Jotrm,r.,~x oF ~rTATION AND BIOENGINEERING Vol. 77, No. 4, 425--427. 1994

Production of Poly-D-3-Hydroxybutyric Acid from Carbon Dioxide by a Two-Stage Culture Method Employing

Alcaligenes eutrophus ATCC 17697 T KENJI TANAKA AND AYAAKI ISHIZAKI*

Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan

Received 27 October 1993/Accepted 10 December 1993

A two stage culture was examined as a new method for the cultivation of Alcaligenes eutrophus to produce puly-D-3-hydroxybutyric acid, P(3HB), from H2, Oz and COz without danger of gas explosion. In this culture method, the microorganism was grown beforehand in an organic medium under heterotrophic conditions for exponential growth, and the cells were then cultivated for P(3HB) accumulation under autotrophic conditions in which the Oz concentration in the substrate gas was below the explosion limit of 6 .9~ . Although the KLa of the fermentor used was not high (ca. 340 h-l), it was possible to obtain P(3HB) at a relatively high production rate (0.56-0.91 g.dm -3. h -1) and concentration (15.23-23.91 g.dm -3) in the autotrophic stage, even under such a low 02 concentration.

Polyhydroxyalkanoic acid (PHA) is a potential raw material for biodegradable plastic (1-3). A hydrogen-ox- idizing bacterium, Alcaligenes eutrophus, is able to grow using H2, 02 and CO2 in an autotrophic culture and utilizing various organic carbon sources under in hetero- trophic conditions. The microorganism accumulates poly- (D-3-hydroxybutyric acid), P(3HB), which is a kind of PHA in the cells under nutrient- or oxygen-limited condi- tions (4). Production of P(3HB) from CO2 may simulta- neously contribute to solving two environmental pollu- tion problems; the elevation of CO2 concentration in the atmosphere and the disposal of non-degradable plastic waste. However, for the practical application of P(3HB) production from CO2 employing A. eutrophus, the dan- ger of substrate-gas explosion needs to be eliminated from the fermentation system. To avoid a gas explosion, 02 concentration in the gas phase should be maintained below 6.9% by volume (5). Unfortunately, the 02 trans- fer rate in the culture system becomes very small under such low 02 condition, which causes a serious decrease of biomass productivity. Furthermore, a high P(3HB) concentration cannot be attained if exponential cell growth ceases at a low cell concentration due to 02 limi- tation because the P(3HB) production rate decreases with increased P(3HB) accumulation in the cells and eventually ceases when the cellular content of P(3HB) reaches about 90% by weight (6, 7). Therefore, produc- tion of P(3HB) becomes very poor in a cultivation in which the 02 concentration in the gas phase is main- tained below 6.90//00 (v/v) to minimize the hazard of an ex- plosion. In fact, a fermentor with a KLa of 2,970h -~ was found to be necessary to obtain a P(3HB) produc- tion rate of 3 .0g .dm-3.h -~ in a high-cell-density batch culture of A. eutrophus using an explosion-proof type of fermentation system in which the 02 concentration was maintained below 6.9% (v/v). A fermentor with such a high KLa is not practical in industrial-scale fermentation processes because the power consumption for agitation

* Corresponding author.

would be very large. Hence, in this study, we investigat- ed a new culture method, which consists of a heterotro- phic culture for exponential growth and an autotrophic culture for P(3HB) accumulation, in order to obtain a high P(3HB) concentration using a fermentor with nor- mal KLa under safe culture conditions.

