Journal of Biotechnology, 27 (1993) 317- 329

:~, 1993 Elsevicr Science Publishers B.V. All rights reserved 0168-1656/93/$06.0{~

317

BIOTEC 0{)816

Microbial production and downstream processing

of 2,3-butanediol
A.S. A f s c h a r ", C.E. Vaz Rosscli b, R. J o n a s ", A. Q u e s a d a C h a n t o ;'
and K. Schaller "
" GBF-Gesellschaft fiir Biotechnologische Forschung mbtt, Mascheroder Weg 1. W-3300 Braunschweig,
Germany, and b Centro de Technologia Copersucar, Caixa Postal 162, Sdo Paalo, S.P., Brazil
(Received 11 November 1991; revision accepted 26 April 1992)

Summary
In thc direct conversions of starch by Bacillus polymyxa a maximum of 38 g l2,3-butanediol is produced with a yield of 0.28 diol pcr starch (g g - l ) . By
preliminary saccharification of starch and then cultivation with Klebsiella o~'toca,
a 2,3-butanediol concentration of 99-100 g ! - t is achieved with a yicld of 0.5 diol
pcr starch (g g-1). Klebsiella oxytoca converts high-test molasses to 2,3-butanediol
in the same concentration and yield. The same diol concentration only at lower
productivity can also be achieved by conversions of black strap molasscs, providcd
it contains less than 2% salts.
2,3-Butanediol can be separated from bioprocess media with very good rcsults
by salting out using water-free potassium carbonate. After precleaning the medium,
from molasses or saccharified starch conversion process, it was possible to separate
94-96% (w w - I ) of the 2,3-butanediol present using 53-56% weight potassium
carbonatc. Thc concentration of the 2,3-butanediol in the diol phase resulting was
in this case 97% weight.
Salting out can be used to separate other diols also produced using microbiological mcthods.
2,3-Butanediol; Klebsiella oxytoca; Starch; Molasses; Downstream process; 1,3-Propanediol; Salting out; Biodegradable solvent

Correspondence to." A.S. Afschar. GBF-Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder
Weg 1, W-3300 Braunschweig, Germany.

318

Introduction
2,3-Butanediol can be used not only to produce a wide range of basic chemicals
but also directly as an extraction agent and solvent. Figure 1 shows the most
important uses of 2,3-butancdiol (Tegtmeier, 1989).
Most interesting here are the further processing of 2,3-butanediol to
polyurethane, diacetyl, octanisomers, and its synthesis, involving chlorination and
nitrification reactions as well as phenylbutyric acid, to produce synthetic fabrics
such as Perlon and Tetralon. Diacetyl, used as additive in foodstuffs, can bc
produced relatively simply from 2.3-butanediol and is definitely a high-price
product (Tegtmeier, 1989).
2,3-Butanediol is micro-biodegradable, whereby even Klebsiella oxytoca, Klebsiella pneumoniae and Enterobacter aerogenes, which all produce 2,3-butanediol in
high concentrations, are capable to consume the 2,3-butanediol produced at high
rates. This biodegradability means 2,3-butanediol can be used as an environmentally friendly solvent or cross-linking agent in the production of dyes and polymers.
It is undoubtedly also the case that the largest potential market volumes exist for
products on a butadienc basis, which arc produced by hydrogenation of thc diol.
Butadiene can, however, bc produced more cheaply petrochemically from low
priced crude oil, such that there is no current rcalistic compctitivc basis (Tegtmeier,
1989).

2,3-Butanediol I
tO

tO

AntifreezeI
agent
1o

I Butadiene I
Extractant [

e,--q

Methyl-Ethyl I
Ketone

'D

"O

Polyurethane ]
Solvent [

I Isomersof I
octane
Fig. I. Uses ~f 2,3-butancdiol.

