Acetic acid production by

Appl Microbiol Biotechnol(1990) 34:344-349

Applied Microbiology Biotechnology © Springer-Verlag 1990

Acetic acid production by Acetogenium kivui in continuous culture - kinetic studies and computer simulations J. von Eysmondt ~ *, Dj. Vasic-Racki 2, and Ch. Wandrey 1 ~ Institute of Biotechnology, Research Center, W-5170 Jtilich, Federal Republic of Germany ~ Faculty of Technology, University of Zagreb, Zagreb, Yugoslavia Received 6 July 1990/Accepted 12 September 1990

Acetogenium kivui

Summary. This work considers the continuous production of acetic acid by the homoaeetogenic and thermophilic bacterium Acetogeniurn kivui. A mathematical model for the growth kinetics has been developed. The unstructured model for growth and product formation includes product and substrate inhibition as well as maintenance energy effects. The associated model parameters have been identified by non-linear optimization and evidenced experimentally in continuous culture as steady-state data. By using a mineral medium with glucose as the energy and carbon source for the bacteria proper carbon balances are available. The model permits good predictions of steady-state concentrations.

~ 1 C6H1206

~ pH 6.4,66 °C

r

-

Offprint requests to: J. von Eysmondt

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' tii7~ iU l I31_.l JLoJ

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fach 800320, D-6230 Frankfurt a. M. 80, FRG

3 mole acetic acid

b

Introduction

* Present address: Hoechst AG, Verfahrenstechnik G 811, Post-

3 CH3COOH

1 mole glucose

~ The homoacetogenic bacterium Acetogenium kivui, first described by Leigh et al. (1981), is an anaerobic, thermophilic and Gram-negative microorganism that is able to convert glucose into acetic acid in a one-step fermentation (Fig. la). Compared to the aerobic production of vinegar, the anaerobic method offers higher acetate yields (nearly 3 mol acetate from 1 ml glucose) and lower energy costs because no aeration with oxygen is required (Klemps et al. 1987). For this reason homoacetogenic conversion of glucose has been the subject of investigations in connection with the production of acetic acid as a feed-stock chemical from renewable resources (Ljungdahl et al. 1985). Growth of A. kivui in batch and continuous culture has been described by different authors (Klemps et al. 1987; Ljungdahl et al. 1985) but no kinetic modelling of acetic acid production by these bacteria has been described in the literature up to now. It is reported that,

I [

Acetogenium kivui

([[[[[~

weighing machine

Fig. 1. Reaction scheme (a) of the anaerobic conversion of glucose to acetic acid by Acetogenium kivui and a schematic view (b) of the equipment used for continuous fermentation

due to strong product inhibition, maximum acetate concentrations are limited to 30-40 g/1. This paper deals with the experimental and theoretical investigation of acetic acid production by A. kivui in continuous culture. A suitable model for product formation kinetics including product and substrate inhibition as well as maintenance energy effects has been developed.

Materials and methods Microorganism and culture conditions. A. kivui was obtained from the German Collection of Microorganisms (DSM 2030). The cup

345 Table 1. Mineral medium for continuous fermentation with Acetogenium kivui

Solution A

Solution B Solution C Solution D Solution E Solution F Substance G

Substance

Concentration

(NH4)zSO4 MgSO4- 7HzO CaClz. 2HzO NaC1 Citric acid

5.0 0.25 0.15 0.5 0.25 50.0

C6H1206-H~O (glucose) KHzPO4 K2CO3 Trace elements A Trace elements B Resazurin (redox indicator)

Na2S~O4

g/1 g/1 g/1 g/1 g/1 g/1

2.0 g/1 0.8 g/l 1.2 ml/1 0.3 ml/1 0.2 ml/1 (0.1% w/v) 0.35 g/1

Table 2. Trace element solutions for continuous culture of A. kivui Substance Trace elements A: Fe(SO4)2(NH4)2.6H20 Co(NO3)~. 6H20 Na2MoO4- 2H20 NiClz. 2H20 ZnC12 MnC12.4H20 CuC12- 2H20 H3BO3 Titriplex 1 Trace elements B: NaSeO3.5H20

