whatman No. 1 filter disc) and drying at 100 °C
until constant weight (dry weight basis). The residual
sugar was determined by method of Frank. A. Loewus
[8].
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of Basic Microbiology 2008, 48, 53 – 58
Fermentation kinetics were calculated by the proce-
dures described by Kim et al. [9].
The specific growth rate, µ (h–1) was calculated from
the equation:
⎛ 1 ⎞ ⎛ dX ⎞
µ =⎜ ⎟⎜ ⎟
⎝ X ⎠ ⎝ dt ⎠
where X is the cell concentration (g l–1) at time t (h). The
specific consumption rate of substrate, QS/X (g g–1 h–1)
was estimated by the equation:
⎛ dS ⎞ ⎛ 1 ⎞
Q S/X = ⎜ ⎟ ⎜ ⎟
⎝ dt ⎠ ⎝ X ⎠
where S is the concentration of starch (g l–1) at time t
(h). The specific production rate of gentamicin, PP/X
(g g–1 h–1) was estimated by the equation:
dP 1
PP/X =
dt X
where P is the concentration of gentamicin (g l–1) at
time t (h).
The yield of gentamicin on substrate, YP/S (g g–1) was
estimated by the equation:
⎛ dP ⎞ ⎛ dS ⎞
YP/S = ⎜ ⎟ ⎜ ⎟ .
⎝ dt ⎠ ⎝ dt ⎠
Results and discussion
Effect of starch on gentamicin production kinetics
in STR
The effect of initial starch concentration on the produc-
tion kinetic aspects of culture Me-22 fermentation was
studied in a STR. The gentamicin concentration in-
creased with the increase in initial starch concentration
from 2.5 to 7.5 g l–1. Further increase in starch concen-
tration resulted in a slight reduction of gentamicin
concentration. The highest concentration of gentamicin
(1.92 g l–1) was obtained at an initial sugar concentra-
tion of 7.5 g l–1 after 5 days of incubation. The decline
in gentamicin production observed at 12.5 g l–1 of
starch concentration. This situation was probably due
to catabolite repression exerted by high carbohydrate
concentration on the synthesis of gentamicin as re-
ported in literature [10]. In previous studies starch con-
centration of 9 g l–1 has been reported optimum for the
production of gentamicin from M. echinospora at shake
flask level however the gentamicin production was
0.8 g l–1 [5]. The increase in gentamicin production in
the present study is because of controlled conditions in
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Journal of Basic Microbiology 2008, 48, 53 – 58 Gentamicin production by Micromonospora echinospora 55

STR in contrast to uncontrolled conditions in shake Effect of soyabean meal on gentamicin

