Production of PoIy(~-3=Hydroxybutyrate) from COP, H2, and O2 by High Cell Density Autotrophic Cultivation of Alcaligenes eutrophus

Kenji Tanaka and Ayaaki Ishizaki* Department of Food Science and Technology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 872, Japan

Toshihisa Kanamaru and Takeharu Kawano Research and Development Institute, Saibu Gas Co., Ltd., Higashihama, Higashi-ku, Fukuoka 8 12, Japan

Received June 23, 1994lAccepted October 7, 1994

Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(o-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible oc- currence of gas explosions from the fermentation pro- cess. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a basket- type agitator, resulting in a K,a value of 2970 h-’. Oxy- gen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g . dm-3 of the cells and 61.9 g . d ~ n - ~ of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. 0 1995 John Wiley & Sons, Inc. Key words: poly(o-3-hydroxybutyrate) P(3HB) Alcali- genes eutrophus gas explosion autotroph hydrogen- oxidizing bacterium * carbon dioxide fixation

INTRODUCTION

Microbial polyesters have recently attracted considerable attention and interest because of their potential use in in- dustrial manufacture of biodegradable plastics. Many bac- terial strains have now been found to possess the ability to accumulate polyhydroxyalkanoic acids (PHAs) in the cells. Among PHA-producing bacteria, Alcaligenes eutrophus is the best characterized and most widely employed strain in studies of PHAs. This microorganism was originally iso- lated as a hydrogen-oxidizing bacterium, which could au- totrophically grow using carbon dioxide, oxygen, and hy- drogen as substrates and accumulates poly-~-3-hydroxybu- tyric acid, P(3HB), in the cell under oxygen- and/or nutrient-limited culture conditions. Production of biode-

* To whom all correspondence should be addressed.

gradable plastics employing carbon dioxide-assimilating A . eutrophus, therefore, has the potential to contribute, to a great extent, in the solution of two environmental pollution problems: (i) increased CO, levels in the atmosphere, and (ii) disposal of nonbiodegradable plastic waste. However, considerable technological challenges need to be overcome before there is practical application of autotrophic cultiva- tion of PHA-producing A. eutrophus on a large and eco- nomical scale. Among them, the problem of loss in utili- zation efficiency as the substrate gases are exhausted in the culture vessel, and the potential of a serious gas explosion occurring, need to be dealt with in particular. This problem of gas exhaustion was solved by using a recycled-gas, closed-circuit culture system for the autotrophic cultivation of A . eutrophus.’ The recycled exhausted gas mixture is reused by the microorganism. We are presently involved in the search for a solution to the other major problem of the possibility of gas explosion occurring.

We report here a strategy and system set up to carry out autotrophic cultivation of a hydrogen-oxidizing bacterium under safe culture conditions, by eliminating the potential for the substrate mixture in the culture vessel exploding.

MATERIALS AND METHODS

Strategy to Prevent Gas Explosion

The best precaution against a combustible gas mixture com- posed of hydrogen, oxygen, and carbon dioxide exploding in a culture system is to maintain the composition of the gas phase in the culture system out of the explosion range. A substrate gas mixture will not explode if the oxygen con- centration is lower than about 6%-referred as the “lower explosion limit” for oxygen. l8 The lower limit for oxygen in the substrate gas mixture used for cultivation of hydro- gen-oxidizing bacteria was determined to be 6.9% in our experiment using the modified Hempel apparatus for anal- ysis of fuel gas and natural gas (Japanese Industrial Stan-

