Biotechnology Letters Vol 13 No i0 733-738 (1991)

Received 27th August

DIRECT FERMENTATION OF STARCH TO LACTIC ACID BY

LACTOBACILLUS AMYLOVORUS

D.X.Zhang and M. Cheryan*

Department of Food Science

University of Illinois at Urbana-Champaign
Agricultural Bioprocess Laboratory
1302 W. Pennsylvania Avenue
Urbana, Illinois 61801, U.S.A.

SUMMARY

Fermentation production of lactic acid directly from starch was

studied in a batch fermentor using Lactobacillus amylovorus. At an
initial concentration of 120 g/L starch, 96.2 g/L of lactic acid was
produced from liquefied starch in 20 hours while 92.5 g/L of lactate
was produced from the raw starch in 39 hours. High initial glucose
levels (100 g/L) in the medium inhibited the organism, unless it had
been adapted by growing it in a low-glucose medium. The direct prod-
uction of lactic acid from starch could reduce overall production
costs significantly.

INTRODUCTION

Refined sucrose, although expensive, is the most commonly used

substrate for producing lactic acid by fermentation (Vick Roy 1985).
Production cost could be reduced if corn starch was used instead, esp-
ecially if the microorganism could produce lactic acid directly from
starch, since it would result in a large savings in liquefaction and/-
or saccharification cost:

Lactobacillus amylovorus is one such organism. Nakamura and

Crowell (1979) and Nakamura (1981) isolated L.amylophilus and L.
amylovorus which were found to have amylolytic activities and there-
fore able to utilize starch directly to form lactic acid. Cheng et al.
(1991) reported similar results using L. amylovorus.

This paper reports on the kinetics of the fermentation of lactic

acid from raw and liquefied starch using L.amylovorus. This has great
potential in the industrial manufacture of lactate, since it would
obviate the need for separate starch liquefaction and/or saccharific-
ation, provided the organism produced lactate in sufficiently high

733
concentrations, and with a low cycle time.

MATERIALS AND METHODS

A 2-liter glass fermenter equipped with temperature control, pH

controller and agitation was used in these experiments. Fermentation
conditions were 40~ and pH 5.5. Sodium hydroxide (10 M) was used to
neutralize the fermentation broth in order to ensure the lactate was
maintained in a soluble form. At this pH, about 95% of the lactate
is in the dissociated form.

The organism, Lactobacillus amylovorus NRRL B4542, was obtained

from USDA Northern Regional Research Center, Peoria, Illinois. It is a
facultative anaerobe, gram-positive, non-motile, non-sporeforming and
rod shaped organism. It possesses an extracellular amylolytic activity
and is apparently able to produce both L(+) and D(-) lactic acids from
starch (Nakamura 1981):

The medium was adapted from Cheng et al (1991). It contained liq-

uefied or raw starch to the required concentration, FeSO 4 (0,03 g/L),
sodium acetate (1 g/L), MgSO4.7H20 (1.23 g/L), MnSO4.H20 (0.034 g/L),
K2HPO4.3H20 (0.65 g/L), KH2PO 4 (0.5 g/L) and yeast extract (30 g/L).

The liquefied starch was obtained by amylolytic degradation of

raw starch. Corn starch powder (A.E. Staley Co., Decatur, 1L) was
suspended in distilled water at a concentration of 30% w/w. Prior to
heating, the pH of the resulting suspension was adjusted with 10% NaOH
to 6.85. Calcium ions were added to the starch suspension using 1M
CaC12, to assure a Ca 2+ concentration of at least 50 ppm for enhanced
enzyme stability. T h e starch suspension was then heated to 9 0 - 95~
and the alpha-amylase (Termamyl, Novo Biochemical Industries,
Franklinton, NC) added at a rate of 0.055% w/w based on the weight of
starch. After liquefying the starch for 2 hours, the enzyme was
inactivated by adding 10% HC1 to adjust the pH to 3.5 and heating for
10 minutes at 95~ The liquefied starch was then freeze-dried and
used as needed.

Lactic acid and residual sugar concentration were measured by

HPLC. An HPX-87H column (BioRad, Tustin, CA) and a refractive index
detector were used with 0.01 N H2SO 4 as the eluant at a flow rate of
0.8ml/min. at 65~

RESULTS AND DISCUSSION

Figure 1 shows the production of lactic acid during batch ferm-

entation of liquefied starch. Fermentation was complete after 10-15

734
hours, depending on the initial substrate concentration in the medium.
The final lactic acid concentration was higher with higher initial
substrate concentration. The highest concentration of lactic acid
(96.2 g/L) was obtained at the initial substrate concentration of 120
g/L.

A similar trend was observed with raw corn starch (Fig. 2).
However, fermentation times were almost double that with liquefied
starch. This is probably due to a reduced rate of starch hydrolysis by
the microorganism. Raw starch usually has a more compact structure and
smaller surface area than the liquefied starch, which has been
"thinned" by the action of alpha-amylase. This probably restricted
access of the amylolytic enzymes of L.amylovorus to the raw starch.
Final lactic acid concentrations with raw starch were about 10-20%
lower and substrate utilization was also lower than with liquefied
starch as the substrate (Fig. 3).

