Bioresource Technology 43 (1993) 19-25
OPTIMIZATION OF A CULTURE MEDIUM FOR
STREPTOMYCIN PRODUCTION USING RESPONSE-SURFACE
METHODOLOGY
S. Saval, a L. P a b l o s b & S. S a n c h e z a
Institute of Biomedical Research, Biotechnology Department, bFaculty of Veterinary Medicine and Zootechnics, Genetics and
Statistics Department, National University of Mexico (UNAM), Apartado Postal 70228, Mexico D.F. 04510
(Received 15 December 1991; revised version received 28 February 1992; accepted 3 March 1992)
Abstract
The effects of all ingredients of a culture medium
designed for streptomycin production were evaluated in
order to select the variables which influenced antibiotic"
production. Among them, glucose, beer-yeast autoly-
zate, sodium chloride and dibasic potassium phosphate
showed a substantial effect on this parameter and were
therefore selected. The optimum concentration for each
variable was determined using an orthogonal-central
composite experimental design and the response-surface
methodology. The determination coefficient (R 2) of the
fitted second-order model was 0.9384. Applying this
model, the optimum concentration of the four variables
was obtained and with this optimized medium the
streptomycin titre was increased by 52% with respect to
the original medium, using an industrial strain of
Streptomyces griseus.
Key words: Streptomycin, optimization of culture
medium, response-surface methodology, experimental
design.
INTRODUCTION
The application of statistical methodologies can be
very helpful in defining the main effects and interac-
tions of the factors which play a fundamental role in
biotechnological processes.
The response-surface methodology (RSM) was
described four decades ago (Box & Wilson, 1951).
Although Calam (1967) suggested its application in
biotechnology, its exploitation has been limited in this
area. The RSM allows determination of the optimum
conditions maximizing a response. Examples of these
applications are optimization of the conditions for the
synthesis of metabolites (Returners & Dunn, 1961;
McDaniel et aL, 1976), microbiological media
Bioresource Technology 0960-8524/92[S05.00 © 1992
Elsevier Science Publishers Ltd, England. Printed in Great
Britain
19
(Maddox & Richert, 1977; Desrochers et al., 1981),
and enzymatic activity (Cheynier et al., 1983; Malty,
1985).
We are presently studying a process for streptomy-
cin production using an industrial strain of Strepto-
myces griseus deposited in the UNAM-48 Culture
Collection, Mexico, D.F. This paper reports on the
optimization of a culture medium for maximum
streptomycin titre in shaken flasks by applying ortho-
gonal and orthogonal-central composite experimental
designs as well as response-surface methodology.
Even though streptomycin is an old product, studies
on its fermentation are important, since little informa-
tion on the details of this process has been published.
Streptomycin fermentation can be herein used as an
interesting model which shows the benefits of the sta-
tistical-optimization methodology applied to a system
with variable production titres. This technique is
apparently used in industry with some frequency, but it
is not commonly reported in research literature.
METHODS
Mircoorgansms
Spores of Streptomyces griseus were suspended and
maintained at - 10°C in a medium containing per liter:
glycerol, 400 ml; malt extract, 10 g; yeast extract, 4 g.
An aliquot (0.1 ml) of this suspension with optical
density of 15 (540 nm) was used to inoculate Eden-
meyer baffled flasks containing the culture medium (50
ml).
Culture medium
The initial medium contained per liter: monohydrated
dextrose (Arancia Comercial, S.A. de C.V., Mexico),
10 g; beer-yeast autolyzate, 25 g, magnesium sulphate,
1 g; sodium chloride, 10 g; sodium nitrate, 5 g; dibasic
potassium phosphate, 1 g, FeSO4, 0"1 rag; ZnSO4, 0-02
mg; CaCI 2, 1 rag. All nutrients were of industrial grade.
Tap water had a total hardness of 130 mg liter-1 and
permanent hardness of 54 mg liter -~, measured as
20 S. Saval, L. Pablos, S. Sanchez
CaCO3, besides traces of sodium chloride, calcium and
magnesium bicarbonates and magnesium and sodium
sulphates.
Culture conditions
Baffled flasks (250 ml) containing 50 ml culture
medium were sterilized at 124°C for 20 rain. After
inoculation, the flasks were incubated for 5 days at
28°C on a rotary shaker at 200 rpm. The experiments
were performed in triplicate.
Streptomycin
This was assayed by the agar-diffusion method with
Bacillus subtilis ATCC 6633 as test microorganism
(Kirshbaum & Arret, 1967). Streptomycin titre was
expressed as relative concentration.
Statistical design and analysis
Response-surface methodology consists of an empiri-
cal modeling technique devoted to the evaluation of
relationships existing between a group of controlled
experimental variables and the observed response. A
prior knowledge of the process to be optimized is
necessary to achieve a realistic model.
The steps to apply this methodology are:
(1) the selection of the variables to be studied and
the definition of the response to be optimized
(in our case, streptomycin titre);
(2) the definition of limits concerning the experi-
mental domain to be explored -- the range can
be as large as possible to obtain a clear
response;
(3) the selection of an experimental design -- we
used orthogonal and orthogonal-central com-
posite designs;
(4) performance of the experimentation according
to the established design;
(5) fitting a mathematical model to obtain optimum
values of each variable and the respective
response-surface. Our results were fitted to a
four-variable linear regression model of
second-order:
~= bo + blXt + bex2 + b3x3 + b4x4 + bl2XiX 2 + b13xlx 3
bl4XlX4 + b23x2x3 + b24x2x4 + b34x3x4 + bllX ~
-I- b22 X2 -1- b33 X2 Jr- b44 X2
where ~= estimated response, b0 = constant,
b i = coefficients for each term, and
xi = variables to be optimized. The mathemati-
cal model was fitted using Statgraphics (Statisti-
cal Graphics Corporation, 1987).
RESULTS AND DISCUSSION
Elimination of non-essential nutrients
The effect of each nutrient on streptomycin titre was
evaluated prior to medium optimization. To achieve
this, a complete experiment was performed always
excluding one of the nutrients at a time. Results shown
in Fig. I(A) indicate that the elimination of various
constituents reduced the streptomycin titre. As
expected, the most drastic effect was caused by the
absence of beer-yeast autolyzate, which abolished
growth and consequently antibiotic production. Upon
glucose elimination, beer-yeast autolyzate supported
growth and streptomycin production at one-third of
the titre obtained in a complete medium (control).
According to Demain and Inamine (1970), sodium
chloride is important for antibiotic release into the
medium and they found that phosphate had a regulat-
ing role in streptomycin synthesis.
The microorganism employed in this study does not
seem to use sodium nitrate as nitrogen source, since its
elimination did not significantly affect streptomycin
production. A similar observation was made by Dula-
ney (1948). The elimination of magnesium sulphate
from the medium did not affect streptomycin titre
significantly. The simultaneous elimination of sodium
nitrate and magnesium sulphate from the medium (Fig.
I(B)) proved that these did not have a noticeable effect
on antibiotic production. These results established the
following nutrients as important for streptomycin
production: glucose, beer-yeast autolyzate, sodium
chloride and dibasic potassium phosphate.
Effect of tap water and trace elements (Fe 2÷, Zn 2÷ and
Ca 2+)
To evaluate the effect of these factors on streptomycin
production, an experiment was performed using four
groups of flasks. Groups A and B contained tap water,
and groups C and D, distilled water, in addition to
glucose, beer-yeast autolyzate, sodium chloride and
dibasic potassium phosphate. Groups B and D also
contained the trace elements. Results were compared
to group A which served as control. Following incuba-
tion, the streptomycin titres showed no significant
differences.
Beer-yeast autolyzate apparently supplies the micro-
organism with all necessary trace elements for growth
and antibiotic synthesis, since addition of the latter to
the medium did not alter streptomycin titre.
P,
~l~ 0.5
noone glucose beer NoNO NaCl M(jSO K2HPO4 NaNO3
( Control ) yeast and
autolysate MgS04
ELIMINATED NUTRIENT
Fig. 1. Effect of each ingredient of the culture medium on
streptomycin production. (A) Indicated ingredient was elimi-
nated alone. (B) Sodium nitrate and magnesium sulfate were
eliminated simultaneously. All cases were compared with a
control containing all ingredients.
 Optimization of streptomycin production 21

