BIOTECHNOLOGY LETTERS Volume 15 No.12 (Dec.1993)pp.1217-1222 Received as revised 26th November

FERMENTATIVE PRODUCTION OF POLY-fJ-HYDROXY- BUTYRJC ACID FROM XYLOSE BY A TWO-STAGE CULTURE METHOD EMPLOYING LACTOCOCCUS LACTIS IO-1 AND ALCALIGENES EUTROPHUS

KENJI TANAKA, KOUJI KATAMUNE, AYAAKI ISHIZAKI* Department of Food Science and Technology, Faculty of Agriculture, Kyushu

University, Hakozaki, Higashi-ku, Fukuoka 812, Japan.

Summary A two-stage culture method employing Lactococcus.lactis IO-1 and Alcafigenes eutrophus was developed for production of poly+hydroxybutyric acid (PHB) from xylose. In this method, xylose was converted to L-lactic acid and acetic acid by Llactis IO-l, and then the organic acids were converted lo PHB by Aeurrophus. When the supernatant of the IO-1 culture broth, containing 10 gdm” L-lactate derived from xylose, was used as medium for A. eutrophus, the concentration of cells increased to 8.5 gdm” in 24 h and 55% of the content in the cells by weight was PHJ3.

Introduction

The microbial polyester PHA (poly+hydroxyalkanoic acid) is a potential raw material

for the manufacture of biodegradable plastic. Many studies have been carried out on PHA from

various points of view. For example, synthesis of new types of PHA, screening and

characterization of producer strains, degradation in soil and sea, and development of a

fermentation process for mass production have been examined. The substrates now used for

production of PHA are glucose, sucrose and fatty acids, as well as other chemical compounds,

such as alkane, chloroalkanoic acids, etc. (Witholt e&al., 1983; Doi et al., 1987 ). For the

practical application of PHA, more economical culture media need to be developed to reduce

production costs. However, it is also important to take into account the origin of possible

substrates because PHA is decomposed to COz or converted to biomass by microorganisms in

the natural environment. Therefore, it is desirable that material derived from agricultural waste

can be the substrate for PHA production. Xylose is one of main components of hemicellulose.

However, at present, few useful products can be obtained from xylose. Laclococcus factis IO- 1, isolated in our laboratory, is able to produce L-lactic acid and acetic acid at a high rate from

xylose and from a mixture of xylose and glucose (Ishizaki er al., 1992b and 1993). The

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hydrogen-oxidizing bacterium Al&genes eutrophus is the most frequently used and best

characterized PHA-producing bacterium and although it is not able to utilize xylose as carbon

source, it is able to utilize lactate. Wb recently succeeded in developing a culture method for

the production of PHA from xylose. This culture method consists of a production stage of L-

lactate from xylose by L.lactis IO-1 and subsequent production of PHA from L-lactate by

A.elclrophus. The PHA produced was confirmed as poly-@hydroxybutyric acid(PHB) by

NMR analysis. We named this method the two-stage culture method. In this paper, we

describe the basic characteristics of growth of A. eurrophus on lactate and show that it is

possible for the microorganism to grow on IO-1 culture broth which contains L-lactate derived

from xylose, at high specific growth rate and accumulation of PHB in cells.

Materials and Methods

Microorganisms The strains used were Lacmoccuslactis IO-1 and Alcaligenes eufruphus KKC 1 7697T. The fermentation properties of L. facris IO-1 with glucose and xylose as substrates have previously been determined in our laboratory(Ishizaki et al., 1992a

and 1992b).

Cultivation Growth characteristics of A. eutrophus on lactate were investigated by test-tube cultivation and pH-controlled jar cultivation. Mineral medium (Siegel ef al., 1984) containing commercially available L- or DL-sodium lactate and CML medium

composed of 5 gdm” yeast extract, Sg*dm” polypepton, 5 gdm” NaCl and DL-sodium lactate were used for test-tube cultivation and pH-controlled jar cultivation. The pH-controlled cultivation was carried out in a glass jar fermenter (total volume 1 dm’; Able Co., Ltd., Tokyo)

under the conditions: temperature, 3O’C; agitation speed, 1,000 rpm; working volume, 500 ml; aeration rate, 0.2 wm; and pH 6.0 maintained by using a pH controller (PHC-2201; Able Co., Ltd.). Twenty-five milliliters of culture broth, prepared in a shaking flask, was used as the seed culture. Two-stage culture for production of PHB from xylose was carried out as follows. First ,pH-controlled jar cultivation of L. lads IO-1 was carried out as described previously (lshizaki et al., 1992b). The medium was composed of 5 g of yeast extract, 5 g of polypepton and 30 g of xylose per dm’of distilled water (CMX medium). When xylose in the medium, has almost all been consumed, the culture broth was centrifuged at 8,000 x g for 15 minutes and the supernatant was returned to the fermenter. Then A.eurrophus ceils were inoculated into the supernatant and cultivated under the conditions described above.