Biotechnology and Bioengineering, Vol. 45, Pp. 268-275 (1995) 0 1995 John Wiley i3 Sons, Inc. CCC 0006-3592/95/030268-08

duds K 2301-1980), whereas that for hydrogen was about 4%. Safe autotrophic cultivation of A. eutrophus can, there- fore, be accomplished if the concentration of oxygen or hydrogen is maintained below these lower limits, respec- tively. However, low hydrogen concentration and/or high oxygen concentration in the substrate gas has been found to inhibit seriously the cell growth of A. eutrophus.' Hence, it is desirable to keep the proportion of oxygen in the substrate gas mixture below 6.9% from a growth kinetics point of view. This proportion of oxygen is favorable to the growth of the microorganism, because the maximum specific growth rate of p = 0.42 h-', under the autotrophic con- dition, is obtained at a dissolved oxygen concentration of about 1.7 ppm (equivalent to 4.9% oxygen in the gas phase). The maximum production rate of biomass (grams dry weight dm-3 * h-') in aerobic fermentation depends on the stoichiometry for biomass formation and the oxygen transfer rate in the culture system. Oxygen transfer rate (OTR) depends on the volumetric coefficient of oxygen transfer (KLa) and the driving force of oxygen from the gas phase into the liquid phase, so then the maximum produc- tion rate of biomass is determined by the driving force of oxygen from the gas into the liquid phase and the KLa of the culture system. When the oxygen concentration in the used gas mixture is limited to 6.9%, the maximum production rate decreases to one third that using air. Therefore, to cultivate hydrogen-oxidizing bacterium without the associ- ated risk of the substrate gas exploding through the main- tenance of low levels of oxygen concentration, the KLa of the fermentor should be very high to compensate for the decrease of "driving force" of oxygen. The stoichiometry equation^',^ for the formation of the exponentially growing cells and P( 3HB) in autotrophic culture of A. eutrophus had been determined to be exponential cell growth:

21.4 H, + 6.2 0, + 4.1 CO, + 0.8 NH, + c4.1H7.1N0.8 + 18.7 H2°

and P(3HB) accumulation:

33 H2 + 12 O2 + 4 CO, +- C4H602 + 30 H20.

From the above equations, the KLa value for a fermentor to produce P(3HE3) at a production rate of 1.0 g * dm-3 * h-', while using a gas mixture with an oxygen concentration of 6.9%, has been estimated to be 1940 h-'. Such a high KLa cannot be attained by using a standard type of fermentor. Thus, in this study, a specially designed agitation system was applied for safe autotrophic cultivation of A. eutrophus using a bench plant to gain a very high KLa value without the concurrent increase in energy consumption required for agitation.

Agitation system

The specially designed agitation system used in the set-up was a doughnut-shaped basket-type agitator, already used in several studies of aerobic fermentation in Japan."*12 This agitation system was provided by Biott Co., Ltd. (6-5 Nish-

igokencho, Shinjuku-ku, Tokyo 162). Figure 1 shows a diagram of the agitation system. The agitation system was composed of a doughnut-shaped basket made of stainless- steel net (mesh size 4 mm) and two-propeller-type impellers equipped with four blades, respectively. The diameter and the height of the basket was 70 mm and 80 mm, respec- tively. The position of the propeller-type impellers were variable on the shaft of the agitator. The KLa of the fermen- tor equipped with the agitation system was measured by the sulfite oxidation method while changing the position of the impellers. Several factors associated with the agitation sys- tem and agitation conditions affect the value of KLa. The effects of aeration rate, agitation speed, mesh size of the net, and size of the basket on KLa and the power consump- tion for agitation have already been investigated by re- searchers at Biott Co., Ltd. The agitation system was op- erated used at the optimum levels for the various factors described above.

Cultivation System

The cultivation system consisted mainly of a bioreactor, a gas recycling system, and a gas chamber. A glass jar fer- mentor (total volume 2 dm3) was used as the reactor. The iron-bell-type gas chamber (total volume 2 m3), filled with hydrogen, oxygen, and carbon dioxide gases, was stood in

E E i i 8

1 -113 mm-

Figure 1. Diagram of a fermentor equipped with the basket-type agita- tion system. I-Fermentor vessel; 2-sparger; %impeller; "basket net; 5-DO electrode; b p H electrode; 7-baffle plate; 8-sterile filter; %sampling port; 10-gas inlet; 1 1-gas outlet.