Results also indicated that the use of tap water does

not affect the streptomycin production in either way,
hence it was used throughout the experiments.
I

Optimization of the culture medium

The response-surface methodology was applied to
determine the optimum concentration of ingredients in
the medium for maximum streptomycin titre. The
I
selected variables were identified as follows: glucose x~,
beer-yeast autolyzate x2, sodium chloride x~ and diba-
sic potassium phosphate x4. 7

Experiment 1 7
P
An orthogonal design for first-order models (Box &
Draper, 1987) was explored, as shown in Table 1. Each
variable was evaluated at a high ( + 1) and a low level 7
( - 1 ) , and these were referred to a central point (0),
which was repeated four times to consider the. experi-
mental variability of the system. The concentration at
L
the central point for x~ was 10 g liter- ~ and a distance
between levels of 5 g liter-~ was chosen, hence levels
( - 1 ) and ( + 1 ) were 5 and 15 g liter- l, respectively.
For x2, the central point was 25 g liter-~ and the dis-
tance between levels 10 g liter- ~, which rendered ( - 1 )
at 15 g liter- l and ( + 1 ) at 35 g liter- 1. Variables x 3 and
x4 were treated likewise, and codified as shown in
Table 2. This design considers all possible combina- °~

tions at two levels for the variables, i.e. 2 4 = 16 observa-

tions, which added to the four central points give a total
of 20 runs. A four factors linear regression model was
fitted to the results observed in this experiment. The
determination coefficient (R 2) obtained in this case was m