The organism appears to be inhibited by high glucose concentrat-

ions. Initial attempts to grow the culture on glucose resulted in
little o r nor growth, compared to starch medium. The results of a
typical experiment using glucose as the carbohydrate source is shown in
Fig.4. Little or no lactic acid was produced by L.amylovorus up to 77
hours o f batch fermentation. (The lactate shown in Fig.4. is due to
the lactate in the inoculum). The culture used in this particular
experiment had previously been grown on a starch substrate.

However, a fed-batch technique can overcome the glucose

inhibition, as shown in Fig. 5. The initial glucose was kept at a low
level (5 g/L) and increased step by step by adding glucose into the
fermentation broth as soon as the glucose in the medium was exhausted.
Lactic acid was produced, but at a very slow rate compared to starch.
After about 42 hours of this fermentation, the organism seems to have
adapted itself well to glucose, since even a large addition of glucose
at 42 hours did not appear to seriously affect its growth or lactate
production. Cell concentration increased from about 0.6 g/L to 7.6 g/L
and then declined to 6.5 g/L at the end of the fermentation.

CONCLUSIONS

The production of lactic acid b y fermentation can be greatly

enhanced by utilizing a lactate-producing organism with amylolytic
activity. The most attractive features of L. amylovorus are its
ability to utilize both raw and liquefied starch, the high concentrat-
ions of lactic acid produced in the fermentation broth (which will
keep downstream processing costs l o w ) a n d the relative rapidity of the
fermentation ( e q u i v a l e n t . to a productivity of 5-6 g lactate per liter

735
 120
Liq. S t a r c h , Batch Reactor
100

i 8O
__] 9

G>
6O - []

r~ Starch
D
0 4O (gL-?)
D [] 55
__]
9 85
zo v 100
o 120 1
0
0 5 10 15 20 25 50

Time (h)
F i g . l , F e r m e n t a t i o n of l i q u e f i e d s t a r c h b y L. a m y l o v o r u s in a
0
b a t c h r e a c t o r a t 40 C, pH 5.5

120
Raw Starch, Batch Reactor

100
0
"
I 80
C~

@ 60 Starch
C]
.. (gk- 1)
4~
0 40 [] 50
D
9 85
v
20 v 100
o 120
0 I I I I

0 10 20 30 40 50 60

Time (h)
Fig.3. F e r m e n t a t i o n of r a w s t a r c h by L. a m y l o v o r u s in a b a t c ~
O
r e a c t o r a t 40 C, pH 5.5

736
 120
J l I J 100
II " 9
110
0 []
100 o~
I E
_J 90
On 60.2
c?
9 80 N
9
70
4o
9
C3 9
_2 6O

5O ~ Lactate OD Raw starch

9 9 Liquefied storch
2o o
co
_(D
4O r 1 r I 0 C/?
40 60 80 1 O0 120 40
-1
Initial Starch Concentration (gL )
F ig .3 . Maximum lactate concentration and substrate utilization
b y L.c~m~Zovo~azs i n b a t c h c u l t u r e

120

100 )- o |
I
_J o Glucose

80 -
c-
O
. - -

60 -

c 40 -
@
o
c Lactate
0
L_) 20f [] []
[] []

o t I I I I I I
o 10 20 30 40 50 60 70 80

Time (h)

Fig.4. F e r m e n t a t i o n of glucose (100 gL -I) b y L.c~ylovorus in a

b a t c h reactor. E x p e r i m e n t a l conditions s a m e as Fig.l.

737
 80 , -- , 50
Fed-ba[ch, Glucose, 40~ pH 5 . 5
,I'-4

I Lactate ,r
,-..]
6O I

2O b.b
9 \ [] >.,
..+.n 4o 9 f--'i

qp
. . . . Cells - 10 ~:::~
o 2o
(p
0
LD
r.D

0 0
0 20 40 60 80 100

Time (h)
Fig.5. F e d - b a t c h f e r m e n t a [ i o n of g l u c o s e by L. ctrrt?g~o~oTtts.
E x p e r i m e n t a l c o n d i [ i o n s s a m e as Fig. 1.

per hour with liquefied starch). In addition, a substantial reduction

in upstream processing costs can be realized, by eliminating one (or
perhaps both) the starch conversion steps (i.e., the liquefaction or
the saecharification).

It should be pointed out that the experiments described h e r e used

conventional batch fermentors with relatively low concentrations of
cells. We expect to be able to improve productivity by using high-rate
continuous bioreactors with higher cell concentrations, as we have
demonstrated with lactic acid from whey (Mehaia and Cheryan 1986, 1987).

REFERENCES

Cheng, P.,Mueller, R.,Jaeger, S.,Bajpai R. and Iannotti, G. 1991.

J. Ind. Microbiol. 7: 27-34.
Mehaia,M.A. and Cheryan,M. 1986. Enzyme Microbial Technol. 8:289-292
Mehaia,M.A. and Cheryan,M. 1987. ProcessBiochem. 22: 1 8 5 - 1 8 8 .
Nakamura, L.K. and Crowell, C.D. 1979. Dev.Ind.Microbiology 20:531
Naknmura, L.K. 1981. Intern.J. of System. Bacteriology 31:56
Vick Roy, T.B. 1985. In Moo-Young,M.(ed.), Comprehensive Biotechnology,
Vol. 3, pW69

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