Na2WoO4-2H20

Concentration [g/l] 39.21 8.73 2.42 1.19 2.42 0.20 0.004 0.012 4.78 0.026 0.033

kin-Elmer UV/VIS-Photometer 550 S (~berlingen, FRG). Gravimetric measurements of biomass gave a linear relationship between dry biomass and OD. Quantitative analysis of acetic acid was carried out by Sykam HPLC ion chromatography (Gautingen, FRG) with a Biorad Aminex HPX-87H column (Munich, FRG). Glucose was analysed using a commercial test kit (Boehringer Mannheim, FRG) with hexokinase and glucose-6-phosphate-dehydrogenase. The detection of NADH as the result of glucose oxidation was carried out at 365 nm in the same photometer as described above. Data handling. The resulting two models of yield coefficients (Eqs. 3 and 4) contain two parameters respectively, whereas Eq. 2 (growth rate p~) depends on five parameters and two constant values calculated earlier from Eq. 3. The parameters were estimated by means of the non-linear Nelder and Mead regression method (Nelder and Mead 1965). The steady-state concentrations in the bioreactor were estimated by solving the first order differential mass balances with the help of a simulation programme that calculates the zero values of these equations. The three steady-state parameters substrate (S), product (P) and biomass (X) are found by regression analysis. This method permits faster calculations than classical numerical methods. For all calculations an IBM (Boca Raton, Fla, USA) personal computer AT was employed. The software programme STESTA (STEady STAte), which was developed in our Institute, was used for the calculations.

Results and discussion I n the case o f a c o m b i n e d i n h i b i t i o n b y s u b s t r a t e a n d p r o d u c t the c o n v e n t i o n a l M o n o d e q u a t i o n (Eq. 1), w h i c h d e s c r i b e s g r o w t h o n l y as a f u n c t i o n o f t h e limiting s u b s t r a t e c o n c e n t r a t i o n (S), has to b e m o d i f i e d : S ~t=~max. S + / ~ ~

~ ~---~max S -~-Ks + s 2 / g i ture medium (batch) was prepared as described by Klemps et al. (1987). The fermentation medium (continuous culture) used in this investigation was previously developed by optimization of the trace element requirement and contained only mineral components (no yeast extract or peptone). The compounds are listed in Tables 1 and 2. Solutions B to F were autoclaved separately as stock solutions and added to the main solution A. Sodium dithionite (Na2S204) was used as a reducing agent. Bioreactor. The bioreactor (Fig. lb) was composed of a glass vessel (0.5-1 volume) equipped with a pH electrode, a pH titrator using 5.0 M NaOH, a level control system with a conductivity sensor, a thermostat and a weighing machine for flow-rate control. Cultures grown overnight were suitable for inoculation (10% v/v). The sterile feed flow was passed through the reactor at a constant dilution rate. Approximately five to ten residence times were required for steady-state conditions in the bioreactor. Wall growth of the bacteria could be avoided by intensive stirring at 300 rpm with a special magnetic stirrer. A temperature of 66 ° C and pH 6.4 were kept constant. Feed concentrations of 50 g/l and 5 g/1 glucose were used for these experiments. Dilution effects caused by added alkali were taken into account. Analytical methods. Bacteria growth was determined by measuring the optical density (OD) of the culture broth at 660 nm in a Per-

(1)

1

--

Pmax

- - m s . Y(x/s)max (2)

T h e p r o d u c t i n h i b i t i o n t e r m (Eq. 2) was first i n t r o d u c e d b y L e v e n s p i e l (1980). It p r e d i c t s the e x i s t e n c e o f a m a x i m u m p r o d u c t c o n c e n t r a t i o n (Pmax) w h e r e n o f u r t h e r b a c t e r i a l g r o w t h is p o s s i b l e . F o r this r e a s o n Eq. 2 is o n l y v a l i d f o r c o n c e n t r a t i o n s l o w e r t h a n Pmax. T h e inhib i t i o n c o n s t a n t (n) as a n e x p o n e n t i a l v a l u e a l l o w s a more general description of different types of inhibitions. F o r e x a m p l e the m o d e l c a n d e s c r i b e l i n e a r (n = 1), h y p e r b o l i c (n < 1) a n d p a r a b o l i c (n > 1) t y p e s o f i n h i b i t i o n ( L u o n g 1985). T h e last t e r m (ms. Y(x/S)max) c o n s i d e r s t h e m a i n t e n a n c e energy, w h i c h c o n s u m e s a constant amount of substrate and reduces the apparent g r o w t h rate (~t) in c o m p a r i s o n to t h e m a x i m u m t h e o r e t i c a l g r o w t h rate (~.~max)o This m a i n t e n a n c e m o d e l for t h e s p e c i f i c g r o w t h rate (Ix) was first i n t r o d u c e d b y Pirt (1975). I n h i b i t i o n b y s u b s t r a t e was t a k e n into a c c o u n t b y a s u b s t r a t e i n h i b i t i o n term. S u b s t r a t e c o n s u m p t i o n a n d p r o d u c t f o r m a t i o n are p a r t l y g r o w t h - l i n k e d p r o c e s s e s , b e c a u s e acetic a c i d is the e n d - p r o d u c t o f the e n e r g y m e t a b o l i s m o f the b a c t e ria. E n e r g y is n e e d e d f o r g r o w t h as well as for m a i n t e n a n c e p r o c e s s e s . S o m e e x a m p l e s for t h e l a t t e r a s p e c t

346 are turnover of cell wall substances and other cell components and preservation of osmotic gradients: Ix q + - - -

