Journal of Biotechnology,

18 (1991)
0 1991 Elsevier Science Publishers
ADONIS
0168165691000898

BIOTEC

255-264
B.V. 0168-1656/91/$03.50

255

00607

Production of acetone and butanol from starch

by continuous bioprocess
A.S. Afschar
GBF-Gesellschaft

fir

Biotechnologische

(Received

and K. Schaller
Forschung

26 July 1990; accepted

mbH,

Braunschweig,

21 October

1990)

F.R. G,

Conversions of saccharified and non-saccharified starch to acetone, butanol and

ethanol (ABE) with a continuous culture of Clostridium acetobutylicum were investigated and compared with regard to products and productivity. It was found,
that compared with the microbial process using saccharified starch, the process
using non-saccharified starch produced ABE in considerably lower concentrations
at dilution rates of > 0.15 h-. Two-stage continuous bioprocess using non-saccharified starch resulted in no appreciable increase in acetone and butanol concentrations. For the reasons given above, preliminary saccharification processing is
recommended for the production of ABE from starch using a continuous bioprocess. A two-stage process was developed for continuous production of ABE from
saccharified starch. By this process, 15 g 1-l ABE are produced at the rate of 3.3 g
1-l h-1
Conversion of saccharified starch; Conversion of non-saccharified starch; Acetone,
butanol, ethanol production; Continuous multistage bioprocess; Cell recycling;
Shear activation

Introduction
The microbial conversion of agricultural and industrial wastes and residues into
chemically useful substances is attracting increasing interest. The reasons for this
Correspondence
Weg 1, D-W

to: AS. Afschar,

3300 Braunschweig,

GBF-Gesellschaft
F.R.G.

!iir Biotechnologische

Forschung

mbH,

Mascheroder

256

are partly economic, but ecological considerations are also important. Apart from
surplus agricultural products, suitable source materials are now readily available in
the form of wastes from the starch producing and processing industry and potato
processing factories. In early microbial batch processes for the production of ABE
(solvents) on an industrial scale, cereals (mainly maize) were used as a source of
carbohydrate and potato juice as a source of minerals and nitrogen (Beesch, 1953).
For every 100 kg starch, this process yielded 22 kg butanol, 10.5 kg acetone, 5.3 kg
ethanol, 1.7 kg hydrogen and 62.4 kg carbon dioxide. The dry mass contained 33 to
40% protein and 40 to 70 pg riboflavin (Beesch, 1953; Spivey, 1978). Solvent
productivity amounted to around 0.3 g 1-i h-l, for a cultivation period of 50 to 60
h.
Very little data is available on continuous microbial conversion of starch to ABE.
Dyr et al. (1958) reported on a continuous cascade to produce ABE from a mixture
of flour, molasses and saccharified wood. In this continuous culture, solvents were
produced for 90 h only. Cultivation had to be interrupted due to infection and acid
formation. In 1964, Yarovenko reported the results of continuous cascade bioprocess on a semi-industrial scale in the USSR. This process ran without problems for 8
d in 11 bioreactors connected in series, each with a volume of 3..- m3, and produced
20.9 g 1-l ABE from a mixture of maize, wheat and rye, with solvent productivity of
0.69 g 1-l h-l.
The purpose of this investigation is to examine the continuous culture techniques
previously developed for glucose only, to determine their suitability for conversion
of starch and to propose a continuous bioprocess to produce ABE from starch.

Materials and Methods

Microorganisms
Clostridium acetobutylicum DSM 792 (ATCC 824) was used for the investigations.
This strain is known to be one of the Clostridia strains which converts glucose and
starch to ABE. By repeated pre-culturing, the culture was first adapted either to
starch (NSS) or to saccharified starch (SS).
Medium
The cultivation medium contained the following, per litre of deionised water:
KH,PO, 2 g, (NH&SO,
4 g, MgSO, +H,O 0.02 g, CaCl, .2H,O 0.02 g, FeSO, .
6H,O 0.038 mg, CuCl, .6H,O 0.048 mg, yeast extract 0.5 g, starch or saccharified
starch 50 g for batch cultivations and 40.5 g for continuous cultures. Industrial
amylases BNA and AMG (Novo Industri AS, Denmark) were used to saccharify the
starch. For pre-cultures, CaCO, was used as a buffer.