TANAKA ET AL.: PRODUCTION OF P(3HB) FROM CARBON DIOXIDE 269

saline solution. The purity of the gases employed was 99.9%. The gases were supplied from compressed gas cyl- inders (volume size 7 m3). Gases in the chamber were mixed by a pump (BA-106 type, Iwaki Co., Ltd., Japan) to homogenate the gas composition within the chamber. The gas mixture was then fed into the jar fementor through a sterile filter (LS Filter, Pall Co., Ltd., Japan) by a circu- lating pump (BAS-106 type). The gases were utilized by the microorganism in the fermentor for metabolic purposes. Unconsumed gas in the fermentor was returned to the cham- ber through a gas return line and was resupplied to the fermentor. The pressure of the gas phase within the cham- ber was kept constant by the sinking of the chamber in the saline water as gas was consumed by the cells.

To improve safety in the cultivation system, several se- curity devices were incorporated into the bench plant. Pres- sure sensors were installed at the gas feed line so that gas in the system could be vented outdoors when the internal pres- sure exceeded 0.5 kg/cm2. All of the electric equipment was explosion-proof. The gas chamber and all associated equip- ment used was installed outdoors and the stainless-steel pipes serving as gas recycling lines were treated with oil insulation. A combustible gas detector was interfaced with a shutdown device in the gas feed line so that gas feeding was interrupted when gas leakage from the system was de- tected.

Microorganism and Medium

The strain used was Alcaligenes eufrophus ATCC 17697=. A mineral medium5 was used for the preculture and main culture.

Culture Condition

Seed culture was prepared using a laboratory-scale, recy- cled-gas, closed-circuit culture system equipped with a 200- cm3 scale jar fermentor under autotrophic culture conditions according to a previous study. One hundred milliliters of the culture broth was used to seed the main culture in the bench plant. Cultivation was carried out under the following con- ditions: working volume was 1 dm3 and pH was maintained at 7.0 by automatic addition of 12% NH,OH using a pH controller (PHC-2201, Biott Co., Ltd.) connected to a peri- staltic pump. Temperature was kept at 30°C, agitation speed was 700 rpm, while the feeding rate of substrate gas mixture was 2 dm3 * min-' (equivalent to 2 vvm).

An a I ysis

The composition of the gas phase in the chamber was de- termined by a Shimadzu Type GC-8A gas chromatograph equipped with two stainless-steel columns (i.d. 4 mm X 6 m) in which a molecular sieve 5 A and Porapack Q were packed. The dissolved oxygen concentration in the culture liquid was monitored with a membrane-type DO electrode (S-type DO electrode, Biott Co., Ltd.). Cell concentration

was determined by measuring the optical absorbance at 562 nm of the culture broth with a spectrophotometer (UV- 160A, Shimadzu Co., Ltd., Japan), and converted to dry cell weight according to a calibration curve prepared earlier during the non-oxygen-limited-culture phase (exponential growth phase). However, during the oxygen-limited culture phase [P(3HB) accumulation phase], cell concentration was determined by measuring the weight of the harvested cells after centrifugation and drying at 60°C. The concentration of P(3HB) was determined by the gas chromatographic method after methanolysis of the dried cells with chloro- form and 4% H,SO, methanol according to the report of Sonnleitner et al." Gas flow rates in the gas circulating lines were monitored by a differential-pressure-type digital flow meter (DM-3300, Cosmo Co., Ltd., Japan). KLa was determined by the sulfite oxidation method under the fol- lowing conditions: working volume 1 dm3, aeration rate 2 vvm, agitation speed 700 rpm, and temperature 30°C.,

