초호열성 미생물의 고농도배양을 위한 정적 유가식배양기술

박 찬범, 이 선복

포항공과대학교 화학공학과

Constant-Volume Fed-Batch Operation for High Density

Cultivation of Hyperthermophilic Microorganism

Chan Beum Park, Sun Bok Lee

Department of Chemical Engineering,

Pohang University of Science and Technology

INTRODUCTION

Hyperthermophiles isolated in the last decade have attracted many interests due to their potential use as an important resource for biotechnologically useful products [1,2]. However, cultivation of the hyperthermophiles with controlled bioreactors are rare and culture techniques at high temperatures are still lacking [3-5].

One of the problems encountered in the cultivation of thermophiles is evaporation of the liquid medium. Rapid decrease of the culture volume during fermentation often adversely affects the cell growth due to increased concentration of nutrients and/or inhibitory products in the medium [6].

Fed-batch culture techniques, on the other hand, have been widely adopted for high density cultivation of various microorganisms [7]. In a conventional fed-batch system, culture volume varies with time and this transient condition makes it difficult to analyze the growth kinetics in a fed-batch culture system. In some fed-batch operations, very concentrated feed solution or powder form of nutrient was supplied to the culture to reduce the volume increase.

In this work we have proposed a new fed-batch strategy, called a constant-volume fed-batch (CVFB) system, for hyperthermophile cultivation. In this system, culture volume was maintained at a constant level by compensating the loss of culture volume with feed medium and water. This method not only eliminates the difficulties caused by evaporation of the medium but also resolves the volume-change problem. To explore the applicability of the proposed scheme, we have investigated high density cultivation of Sulfolobus solfataricus, a hyperthermophilic aerobe which normally grows at 75 to 85 oC. We also analyzed cell culture kinetics in the CVFB system.

EXPERIMENTAL METHODS

S. solfataricus (DSM 1617) isolated from volcanic hot springs in Italy  was obtained from the Deutsche Sammlung von Mikroorganismen.  GY3M medium was used as base medium [8]. Cultivation was carried out in a 19-L fermentor (L1523, Bioengineering AG, Switzerland). The inoculum size was 0.6 L and working volume of main cultivation was 13.8 L. Culture temperature and aeration rate were 78 oC and 1.0 vvm, respectively. The exhaust gas was passed through a condenser (surface area: 0.032 m2) and tap water (17-20 oC) was used as a coolant.

Schematic of a constant-volume fed-batch culture system is shown in Fig. 1. Feed medium (reservoir 1) and sterile water (reservoir 2) were fed into the fermentor by peristaltic pump. Feed medium was composed of glucose (360 g/L) and yeast extract (90 g/L). Evaporation rate of the medium (Medium GY3M) was determined in separate experiments, using the same setup as for the main cultivation (19-L fermentor). The loss of culture volume was measured at predetermined time intervals.

RESULTS & DISCUSSION

Evaporation rate

When the exhaust gas was not condensed at 78oC, the evaporation of water due to aeration became significant and its rate was approximately proportional to the aeration rate.  The rate of water loss at the aeration rate of 1.0 vvm was 0.430 liter per liter of medium per day(LLD) at 78 oC. Evaporation rate was reduced with condensing the exhaust gas. When the aeration rates were greater than 1.0 vvm, however, evaporation rate was sharply increased and approached to the value obtained without operating a condenser. This indicates the condensation of water vapor is inefficient at higher aeration rates. On the other hand, evaporation rate at 750 rpm was almost identical to that at 350 rpm, implying that evaporation rate is little influenced by the agitation speed.

Without aeration, evaporation of the liquid medium was insignificant even at 98oC. However, when the air was sparged into the fermentor, evaporation rate was markedly enhanced with increasing temperatures and aeration rates. At 98oC, for example, entire liquid medium was evaporated within 12 h at the aeration of 0.5 vvm.

Analysis of constant-volume fed-batch system

When hyperthermophiles are cultivated in a fed-batch mode of operation, the culture volume can be fixed at a constant level by adding the same amount of water that is evaporated during cultivation. To determine the feed rates of medium (F1) and distilled water (F2), the performance of the CVFB system is analyzed.