251

Method
A 2 1 and a 4 1 bioreactor (Setric Genie, Toulouse, France) were used for the
investigations. The bioprocess was initially run as batch system after 5 to 10%
inoculation. At the end of the acid phase, at a butanol concentration of around 4 g
l-, the batch cultivation was changed to continuous operation. For cell recycling or
shear activation of the culture (Afschar et al., 1986), the medium containing the cells
was pumped at a speed of 0.4 cm s- through small capillaries of a tubular
cross-flow microfiltration
module (1.8 mm diameter, Enka, Wuppertal). The circulated medium was fed onto the existing foam in the bioreactor so that, as well as
being freed from gas itself, it also broke down the foam. To prevent the formation
of a layer of microorganisms at the surface of the filter, the filtration module was
backwashed hourly. Daily microscopic examination of the culture showed that the
cells remained intact in spite of rapid circulation and constant friction against the
surface of the capillary, showing no signs of lysis. At each dilution rate, to make
sure that the steady state had actually been attained, the operational conditions
were kept constant for an additional 7 to 8 mean residence times. All cultivations
were carried out at 37 C and pH 4.5.
To saccharify the starch, it was first dissolved in BAN (Novo Industri AS,
Denmark) at 80C and pH 7.0 and then saccharified with AMG (Novo Industri
AS, Denmark) at 70 o C and pH 4.2.
Analytical methods
The cell mass concentration was determined by measuring the optical density at
578 nm as well as by the gravimetric method after centrifugal separation at 15,000
rpm and having been dried at 80C for 24 h.
The glucose, ammonia and phosphate concentration content of the cell-free spent
medium was measured on-line by an autoanalyser. Residual glucose was measured
with p-hydroxybenzoic acid hydrazide (Schmidt et al., 1985), the residual ammonia
concentration using the Berthelot reaction with phenol, Na-nitroprusside, Na-hypochlorite and NaOH solution and residual phosphate concentration by the phosphorus molybdate method. The starch content of the cell-free spent medium was
measured on-line with an autoanalyser (iodine starch method) and also enzymatitally as a control (Kipp und Zonen). Butanol, acetone, ethanol, acetic acid, butyric
acid, 3-hydroxy-2-butanone
(acetoin) were determined using a gas chromatograph
(Shimadzu, GC 9A), fitted with a flame ionisation detector (FID) and a 2 m long
glass column, filled with Chromosorb 101.

Results
Batch culture
To investigate the substrate consumption capability of Clostridium acetobutylicum, strain DSM 792 in the conversion of non-saccharified starch (NSS) compared

52;
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a-

.- /67 -y;.;
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_ _ . ._
.,....,...I
10
20
30

10

20

.--

o----s_
.-.-.-._. . ...-. .._._.
. . .. . ... .y,F,3n
I I IT1 _ _..r-n-L&
.40
50

30

40
Time

-.<
,
60

50

(
70

60

70

(h)

Fig. 1. The results of a microbial batch process using non-sacchaxified starch.

with saccharified starch (SS), a number of batch processes were carried out under
the same conditions. Figs. 1 and 2 show the results of these batch cultures.
No clear difference can be found between the kinetics of the microbial batch
process using NSS in comparison with SS. The acid production phase ends in both

co
......*. .. ... .. .. . . .. . ... .. ... . .. .. .. .. .. ... . .. .. .... . .... .. . ... . .. .._..........................
. ... .. . _.
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./*
-.#~-----
O-IrlF~~rI . .
0

.4-$cetatc

.-.-...

.. n~-~-n~.~._.~.~:,,
...
10
20

10

20

_._._.-a--.. . ... :y,u.Y-I~.:.J..~~~~~~-.

-i-*-*
T
, ,

P
30

30

.,

40

50

, ,
60

40

50

60

Time(h)

Fig. 2. The results of a microbial batch process using saccharified starch.