RESULTS

The Effect of the Basket-type Agitator on K,a in the Culture System

KLa of the fermentor equipped with the basket-type agita- tion system, measured by the sulfite oxidation method at various positions of the two propeller-type impellers on the agitation shaft, was compared with that of a conventional turbine-type impeller equipped with six plane blades. The positions of the propeller impeller and measured values of KLa are shown in Figure 2. The highest KLa of 2970 h- ' was obtained when the propeller impellers were located above and below the basket, respectively; namely, in posi- tion "A" in Figure 2. This value was several times higher than that of a turbine-type impeller at the same agitation speed. According to the manufacturers, the effect of the agitation system on KLa is explained as follows: The air sparged into culture liquid is enfolded in the basket net by the rotation of the impeller and the water including air bub- bles are vigorously blown out from the basket. When the air bubbles pass through the net, very small bubbles are formed and, as a result, the contact area between air and water increases. In other impeller positions, such an effect is small, because the air bubbles passes directly through the basket.

Foaming of Culture Broth Caused by Using a Basket-Type Agitator

The use of such a high-performance basket-type agitation system, however, caused much foaming of the culture broth. In batch culture of A. eufrophus using a 2W-cm3 scale fermentor with a magnetic stirrer bar (KLa = 720 h- ' at an agitation speed of 1400 rpm and aeration rate of 2 vvm), foaming occurred at a cell concentration of 15 to 20 g ~ m - ~ . In the cultivation using the basket-type agitation system, in contrast, foaming occurred at a cell concentra-

270 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 45, NO. 3, FEBRUARY 5, 1995

KLa4608 h-1 KLa= 1680 h -' KLa=1890 h-I KLa= 680 h-I

Figure 2. at the agitation speed of 700 rpm, temperature of 30°C, and aeration rate of 2 vvm.

&a values for the fermentor at when various positions of the impeller on the shaft of the basket-type agitator. The experiment was carried out

tion of 3 to 5 g dm-3. The foaming caused loss of broth which overflowed into the gas return line via the outlet of the fermentor, interfering with the supply of substrate gas by the gas line. To prevent foaming, an antifoaming reagent is usually added during cultivation, unfortunately, au- totrophic growth of the A. eutrophus is completely sup- pressed by addition of antifoaming reagents such as silicon oil. The usual antifoaming reagents, even when used in small amounts, drastically decreased the dissolved oxygen concentration in the culture liquid of A. eutrophus under autotrophic conditions. Thus, an antifoaming reagent, A-no1 (Biott Co., Ltd), which does not reduce dissolved oxygen concentration of the culture liquid, was used in this study. The use of A-no1 enabled high density cultivation of A. eutrophus without foaming of the broth.

g dmP3 and 10.8 g - dmP3, respectively. The percentage of P(3HB) content in the cell was 52.2% by weight. The time courses for the production rates of cell and P(3HB) and the specific growth rate during cultivation are shown in Figure 4. Both production rates gradually decreased in the latter half of cultivation. The decrease of the production rate was due to the decrease of oxygen concentration in the gas phase. The oxygen concentration in the gas phase was 6.3% at the start of cultivation and gradually decreased during cultivation, leading to a reduction in oxygen transfer rate in the culture system and, as a result, the production rates also decreased with time (Fig. 5). Hydrogen concentration in the gas phase, on the other hand, gradually increased as the fermentation proceeded. In the closed autotrophic culture system of A. eutrophus, the concentrations of oxygen and

Fermentation Results

The initial composition of gas mixture was H,:O,:CO, = 85.2:6.3:8.3, and the volume of the headspace within the system including the gas chamber was 500 dm3. Figure 3 shows the fermentation time course. After inoculation, the microorganism grew exponentially at a specific growth rate of 0.25 h-' without lag phase. When the cell concentration increased to about 10.1 g * dmP3 after 8-h cultivation, the dissolved oxygen concentration detected by a membrane sensor decreased to about 0 ppm. The critical dissolved ox- ygen concentration for A. eutrophus, under the autotrophic condition, is 1.13 ppm and the microorganism accumulates P(3HB) in the cells when the dissolved oxygen concentra- tion is lower than this level.17 The microorganism accumu- lates P(3HB) in the cells under the oxygen-limited culture condition. During the P(3HB) accumulation phase, residual biomass concentration was almost constant and only the P(3HB) concentration increased. After 15-h cultivation, the concentrations of cell and P(3HB) increased to 20.7

z - E * O i 4c

I 0 10 20

Cultivation time (h)

Figure 3. Time course for autotrophic batch cultivation of A. eutrophus using the bench plant equipped with the basket-type agitator. Symbols: (O), cell concentration; (0), P(3HB) concentration; (U), residual biomass concentration; and (-), dissolved oxygen concentration in culture liquid.