Analyses Concentrations of xylose, L-lactate, cells and PHA were determined by the methods used in the previous study (Ishizaki ef al., 1992b and 1991). Accumulated PHA in the cells was extracted with chloroform after lyophilization and purified by precipitation with 5 volumes of diethyl ether. PHA was also dissolved in CD& and nuclear magnetic resonance (NMR) measurements were recorded on a JEOL GSX400 spectrometer operated at 400 MHz for ‘H and 100 MHz for 13C. All the peaks that appeared on the NMR spectrum originated from D-3-hydroxybutyric acid. Thus, PHA produced from lactate was a homopolymer of D-3- hydroxybutyric acid (PHB).

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Results and Discussion

Growth of A. eutfophus on lactate First, we carried out several

shaking-culture experiments with test tubes lo investigate the basic growth characteristics ofA.

eu~@~ on lactate. Then, to investigate the influence of IacMe concentration on growth of

the microorganism, cultivations at various concentrations of DLlactale were carried out. The

lime courses of fermentation are shown in Figure 1. The concentration of cells decreased as the

OYd 0 10

Cultivation time(h) Fig. 1 Effect of the concentration of DLlactate on

growth of A. euhphus in test-tube culture -4 - s

initial concentration of lactate in the medium

increased, and growth was not observed

when the concentration of DL-lactate was

above 30 g*dmm3. The inhibitory effect of

DLlactate on cell growth was the same as .O 0 5 10 15

that of Llaclate under Ihe same culture Cultivation time (II)

conditions and at the same concentrations Fig. 2 Growth ofA. eutrophus in test-tube culture

in DLlactate and Llactate media

of lactate (Fig. 2).

Production of PHA by A.eutfophus from lactate pH-controlled jar

cultivation was carried out with batchwise operation to investigate production of PHB from

lactate by A. eutrophus. Figure 3(a) shows the time course of fermentation using CMX

medium that contained 5 gdm” DL- lactate. 3 N H2S04 solution was automatically added to the

culture liquid to maintain a constant pH during fermentation. Under such conditions, growth

was rapid and the specific growth rate was 0.61 h”. After 6 hours of cultivation, about 6

gdm-3 of cells were produced. Usually PHB is produced by limiting one nutrient in the culture

medium, such as the nitrogen source, minerals or oxygen. Although neither the nitrogen

source nor oxygen was limiting under our condition(C/N ratio was low and the concentration of

dissolved oxygen was always higher than the critical concentration of dissolved oxygen for the

microorganism), PHB accumulated in the cells to 24% of the dry weight. In the case of

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cultivation using 10 gdm” of lactate, it took 18 hours to consume the lactate in the medium

completely [Fig. 3(b)]. The specific growth rate and the final concentration of cells were 0.42

h” and 7.0 gdm” , respectively. The percentage by weight of PHB in the cells was 30%.

When 20 g-dm-3 of lactate were used, the growth rate was 0.3 h-’ [Fig.3(c)]. After 30 hours of

cultivation, 7 g*dm” of cells and 3.5 g*dm’ of PHB were obtained (the percentage of PHB in

the cells was 50% by weight). However, the yield of cells in terms of consumed lactate was

lower than those obtained in the above two batch cultures. It is not clear why the cell yield for

the culture with 20 gdm” lactate decreased. On the other hand, as described earlier, the cells

accumulated PHB in though nutrient limitation did not occur. The percentage of PHB in the

cells reached 50% by weight at the end of the cultivation.

8

s6 B L-tAclale

i 4 cells

E

z 2 z

PHII

u 0 2 4 6 8 IO

Cultivation lime(h)

Fig. 3-a Fermentation time course of A. eufrophrcs in batch culture using L-lactate (5 &I ) medium

Cultivation time(b)

Fii. 3-b Fermentation time cuurse of A. etirmhus in batch culture using L-lactate (10 g/l )-medium

0 10 i0 30 Cultivation time(h)

B = c ‘0 Cdl z c s u

0 &

PHB

0 IO 2” Cultivation time(h)

Fig. 4 Fermentation time course of A. e&o@us in tbe supernatant of L. &ads IO-1 broth

Fig. 3~ Fermeotaion time course of A. eufrophus in batch culture using L-lactate (20 p/n medium

Production of PHB from xylose by the two-stage culture method

As described above, Aavophus was able to grow on lactate medium at a relatively high rate