Y(x/S)raax

+m+-

. Ix. . . q v - Ycx/P)max -I-mp

( S o - S). Ix

Ix

X

Y(x/s)

. P'Ix X

0.500.40

(3)


. . . . rn~ and mp by non-linear regression, transformation of Eqs. 3 and 4 is necessary. It must be pointed out that under all steady-state conditions in a continuous culture Ix is identical to the dilution rate (D) so that the two values are interchangeable.

o o

0.40-

Y{X/P> =

IX" Y(X/S)max Ix + m~. Y(X/S)max Ix"

Y(X/P)max

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=

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(5)

015

012

= D*Y(X/P)mox/(D+

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s 0.50-

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0.5

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~x/e)max

,1

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o:s

d i l u t i o n rate D

(7)

The yield coefficient Yw/s) (Eq. 7) is identical to the product selectivity and is defined as the ratio of product formed to substrate consumed. As Ycx/s) and Y is also a function of Ix. A comparison of experimental and calculated data of the yield coefficients depending on D is demonstrated in Fig. 2. The optimal model parameters in Eqs. 5 and 6 are listed in Table 3. The resulting acetate production by maintenartce processes (rnp) was found to be lower than the corresponding substrate demand (m+). The reason is that glucose is not only serving as an energy source for growth and maintenance (end-product acetate) but also as a carbon source, resulting in products other than acetate. A more detailed description of this context is given in the literature (Haughney et al. 1988). A plot of tx in relation to product and substrate concentrations under steady-state conditions is shown in Figs. 3 and 4. The parameters from Eqs. 3 and 4 are used as constant values in model Eq. 2, whereas the parameters k~, k~, n, P~,~ and Ixm~ are found by non-linear regression. The values are listed in Table 4. Both tx and the specific acetate production rate showed a hyperbolic decline with increasing acetate concentration as the result of the product inhibition. The maximum acetate concentration permitting positive growth and dilution rates is calculated as 34.5 g/1 (575 m ~ ) and is in agreement with the experimental data. As the model

0 I

I

ols [h -1]

Fig. 2. Yield coefficients of biomass and acetate production from glucose

Table 3. Kinetic parameters of the yield coefficients Y~x/s) and Y~x/P) for growth and product formation fitted by non-linear regression methods (Nelder and Mead 1965) Constant

Value

Dimension

rns rnp

0.423 0.333 0.276 0.426

[g/(g-h)] [g/(g. h)] [g/g] [g/g]

Y~x/s)~,x Y~x/P)m,x

even allows negative values for ~t, the calculated Pmax has a more theoretical character. In continuous culture without biomass retention only positive values for tx are possible for reaching steady-state conditions for biomass and other compounds. Apart from limitations and inhibitions the theoretical Ixm,x is lowered by a constant maintenance rate (m~'Y(x/S)max) that consumes biomass. The maximum apparent value for Ixmax without the influence of inhibitions and limitations is therefore computed as 0.678 h - ' . As low steady-state product concentrations (low conversion of glucose) correspond to high glucose concentrations the maximum value for Ix in Fig. 3 is 0.55 h - ' . The reason for this additional

347 1.01

/ ~ = f f r n a x * S / ( K s + S * ( S + 1/Ki))*(1 - P / P m a x ) "

substrate inhibition the single types of inhibition are also plotted. The resulting mass balances for the continuous stirred tank reactor are listed below:

2.0

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q(p) = p . / Y ( X / P ) + m p

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[g/I]

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Fig. 4. Influence of the steady-state substrate concentration on Ix

~

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Talfle 4. Kinetic parameters of the specific growth rate IXfitted by non-linear regression methods (Nelder and Mead 1965) Value

Dimension

~max k~

0.795 0.176 250.0 4.17 94.8

[h - ~] [g/l] [g/l]

k, n Pmax

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reduction is an additional substrate inhibition caused by glucose, although it is much smaller than the corresponding product inhibition. Calculating the influence of S on ~t demonstrates that product inhibition is dominating over the much smaller substrate inhibition. In addition to the calculated and measured curve including both product and

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,____~____*-. . . . . . £____'_.:, -

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Fig. 5. Steady-state data: calculated and measured values of glucose, acetate and biomass at varying D in a continuous culture of ,4. kivui