TANAKA ET AL.: PRODUCTION OF P(3HB) FROM CARBON DIOXIDE 271

“ I

0 10 20 Cultivation time (h)

Figure 4. (a) Time course for the specific growth rate during cultivation. Symbol: (O), Specific growth rate. (b) Time courses for the production rates of cell and P(3HB). Symbols: (a), cell production rate; and (O), P(3HB) production rate.

carbon dioxide in the gas phase decrease as the fermentation proceeds unless fresh substrate gas is supplied, but hydro- gen concentration increases due to gas consumption by the cells, as shown by the stoichiometry of cell g r ~ w t h . ~ , ~ Max- imum cell production rate during cultivation was 2.47 g * dm-3 - h-’, almost equal to the estimated value from the data of K,a, the concentrations of oxygen in gas phase and liquid phase, and the stoichiometry for cell formation. The overall productivity of P(3HB) of this batch culture was 0.72 g * dm-3 * h-’.

Cultivation by Separate Feeding of Oxygen Gas

Concentrations of cell and P(3HB) and the percentage of P(3HB) content in the cells obtained in the above experi-

100

l0A I

10 20 Cultivation time (h)

Figure 5. Change of gas composition in the chamber during cultivation. Symbols: (A), hydrogen concentration; (m), carbon dioxide concentration; and (D), oxygen concentration.

ment were not very high. Substrate gases were not supple- mented to the culture system during fermentation, and the cell growth and P(3HB) accumulation were suppressed by the shortage of substrate gas, especially oxygen. To over- come this, fresh gas was supplemented with an aim of achieving high cell and P(3HB) yield. Pure oxygen gas was fed directly into the culture liquid separately from hydrogen and carbon dioxide. The method of feeding the gas to the system is shown in Figure 6. Oxygen gas was intermittently supplied through an explosion-proof-type solenoid operated valve regulated by a dissolved oxygen (DO) controller (DOC-2201, Biott Co., Ltd.). The set value of the DO controller was 1.8 ppm at the start of fermentation (equiv- alent to about 5% of oxygen concentration). This was re- duced by manual operation as the fermentation proceeded so that the oxygen concentration in the return line would not exceed the lower limit for an explosion. The time course for this cultivation is shown in Figure 7. After inoculation, the microorganism grew exponentially without a lag phase. When cell concentration increased to approximately 28.2 g - dm-3 after 18 h of cultivation, the dissolved oxygen concentration decreased to 0.4 ppm, lower than the critical value for A. eutrophus, and P(3HB) accumulation in the cells was observed. After that, complete oxygen limitation was achieved by lowering the set point of dissolved oxygen concentration to 0.2 ppm. During the DO-limited culture

1

2 II 3

11

Figure 6. Diagram of the cultivation system and method of feeding the substrate gas. l-oxygen cylinder; 2-hydrogen cylinder; L a r b o n di- oxide cylinder; &gas chamber; 5-fermentor equipped with a basket- type agitator; &DO controller; 7-solenoid valve; &pH controller; %ammonia solution; 10-peristaltic pump; 1 l-circulating pump; and 12-saline water.

272 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 45, NO. 3, FEBRUARY 5, 1995

0.5

0.0

experiment was lower than that of the heterotrophic fed- batch culture of the mutant strain of A. eutrophus using an on-line glucose analyzer, our results showed that a rela- tively high P(3HB) concentration can be obtained by the use of gaseous substrates.