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and to accumulate PHB in the cells. Next, production of PHE! from xylose by the two-stage

culture method was investigated. First, pH-controlled batch culture of L. lacfis IO-1 in CMX

medium that contained 30 gdm” xylose were carried out and about 13 gdm” of Llactic acid

was produced (first culture). The centrifuged supematant of the IO-1 broth was returned to the

fermenter, and then A. eufrophus cells that had been grown on lactate medium were inoculated

and the second-stage cultivation for accumulation of PHI3 was carried out aerobically . The

initial concentration of L-lactate in the second-stage culture was about 10 gdm’3. The time

course of fermentation is shown in Fig.4. A. eucrophus grew at a specific growth rate of 0.30

h” and concentration of cells reached 8.5 g*dm” after 2.4 hours of cultivation. Although this

cultivation was carried out under non nutrient-limiting conditions, the percentage by weight of

PHB in cells reached 55%. From this result, it is clear that A. eufrophus grew at a relatively

high specific growth rate on IO-1 culture broth and accumulated PHI3 to a high level in the

cells.

The results of batch fermentation obtained in this study are summarized in Table 1. In

standard fermentations, a microorganism’s growth is inhibited by the presence of lactate.

However, relatively high specific growth rates were obtained for A. eulrophus in our

experiments with lactate as substrate. The growth rate increased as the initial concentration of

lactate in the medium decreased. According to previous studies, optimal growth of

A.eurrophus is obtained when fructose is used as the carbon source among various tested

organic compounds,such as lactate and xylose. The specific growth rate of A. eutrophus in

fructose medium was reported to be 0.12-0.16 h”(Mulchandaniefal., 1989).

Table 1 Growth and PEB accumulation of A.eutrophus on L-lactate media

Medium Specific growth (L-lactate (g/o ) rate(h-I) Cell (g/f) PHB(g/f) ky($

5 0.61 5.0 1.2 24

10 0.42 7.0 2.1 30

20 0.30 7.0 3.5 50

IO-1 broth (101 0.30 8.5 4.7 55

We also obtained a specific growth rate of about 0.2 h” for fructose (data not shown).

According to our results, higher specific rates were obtained with lactate and, therefore, lactate

is a good substrate for production of PHB as compare to other carbon sources. Linko et at.

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also reported that the highest PHB volumetric productivity, about 0.22g=dm.‘h”, was obtained

with 10 gdm” of lactate in 6 h and with 20 gdm” of fructose at 24 h(Linko ef al., 1993). Note

that , in the case of cultivation in the supernatant of IO-1 broth, the growth rate was a little

lower than that in CML medium at the same concentration of lactate. However, a higher

concentration of cells and PHB was obtained in the two-stage cultivation with IO-1 culture

broth. The lower growth rate and the higher biomass yield for the IO-1 culture supernatant may

be due to exhaustion of nutrients and acetic acid accumulated in xylose medium. Thus, we

need now to investigate the effects of nutrients and acetic acid on growth of and accumulation

of PHI3 by A. eutrophus. Growth of A. eulrophus was inhibited at a high concentration of

lactate, so that high-cell-density cultivation ofA. eutrophus using lactate is hard to accomplish

in standard batch culture. However, under non-limiting conditions with lactate as carbon

source, PHB was accumulated at a relatively high level in cells grown with lactate, even under

culture conditions wherein nutrients were not limited. This result presents a very interesting

phenomenon from the standpoint of the mechanism of synthesis of PHB. We are now

investigating utility of the pH-auxostat continuous or fed-batch cultures under nutrient-limiting

conditions and non-limiting conditions in an attempt to achieve high PHB productivity by

maintaining the lactate concentration at a low level.

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Doi, Y, Tamaki, A., Kunioka, M.,. & Soga, K. (1987). Makromol. Chem. Rapid Commun., 8, 631

Ishizaki, A. & Tanaka, K. (1991). J. Ferment. Bioeng., 71, 254. Ishizaki, A., Ohta, T. & Kobayashi, G. (1992a). J. Biotechnology, 24, 85. Ishizaki, A., Ueda, T., Tanaka, K. & Stanbury, P.F. (1992b). Biotechnology Letters,

14, 599. Ishizaki,A., Ueda,T., Tanaka,K. & Stanbury, P.F. (1993). Biotechnology Letters, 15, 489. Linko, S., Vaheri, H., & SepllB: (1993). Appl. Microbial. Biotechnol., 39, 1 1. Mulchandani, A., Luong, H. T. & Groom, C. (1989). Appl. Microbial. Biotechnol., 30, 11; Siegel, R.S. & Ollis, D. F. (1984). Biotechnol. Bioeng., 26, 764. Witholt, B., Smet, M.J., Eggink, G., Kingma, J.& Wynberg, H.(1983). J. Bacterial.,

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