Effect of Yeast Extract on a-Amylase
Synthesis by Bacillus am yloliquefaciens
Shahriar Alam, Juan Hong,* and William A. Weigand
Department of Chemical Engineering, Illinois Institute of Technology,
Chicago, Illinois 60616
Accepted for publication March 7, 1988
INTRODUCTION
Rapidly metabolizable complex medium containing high
levels of yeast extract (i.e., 5 g/L) are widely used for
studying growth and product formation characteristics of
microorganisms in the laboratory. Yeast extract provides a
cheap source of various amino acids, vitamins, minerals,
and other growth factors to sustain a good growth of the
microorganisms. Nutritional requirements for a-amylase
synthesis by Bacillus amyloliquefaciens have been studied
by many investigator^.^-^ The a-amylase synthesis by B .
amyloliquefaciens has been correlated to the presence or
absence of various amino acid^,'.^,^ complex nitrogenous
sources,8-10and most importantly, the rate of growth of the
microorganism on various carbon Starch sup-
ported the highest doubling time for B . amyloliquefaciens
followed by glycerol and glucose re~pectively.~ The rate of
a-amylase synthesis was found to relate inversely with the
growth rate in these batch experiments. In continuous cul-
ture, a-amylase synthesis followed essentially the same
pattern." The lower a-amylase synthesis rates in the pres-
ence of high glucose concentrations were accepted as evi-
dence of catabolite repression for B . amyloliquefaciens by
Ingle and Boyer.6 In Bacillus fermentation the majority of
the carbon is directed to the formation of carbon dioxide,
butanediol, glycerol, ethanol, acetate, and lactate depend-
ing on the species. 12213 Bacillus arnyloliquefaciens mostly
produces carbon dioxide, butanediol, acetate, and a small
amount of lactate.', The medium composition, mostly the
carbon and nitrogen sources, is known to influence the
metabolite formation in B . amyloliquefaciens, l 5 which in
turn can have a modulating effect on a-amylase synthesis
by changing the pH of the system. Most of the previous
studies on a-amylase fermentation ignored the role of
these metabolites on the synthesis of this enzyme and have
scarcely reported the concentration levels of the various
metabolites present.
In a recent study,' the concentration of yeast extract was
found to be an important factor in the a-amylase synthesis
* To whom all correspondence should be addressed. New address: Bio-
chemical Engineering Program, School of Engineering, University of
California, Irvine, CA 92717.
Biotechnology and Bioengineering, Vol. 33, Pp. 780-785 (1989)
0 1989 John Wiley & Sons, Inc.
by B . amyloliquefaciens. The purpose of this study was to
investigate the effect of yeast extract on a-amylase synthe-
sis by B . amyloliquefaciens F under controlled and uncon-
trolled pH conditions. In addition the by-product formation
during fermentation and its influence on a-amylase is also
reported.
MATERIALS AND METHODS
Microorganism and Culture Medium
The a-amylase producing strain, Bacillus amyloliquefa-
ciens F (ATCC 23350) was used in the study. The stock
culture was maintained in litmus milk added medium at
- 80°C.
The composition of the medium for innoculum prepara-
tion was 10 g glucose, 5 g yeast extract (Difco) and 2 g
NaCl in 1 L water. The modified' a-amylase fermentation