I Diacetyl I

319
Some investigations on the application of molasses for producing 2,3-butanediol
were performed between 1943 and 1954. In all these investigations the product
yield achieved was very low: 42-67% of the theoretical yield of 0.5 diol per
carbohydrate (g g-1) (MacCall and Georgi, 1954). This represented a production
of 20-34 g 1-1 2,3-butanediol with a productivity of 0.51-0.84 g 1-1 h-I. Wheat et
al. (1948) examined the production of diol by Bacillus polymyxa grown on wheat
starch. The bioprocess medium contained 23.2 g 1-1 2,3-butanediol and 15.4 g lethanol.
In recent years, biochemical methods have been developed which enable 2,3butanediol to be produced in concentrations of approx. 100 g 1-~ with a yield of
almost 100% of the theoretical value and a diol productivity of 1.0-1.1 g I-i h(Afschar, 1990; Afschar et al., 1991).
The separation of 2,3-butanediol from the bioprocess medium and subsequent
cleaning are the main problems associated with technical-scale production. Since
butanediol has a high boiling point of approx. 180C, it cannot be separated as a
distillate from the mash by distillation because it always fails to the sump of the
distillation column together with the liquid phase. Since the entire liquid fraction
of the mash has to be evaporated several times, this downstream processing
method is expensive. Adding chemicals with high boiling points, e.g. glycerol, or
using spray-drying methods for the cell-frec medium with inert gascs (Leibmann,
1945) does not make the separation method appreciably cheaper. Chemical separation of 2,3-butanediol from the bioprocess medium has also been studied several
times. The chemical conversion of 2,3-butanediol into formate (Senkus, 1946) or
into butyraldehyde (Ledingham and Neish, 1954) with subsequent distillation is
also not substantially less expensive.
No reports are available on downstream processing of 1,3-propancdiol since the
bioproduction thereof is only a relatively recent area of study.
The use of inorganic salts for the dehydration of organic media is current
practice. These salts can, however, also be used for the cleaning and dehydrating
of organic liquids, e.g. ethyl alcohol (Schmitt, 1889). Potassium carbonate is used
for this purpose. Literature also states that such inorganic salts may bc used for
the separation of organic liquids having limited solubility from aqueous media.
Russell (1985) demonstrated that the salting out of organic liquids with unlimited
solubility is also possible using inorganic salts, provided that the weak organic acid
has a pKa value greater than 14 and that the salt has minimum possible solubility
in the weak organic acid. In this patent, potassium carbonate was again used for
salting out univalent aliphatic alcohols (up to C5) from aqueous media. The
separation of diols from bioprocess media using salting out methods has not yet
been investigated.
The purpose of this investigation was the microbial production of 2,3-butanediol
from starch-containing waste and natural materials as well as from blackstrap
molasses using in recent time developed bioprocesses. In addition, we introduce a
new method for the separation of 2,3-butanediol and other diols from the bioprocess medium, which is possibly less expensive than the separation methods mentioned above.