0.32 due to the interaction among variables. It was

therefore considered necessary to amplify the experi- °~

mental design in order to observe the pure effect of

each variable. ~ T ~
r~

Experiment 2
The experimental design was amplified to an ortho-
gonal-central composite for four variables (Box &
Draper, 1987). This design included two star points
( + a and - a ) for each of the variables and a distance
between levels of 1.414. These additional eight star
points allowed the evaluation of five concentration 7~ T
levels for each variable. The amplified codification of 77""
these variables is shown in Table 2. The additional
eight star points increased the number of runs to 28 as -77T
shown in Table 1.
However, once these results were fitted to a linear 7"77
regression model of second-order, it was detected that ~ 7 7 " -r
the exploration range for Xl was not enough. The
experiment was therefore complemented with an assay TTT-
which employed glucose concentrations up to 30 g
liter-1. All other ingredients were maintained in the - TTT7
central point of the same design. The streptomycin titre
obtained from the increase in glucose concentration is
shown in Fig. 2. Optimum glucose concentration was
close to 20 g liter-
22 S. Saval, L. Pablos, S. S a n c h e z

Table 2. C o n c e n t r a t i o n and coded levels of nutrients

Experiment Coded Glucose x~ Beer-yeast NaCI x 3 K2HPO 4 x 4

level (g liter- I) autolyzate x 2 (g liter- ~) (g liter - j )
(g liter- i)

- 1 5 15 5 0.5
0 10 25 10 1.0
+ 1 15 35 15 1.5

- 1.414 3 11 3 0.3
- 1 5 15 5 0-5
0 10 25 10 1.0
+ 1 15 35 15 1.5
+ 1.414 17 39 17 1.7

- 1.414 13 11 3 0.3
- 1 15 15 5 0.5
0 20 25 10 1.0
+ 1 25 35 15 1.5
+ 1.414 27 39 17 1.7

Distance between levels ( a ) 5 10 5 0.5

Table 3. Coefficients for fitted quadratic model

Variable a Coefficient Sum of Student's Significance

squares (%) t-values (P < 0.05)

Constant b0 1"317 39"94

xI b! 0'145 33"85 8"19
x, b2 0'083 11.05 4'68
x3 b 3 - 0"051 4.21 - 2"89
X4 b 4 -0"017 0'46 -0"96
x~ bll --0"116 19"74 --4'40
x~ b22 - 0"154 21"19 - 5"84
x3 b33 - 0'065 3"44 - 2'47
X42 b44 - 0-023 0"40 - 0"89
x~x 2 b l 2 - 0"040 2"06 - 2"02
xlx 3 bl3 0"028 0"98 1-39
XIX4 b14 0'014 0"24 0'69
x2x 3 b23 0"026 0'85 1"30
x2x 4 b24 0"034 1-49 1"72
x3x 4 b34 - 0"006 0"04 - 0"29
Determination coefficient, R 2 = 0.9384
Residual variance = 0"006 29

"Identified as follows: glucose x~, beer-yeast autolyzate x2, sodium chloride x 3 and dibasic potassium phosphate x 4.

Experiment 3
W i t h t h e i n f o r m a t i o n o b t a i n e d in t h e p r e c e d i n g e x p e r i -
1.0 m e n t , a t h i r d o n e was p e r f o r m e d with t h e s a m e o r t h o -
g o n a l - c e n t r a l c o m p o s i t e design, b u t w i t h t h e c e n t r a l
p o i n t o f x~ at 20 g l i t e r - ]. T h e v a l u e s for x2, x 3 a n d x4
w e r e k e p t c o n s t a n t as s h o w n in T a b l e 2. T h e results
Z
w e r e fitted to a f o u r - f a c t o r l i n e a r r e g r e s s i o n m o d e l .
Linear and quadratic terms and their first-order inter-
F- 0.5 actions were considered. The estimated coefficients of
a.
klJ t h e fitted q u a d r a t i c m o d e l a r e s h o w n in T a b l e 3. T h o s e
I-- m a r k e d with (*) s h o w e d a significant effect o n s t r e p t o -
0")
m y c i n p r o d u c t i o n . T h e e s t i m a t e d m o d e l was:
I I I )3= 1.317 + 0 . 1 4 5 x I + 0 . 0 8 3 x 2 - 0.05 l x 3
IO 20 30
- 0 . 1 1 6 x 2 - 0 . 1 5 4 x 2 - 0.065x32 - 0 . 0 4 0 x ] x 2
GLUCOSE ( gl- J )
Fig. 2. Effect of glucose concentration on streptomycin T h e o b s e r v e d results a n d p r e d i c t e d v a l u e s b a s e d o n
production by S. griseus. t h e fitted m o d e l a r e c o m p a r e d in Fig. 3. T h e d e t e r m i -
 Optimization of streptomycinproduction 23

i'-sf
-7.