0 1 0 2 0 3 0 4 0 5 0 Cultivation time@)

Figure 7. Time course for autotrophic batch cultivation of A . eutrophus using the bench plant with separate feeding of the substrate gas. Symbols: (O), cell concentration; (O), P(3HB) concentration; (m), residual biomass concentration; and (-), dissolved oxygen concentration in culture liquid.

phase, the residual biomass concentration was almost con- stant. After 40 h of cultivation, cell concentration increased to approximately 9 1.3 g * dm - 3, and P(3HB) concentration was 61.9 g . dmP3. A major proportion of biomass pro- duced under oxygen-limited condition was P(3HB). The final percentage of P(3HB) content in the cell by weight was 67.7%. In this cultivation, although the oxygen concentra- tion in gas phase was maintained at a very low level, the overall productivities of biomass and P(3HB) were 2.28 g . dm-3 * h- ' and 1.55 g . dm-3 * h-', respectively, much higher than those reported for other autotrophic cul- tivations of hydrogen-oxidizing bacteria (Table I). Al- though the overall productivity of P(3HB) obtained in our

DISCUSSION

Figure 8a and b show the time courses for the specific growth rate and the production rates of cell and P(3HB) during the cultivation with separate feeding of oxygen gas to the fermentor. A maximum cell production rate of 5.23 g - dm-3 h-' was observed after 23 h of cultivation. The maximum P(3HB) product ion r a t e was 5 . 0 2 g * dm-3 * h-' after 25 h. These production rates obtained in our experiment were very high compared with those re- ported for biomass production employing hydrogen- oxidizing bacteria. Under oxygen-limited culture of hydro- gen-oxidizing bacteria, maximum biomass production rate depended on the OTR in the culture system and the stoi- chiometry of biomass formation. According to the result of our study, cell production rate of A. eutrophus (ycell; g * dm-3 * h-'), under the autotrophic condition, follows the equation:

ycell = 0.26 OTR (1)

OTR is a product of KLa, Henry's constant, and the differ- ence in partial pressure of oxygen between gas phase and liquid phase (pg and pL respectively); hence, ycell can be expressed as:

(2) ycell = 0.26 - KLa H * (pg - pL)

and the K,a of the fermentor in our experiments was 2970

Table I. Autotrophic cultivations of hydrogen-oxidizing bacteria and fermentative production of P(3HB) using various microorganisms and substrates.

Cell Cell P(3HB) P(3HB) Culture Cultivation concentration productivity concentration productivity Ref.

Strains Substrate method time (h) (g . dm-3) (g . dm-3 . h-') (g . dm-3) (g . dm-3 . h-I) no.

Alcaligenes eutrophus

A . eutrophus Pseudomonas

hydrogenovora A . eutrophus

ATCC 17691 A. eutrophus HI6 A . hydrogenophilus P . hydrogeno-

thermophila A . eutrophus Recombinant

E . coli Protomonas

extroquens A . eutrophus Arotobacter

vinelandii UWD

H2/02/C02

H21021C02 H2102/C02

Hz/02C02

H2/02/C02 H2/02C02 H2102/C02

H2/O2/CO2 Glucose + Methanol

Glucose 5% molasses

LB medium

Batch

Batch Batch

Continuous

Batch Continuous Continuous

Batch Fed-batch

Fed-batch

Fed-batch Fed-batch

25

40 48

70

60 42

121

50 36

25.0

91.3 24.0

18.0

60.0 117.0

223.0

164.0 -

1 .OO

2.28 0.50

0.40

0.26 0.33 3.00

1 .oo 2.19

1.84

3.28 -

-

61.9 -

-

16.0 - -

36.0 89.0

136.0

121 .o 22.0

-

1.55 -

-

0.23 - -

0.60 2.11

1.12

2.42 1.10

14

This study 3

10

15 9 4

6 I

16

8 13

TANAKA ET AL.: PRODUCTION OF P(3HB) FROM CARBON DIOXIDE 273

0.4 4

0 1 0 2 0 3 0 4 0 5 0 Cultivation time(h)