The microorganism used in this study was A. eutro- phus ATCC 17697 T. Mineral medium (8) containing fructose and a second mineral medium containing no carbon source were used for heterotrophic growth and autotrophic P(3HB) accumulation, respectively, in the two-stage cultivation, which was carried out as follows; Heterotrophic cultivation was first performed aerobically for exponential growth of A. eutrophus. The seed was prepared in a test-tube shaking culture with 5 ml of the fructose medium at 30°C for 16 h. The seed was then in- oculated into 100 ml of the fructose medium in a micro- jar fermentor (total volume, 210ml; Biot Co. Ltd., Tokyo) and air was fed into the medium at a rate of 2 vvm. The agitation speed and temperature were main- tained at 1,400 rpm and 30°C, respectively. The pH was automatically controlled at 7.0 by feeding 4% (w/v) NH4OH solution using a pH controller (PHC-2201, Blot Co. Ltd., Tokyo). After the fructose in the medium was exhausted, the culture broth was centrifuged at 8,000 x g for 10 min. The harvested cells were suspended in 100 ml of sterilized mineral medium and then the suspension was transferred into a fermentor equipped with a recy- cled gas closed-circuit culture system (9). A gas mixture of H2, O 2 and CO2 in which the 02 concentration was below the lower limit for explosion, was supplied to the fermentor and autotrophic cultivation was performed (second culture stage). The temperature, pH, agitation speed and aeration rate of the substrate gas mixture in the second culture stage were the same as those in the heterotrophic stage. Under the above conditions, the DO2 concentration in the culture liquid is too low to be detected by a membrane electrode, and the microorgan- ism accumulates P(3HB) in the cells under the O2-limita- tion (10). Analyses were carried out according to the

425

426 TANAKA AND ISHIZAKI

TABLE 1. Results of two-stage culture for PHB production from CO 2

J . FERMENT. BIOENG.,

Heterotrophic stage Autotrophic stage Concentration Initial fructose Cell ~m~x Gas composition PHB PHB content

Experiment concentration concentration Cells PHB productivity in cells no. (g.dm -3) (g.dm 3) (h-l) H2102:CO 2 (g.dm_3) (g.dm_3) (g.dm-3.h l) (~oo)

1 10 4.3 0 . 1 9 86.5:4.9:9.8 26.3 21.6 0.556 82.1 2 20 9.8 0.34 83.0 : 5.3 : 10.6 27.3 15.2 0.684 55.7 3 30 14.9 0.12 84.1 : 6.7 : 10.3 42.5 23.9 0.906 56.3

methods described in our previous papers (9-11); the gas mixture composition was determined by a gas chro- matographic analyzer equipped with a TCD sensor (GC- 8A, Shimadzu Co. Ltd., Tokyo). The DO2 concentration was measured by a membrane-type DO2 electrode (Biot Co. Ltd., Tokyo). The cell concentration was deter- mined by measuring the optical absorbance of culture broth at 562 nm and conversion to g-dry cell weight per 1-dm 3 broth. The P(3HB) concentration was determined by using the GC-8A sensor with a flame ionization de- tector after methanolysis treatment. The fructose concen- tration in the culture liquid was determined by the method of Somogyi-Nelson (15).

Figure 1 shows the fermentation time course of A. eu- trophus in the two-stage culture system, in which 10 g. dm -3 of fructose was used as the carbon source for heterotrophic growth. The highest specific growth rate observed during heterotrophic growth was 0.19h - ' , which was almost equal to the highest growth rate of the microorganism in fructose medium reported in a previ- ous study (12). Since the DO2 concentration in the cul-

0.20 . .... o .... .°° °°°°°°.

~ °°°°

0.i0

Partial pressure of dissolved oxygen

% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00

30 --

-~ ---A-- B- ) , 4A 2 5 - - /

/ Cells

0

_ PItB

¢/1

~'{ i 0 Fructose

~ ~ $~ / / P r O t e i n

~ 0 0 i0 2 0 8 0 40 5 0 6 0 ' 7 0

Cultivation time (hi

FIG. 1. Time course of a two-stage culture of A. eutrophus using 10 g. dm -3 fructose medium in the heterotrophic stage (indicated by "A") and a substrate gas mixture composed of H2:O2:CO2 = 86.5:4.9:9.8 in the autotrophic stage (indicated by "B'). Sym- bols: concentrations of cells (e), P(3HB) (©), protein (4), fructose (t3) and dissolved oxygen in the culture liquid (o).