32O
Materials and Methods

Microorganisms and media

Klebsiella oxytoca DSM 3539 and Bacillus po6'mvxa DSM 356 were used for all
tests. T h e culture was stored in tilted agar tubes containing a mixture of 1 g
glucose, 5 g yeast extract, 5 g tryptone, 1 g K_,HPO 4 and 25 g agar in 1 litre
dcionized water. The m e d i u m was autoclaved for 15 min at 121( ` . The cultures
were a d a p t e d for a high substrate concentration in a shaker. The medium in the
shakcr contained per litre deionizcd water: 5 g yeast extract, 5 g tryptonc, 2 g
K z H P O a, and 30 g, 60 g or 100 g of saccharificd starch or the carbohydrates in the
molasses. The medium in the bioprocess contained per litre deionized water: 5 g
yeast extract, 5 g tryptonc, 2 g K 2 H P O 4, and 200 g starch, saccharified starch or
the carbohydrates in the molasses. Starch (B) was used in this investigation
(Emsland-St/irke G m b H , Emlichhcim, FRG), with BAN or "Fermamyl (Novo
lndustri A/S, C o p e n h a g e n . D e n m a r k ) to liquefy the starch and Fungamy or A M G
(Novo lndustri A/S, C o p c n h a g e n , D e n m a r k ) to saccharify the starch. High-test
molasses or blackstrap molasses (Copersucar, Sao Paul(), Brazil) were used for this
investigation. The blackstrap molasses used contained 29'~4 weight solids, 9.6%
weight ash, 0.5,} weight nitrogen-containing compounds, 0.55C4 wcight sulphurcontaining c o m p o u n d s and approx. 55% weight sugar. The high-test molasses
contained 16c/c weight saccharosc, 2 7 ~ weight glucose and 25C~ weight fructose.
The model media for salting-out experiments contained 1()(1 g 1 ~ 2,3-butancdiol
or 100 g I i 1,3-propanediol or 100 g I t 1,3-propanediol and 100 g I i ethanol.
The m e d i u m tk)r salting-out experiments p r o d u c e d from high-test molasses contained 101.6 g I ~ 2,3-butancdiol, 2.5 g I-~ acetoin and 23) g I ~ ethanol, that
p r o d u c e d from blackstrap molasses 93.2 g I i 2,3-butancdiol, 4.2 g I ~ acetoin and
0.79 g I ~ ethanol, and that from saccharified starch 99.8 g I " ~ 2,3-butanedio[, 1.7
g 1-~ acetoin and 4.3 g 1 ~ ethanol.
Method,s
All bioprocesses were carried out as batches with substrate shift in 3 and 10 I
bioreactors (Applikon Biotek G m b H & Co. Vertriebs KG, Kniillwald-Remsfeld,
F R G ) at 350(7'. T h c pH-values were controlled at 6.8 during the microbial conversion of saccharified starches and at between 5.5 and 6.5 for the microbial
conversion of molasses, using 2 molar KOH. The aeration was carried out using
0.35 w m air at an agitator spced of 200 rpm. The oxygen input rate had to bc
repeatedly reduced such that the acetoin concentration did not exceed a value of 7
g I-~. After the consumption of the available sugar, the aeration rate was also
considerably reduced to 0.03 w m at 50 rpm.
Cell separation was done using a capillary type cross-flow microfiltration module (Frcsenius AG, Bad H o m b u r g , FRG).
In order to p r o d u c e 2,3-butanediol of maximum purity from the bioprocess

321
medium and allow repeated use of the salt, an additional cleaning of the medium
is recommended. We proceeded as follows in this:
The pH-value of the cell-free medium is adjusted to a value of approx. 7.2 using
calcium hydroxide Ca(OH) 2.
- Potassium carbonate is added to increase the pH-value to approx. 9-9.5.
- The bioprocess medium is boiled for approx. 2 rain.
- 1-3 ppm flocculating agent, e.g. Bozofloc or Labufloc (Benckiser-Knapsack
Ladenburg GmbH, FRG), is added while mixing intensively.
After settling of the flocculated solids the clear medium is separated.
The preclcaned bioprocess medium is cooled and agitated and K2CO 3 added.
After dissolution of the K2CO 3 the medium is gently warmed (39-40). After a
short time two phases form. The upper phase (diol phase) consists primarily of the
diol, the lower phase (salt phase) of water and salt.
The salt present in the salt phase can be separated using vacuum distillation
and used again for diol recovery.
When separating 1,3-propanediol it is first necessary to add ethanol (approx.
100 g I-1) prior to adding the potassium carbonate.

Analytical methods
The cell mass concentration was determined by measurement of the optical
density at 578 nm as well as the gravimetric method after centrifugal separation at
150(X) rpm and having been dried at 80C for 24 h. The glucose, maltose,
saccharose and fructose contents were estimated by HPLC using a Sugar-Pak
column and refractomcter (Waters, Division of Millipore, USA). The dctermination of 2,3-butanediol, ethanol, 3-hydroxy-2-butanone (acetoin), acetic acid and
1,3-propanediol were carried out by a gas chromatograph (Shimadzu, Kyoto,
Japan), fitted with a flame ionisation detector and a 2-m glass column, filled with
Chromosorb 101. The exhaust gas analysis was carried out for the CO2-molar
fraction by an infrared method (Maihak, Hamburg, FRG) and for the O~-molar
fraction using a thermomagnetic method (Maihak, Hamburg, FRG).

