Appl Microbiol Biotechnol (1991) 34:582-585
0175759891000301
The production of 2,3-butanediol by fermentation
of high test molasses
A. S. Afschar 1, K. H. Bellgardt 2, C. E. Vaz Rossell 3, A. Czok 1, and K. Schaller ~
1 GBF-Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, W-3300 Braunschweig, Federal Republic of
Germany
2 Institut fiir Technische Chemie der Universit~it Hannover, Callinstrasse 3, W-3000 Hannover, Federal Republic of Germany
3 Centro de Technologia Copersucar, Caixa Postal 162, S~o Paulo, Brasil
Received 27 June 1990/Accepted 24 September 1990
Summary. Klebsiella oxytoca fermented 199 g . l - 1 high
test or invert molasses using batch fermentation with
substrate shift to produce 95.2-98.6 g 2,3-butane-
diol. 1-1 and 2,4-4.3 g acetoin. 1-1 with a diol yield o f
96-100% o f the theoretical value and a diol productivity
of 1.0-1.1 g.1-1 .h -1. Fermentation was performed nu-
merous times with molasses in repeated batch culture
with cell recovery. Such repeated batch fermentation, in
addition to a high product yield, also showed a very
high product concentration. For example, 118 g 2,3-bu-
tanediol. 1-1 and 2.3 g acetoin. 1-1 were p r o d u c e d from
280 g-1-1 of ~high test molasses. The diol productivity
in this fermentation amounted to 2.4 g. 1-1. h -1 and
can u n d o u b t e d l y be further increased by increasing the
cell concentration. Because the Klebsiella cultures fer-
ment 2,3-butanediol at an extremely high rate once the
sugar has been consumed, the culture was inhibited
completely by the addition of 15 g ethanol.1-1 and
switching off aeration.
Introduction
2,3-Butanediol is characterized by interesting proper-
ties and a wide range of applications (Rehm 1980). This
c o m p o u n d could be a valuable c o m p o n e n t various po-
lymers which are probably easily biodegradable~ The
possible applications o f 2,3-butanediol mean that the
substance is suitable for a number o f developments
with anticipated future industrial production potential.
This type o f production will however have to be a mi-.
crobiological process, as the current known chemical
processes are not economically competitive (Rehm
1980).
Some investigations on the application o f sugar
beet molasses and blackstrap molasses for producing
2,3-butanediol were performed between 1943 and 1954.
In all these investigations the product yield achieved
Offprint requests to: A. S. Afschar
App//d
o., Microbiology
Biotechnology
© Springer-Verlag 1991
was very low (approx. 42-67% of the theoretical yield
o f 0.5 g diol. g sugar). This represented a production of
20-34 g 2,3-butanediol.1-1 with a productivity o f 0.51
to 0.84 g . l - l . h -1 (MacCall and Georgi 1954). Wheat
(1953) even performed batch fermentation on a pilot
plant scale (4000-1 fermentor). In this case 182.2 kg ace-
toin and 2,3-butanediol and 25.5-65.1 kg ethanol were
recovered from 1000 kg molasses. The economic pro-
duction o f 2,3-butanediol from high test molasses re-
quires the highest possible product yield due to the re-
latively high price of the substrate. Moreover, due to
the high down-stream processing costs, the highest pos-
sible product concentration is required.
Materials and methods
Culture and media. Klebsiella oxytoca strains NRCC 3006, DSM
5175, DSM 3539 and DSM 30107 were used for continuous and
fed-batch fermentations and K. oxytoca strain DSM 3539 in batch
fermentation. The strains were maintained on agar slopes contain-
ing 1 g glucose, 5 g yeast extract, 5 g tryptone, 1 g K2HPO4 and
25 g agar in 11 distilled water. This was autoclaved at 121°C for
15 rain. The media for the fermentation experiments contained
glucose or high test molasses (HTM) at the specified concentra-
tion, and 5 g yeast extract, 5 g tryptone and 2 g K2HPO4per litre.
Because the K. oxytoca cultures ferment 2,3-butanediol at a very
high rate immediately after complete sugar uptake, once the max-
imum of 2,3-butanediol concentration was reached, 15 g etha-
nol, 1-1 was added as an inhibitor and aeration was stopped (Af-
schar 1990). In repeated-batch fermentation, the most of the etha-
nol was removed in the process of separating the extracellular
product 2,3-butanediol, thereby enabling recycling of the cul-
ture.
Fermentation. All continuous fermentations were carried out in a
4-1 fermentor (Setric Grnie Industrial, Toulouse, France) at 35°C
and a pH value of 5.5. For repeated-batch experiments a 20-1 fer-
mentor with a 16-1working volume and integrated ceramic cross-
flow microfiltration module (Ultrafermentors from Setric Grnie)
was used. All fed-batch and batch fermentations were carried out
in a 20-1 fermentor (Setric G6nie) at 35° C. In the fed-batch and
batch fermentation continuous and accurate pH regulation was
not absolutely necessary. If the pH dropped below 5.5, it was ad-
justed to 6.5 with 2 M KOH. Initially batch cultures with substrate
shift were fermented with a sugar concentration of approx. 120
 583