flask experiments. and biomass production
The biomass concentration was increased (from 3.1 In order to study the relation between ratio of carbon
to 8.2 g l–1) with corresponding increase in initial starch and nitrogen source for gentamicin production experi-
concentration from 2.5 to 12.5 g l–1. Maximum biomass ments were carried out at different starch (carbon) and
concentration (8.2 g l–1) was attained in medium con- soyabean meal (nitrogen) concentrations.
taining 12.5 g l–1 at day 4 by Me-22 strain. The effect of initial soyabean meal concentrations
During fermentation, the concentration of residual (2, 3 and 4 g l–1) on the production of gentamicin by
sugars continuously declined, following an inverse culture Me-22 was studied at two levels of initial starch
trend to those of biomass and gentamicin production. concentration (7.5 and 10 g l–1) in the STR. At low
After 5 days of incubation (where maximum concentra- starch concentration of 7.5 g l–1, the gentamicin pro-
tion of gentamicin was achieved), almost complete duction was increased significantly to 2.68 g l–1 when
sugar depletion was observed for the culture grown at the soyabean meal concentration was increased from
an initial sugar concentration of 2.5 g l–1 and 5 g l–1. 3 to 4 g l–1. Whereas, higher and lower starch concen-
Whereas, 4.78 g l–1 of residual sugars remained in the trations with lower soyabean meal concentration (2 and
fermentation broth for the culture grown at 12.5 g l–1 3 g l–1) has resulted in lesser gentamicin production. On
of initial starch concentration. At high carbohydrate the other hand, an increase in soyabean meal concen-
concentration (12.5 g l–1) the biomass concentration tration from 3 to 4 g l–1 at high starch concentration
reached maximum (8.6 g l–1). In the medium having (10 g l–1) resulted in decreased gentamicin production.
high carbohydrate concentration most of the carbohy- This might be because of catabolite repression exerted
drate was diverted to biomass formation rather than on the microorganism at high carbon source [10]. Gon-
gentamicin synthesis might results in decrease of gen- zalez et al. [11] in his shake flask experiments showed
tamicin concentration. The production kinetic data at the stimulatory effect of ammonium ion (nitrogen
different initial starch concentrations are illustrated in source) on the synthesis of gentamicin and reported
Table 1. The yield of gentamicin from substrate (YP/S) that ammonium increases antibiotic formation through
and the specific growth rate of the cells ( µ) in medium its conversion to glutamine. A similar effect of nitrogen
having 10 g l–1 of starch were 0.331 g g–1 and 0.0096 h–1, has been reported for neomycin [12] and streptomycin
respectively. The fermentation medium having starch [13] biosynthesis i.e. glutamate, glutamine and gluco-
concentrations of 7.5 and 10 g l–1 were selected and samine stimulated antibiotic formation.
further tested for optimization of soyabean meal con- At low initial starch concentration (7.5 g l–1) and at
centration for maximum production of gentamicin. soyabean meal concentration of 4 g l–1 the biomass
concentration increased to 6.8 g l–1. Maximum biomass
Table 1. Fermentation kinetics of Me-22 at different starch concentration (7.2 g l–1) was attained in medium con-
concentrations. taining starch 10 g l–1 and soyabean meal 3 g l–1 at day
4, but production of gentamicin was less this might be
Kinetic 2.5 g 5g 7.5 g 10 g 12.5 g
parameters due to high carbohydrate concentration. At low soya-
bean meal concentrations the biomass production was
Maximum bio- 3.1 4.2 6.1 7.2 8.2
mass concentra- less irrespective of initial starch concentration. It shows
tion x (g l–1) M. echinospora depends on nitrogen source for growth at
Maximum genta- 0.8 1.48 1.92 1.52 1.02 low carbohydrate sources.
micin concentra- Complete utilization of carbohydrates was observed
tion P (g l–1)
Specific growth 0.0076 0.0086 0.0096 0.0096 0.0075 at low initial starch and low initial soyabean meal con-
rate µ (h–1) centrations. As the concentration of initial starch and
Specific con- 0.215 0.140 0.120 0.120 0.160 initial soyabean meal increases high concentrations of
sumption rate
of substrate Qs/x
residual sugars are remained in the fermentation me-
(g g–1 h–1) dium. On the other hand at low initial starch (7.5 g l–1)
Specific produc- 0.021 0.0020 0.0023 0.0023 0.0020 and high initial soyabean (4 g l–1) concentration the
tion rate of high residual sugars were observed indicating that
gentamicin Pp/x
(g g–1 h–1) carbohydrates consumption was less at high nitrogen
Yield of genta- 0.147 0.313 0.331 0.331 0.307 concentration. This might be due to decrease in the
micin on substrate carbon uptake rate by the Micromonospora and this
Yp/s (g g–1) situation is because organic nitrogen components pre-

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56 H. Meenavilli et al. Journal of Basic Microbiology 2008, 48, 53 – 58