R e s u l t s

Production of 2,3-butanediol from starch. Bacillus polymyxa can produce 2,3butanediol directly from starch (Ledingham et al., 1945; Fratkin and Adams, 1946).
However, compared with Klebsiella oxytoca, this microorganism produces much
lower concentrations of 2,3-butanediol and much higher concentrations of ethanol
(Neish, 1945). In addition, Bacillus polymyxa is very sensitive to bacteriophages
(Katznclson, 1944). This fact was confirmed in our investigation for the direct
conversion of starch to 2,3-butanediol. We were not able to produce morc than 38
g 1-t 2,3-butanediol with a product yield of 0.28 diol per starch (g g - t ) in batch
process. For the reasons stated above, the saccharification of starch prior to
cultivation and the microbial conversion of the saccharified starch to 2,3-butanediol
by Klebsiella oxytoca are more advantageous, in cases where the saccharificd starch

322

Adding

enzym

3-ButonedLol

T
'\
~..

tO
0

L
0

0tO
0
L
0

"~'q RoeIdual sugar

~i~-

"\

II)'~.

Aceto|n
40

~.

~e. "

-~thanol

...~ ......... ~ ........................

~4b
A.~__"~ ....
~I~

b._~ . . . .
80

tl~

Thin (h)
Fig. 2. Produc! c o n c e n t r a t i o n and residual s u g a r as a function of time in a batch c u l t u r e with addition
of e n z y m e s using s t a r c h saccharified up to m a l t o s e as st,hstrat.

is primarily present as maltose, the high sugar concentration (approx. 180 g I I

maltose and approx. 20 g l i glucose) is less growth-rate inhibiting than purc
glucose. In thc case of maltose conversion the culture is not able to consume the
existing maltose completely. As a result, after reaching a concentration of approx.
60 g 1- t 2,3-butanediol (residual concentration of maltose approx. 57 g 1-I ) in thc
hioprocess, 2,3-butanediol is c o n s u m e d instead of the highly available maltose. By
adding amyloglucosidase directly into the bioprocess, a higher product concentration can be achieved by conversion of maltose (Fig. 2). In this process 151) A G U
amyloglucosidase (corresponds to I).5 g A M G 31)0L) was addcd per litrc culture.
after a 2,3-butanediol concentration of approx. 50 g I ~ was rcached. Because the
enzyme p r o d u c e d glucose, the culturc stopped the consumption of 2,3-butanediol.
After approx. 150 h a product concentration of 98.9 g 1 ~ 2,3-butanediol and 3.5 g
1- ~ acctoin is reached, with a yield of 0.5 diol pcr starch (g g ~).
Starch can also be converted even after complete saccharification to glucose to
produce 2,3-butancdiol (Fig. 3). In o r d e r to avoid inhibiting thc growth-rate of the
culture to any large extent, and also to achieve a high product yield using high
substratc limitation, it is very important to obscrvc exact suhstratc dosing. The
batch culture must not only be o p e r a t e d with an oxygen-limitation regime but also
with a growth-rate inhibitor, like high substrate or product concentration, to
achieve a higher product yield. By this means after approx. 70 h a 2,3-butanediol
concentration of 99.8 g I ~ is achieved with a yield of 1).5 diol per starch (g g-~).

323

8-

A
T

t"
O

! "%
I
%.

I
I
I

8~

i
\ it

'~.

2.3-Butanedlol
J

%.

"\

i
!

%.

Residual

euga~

\
\
Q

\ i Acetoln

Ethano!

40

Tim (h)
Fig. 3. Product c o n c e n t r a t i o n a n d residual s u g a r as a function of time in a batch culture with s u b s t r a t e
shift using starch s a c c h a r i f i e d up to glucose as substrate.