g.l -t at an optimized aeration rate of 0.5 vvm at 200 rpm to 0.5+

achieve the highest possible cell concentrations. Subsequently,
after approximately 15 h, the remaining 40% of the total sugar was
added and aeration changed to 0.3 w,m at 150 rpm. The oxygen
~xi~/~/~Cell (dry) mass
0.4
input rate had to be repeatedly reduced such that the acetoin con- OO
centration did not exceed 7 g-1-~. After complete fermentation of
the available sugar the aeration rate was also considerably re- • Diol
duced to 0.03 vvm at 50 rpm. 7c~ 0.3

Analysis. Sugar concentrations were estimated by HPLC using a

Sugar-Pak 1 (Waters, Division of Millipore, USA) column with a .~- 0.2
>.
refractive index monitor. The column temperature was 75° C and
mobile phase (0.5 mg Ca EDTA-1 -~ in deionized water) flow was
0.4 ml-min -~. The determination of 2,3-butanediol, ethanol, 3-
hydroxy-2-butanone (acetoin) and acetic acid was carried out with 0.1
a gas chromatograph (GC 9A, Shimadzu, Kyoto, Japan), fitted 0.2 0.4 0.6 0.8
with a flame ionisation detector and a 2-m long glass column, Dilution rate (h - ~)
filled with Chromosorb 101. The cell biomass was determined by
measuring the optical density at 578 nm and by the gravimetric Fig. 1. Product yield as a function of the dilution rate in a glucose
method after centrifugal separation at 15000 rpm and drying at chemostat culture of Klebsiella oxytoea
80°C for 24 h. The measured CO2-mole fractions in the exhaust
gas were carried out by an infrared method (Defor gas analyser
from Maihak, Hamburg, FRG). tion, in particular of fructose, could not be achieved in
the continuous fermentation of H T M .