Table 2. Fermentation kinetics of Me-22 at different soyabean aeration rate of 2 vvm. Where as a further increase in
meal concentrations.
aeration rate at values over 2 vvm or below, resulted in
Kinetic Starch 7.5 (g l–1) Starch 10 (g l–1) a decrease in gentamicin production. The maximum
parameters Soyabean meal (g l–1) Soyabean meal (g l–1) concentrations of gentamicin at aeration rates of 0.5, 1
2 g l–1 3 g l–1 4 g l–1 2 g l–1 3 g l–1 4 g l–1 and 4 vvm were 2.68, 2.95 and 3.04 g l–1, respectively.
Laznikova et al. [16] have reported that the decreased
Maximum 5.6 6.1 6.8 6.4 7.2 6.3
biomass con- aeration lowered the mycelium productivity with re-
centration X spect to the gentamicin production without changing
(g l–1) the ratio of the components in the gentamicin complex.
Maximum 1.46 1.92 2.68 1.21 1.52 1.03 The concentration of residual sugar sharply de-
gentamicin
concentration P creased during the fermentation with corresponding
(g l–1) increase in biomass and gentamicin production. Almost
Specific growth 0.0094 0.0095 0.0096 0.0096 0.0097 0.0095 complete sugar depletion was observed for the culture
rate µ (h–1)
grown at aeration rates of 2 and 4vvm, whereas 1.02
Specific con- 0.0026 0.0022 0.0029 0.0013 0.0023 0.0026
sumption rate and 1.15 g l–1 of sugar remained in the fermentation
of substrate Qs/x broth in cultures grown at aeration rates of 0.5 and
(g g–1 h–1) 1 vvm for the same fermentation period. The produc-
Specific pro- 0.0021 0.0026 0.0033 0.0016 0.0018 0.0014
duction rate of tion kinetic data at different aeration rates are illus-
gentamicin Pp/x trated in Table 3. The yield of gentamicin from sub-
(g g–1 h–1) strate (YP/S) and the specific growth rate of the cells ( µ)
Yield of gen- 0.289 0.407 0.819 0.150 0.262 0.204 at 2 vvm were 0.587 g g–1 and 0.0098 h–1, respectively.
tamicin on
substrate Yp/s The DO levels at all tested aeration rates were de-
(g g–1) clined from 140% to around 7 – 10% at the end of 3 – 4
days of bioreactor operation. At an aeration rates of
2 and 4 vvm, the DO level increased rapidly as the
sent in the medium supplements the carbon source growth entered a stationary phase, thereafter high DO
[14]. Recently Voelker and Altaba [15] showed an intrin- levels (over 50% and 60%, respectively) were main-
sic link between carbon and nitrogen metabolism by tained towards the end of fermentation, suggesting
demonstrating that in Streptomyces pristinaespiralis that an aeration rates of 2 and 4 vvm provide excessive
amino acids (nitrogen source) provide significant car- aeration for the strain in this study. This result sug-
bon to the organism. They concluded that certain gests that maintenance of high DO levels is important
amino acids (alanine and glutamine) when present in
the medium ‘spared’ the utilization of glucose, the Table 3. Fermentation kinetics of Me-22 at different aeration
primary carbon source. It remains seems to be common conditions.
phenomenon in actinomycetes. The present results
Kinetic 0.5 vvm 1 vvm 2 vvm 4 vvm
indicate the stimulatory effect of soyabean meal on parameters
gentamicin synthesis at low carbohydrate concentra-
Maximum 6.8 7.6 8.2 8.4
tion. The production kinetic data at different initial biomass concen-
soyabean meal concentrations are illustrated in Table 2. tration X (g l–1)
The yield of gentamicin from substrate (YP/S) and the Maximum genta- 2.68 2.95 3.57 3.04
specific growth rate of the cells ( µ) in medium having micin concentra-
tion P (g l–1)
7.5 g l–1, starch and 4 g l–1 of soyabean meal were Specific growth 0.0096 0.00974 0.00981 0.00973
0.819 g g–1 and 0.0096 h–1 respectively. rate µ (h–1)
Specific consump- 0.0029 0.0022 0.0046 0.0054
tion rate of sub-
Effect of aeration rate in STR
strate Qs/x
One important factor that influences the production of (g g–1 h–1)
gentamicin in stirred tank reactor is aeration rate. The Specific produc- 0.0033 0.0032 0.0036 0.0030
purpose of this experiment was to determine the opti- tion rate of genta-
micin Pp/x
mum aeration rate that would result in the highest (g g–1 h–1)
gentamicin product formation. Yield of genta- 0.819 0.709 0.815 0.623
The highest yield of gentamicin (3.57 g l–1) was ob- micin on substrate
tained at day 5 from the culture Me-22 culture with Yp/s (g g–1)

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Journal of Basic Microbiology 2008, 48, 53 – 58 Gentamicin production by Micromonospora echinospora 57