Production of 2,3-butanediol from molasses. High-test molasses ( H T M ) or invert

molasses is an excellent raw material for the production of 2,3-butancdiol. In a
batch process with substrate shift, 200 g 1-l of the carbohydrates present in thc
H T M can bc converted in approx 80 h to produce 95-99 g 1-l 2,3-butanediol and
25-43 g 1-l acetoin with a yield of 0.5 diol per carbohydrate (g g - i ) (Afschar,
1990; Afschar ct al., 1991) In the microbial production of 2,3-butanediol, one has
on the one hand the high substrate concentration limiting the growth of the culture
(Magee and Kosaric, 1987) and on the other hand it is necessary to have a
maximum substrate concentration at the start of bioprocess to improve product
yield Due consideration concludes that following completion of batch proccss the
cell mass generated should be recycled and used in subsequent cultivation It is
possible to separate the cells from the wine using either centrifugal methods or
microfiltration modules In these repeated batch experiments, batch cultivations
with substratc shift were carried out repeatedly in sequence, and after the
completion of bioprocess in each case the cells were recycled and uscd in the next
bioprocess cycle. This enabled the enrichment of the ccll mass up to a concentration of 16 g dry cell mass per litre (Fig. 4).
In this batch process 200 g ! -1 carbohydratc was converted to 118 g I

324

2 , 3 - B u t a n e d 1o

#-%

"%
\

v
C

4-o

"lID

0
I.
4J
f-

~,.

0
U

I::

0
0

1..

L.

0
Ob

:3
(n

.q.
lk
*~o

Acetoln
.~ ~.--A-_
0

10

"~..Resldual
"'o..
~__ _~._.~.__~__~_

sugar

"~...~

20

Tim

3o

4O

(h)

Fig. 4. Product concentration and residual sugar as a function of time in a repeated batch culture with
increased cell concentration.

2,3-butanediol and 1.8 g I-~ acetoin with a yield of 0.5 diol per carbohydrate
(g g-~). The diol productivity had a value of 2.4 g 1 ] h ]
Klebsiella oxytoca reacts sensitively to a reduction in water activity (Esener ct
al., 1980). The growth-rate decreases and the substrate consumption due to
maintenance increases. This behaviour is very apparent in the conversion of
blackstrap molasses. At a salt concentration of 2 - 3 % weight, in this waste
molasses the lag phase is extremely long, the cell growth-rate very slight and the
diol yield substantially below the theoretical value (Fig. 5). In this case over a 120 h
period 90 g 1 ] 2,3-butanediol was produced with a productivity of 0.42 diol per
carbohydrate (g g - ] ) . As a result of the higher salt concentration and other
chemical impurities of the blackstrap molasses, the diol productivity in this case is
substantially below that of the H T M conversion.
It is thought likely that precleaning the blackstrap molasses, mixing with H T M
and running a very short sterilization time could bring substantial cuts to thc
bioprocess period.

Salting out of 2,3-butanediol

In a series of salting-out experiments the effect of various salts on the separation of dio[s from aqueous phases was investigated. The best salting out results

325

8-

"\

z-k

~" ~-

",,

i
i

",,

II

I\

",.

\1t

2.,,T~Butonodlol ~' k

8-

~,

Reslduol eugor'~

"

~-

\i
"~

..~1, . . . . . . . . .
,.A .........

i~..

. A "'A" . . . . . . . . . . . . .

.....

".,..

A'~" . . . . . . . . . . . . .

""

,~...
I

T[~

(h)

Fig. ~. Product c o n c e n t r a t i o n and residual sugar as a function of time in a batch culture with substrate
shift using blackstrap m o l a s s e s as substrate.