medium consisted of 9 g K,HPO,, 3 g KH,PO,, 5 g
(NH,),SO,, 1 g Na3C6H50,. 2H,O, 0.2 g MgSO, . 7H,O,
0.14 g FeC1, . 6H,O, 0.05 g CaCl, . 2H,O, 0.01 g
-
MnSO, 4H,O, 0.01 g FeSO, 7H,O, 0.001 g ZnSO, .
7H,O, and desired amounts of glucose and yeast extract in
1 L water.
Fermentations
Fermentations were carried out in a l-L Bellco fermen-
tor with a working volume of 750 mL at 37°C. The agita-
tion and air flow were maintained constant at 500 rpm and
1.8 vvm. The pH was controlled by the addition of acid
(5N HCl) or alkali (5N NaOH) as required. One con-
trolled pH experiment was carried out in a 20-L New
Brunswick microprocessor-controlled fermentor with a
working volume of 8 L. Agitation and aeration were kept
the same as in the preceding.
Amylase Assay
Spectrophotometric liquefying a-amylase activity assay
(at pH 6.8) of Miles Laboratories was used in this investi-
gation. One liquefying a-amylase unit (LAU) is that activ-
CCC 0006-3592189/060780-06$04.00
ity that will hydrolyze 1 p g soluble starch/min under the
conditions of the assay.
Protease Assay
Detergent alkaline Protease activity assay (at PH 8.5) of
Miles Laboratories was also used. One detergent alkaline
protease unit (DAPU) is that activity that will liberate
four nmol tyrosine/min under the conditions of the assay.
Amylase Deactivation Study
The a-amylase deactivation studies were carried out by
measuring the activity of the enzyme solutions kept at
40°C over an interval of several hours. The systems stud-
ied were as follows: system A, centrifuged broth from
1 g/L yeast extract fermentation with a-amylase present;
system B, centrifuged broth from 5 g/L yeast extract sys-
tem and "system A" in equal volumes; system C, cen-
trifuged broth from 5 g/L yeast extract fermentation added
to this was a-amylase from Sigma Chemical Co; sys-
tem D, fresh medium containing a-amylase from Sigma.
Levels of enzyme used were 10,000 LAU/mL in all experi-
ments except system B. The level of enzyme in system B
was -5000 LAU/mL as broth from 5 g/L yeast extract
fermentation contained a negligible amount of the enzyme.
Other Methods
The glucose concentration was measured by a YSI
model 27A automatic glucose analyzer. The acid and sol-
vent analysis were carried out by a Waters HPLC system
in an Aminex HPX-87H organic acid analysis column
(Bio-Rad). Cell concentration was determined from the
turbidity measurement at 620 nm.
RESULTS AND DISCUSSION
Batch experiments were carried out with several initial
glucose concentrations of between 0.20 to 20.0 g/L and
yeast extract concentration of 1 g/L. Figure 1 represents a
typical batch fermentation for the initial glucose concentra-
tion of 19 g/L, characterizing an enzyme synthesis in the
exponential phase that continued at the same rate even in
the death phase before reaching the plateau. The enzyme
productivity with various initial concentrations of glucose
is given in Figure 2. The enzyme productivity is defined as
the amount of enzyme produced per unit time per unit cell
mass (LAU/hr g). Here the cell mass represents the maxi-
mum cell mass produced during the exponential phase of
growth. The productivity was found to decrease with in-
creasing initial glucose concentration.
To investigate the effect of yeast extract, we varied its
concentration in the fermentation medium between 1 and