348 o •

100-

50 g/I glucose 5 g/I glucose

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and Y~x/P) are influenced by ~t. Consequently, selectivity of product formation (Y~P/S)) at lower D is higher, because less glucose is converted to biomass. At 5 g/1 glucose as feed concentration, the measured and computed values look like a conventional continuous culture fermentation (Pirt 1975). As only a little acetate can be produced from the low feed concentration, product and substrate inhibition is low, li is nearly exclusively controlled by the limiting substrate and therefore contrasts with the situation at a higher feed concentration. The calculated data for qp, qs, Y~x/s) and Y~x/~,) give the same values for both feed concentrations because D is the only independent parameter in Eqs. 3-7. The good agreement between calculated and measured data indicates that the model asumptions are appropriate to describe the continuous fermentation process with A. kivui bacteria. By using a mineral medium it could be demonstrated that homoacetate fermentation produces lower acetate yields than the theoretical value of 1 kg acetic acid (3 mol)/kg glucose (1 mol) (Gottschalk 1986). The maximum product yield observed in this investigation was 78.7% (2.35 mol acetic acid/mol glucose). The main reason for this phenomenon was the fact that biomass production requires a considerable part of the converted glucose. An additional effect is caused by the maintenance metabolism, in which consumed glucose is not only converted to acetate but also to other products that are needed to keep the bacterial cells alive. As a consequence, product selectivity decreases with increasing dilution rate and biomass production.

._©

~.4~

-0.40 ~

v(~/~)

~-~ ~.2~ ~1_ >-

~

~

0.00

o.o

•

_

, ~ _ ~ - ~ - ~ o ~

oi ~

o~ dilution

o~ rote

-

~

-

...........

-0.20 ~. >( >-

oX D

o'.5

o'.~

0,00

[h -1]

Fig. 6. Steady-state data: calculated and measured values of space-time yield, qp, qs, Yx/s and YP/s at varying D in continuous culture of A. kivui

strated in Figs. 5 and 6. At higher feed concentration (50 g/l), conversion of glucose was incomplete even at very small D. For this reason ti and qp were influenced mainly by the inhibiting product and not by the limiting substrate. As the result of decreasing product inhibition, the biomass concentration was nearly linear, falling off with increasing D. At low D the maintenance energy is responsible for decreasing biomass concentrations (Pirt 1975). Another consequence of the opposite trends of biomass concentration and qp is the fact that the spacetime yield has a clear maximum at medium D. As mentioned before, maintenance metabolism favours the production of acetate in comparison to biomass especially at low D. So the yield coefficients Y~x/s)

Acknowledgements. The authors gratefully acknowledge the assistance of Mr. B. Joksch and Mr. U. Giesecke, Research Center, Milich, for the supply of software concerning the mathematical calculations.

Symbols: D [h-1], dilution rate; K~ [g/l], substrate inhibition constant; K~ [g/l], substrate saturation constant; mp [g/(g-h)], maintenance constant for product formation; ms [g(g-h)], maintenance constant for substrate consumption; ~t [h-l], specific growth rate; ~max [ h - 1 ] , maximum specific growth rate; n, product inhibition constant; P [g/l], product concentration; Pm,x [g/1], maximum product concentration; qp [g/(g.h)], specific product formation rate (g acetate/(g biomass-h)); qs [g/(g" h)], specific substrate consumption rate (g glucose/(g biomass.h)); S [g/l], substrate concentration; So [g/l], influent substrate concentration; t [h], time; X [g/l], biomass concentration (dry weight); Y(e/s) [g/g], yield coefficient defined as mass of product formed per mass of cells grown (product selectivity); Y~x/e~ [g/g], yield coefficient defined as grown cells per mass of product formed; Y~x/s) [g/g], yield coefficient defined as cells grown per mass of substrate consumed (biomass selectivity); Y~x/mm,x [g/g], maximum yield coefficient Y~x/ P)'~ Y(x/S)max [g/g], maximum yield coefficient Y~x/s)

References Gottschalk G (1986) Bacterial metabolism. Springer, Berlin Heidelberg New York Haughney HA, Dziewulski DM, Naumann EB (1988) On material balance models and maintenance coefficients in CSTRs with various extents of biomass recycle. J Biotechnol ~: 113-130

349 Klemps R, Schoberth SM, Sahm H (1987) Production of acetic acid by Acetogenium kivui. Appl Microbiol Biotechnol 27: 229234 Leigh JA, Mayer F, Wolfe RS (1981) Acetogeniurn kivui, a new thermophilic hydrogen-oxidizing, acetogenic bacterium. Arch Microbiol 129:275-280 Levenspiel O (1980) The Monod equation: a revisit and a generalization to product inhibition situations. Biotechnol Bioeng 22:1671-1687 Ljungdahl LG, Carreira LH, Garrison RJ, Rabek NE, Wiegel J

(1985) Comparison of three thermophilic acetogenic bacteria for production of calcium-magnesium acetate. Biotechnol Bioeng Symp 15:207-223 Luong JHT (1985) Kinetics of ethanol inhibition in alcohol fermentation. Biotechnol Bioeng 27:280-285 Nelder JA, Mead R (1965) A simplex method for function minimization. Comput J 7:308-313 Pirt SJ (1975) Principles of microbe and cell cultivation. Blackwell Scientific Publications, Oxford, pp 63-81