were achieved using water-free potassium carbonate K2CO 3. This salt is also well
suited for salting out diols due to its high solubility in water and its low solubility in
diols.
2,3-Butanediol can be separated almost quantitatively from an aqueous medium
by salting out with potassium carbonate. In order to achieve maximum separation
of the 2,3-butanediol present in the aqueous medium, it is first necessary to
investigate the dioi concentration in the resultant phases for various K2CO ~
concentrations in a model medium (Table 1).
As Table 1 shows, the addition of 46% weight K2CO 3 allowed the separation of
up to 96% (w w -1) of the 2,3-butanediol present from the aqueous medium. The
separated diol phase contained 93.3% weight 2,3-butanediol. The separation of
2,3-butanediol from a cell-free and not precleaned medium requires, compared to
a model medium, higher potassium carbonate concentrations and at the same time
results in a diol phase with higher water content. By salting out the bioprocess
medium with 67.9% weight K2CO3, 94.6% (w w 1) of the 2,3-butanediol present
were separated (Fig. 6). The concentration of the 2,3-butanediol in the diol phase
was 73.6% weight and much lower than the 2,3-butanedio[ separated from a model
medium (Table 2). By precleaning the medium and removing the ethanol by brief
boiling, it was possible to separate 94% (w w - I ) of the 2,3-butanediol present

326
TABLE I

Percentage of 2,3-butanediol recovered and its concentration in the phases created by salting out as a
function of concentration of potassium carbonate added to a model medium containing 2,3-butanediol
K 2C0.~

2,3-Butanediol

( ~ weight)

diol phase

salt phase

recovered

(~Y~weight)

(c,~ weight)

("~ w / w )

67.10
67.21)
68.00
76.1()
81.60
93.20
93.90
93.30

3.63
3.71
2.26
1.40
1.(}0
0.30
0.30
0.20

36.04
37.49
41.14
44.50
45.88
46.63
50.79
53.71

2,3-Butanediol

58.10
56.82
72.79
81.44
87.81
96.02
96.28
97.06

using 53% weight potassium carbonate. The concentration of the 2,3-butanediol in

the diol phase increased up to 97% weight 2,3-butanediol. Precleaning by flocculation is particularly recommended in the case of molasses conversion, because
otherwise the salt is no longer of suitable quality for salting out after a few charges
due to strong coloration and contamination. By precleaning it prcwed possible to
recover up to 96% (w w -1) of 2,3-butanediol present in a bioprocess medium
produced by conversion of saccharified starch. For the salting out 56% weight
potassium carbonate was added.

100

90
"D

80

'70
60
50
I
T]

4O
20
20
:.30

40

F,(]

60

70

80

90

0,%

Fig. 6. 2,3-Butanediol recovered as a function of concentration of potassium carbonate added to a

bioprocess medium produced by conversion of molasses.

327
TABLE 2
2,3-Butanediol concentration in the phases created by salting out as a function of concentration of
potassium carbonate added to a bioprocess medium produced by conversion of molasses
K 2CO3
(% weight)

2,3-Butanediol
diol phase
(% weight)

salt phase
(% weight)

46.0
50.1
53.6
57.2
60.8
67.9
89.4

52.0
53.8
56.0
59.0
62.0
73.6
76.8

5.80
5.20
3.60
2.30
1.40
0.40
o. 10

Separating other diols by salting out

Salting o u t can be used to s e p a r a t e o t h e r diols also p r o d u c e d using microbiological m e t h o d s . A s an e x a m p l e , we c o n s i d e r e d thc s e p a r a t i o n o f 1,3-propancdiol.
T h e s e p a r a t i n g effect o f K 2 C O 3 for r e c o v e r i n g 1,3-propanediol from the m o d e l
m e d i u m is substantially less than for 2,3-butanediol. This result is probably d u e to
the g r e a t e r solubility in w a t e r and the relative location of the O H - g r o u p s in
1,3-propanediol m o l e c u l e s . A d d i n g e t h a n o l to the m o d e l or process m e d i u m raises
s e p a r a t i o n to 93.6% (w w - l ) of the 1,3-propanediol p r e s e n t from t h e a q u e o u s
m e d i u m . T h e diol p h a s e p r o d u c e d by salting out c o n t a i n s a r o u n d 20 weight %
water. T a b l e 3 s u m m a r i s e s the results of salting out an a q u e o u s m e d i u m c o n t a i n i n g
100 g !-~ 1 , 3 - p r o p a n e d i o l and 100 g 1-n ethanol.