TIME IN HOURS
Figure 1. Effect of yeast extract on a-amylase fermentation by B . urnyloliquefu-
ciens. Open and filled symbols represents fermentation in 1 and 5 g/L yeast extract,
respectively (see text for medium composition). Symbols: a-amylase fV,V); biomass
(A& glucose (El,=); pH (0,O).

COMMUNICATIONS TO THE EDITOR 781

 0
2
'X
200
. V
175 -
150 -
v\
125 - \
I00 -
\v
75 -
GLUCOSE CONCENTRATION, g/l
Figure 2. Change in overall productivity with increasing glucose concentration
in batch fermentation with 1 g/L yeast extract.
5 g/L keeping the glucose concentration constant around
19 g/L. Figure 1 represents a typical batch fermentation
data with 1 and 5 g/L concentration of yeast extract in the
medium. In presence of 5 g/L yeast extract the a-amylase
activity in the medium was found to be extremely low
(hereafter to be referred to as the nonproducing system).
Analysis of batch data with 1-5 g/L yeast extract concen-
tration in the fermentation medium shows that the overall
productivity of fermentation initially increased slightly be-
tween 1 and 2 g/L yeast extract concentration and then fell
very rapidly beyond this point. The results are shown in
Figure 3. To further investigate if the cells had lost their
ability to produce a-amylase in the medium containing
yeast extract at 5 g/L, a 10-mL sample was withdrawn
from the fermentor after 18 h (shown by the arrow in
Fig. 1) and was incubated in a medium containing 1 g/L
yeast extract without any glucose. In 12 h, the cell concen-
tration increased by 164%, with enzyme production level
reaching 1647 LAU/mL. This indicates that the cells
grown in the medium of yeast extract at 5 g/L were not
physiologically different from the cells grown in the
medium containing yeast extract of 1 g/L.
Bacillus amyloliquefaciens F is known to produce
mostly alkaline protease in addition to a-amylase and
small amounts of neutral p r ~ t e a s e . The
~ . ~ activity ratio of
alkaline protease to neutral protease produced by the
present organism was reported to be over 111.6 It is well
established that free amino acids repress protease biosyn-
782 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 33, FEBRUARY 1989
. W
i
thesis but may stimulate a-amylase synthesis .' Commer-
cially complex nitrogenous sources are used to sustain
synthesis of both a-amylase and pr~tease.~." A complica-
tion concerning the effect of complex nitrogenous sources
is the likelihood of a-amylase destruction by inducible
pro tease^.^ The protease synthesis characteristics were
thus studied in a medium containing 1 g/L yeast extract
at a constant pH of 7.0 (in the New Brunswick fermentor).
The protease production was found to be concomitant with
the a-amylase synthesis but reached a plateau in 10 h
compared to 28 for a-amylase. About 2000 DAPU/mL
alkaline protease and 6500 LAU/ml a-amylase were syn-
thesized from 10 g/L glucose. The a-amylase produced
during the fermentation may be degraded by this protease.
The possibilities of the protease degradation of a-amylase
will be discussed later.
The effect of enzyme deactivation in the fermentation
systems were investigated next. The strain B. amylolique-
faciens is known to produce considerable amounts of 2,3-
butanediol, acetate, and lactate.I4 Under uncontrolled pH
experiments, increasing the yeast extract concentration re-
sults in increased acid production. The total acid produc-
tion in 5 g/L yeast extract fermentation was 4 times higher
in the uncontrolled pH system over the same fermentation
at a constant pH of 7. The levels of acid and solvent pro-
duced in various experiments are presented in Table I.
These metabolites are likely to deactivate the a-amylase
produced in the system. Hence, deactivation of a-amylase
 L '

iin

100:

90 :

80 :

70 :

60 :

50 :

40 :

30 :

20 7 v\ V

10 r
\

YEAST EXTRACT CONCENTRATION, g/I

Figure 3. Effect of yeast extract concentration on productivity of a-amylase
fermentation.

was studied in the cell-free fermentation broth (systems A ,
B, and C) as well as fresh medium (system D). Details of
these systems are given in the Materials and Methods
section. The results of the stability tests are presented in
Figure 4. It appears from Figure 4 that the extent of de-
activation in 25 h was between 20 and 30% in all four dif-
ferent systems studied, indicating that the deactivation of
a-amylase by the metabolites cannot account for the al-
most zero level of a-amylase in the medium with yeast ex-
tract at 5 g/L. It also indicates that the degradation of
a-amylase by protease produced in the medium containing
yeast extract at 5 g/L (systems B and C) was not signifi-
cant. Even though the rate of deactivation of a-amylase in
Table I. Production levels of acids and solvents."
Yeast extract Hours of Acetic acid
(g/U PH fermentation (g/L)
~
the cell-free broth from the 5 g/L yeast-extract-containing
system (system C) was higher compared to the deacti-
vation in the broth from 1 g/L yeast extract fermenta-
tion (system A), it is obvious that it cannot account for
lower levels of a-amylase synthesis in high-yeast-extract-
containing systems ( 5 g/L yeast extract).
The mode of glucose utilization in the a-amylase pro-
ducing and nonproducing systems was essentially the
same, except the presence of additional yeast extract in the
nonproducing system supported a somewhat higher growth
rate. The higher growth rate with increasing yeast extract
concentration was associated with a more rapid fall in the
pH of the system, as shown in Figure 1. The decrease in
pH was the result of high acid concentration in uncon-
trolled conditions. For this reason we further investigated
Butanediol the effect of keeping the pH constant throughout the fer-
(g/L) mentation (5 g/L yeast extract). The fermentation data is

