Appl Microbiol Biotechnol (1991) 34:582-585 0175759891000301 App// d

o., Microbiology Biotechnology © Springer-Verlag 1991

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. Klebsiel la 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 of 96-100% of 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 produced 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 undoubtedly 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 compound could be a valuable component various po- lymers which are probably easily biodegradable~ The possible applications of 2,3-butanediol mean that the substance is suitable for a number of developments with anticipated future industrial production potential. This type of 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 of 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

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was very low (approx. 42-67% of the theoretical yield of 0.5 g diol. g sugar). This represented a production of 20-34 g 2,3-butanediol.1-1 with a productivity of 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 of 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 K2HPO4 per 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 16-1 working 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

g.l - t at an optimized aeration rate of 0.5 vvm at 200 rpm to 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 input rate had to be repeatedly reduced such that the acetoin con- centration did not exceed 7 g-1-~. After complete fermentation of the available sugar the aeration rate was also considerably re- duced to 0.03 vvm at 50 rpm.

Analysis. Sugar concentrations were estimated by HPLC using a Sugar-Pak 1 (Waters, Division of Millipore, USA) column with a 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 a gas chromatograph (GC 9A, Shimadzu, Kyoto, Japan), fitted with a flame ionisation detector and a 2-m long glass column, filled with Chromosorb 101. The cell biomass was determined by measuring the optical density at 578 nm and by the gravimetric method after centrifugal separation at 15000 rpm and drying at 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).

Results

Fermentat ions of H T M to 2,3-butanediol with contin- uous, fed-batch and batch cultures of K. oxytoca were investigated and compared With regard of product yield and product concentration. A preliminary experiment was carried out with glucose as the substrate+

Continuous fermentation

The product ion of 2,3-butanediol by s tandard contin- uous fermentat ion is not favourable, because only rela- tively low product concentrations (maximum 40 g. 1-1) and produc t yield levels can be achieved in continuous cultures despite the high levels o f productivity. In our investigations on continuous fermentat ion of glucose, product yield varied between 34 and 80% of the theore- tical value at full uptake of available glucose depending on the dilution rate (Fig.l). Complete sugar consump-

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tion, in particular of fructose, could not be achieved in the continuous fermentat ion of HTM.

Fed-batch fermentation

The product yield and product concentrat ion of 2,3-bu- tanediol could be increased using the fed-batch proc- ess. The development of a pulsed substrate feeding strategy in fed-batch fermentat ion (Afschar et al. 1990) led to a m a x i m u m 2,3-butanediol concentrat ion of 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 product ion 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 concentrat ion of 68 g. 1-1 with a produc- tivity of 1.1 g . l - t . h -1 and a diol yield of 84% of the theoretical value. The available sugars in H T M (fruc-

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584

tose, 25%; saccharose, 16%; glucose, 27%; by weight) were fermented up to a fructose and saccharose con- centration of 1.8 g. 1-1.

Batch fermentation The batch culture must not only be operated with an oxygen-limitation regime but also with a growth-rate inhibitor to achieve a higher product yield. These growth-rate-inhibiting effects can be achieved by high substrate concentrations (Magee and Kosaric 1987) or the addition of an inhibitor such as lactic acid (Qureshi and Cheryan 1989) or acetic acid (Yu and Saddler 1983). In the case of high substrate concentration, to achieve a sufficient cellular growth rate despite high in- hibition, the substrate must be added in two shifts. Fig- ure 3 shows the batch fermentation process with sub- strate shift for the production of 2,3-butanediol from HTM.

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 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 (2.6 g. 1-1) on the fermentation of HTM to 2,3-butane- diol in a batch process. Diol productivity was reduced by more than 50% at the same diol concentration and diol yield.

In the batch fermentation with substrate inhibition in addition to oxygen limitation, high substrate concen- trations limited the growth rate of the cultures. On the other hand, a high substrate concentration at the start of fermentation was necessary to achieve increased product yield. Therefore the concept of using the sepa- rated cell mass after completion of a batch fermenta- tion process for subsequent fermentation processes was obvious. Centrifuges or microfiltration modules can be used for separating the cells from the fermentation me- dium.

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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- richment of the cell mass up to a concentration of 16 g dry cell mass. 1 -~. Subsequently the fermentation was performed as a batch process, i.e. by the addition of the total substrate (280 g HTM- 1- ~) at the start of fermen- tation. Figure 5 shows such a batch fermentation with increased cell concentration. In the final stage of this fermentation process l18g 2,3-butanediol.1 -~ and 1.8 g acetoin-1-1 were produced with a diol yield of ap- prox. 100% of the theoretical value. The diol productiv- ity here was 2.4 g - l - l . h -1 (Afschar 1990). Therefore the application of batch processing in the fermentative production of 2,3-butanediol is advantageous because this process enables high product yield in addition to high product concentration.

585

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

Magee RJ, Kosaric N (1987) The microbial production of 2,3-bu- tanediol. Adv Appl Microbiol 32:89-161

Qureshi N, Cheryan MJ (1989) Production of 2,3-butanediol- by Klebsiella oxytoca. Appl Microbiol Biotechnol 30:440-443

Rehm HJ (1980) Industrielle Mikrobiologie, 2nd edn. Springer- Verlag Berlin Heidelberg New York

Wheat JA (1953) Production and properties of 2,3-butanediol. Can J Technol 31:73-84

Yu EKC, Saddler JN (1983) Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae on high substrate concentration. Appl Environ Microbiol 46, 3:630-635