TABLE 3
Percentage of 1,3-propanediol and ethanol recovery and their concentrations in the phases created by
salting out as functions of the concentration of potassium carbonate added to a model medium
K ~CO 3

1,3-Propanediol

(% weight)

diol phase
(% weight)

30.7
31.5
32.7
40.9
44.9
49.0
53.1
61.3
65.8

salt phase
(% weight)

recovered
(% w/w)

ethanol

diol

ethanol

diol

ethanol

diol

23.1
25.9
32.9
33.3
34.7
36.1
37.8
39.4
49.6

5.30
18.0
24.4
28.1
30.0
32.7
34.3
39.0
46.5

1.80
1.30
1.10
0.50
0.40
0.30

2.30
1.70
1.40
0.80
0.60
0.50

0.20

0.40

0.10
0.15

0.16
0.17

25.9
46.6
53.9
79.4
84.6
88.2
90.8
94.8
94.0

6.00
32.4
40.1
62.9
73.3
79.8
82.3
93.6
93.2

328

Discussion

In industrialized countries agricultural natural resources do not represent a

cheap source of raw materials even when mass produced because of" the inherent
cost structure. Thus, any alternative products from these natural resources can
only gain market penetration if the processing technologies applied allow economic production. Enhanced processes can represent an important contributory
factor to achieve this. The major factors in process improvement are:
- increased exploitation of the natural resource (yield)
- improved product concentration and reduced levels of by-product
- viable product purification.
In the production process for 2,3-butanediol from starchy raw and waste
materials, including molasses from the sugar industry, it was attempted to take the
above named factors into account in the industrial development of the process. By
applying the described arrangement for substrate dosing and the control of oxygen
levels depending upon the concentration of a by-product (acetoin), the yield of
2,3-butanediol was maximized and production realized at a relatively high concentration for this biological process. Because Klebsiella oxytoca does not produce
amylase when degrading starch and since Bacillus polymaxirna digests starch at
very low concentrations and yield to 2,3-butanediol, it was decided to use saccharifled starch for the biological production of 2,3-butanediol.
Bearing in mind that in the starch processing industry saccharified starch
a n d / o r its wastes - in the form of maltose-glucose mixes or glucose - predominate, these two forms of starch saccharification were considered. The results above
show that in the microbial production of 2,3-butanediol by Klebsiella oxytoca the
glucose is consumed at a considerably faster rate than the maltose-glucose
mixture.
The quality of the blackstrap molasses with respect to salt content depends
upon the sugar production process itself. The salt concentration fluctuates between 4 and 14% (weight) while the sugar concentration varies between 45 and
55% (weight). For blackstrap molasses with a sugar content between 45 and 5 5 ~
(weight) and a salt content of around 4% it is possible, after dilution (approximately 1: 2), to implement direct processing to 2,3-butanediol. For salt concentrations above 4% (weight) the blackstrap molasses must be mixed with other
molasses of lower salt content (e.g. high-test or invert molasses). Only by reducing
the water activity of the substrate is it possible to produce at higher yields
(between 0.4 and 0.5 g g - ~ (diol per carbohydrate) and a concentration of approx.
100 g I ~ 2,3-butanediol.
In the past, attempts have been made to use concentration plants to produce
the major fraction of the 2,3-butanediol as condensate, passing the product
through several columns for purification. It is obvious that to achieve this end
almost the entire water fraction of the mash has to be evaporated, several times.
This very expensive technique resulted in uneconomic production plants in the
USA. The salting out process investigated by us to separate 2,3-butanediol from
the medium is probably less expensive than the thermal process described, because

329

the recovery of the salt only involves a single evaporation of the water phase. In
the thermal process the medium has to be evaporated several times (Magcc and
Kosaric, 1987). The recovery rate of 2,3-butanediol in both methods is approx. 95%
(weight/weight), with the 2,3-butanediol produced, again for both methods, containing around 5-1(1% water.

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