Results
Fermentations o f H T M to 2,3-butanediol with contin-
uous, fed-batch and batch cultures o f K. oxytoca were
investigated and c o m p a r e d With regard of product yield
and p r o d u c t concentration. A preliminary experiment
was carried out with glucose as the substrate+
Continuous fermentation
The p r o d u c t i o n o f 2,3-butanediol by standard contin-
uous fermentation is not favourable, because only rela-
tively low product concentrations ( m a x i m u m 40 g. 1-1)
and p r o d u c t yield levels can be achieved in continuous
cultures despite the high levels o f productivity. In our
investigations on continuous fermentation of glucose,
product yield varied between 34 and 80% of the theore-
tical value at full uptake o f available glucose depending
on the dilution rate (Fig.l). Complete sugar c o n s u m p -
Fed-batch fermentation
The product yield and product concentration o f 2,3-bu-
tanediol could be increased using the fed-batch proc-
ess. The development of a pulsed substrate feeding
strategy in fed-batch fermentation (Afschar et al. 1990)
led to a m a x i m u m 2,3-butanediol concentration o f 72
g.1-1 with a productivity of 0.5 g.1-1 .h -1 and a prod-
uct yield of 88% of the theoretical value. Substrate ad-
dition was controlled via the carbon dioxide content in
the exhaust gas. In this investigation the glucose used
was fermented up to a final concentration of 0.8 g. 1-1.
Figure 2 shows a fed-batch process for the production
of 2,3-butanediol regulated with the E X P C O N (Af-
schar et al. 1990) process control system.
The utilization of molasses in fed-batch cultures led
to a product concentration of 68 g. 1-1 with a produc-
tivity o f 1.1 g . l - t . h -1 and a diol yield o f 84% of the
theoretical value. The available sugars in H T M (fruc-

80- 8-1~ 207

70- 1
6-1 ~ 16 2,3-buta~EJ~
60- RQ
E .
,I 44_ =
-'. 50+
>°t2-
e- 40-:
~£

~ 30 2 ~ 82
04 ~
28
~" 20 1
t0 -a] 4-
. Fig. 2. Fed-batch culture controlled by the
EXPCON process control system (Afschar et al.
0 -4 O 2 1990) for the production of 2,3-butanediol:
0 10 20 30 40 50 60 70 80 2,3-butanediol concentration, respiration quotient
-time (h) (RQ), and CO2 concentration in the exhaust gas
 584

tose, 25%; saccharose, 16%; glucose, 27%; by weight) 200 00 n

were fermented up to a fructose and saccharose con-
centration of 1.8 g. 1-1. ~ ~" ] . . . . ~
,__ 80 4 !t /
~. 150 .~ 4 I\ ~ 2,3-butanediol
Batchfermentation : I i °', /
.~. ~ eo ~ I t ~o~ua,,u~:, ~"
The batch culture must not only be operated with an ~00 ~ t~ i t .~'~
oxygen-limitation regime but also with a growth-rate /
inhibitor to achieve a higher product yield. These o=
O
~ 40 1~
O
,~./
'o
growth-rate-inhibiting effects can be achieved by high o s
substrate concentrations (Magee and Kosaric 1987) or ~' 50. ~ ~ Xo~,
m £ 20 ~ ," "~
the addition of an inhibitor such as lactic acid (Qureshi 4 / ~--~-~--2"~=-~-_~ ,cotoi~
and Cheryan 1989) or acetic acid (Yu and Saddler ~ ~ , /.c~" -o-o_.~-~-._
~ & i~-" ~ - %~. ~Z--Z-~ --~
1983). In the case of high substrate concentration, to 0 0 ~ ............... " ? - ~ , ,
achieve a sufficient cellular growth rate despite high in- 0 50 100 150 200 250
hibition, the substrate must be added in two shifts. Fig- ~me(h)
ure 3 shows the batch fermentation process with sub- F~. 4. Residual su~aL 2,3-butanedJo~, and acetoJn concentration
strate shift for the production of 2,3-butanediol from as a function of time ~n a lactic acid (2.6 $.]-~) inhibited batch
HTM. culture with substrate
This batch fermentation with substrate shift pro-
duced 95.2-98.6 g 2,3-butanediol.1-1 and 2.4 to 4.3 g
acetoin. 1-1 from 200 g carbohydrate.1-1, with a diol 100 - 120 -
yield of 96-100% of the theoretical value and a diol ~ ~ ~-----~ ~%
productivity of 1.0-1.1 g. 1- 1. h - 1 (Afschar 1990). ~ ~,. ~
Figure 4 shows the influence of lactic acid ~C 80- ~ "\\., o/
133
(2.6 g. 1-1) on the fermentation of HTM to 2,3-butane- "\ \ ' \ / 2,3-butanediol
diol in a batch process. Diol productivity was reduced
t-
O 60.
~
-
80-
\ \ /./°
,~ \ \ ,.
by more than 50% at the same diol concentration and
t--
diol yield. 0
~o
•= X:% °-*)~o"
In the batch fermentation with substrate inhibition = 40. o= ~.~" %-%
O
in addition to oxygen limitation, high substrate concen- O o40
~ "o,. fro.
trations limited the growth rate of the cultures. On the = ".~ & glc. ~o~
~ ao. o /~. sac. X ~.~
other hand, a high substrate concentration at the start ~ / ~ & -o.
of fermentation was necessary to achieve increased ...-~- " ~ - ? ~ p ~ - - ~ - . ~ _ _.~__Acetoin % 0,%
product yield. Therefore the concept of using the sepa- 0- 0 ~ 7", . . . . . . . ""p-;~--~ . . . . . . . . . . . . . ,,,,
rated cell mass after completion of a batch fermenta- 0 10 20 30 40 50 60
tion process for subsequent fermentation processes was ~me (h)
obvious. Centrifuges or microfiltration modules can be Fig. 5. Residual concentration of available sugars in H T M [sac-
used for separating the cells from the fermentation me- charose (sac), glucose (glc) and fructose (frc)], 2,3-butanediol, and
dium. acetoin concentration as a function of time in a repeated batch
fermentation with increased cell concentration