given in Figure 5. Under a controlled pH condition the a -

1.o uncontrolled 48.0 0.75 6.61 amylase synthesis was found to revive but the total enzyme
2.0 uncontrolled 50.0 0.67 5.99
3.0 uncontrolled 50.0 0.82 6.86 synthesis was less than the total enzyme produced in the
4.0 uncontrolled 50.0 1.28 7.54 fermentation containing 1 g/L yeast extract. It should be
5.0 uncontrolled 48.0 2.99 6.30 noted that the a-amylase synthesis under the uncontrolled
1 .o 7.0 48.0 0.59 5.84 pH condition was completely repressed with yeast extract
5.0 7.0 48.0 0.70 6.16 at 5 g/L. On the other hand, for medium with yeast ex-
a Initial glucose concentrations of glucose in these experiments were tract at I g/L, a strict pH control did not result in a signifi-
between 19 and 20 g/L. Levels of lactate produced were very low (cO.01). cant increase in the enzyme synthesis.

COMMUNICATIONS TO THE EDITOR 783

4
m
P

m
0
m
0

B< ENZYME x LAU/mI: BIOMASS, g/l RELATIVE ACTIVITY %

GLUCOSE x g/l u? m u 03 cc
0 0 0 0 0

4
(D
03
(D
CONCLUSIONS
In previous studies yeast extract was found to be essen-
tial to obtain a good growth of B . amyloliquefaciens. Our
studies indicate that there is an optimum yeast extract con-
centration for a-amylase production. For uncontrolled pH
fermentations, increasing the yeast extract concentration to
a level of 5 g/L lowers the pH significantly. This results in
the complete repression of the enzyme synthesis. Under
controlled pH condition, the a-amylase synthesis with the
medium containing yeast extract at 5 g/L was comparable
to that with yeast extract at 1 g/L. This study suggests that
a strict pH control is required in complex media containing
high levels of yeast extract for studying the a-amylase syn-
thesis by B . amyloliquefaciens.
The authors would like to thank Mr. John Ponzo for valuable dis-
cussion on culture maintenance.
References
1. T. Maniatis, E. F. Fritsch, and J. Sambmok, Molecrrtar Cloning,Cold
Spring Harbor, N.Y., (Cold Spring Harbor Laboratory, 1982).
2. G. Coleman, J . Gen. Micrbioi., 49, 421 (1967).
3. N. E. Welker and L. L. Campbell, J . Bacteriol., 86, 681 (1963).
4. G. Coleman and M. A. Grant, Nature. 211, 306 (1966).
5. Q. Zhang, N. Tsukagohi, S . Miyashiro, and S . Udeka, Appl. Envi-
ron. Micrbiol., 46, 293 (1983).
6. M. B. Ingle and E. W. Boyer, in Microbiology, D. Schlessinger,
Ed.,Washington D.C. (American Society of Microbiology, 1975),
p. 420.
7. W. M. Fogarty and C. T. Kelly, in Microbial Enzymes and Biocon-
versions, A. H. Rose, Ed. (Academic, New York, 1980), p. 115.
8. B. K. May and W. H. Elliot, Biochem. Biophys. Acta, 157, 607
(1968).
9. Y. J. Yoo, Ph.D. Dissertation, “Kinetics and Optimal Control of
a-amylase Production from Bacillus arnyloliquefaciens” University
of Maryland, Maryland, 1986.
10. L. Nyiri, Inr. Chem. Eng., 11, 447 (1971).
1 1 . F. G. Heineken and R. J. O’Connor, J . Gen. Microbiol., 73, 35
(1972).
12. A. C. Blackwood, A. C. Neish, W. E. Brown, and G. A. Ledingham,
Can. J . Res., 25B, 56 (1947).
13. E. T. Papoutsakis and C. L. Meyer, Biotechnol. Bioeng., 27, 50
(1985).
14. S. Alam, Ph.D. Dissertation, “Fermentation and Separation in Aque-
ous Two-Phase System,” Illinois Institute of Technology, Chicago,
IL, i988.
15. R. J. Magee and N. Kosaric, Adv. Appl. Microbiol, 32, 89 (1987).
COMMUNICATIONS TO THE EDITOR 785