Figure 8. The time courses of the specific growth rate (a); the production rates of cell and P(3HB) (b); and the oxygen concentration in the feed line and the return lines during the cultivation with separate feeding of the substrate gas (c). Symbols: (O), specific growth rate; (O), cell production rate; (0), P(3HB) production rate; (A), oxygen concentration in the gas feed line; and (A), oxygen concentration in the retum line.

h-'. Henry's constant at 30°C is 0.037 g * dm-3 * atm-'; thexfore, Eq. (2) can be rewritten as:

(3)

According to Eq. (3), partial pressure of oxygen in the gas phase under the oxygen-limited condition must be 0.093 atm (equal to oxygen concentration of 9.3%) to obtain a cell production rate of 5.23 g * dm-3 * h-'. However, oxygen concentration in the gas phase of the system was maintained below 6% throughout the cultivation. Pure oxygen gas was directly fed into the medium where it was mixed with the other substrate gases; hence, OTR in the culture system would be higher than that estimated from the KLa value of the fermentor and the oxygen concentration in the gas feed line. In the cultivation in which the gas mixture of hydro- gen, oxygen, and carbon dioxide was fed (without separate feeding of pure oxygen gas), OTR was not very high and the maximum production rates of cell and P(3HB) were 2.47 g - dm-3 - h-' and 2.30 g * dm-3 - h-', respec- tively [maximum concentrations of cell and P(3HB) were 20.7 g - dm-3 and 10.8 g * dm-3, respectively]. Separate

Ycell = 28.6 * (P* - P L )

feeding of oxygen gas to the fermentor is favorable for the achievement of a high biomass production rate in au- totrophic cultivation of hydrogen-oxidizing bacteria, whereas the oxygen concentration in the gas phase is main- tained at very low level.

As shown in Figure 8b, production rates of cell and P(3HB) were very high between 20 and 30 h, but gradually decreased after 30 h. The decrease of production rate was thought to be due to the decrease of OTR in culture system. Figure 8c shows the changes in oxygen concentration in the gas return line and the feed line. Oxygen concentration in the gas return line gradually increased as the fermentation proceeded and it increased to 6% after 30 h of cultivation. The setpoint of the DO controller was reduced so that ox- ygen concentration in the gas phase could not exceed the lower limit. DO concentration in culture liquid decreased to 0 ppm after 30 h of cultivation; therefore, the feed rate of pure oxygen gas could no longer be controlled by lowering the DO setpoint. Because of this, separate feeding of pure oxygen was stopped after 30 h of cultivation. After cessa- tion of oxygen feeding, oxygen concentration in the gas phase decreased, resulting in decreased OTR. This is the cause of the decrease in P(3HB) production rate halfway through the cultivation period, nevertheless P(3HB) accu- mulation continued. After 40 h of cultivation, oxygen con- centration in the feed line decreased to 1.8% and P(3HB) formation stopped. If feeding of oxygen could be controlled so as to maintain oxygen concentration in the gas phase at a constant level, cultivation could be maintained for a longer period of time near the maximum production rate.

Our study showed that P(3HB) could be produced at a high production rate from carbon dioxide by autotrophic cultivation of A. eutrophus using an agitation system, which achieved very high KLa values while maintaining oxygen concentration in the gas phase at levels below that for an explosion. However, the question for scale-up of the special agitation system used in this study, for commercial the pro- cess, is unsolved at present. We are studying the develop- ment of an airlift-type bioreactor achieving high KLa with- out high energy consumption. On the other hand, it was also shown that the composition of the gas phase must be strictly controlled to maintain a high biomass production rate. This can be accomplished by on-line measuring devices coupled with a computer control system.

A part of this study was supported by a Grant-in-Aid for Scien- tific Research from the Ministry of Education, Science and Cul- ture, Japan.

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