160

-:" 120
,/°°l
~801 \
2,3-bu:a.:e__':'/ /
A In the repeated batch experiment numerous succes-
sive batch fermentations with substrate shift were per-
formed initially, whereby the cells were retained at the
~ end of each fermentation cycle. This enabled the en-
i-
.£ \ // richment of the cell mass up to a concentration of 16 g
~ ~\ dry cell mass. 1-~. Subsequently the fermentation was
~~- a0 ,.\k~~/° performed as a batch process, i.e. by the addition of the
e- ~4oi \ total substrate (280 g HTM- 1- ~) at the start of fermen-
0
0
g
o /-- ,2e~,~o~,~o~ tation. Figure 5 shows such a batch fermentation with
~
I~
::~
40 o
-~ 20-
./ ~ "~ ~

&'o
increased cell concentration. In the final stage of this
~_ • ~.
r.~
n /~ "~.~.~._.~ Acetoin
fermentation process l18g 2,3-butanediol.1 -~ and
~ _ ~ ~--&~ .~ ~ ~ 1.8 g acetoin-1-1 were produced with a diol yield of ap-
/~ - -~--&--A .... o~ o
0 0~ ............. o ...... prox. 100% of the theoretical value. The diol productiv-
0 20 40 ~0 80 100 ity here was 2.4 g - l - l . h -1 (Afschar 1990). Therefore
~ m ~ (h) the application of batch processing in the fermentative
~i~. ~. Residual sugar, 2,3-butanediol, and acetoin concentration production of 2,3-butanediol is advantageous because
as a funcdon of ti~e in a batch fermentation of high test molasses this process enables high product yield in addition to
( H T ~ ) with substrme shift high product concentration.

References
Afschar AS (1990) German patent application no. P 4017113.2
Afschar AS, Bellgardt KH, Bartzke U, Nothnagel J, Schaller K
(1990) EXPCON. A new approach for automatic control of
the substrate flow rate in chemostat and fed-batch processes.
Food Biotechnol 4:113-122
MacCall KB, Georgi CE (1954) The production of 2,3-butanediol
by fermentation of sugar beet molasses. Appl Microbiol
2:355-359
585
Magee RJ, Kosaric N (1987) The microbial production of 2,3-bu-
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Qureshi N, Cheryan MJ (1989) Production of 2,3-butanediol- by
Klebsiella oxytoca. Appl Microbiol Biotechnol 30:440-443
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