for both cell growth and gentamicin formation in Me-22 however Guo et al. [20] found that the shearing has a
fermentation. Several investigators have reported simi- remarkable effect on mycelial morphology and biosyn-
lar results during secondary metabolite production in thesis of gentamicin. During the gentamicin fermenta-
batch fermentations [17, 18]. The DO levels at 2 and tion process, mycelial morphology has a close relation-
4 vvm where Maximum gentamicin concentration ship with gentamicin. Hypha could be hurt mechani-
achieved were 34 and 42 respectively. Zhang et al. [19] cally and shortened by high shear, which resulted in
also reported similar results where the synthesis and the decrease of the production.
secretion of gentamicin were increased when DO was The concentration of dissolved oxygen fell rapidly
40%. during the first 72 h of fermentation. This might be
due to the rapid increase of biomass concentration
Effect of agitation speed in STR observed in this period. In all cultures, the concentra-
The agitation speed plays an important role in the bio- tion of dissolved oxygen from 48 to 72 h fluctuated at
reactor cultivation for the production of secondary 10 – 22% of the initial saturation level. The DO level was
metabolites. At low agitation speed the mixing may increased gradually as the growth shifted around sta-
either be inadequate, leading to cell sedimentation tionary phase. The dissolved oxygen levels showed a
while at high speed cell shearing may been enhanced. slender increase in the later stage of fermentation (day
To determine the optimum rotational speed, Me-22 cells 5 – 7) at all agitation rates and maintained these levels.
were cultivated under different agitation speeds in the An increase in gentamicin production was observed
STR. A set of batch experiments were carried out in a with the increase of agitation rate up to 100 rpm and
1.5L STR at various agitation speeds in the levels of thereafter, a decrease in gentamicin concentration was
100, 200, 300, and 400 rpm. Biomass production was recorded. Agitation rate not only affects the oxygen
enhanced as agitation speed increased from 100 to availability, but it also exerts influence on the availabil-
300 rpm at 2 vvm of airflow rate. The highest produc- ity of other nutrients in the medium. Low gentamicin
tion of biomass (8.7 g l–1) was obtained at day 4 an agi- production at higher agitation rates might be attrib-
tation speed of 200 rpm. At a higher stirrer speed of uted to the effect of shear stress on M. echinospora cells.
400 rpm, biomass production indicated a lower value Agitation rate of 200 rpm has been observed optimum
(7.7 g l–1). for the production of gentamicin from M. echinospora.
The highest concentration of gentamicin (4.12 g l–1) High agitation rates create shear forces that may affect
was obtained at day 5 from the Me-22 culture with a microbial growth in some other ways, including mor-
relatively mild agitation rate of 200 rpm. The maxi- phological changes of the culture, cell damage and
mum concentrations of gentamicin in cultures at 100, formation of metabolites [4]. The growth kinetic data of
300, and 400 rpm were 3.57, 3.28, and 2.96 g l–1, respec-
tively. Table 4. Fermentation kinetics of Me-22 at different agitation
Agitation results in a better mixing of the culture conditions
broth, allowing it to maintain a satisfactory supply of
Kinetic 100 rpm 200 rpm 300 rpm 400 rpm
sugars and other nutrients to the cells, while it facili- parameters
tates the removal of gases and other byproducts of ca-
Maximum 8.2 8.7 8.5 7.7
tabolism from the microenvironment of the cells. The biomass concen-
1
assimilation of sugars also increased with agitation tration X (g l– )
speeds shifting from 100 to 300 rpm. As the fermenta- Maximum genta- 3.57 4.12 3.28 2.96
tion progress, the concentration of residual sugar de- micin concentra-
tion P (g l–1)
creased with corresponding increases in biomass and Specific growth 0.00981 0.00983 0.00985 0.00968
gentamicin production. Almost complete sugar deple- rate µ (h–1)
tion was observed at the end of culture at agitation Specific consump- 0.0046 0.0042 0.0043 0.0031
tion rate of sub-
speeds of 100, 200, and 300 rpm, whereas 3.78 g l–1 of strate Qs/x
glucose still remained in the culture broth at a high (g g–1 h–1)
agitation speed of 400 rpm. For all agitation conditions, Specific produc- 0.0036 0.0039 0.0032 0.0032
gentamicin production ceased when the carbon source tion rate of genta-
micin Pp/x
(starch) was exhausted. (g g–1 h–1)
Pfefferle et al. [18] showed a 2 fold increase in secon- Yield of genta- 0.815 0.967 0.775 0.857
dary metabolite formation by increasing the stirrer micin on substrate
speed from 500 rpm to 750 rpm using Streptosporangium Yp/s (g g–1)

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58 H. Meenavilli et al.
Me-22 with different agitation rates are illustrated in
Table 4. The yield of gentamicin formation from sub-
strate (YP/S) and the specific growth rate of the cells ( µ)
at 200 rpm were 0.531 g g–1 and 0.00983 h–1, respec-
tively.
Conclusion
In the present study, the optimized submerged fermen-
tation conditions for mycelial growth and gentamicin
production by mutant strain of Micromonospora echino-
spora-22 in stirred tank reactor were addressed. From
the series of experiments we determined that the initial
concentrations of the starch (carbon) and soyabean
meal (nitrogen) are the factors significantly affecting
the production of gentamicin. An understanding of the
relationship between carbon and nitrogen sources dur-
ing fermentation should benefit the commercial exploi-
tation of studied organism for gentamicin production.
Low initial carbon and high initial nitrogen sources are
most favorable conditions for the enhanced production
of gentamicin. Besides medium composition, fermenta-
tion conditions such as air flow (effecting the available
oxygen) and agitation rates (effecting shear forces) play
an important role in the case of gentamicin production.
Acknowledgements
The authors are thankful to Dr. J.S. Yadav director,
I ICT for his co-operation and one of the authors, M. Hi-
mabindu is thankful to Council of Scientific and Indus-
trial Research, New Delhi, for award of senior research
fellowship.
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