ture liquid was always above the critical concentration for the microorganism, P(3HB) was not accumulated in the exponentially growing cells. When the fructose was almost consumed, the cell concentration was about 4.3 g-dry cells.dm-L The autotrophic culture for P(3HB) accumulation was carried out by feeding a gas mixture of H2 : 02 : CO2=86.5 : 4.9 : 9.8. After 1.5 h of autotro- phic cultivation, the concentration of the cells and that of P(3I-IB) began to increase. In the autotrophic stage it was not possible to determine the DO2 concentration with a membrane electrode because the concentration fell below 0.36ppm. The cell and P(3HB) concentra- tions were 26.3g-dm -3 and 21.6g.dm -3 respectively, [the P(3HB) content in the cells was 82.1% (w/w)], after 40 h cultivation. The increase of biomass concentra- tion in the autotrophic stage was found as linear kinetics due to oxygen limitation. The protein concentration scarcely increased during the autotrophic stage, and slightly decreased at the end. In previous reports (5, 10- 14) on the fermentative production of P(3HB), the pro- ductivity of P(3HB) was 0.08-2.11 g. dm -3. h -1 in hetero- trophic culture and 0.04-1.54 g. dm -3. h-1 in autotrophic culture. The highest growth rates in the heterotrophic and autotrophic cultures were obtained by using recombi- nant E. coli and glucose-LB medium and by using a fer- mentor with a very high KLa, respectively. In this ex- periment, the fermentor used was a conventional type (sulfite KLa=340h - ' ) and the 02 concentration in the gas mixture was much lower than that of air. Never- theless, a relatively high P(3HB) production rate was obtained. The average productivity of P(3HB) in the autotrophic stage was about 0 .56g-dm-3 .h-L From the fermentation result, it is thought that almost the same P(3HB) production rate could be obtained by using a fer- mentor with a KLa of about 700 h - ' with the 02 concen- tration in the gas phase kept at around 2-3% (v/v). Cul- ture experiments using 20 and 30 g-dm -3 fructose media in heterotrophic stage were carried out (Table 1). In all the culture experiments, A. eutrophus accumulated P(3HB) in the cells at relatively high production rates under autotrophic conditions after heterotrophic growth. The specific growth rate ( p = 0 . 3 4 h - ' ) was obtained when using 20g.dm -3 fructose medium. A fructose concentration in the medium above 20g-dm -3 resulted in a decrease of the growth rate in the heterotrophic stage, with increasing fructose concentration (data not shown). Mulchandani et al. reported that the growth rate of A. eutrophus in a fructose medium was affected by the C/N ratio in the medium. Growth is inhibited under high fructose concentration conditions (12). As shown in Table 1, P(3HB) productivity during the autotrophic stage increased as the fructose concentration in the heterotrophic stage and the 02 concentration during the autotrophic stage increased. However, in these cultivations the final P(3HB) concentration did not

VoL 77, 1994 NOTES 427

increase, and even decreased in the cult ivation using 20 g . d m -3 fructose medium. As a result, in the cultiva- tions using 20 and 3 0 g . d m -3 fructose media in the heterotrophic stage, the final P(3HB) contents o f the cells were not as high as 82.1%. This result cannot be explained well for the present. Generally, growth and P H A accumulat ion in A. eutrophus is damaged under the condi t ion o f a high organic-carbon source concentra- tion. The growth abil i ty of the microorganism under autot rophic condit ions might be damaged during long heterotrophic cult ivation. The reason for the decrease in the final P(3HB) content in the cells with increasing fruc- tose concentrat ion in the medium is now being investigat- ed. COz was evolved f rom fructose during the heterotro- phic stage in the culture method. However, the two-stage culture system works as a COz absorpt ion system as a whole because the amount o f CO2 consumed during the autot rophic stage was about two times larger than the amount o f COe generated during the heterotrophic stage to obtain the same amount o f biomass. We used fruc- tose as carbon source during the heterotrophic stage so that a high growth rate could be obtained. However since fructose is expensive in the fermentat ion industry, it is possible to use some other more economical carbon source instead of fructose for the heterotrophic growth o f the microorganism.

In this study, we showed that a two-stage culture method enabled the product ion o f P(3HB) f rom CO2 with a relatively high level o f product ivi ty while keeping O2 concentrat ion in the gas phase below the lower gas- explosion limit using a conventional type o f fermentor. We are now investigating the continuous product ion o f P(3HB) by the use o f this culture method.

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

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