A material balance on the cell concentration in the fermentor (see Fig. 1) yields:

d(VRX)/dt = FIN XIN - FEVAPXEVAP + VRRX = VINRIN          (1)

In Eq (1) VR, X and RX represent the reactor (culture) volume, cell concentration and cell growth rate, respectively, and the subscripts IN and EVP denote the inlet stream and the evaporate, respectively. If VR remains constant during the culture, Eq (1) becomes (XIN = XEVP = 0)

dX/dt = RX	(2)

Eq (2) indicates that the cell growth kinetics of a CVFB system is apparently identical to that of a batch culture.

The balance on the rate-limiting substrate is

d(VRS)/dt = FINSIN - FEVAPSEVAP - (1/YX/S) d(VRX)/dt	(3)

where S and YX/S are substrate concentration and the yield coefficient, respectively. Since FINSIN = F1S1 and SEVP ≒ 0, Eq (3) becomes

d(VRS)/dt = F1S1 - (1/YX/S) d(VRX)/dt	(4)

If VR is constant,

dS/dt = F1S1/VR - (1/YX/S) dX/dt	(5)

or

F1 = (VR/S1) [dS/dt + (1/YX/S) dX/dt	(6)

For high density cultivation of microorganisms, it is necessary to keep the limiting-substrate concentration constant at a low level. The residual substrate concentration in the fermentor remains constant when nutrient consumption rate is nearly equal to nutrient feed rate. This is called the quasi-steady state. Since dS/dt ≒ 0 at quasi-steady state, then

F1 = (VR/YX/SS1) RX	(7)

If RX is expressed in terms of the cell mass increment

RX = dX/dt  ≒ □X/□t	(8)

and if YX/S is assumed to be constant, Eq (7) can be rearranged to

F1 = K (□X/□t)	(9)

where K = VR/YX/SS1. The feed rate of the medium can therefore be estimated from cell growth measurement once K is known.

Assuming that the change in the culture volume is mainly attributed to the water in the feeds and the loss of water due to evaporation, the water balance is given by

dVR/dt = FINWIN - FEVAPWEVAP = F1W1 + F2W2 - FEVAPWEVAP	(10)

In order to maintain the culture volume at a constant level, the following constraint should be satisfied.

0 = F1W1 + F2W2 - FEVAPWEVAP	(11)

Since the water content of WEVAP does not significantly differ from that of pure water (WEVAP ≒ W2),

F2 = (FEVAPWEVAP - F1W1)/W2 ≒ FEVAP -□F1	(12)

where □ = W1/W2. Eqs (9) and (12) are the relationships used for the determination of F1 and F2 in cell culture experiments.

Constant-volume fed-batch cultivation of S. solfataricus

A constant-volume fed-batch culture of S. solfataricus was carried out in a 19-L fermentor at 78 oC and 1 vvm. Under culture conditions, the evaporation rate (FEVP) was 0.095 LLD. By cultivating S. solfataricus in a CVFB system, it was possible to obtain maximum cell density of 22.6 g dry cell weight per liter. To our knowledge, this is the highest value reported for hyperthermophiles in fed-batch cultures.

The advantage of the CVFB system over the conventional variable- volume fed-batch system lies in its simplicity; all works required are the monitoring of cell concentration and the controlling of the feed rates based on Eqs (7) and (12). It is expected that the CVFB system may also find its application to the cultivation of other hyperthermophiles including anaerobes. Most anaerobic hyperthermophiles have their optimum temperature around 100oC, and produce inhibitory gaseous products (such as H2 and H2S) that must be removed with inert gas. In such cases, evaporation of the medium can be a serious problem even at lower gas sparging rates due to extremely high culture temperatures and conventional fed-batch operations may result in low cell densities due to increased concentration of inhibitory products in the medium.

REFERENCES

1.	Herbert, R. A. : Trends Biotechnol. 10, 395-401 (1992)

2.	Cowan, D. A : Trends Biotechnol. 10, 315-323 (1992)

3.	Park, C. B. and Lee, S. B. : Theories and Applications of Chem. Eng.,  2(2), 1837-1840 (1996)

4.	Clark, D. and Kelly, R. : CHEMTECH 20, 554-662 (1990)

5.	Stetter, K. O. : ASM News  61, 285-290 (1995)

6.	Sonnleitner, B. and Fiechter, A. : Trends Biotechnol. 1, 74-80 (1983)

7.	Yamane, T. and Shimizu, S. : Adv. Biochem. Eng. Biotechnol. 30, 147-194.(1984)

8.	Kan, E. S. : Master thesis, POSTECH (1995)

Fig. 1. Schematic representation of a constant-volume fed-batch culture system. F : flow rate, S : substrate concentration, X : cell concentration, W : water content, VR : culture volume.