,
70

70

259

batch processes after about 10 to 15 h. Slightly more acid is produced by the

conversion of NSS. Reconversion occurs in batch processes with SS, the main result
of which is an increase in the butanol concentration.
Continuous culture
Clostridium culture growing on NSS in a product inhibited continuous culture
has no stable steady state at dilution rates below 0.08 h-. Depending on the
composition of the medium and the condition of the culture, acid production
increases with time at the expense of solvent concentration. In most cases, it is
mainly acids which are produced after a few days. We assume that this degeneration
phenomenon is caused by the inhibitory effect of a high concentration of butanol.
The continuous conversion of SS to ABE, at dilution rates of < 0.08 h-, behave
similarly to the conversion of NSS, but has a less pronounced tendency to degeneration. Figs. 3 and 4 compare the relationship between product concentration and
dilution rate in the continuous culture using SS and NSS under similar conditions.
In the continuous microbial conversion of NSS, the ABE concentration falls
considerably more steeply with increasing dilution rates than by conversion of SS
(Fig. 3). The wash-out point of a continuous culture using NSS is reached at a much
lower dilution rate than that of a continuous culture using SS (Fig. 3). A continuous
8

E
L

Butanol

.-+6
E!
+rt
0,
ii
s
-1

Acetone

%
*\\\\\
2 \\

1
Ethanol

Dilution
Fig.

3. The

concentration
( -)

rate

(h-1

of ABE as function
of the dilution
rate in a continuous
compared
with a continuous
culture using SS (-----).

culture

using

NSS

260

I
0.2

0.1
Dilution
Fig. 4. The
continuous

concentration
culture
using

of acids, acetoin and consumed

compared
with
NSS ( -)
starch) (-----).

rate

(h-l

I
0.3

I
0.4

starch as functions
of the dilution
rate in a
a continuous
culture
using SS (saccharified

bioprocess with NSS produces around 2 g 1-l solvent and 2.5 g 1-l acid at a
dilution rate of 0.2 h-r. At these dilution rates the spent medium contains NSS,
KH,PO,, (NH4)2S04 and all other trace elements in surplus.
Above this dilution rate, it is mainly acid which is produced and the culture starts
to become washed out. The butyric acid concentration reaches a minimum at
around 0.1 h-t with increasing dilution rates (Fig. 4). The striking rise in butyric
acid concentration and the considerably reduced solvent production of the culture
growing on NSS above a dilution rate of approximately 0.2 h- (Fig. 3) clearly
indicates substrate limitation above this dilution rate. The reasons for this are
probably the low specific amylase production rate and/or the fact that starch is
hydrolysed too slowly under these microbial conditions (pH 4.5 at 37 o C).
Two-stage continuous culture
Continuous bioprocess using NSS in a two-stage system, consisting of two
bioreactors of equal volume connected in series gave almost twice the acid concentration (increased from 1.9 to 4.8 g 1-l) without any marked increase in the
solvent concentration (from 8.5 to 9.3 g 1-t) in the second stage.

261

Shear activation
In previous experiments (Afschar et al., 1986) we had found that glucose-consuming continuous cultures of Clostridium acetobutylicum DSM 792 are shear activated
when pumped through capillaries. This increases the specific growth rate and
specific product formation rate as compared with normal continuous cultures. In
order to determine the effect of shear activation by conversion of SS and NSS, the
process was used in the continuous cultures under investigation. In the continuous
culture using SS, product formation and specific growth rate increased under shear
activation as in the glucose-consuming cultures. For the continuous culture converting NSS, no changes in growth and product concentration were achieved by shear
activation. The following reasons may explain this difference in behaviour: (a)
because of the higher viscosity of the soluble starch medium than the soluble
saccharified starch medium, the turbulence required for shear activation was not
reached and degassing of the culture was incomplete; (b) at higher dilution rates an
increase in activity and productivity of the culture was no longer possible due to
substrate limitation.