,
IV II x,,;
I/ !° II/..
.
~ N kel. eor~)predicted values(-) 20-
Fig. 3. Comparison of observed response and predicted
values from the fitted quadratic model obtained from Experi- --" 15-
ment 3 results. Numbers correspond to each run of the I

experimental design.
10-

5-

0-

8
I0 15 20 30
1.0- GLUCOSE ( ol -~ )
Fig. 5. Response-surface and contour graphic of strepto-
0.% mycin production as a function of glucose (x~) and sodium
chloride (x3) concentrations. Other variables, beer-yeast
autolyzate (x2) and dibasic potassium phosphate (x4), at zero
,s I, ~z. level.
GLUCOSE(fl.~) q~.
(for x~ versus x4). The three-dimensional umbrella-
shaped figure visualizes the main effects and interac-

-"I tions of the variables, besides locating the point of

,; ,; 2'0 15
GLUCOSE (gl -~)
Fig. 4. Response-surface and contour graphic of strepto-
mycin production as a function of glucose (Xl) and beer-yeast
autolyzate (x2) concentrations. Other variables, sodium
chloride (x3) and dibasic potassium phosphate (x4), at zero
level.
nation coefficient of 0.9384 indicated that only 6% of
the total variation was not explained by the model
according to the variance analysis.
The response-surfaces and contour graphics gene-
rated by the interaction of each pair of variables, keep-
ing the levels at optimum values, are shown in Fig. 4
(for xl versus x2), Fig. 5 (for x] versus x3) and Fig. 6
maximum production. The contour graphs, which
represent a superior view of the response-surface, also
allowed identification of the point of maximum pro-
duction with the help of the contour lines which are
smaller as they approximate the peak of the surface.
Based on the coefficients of the mathematical model,
and using an equations system, the optimum values for
the variables were determined. Concentrations of the
3o ingredients for the optimized medium are shown in
Table 4.
Taking the values for the sum of squares expressed
as percentages in Table 3, we may only consider the
variables x], x 2 and their quadratic values, x~ and x~, as
terms of the model, since they have values above 10%.
This means that they exert the greatest influence on
streptomycin production. Then the model can be
summarized to the following expression:
~= 1.137 + 0.145x I + 0.083x 2 - 0.116x~
-0.154x 2
The sum of the four variables adds up to 85.83% of
the sum of squares of the proposed model; that is, the
determination coefficient is 0.8583.
24 S. Saval, L. Pablos, S. Sanchez

glS-

O5 ~

g
', 20 T M e o.,
GLUco$£ ( ; 7- t 4,

• , , , ,
I 2 3 4 5 f 2 3 4 5

.ol FERMENTATION DAYS

.~ f.5 J Fig. 7. Comparison of the streptomycin production and

growth of S. griseus between original (o) and optimized (e)
media.
O~ I.O-

W
:z~ 0.5-
O- and pyruvate in the optimized medium (Garner et al.,
1953).
ib I~ i0 2~ 3'0
GLUCOSE (91 "l)
ACKNOWLEDGEMENTS
Fig. 6. Response-surface and contour graphic of strepto-
mycin production as a function of glucose (xt) and dibasic We wish to thank Isabel Perez Monfort for translation
potassium phosphate (x4) concentrations. Other variables, into English.
beer-yeast autolyzate (x2) and sodium chloride (x3), at zero
level.

TabLe 4. Concentration of nutrients in the optimized
medium
Nutrient (g liter-I) Original
medium
Glucose 10
Beer-yeast autolyzate 25
NaCI 10
K2HPO 4 1 1
Comparison between original and optimized media
The streptomycin titre in the optimized medium was
compared experimentally with that in the original
medium. The increment in the optimized medium was
5 2%. In both cases, maximum production was reached
after 3 days (Fig. 7).
Mycelial dry weight showed an increase of approxi-
mately 10%, which gave an increase in specific produc-
tion of 32%. Specific growth rate calculated in the
exponential phase (/~max) was 0-065 h- t in the original
medium and 0.096 h- ~in the optimized medium.
The pH profile was substantially modified due to the
increase in glucose concentration, and values were
consistently higher in the original than in the optimized
medium. This was due to the accumulation of lactate
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