Acetnn~

Dilution
Fig. 5. ABE concentration

as a function
( -)

rate

( h-l

of dilution rate in shear-activated

and using SS (- - - - -).

continuous

cultures

using

NSS

262

Fig. 5 shows the concentrations of products as a function of the dilution rate in a

shear-activated continuous culture using SS and a shear-activated continuous culture using NSS.
The SS-consuming continuous cultures which were shear activated at dilution
rates of less than approx. 0.2 hh have no stable steady state. The level of solvent
concentration probably also plays a decisive role here.
The SS-consuming continuous cultures are capable of producing the same ABE
concentrations as continuous cultures using NSS at considerably higher rates of
dilution (Fig. 5).
Proposed

bioprocess

Whether a bioprocess with conversion of NSS to ABE in a multi-stage process

with long retention times (Yarovenko, 1964) or whether a bioprocess with much
shorter retention times after saccharification is carried out, is an economic problem.
In view of the considerably higher product yield from saccharified starch, the
microbial production of ABE after saccharification of the starch is probably more
economic. For this reason and taking into account data available from earlier
studies (Afschar et al., 1985, 1986) a microbial process was developed for the
conversion of starch to ABE at a higher productivity and with high solvent
concentrations. To increase the space-time yield, tubular cross-flow microfiltration
modules were used for cell recycling. Instead of recycling, cells can also be retained
by immobilization processes. Such processes cannot, in fact, achieve very high cell
concentrations, but the investment costs involved are very low.
In the process described here, starch is first of all saccharified after appropriate
dilution. The saccharified sugar can then be sterilised for a short time at 100C.
Since the pH of the inflow medium is adjusted to 4.2, pH control is unnecessary.
The source medium is then deoxygenated by sparging with exhaust gases of the
bioreactor.
In view of the degeneration phenomena seen for Clostridium
acetobutylicum
cultures under strong inhibitory stress, continuous cultivation is carried out in two
stages. A ratio of 1 to 2 was selected for the volumes of the two bioreactors. The
volume of the first bioreactor could also be considerably smaller, since the task of
the first stage is merely to provide a constant supply of active and productive cells
for the second stage. The investigations described here involved shear activation of

TABLE

The results
Dilution
rate
h-
0.22

of continuous

conversion

Stage

volume
ratio

1
2

a PO
0.39
0.61

of saccharified

starch

by a two-stage

cascade

(dry) cell
mass
gl-

Solvenl
gl-

Acids
gl-

Solvent
productivity
g I- h-

2.80
18.00

5.20
15

3.0
5.8

1.28
3.30

263

the culture in both stages and cell recycling in the second stage. Table 1 shows the
results of conversion of saccharified starch to ABE with a two-stage system of this
kind.

References
Afschar.
A.S., Schaller,
K. and Schbgerl,
K. (1986) Continuous
production
of acetone and butanol
with
shear-activated
Closrridium
occrobury/icum.
Appl. Microbial.
Biotechnol.
23, 315-321.
Afschar,
AS., Biebl, H., Schaller,
K. and Schigerl,
K. (1985) Production
of acetone and butanol
by
CIosfridium
acefobufylicum
in continuous
culture with cell recycle. Appl. Microbial.
Biotechnol.
22,
394-398.
Beesch, SC. (1953) Acetone-butanol
fermentation
of starches. Appl. Microbial.
1, 85-96.
Dyr, J., Protiva,
J. and Prauss, R. (1958) Formation
of neutral solvent
in continuous
fermentation
by
means of Closrridium
acerobufyhcum,
In: I. Malek (Ed.), Continuous
Cultivation
of Microorganisms.
A symposium.
Czechoslovakian
Academy
of Sciences, Prague, pp. 210-226.
Schmidt,
W., Kuhlmann,
W. and Schiigerl,
K. (1985) Automated
determination
of glucose in fermentation broths
with P-hydroxybenzoic-acid
hydrazide
(P.H-BAH).
Appl.
Microbial.
Biotechnol.
21,
18-84.
Spivey. M.J. (1978) The acetone/butanol/ethanol
fermentation.
Process Biochem.
13, 2-5.
Yarovenko.
V.L. (1964) Principles
of the continuous
alcohol and butanol-acetone
fermentation
processes,
In: I. Malek (Ed.), Continuous
Cultivation
of Microorganisms,
2nd Symposium,
Czechoslovakian
Academy
of Sciences, Prague, pp. 205-217.