Biosynthesis and characterization of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Alcaligenes eutrophus

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-4hydroxybutyrate) in Alcaligenes

eutrophus

Yoshiharu Doi*, Atsushi Segawa and Masao Kunioka Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 227, Japan

(Received 1 August 1989; revised 6 September 1989) Copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Alcaligenes eutrophus at 30°C in nitrogen-free culture solutions containing ),-butyrolactone alone or with fructose or butyric acid as the carbon sources. When 7-butyrolactone was used as the sole carbon source, the 4HB fraction in copolyester increased from 9 to 21 mol% as the concentration of T-butyrolactone in the culture solution increased from 10-25 g/l. The addition of fructose to the culture solution of T-butyrolactone resulted in a decrease in the 4 H B fraction in copolyester. The copolyesters produced from 7-butyrolactone and fructose by A. eutrophus were shown to have a random sequence distribution of 3HB and 4HB units by analysis of the 125 MHz 13C n.m.r. spectra. In contrast, a mixture of random copolyesters with two different 4HB fractions was produced by A. eutrophus when 7-butyrolactone and butyric acid were used as the carbon sources. These results are discussed on the basis of a proposed biosynthetic pathway of P(3HB-co-4HB). The copolyester films became soft with an increase in the 4HB fraction, and the elongation to break at 23°C increased from 5 to 444 % as the 4HB fraction increased from 0 to 16 mol%. The P(3HB-co-IO%4HB) film was shown to be biodegradable in an activated sludge. Keywords: Microbial copolyesters;Alcaligenes eutrophus; microstructure

Introduction A variety of micro-organisms produce an optically active poly(3-hydroxybutyrate) (P(3HB)) as an intracellular storage polymer 1. In 1974 it was first observed that poly(3-hydroxyalkanoates) (P(3HA)) incorporating 3hydroxyalkanoate units other than 3-hydroxybutyrate were produced in activated sludge 2.

CH3 I

(. .C H2)n 0II l ---(- O-CH-CH2-CP(3HA)

different types of organic acids. These microbial copolyesters have attracted much attention as environmentally degradable thermoplastics for a wide range of agricultural, marine, and medical applications, since the polyesters are hydrolytically1 7 1 9 and enzymatically2 0 - 2 2 degradable polymers. We have recently found that a new type of microbial copolyester of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) is produced in A. eutrophus from 4-hydroxybutyric acid 2a or ?-butyrolactone 24.

CII

0II

0II

--(- O'CH- CHz-C -')7"-'(-O-CH2 -CH2 -Cl-I - C 3HB

4HB (2)

(1) In addition, P(3HA) has been isolated from environmental samples such as sewage sludge 3'4, marine sediments s, and cyanobacteria 6. Recently, several bacterial strains including Alcaligenes eutrophus 7 ~o,, Bacillus megaterium s, Pseudomonas oleovorans ~t t 3, Rhodospillium rubrum ~4, Pseudomonas extorquens ~5, and Pseudomonas cepacia 16 have been shown to produce P(3HA) from

* To whom correspondenceshould be addressed. Presented in part at BiologicallyEngineered Polymers Conference, Churchill College,Cambridge, 31 July-2 August 1989 0141-8130/90/020106-06 ~) 1990 Butterworth & Co. (Publishers) Ltd 106

Int. J. Biol. Macromol., 1990, Voi. 12, April

The copolyester has been shown to have a statistically random distribution of 3HB and 4HB units 25. The crystalline and thermal properties of P(3HB-co-4HB) have been shown to be regulated by the content of 4HB units in the copolyester 26. In the present work we have carried out a controlled fermentation for P(3HB-co-4HB) production by A. eutrophus from mixtures of ?-butyrolactone with fructose or with butyric acid, and studied the influence of fermentation conditions on the copolyester composition. In addition, we analysed the sequence distributions of 3HB and 4HB units in copolyesters by 13C-n.m.r.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)" Y.Doi et al. spectroscopy. The results are discussed on the basis of the proposed biosynthetic pathway of P(3HB-co-4HB) in A. eutrophus cells. Finally, we studied the physical properties and biodegradation of P(3HB-co-4HB) films. Experimental

Bacterial strain and culture conditions A. eutrophus H 16 (ATCC 17699) was used in this study. Polyester synthesis was carried out by a two-step cultivation of A. eutrophus. A. eutrophus cells were first grown under aeration at 30°C on a reciprocal shaker in ten 500-ml Sakaguchi flasks containing 100 ml of nutrient-rich medium. The medium contained l0 g of polypeptone, 10 g of yest extract, 5 g of meat extract, and 5 g of ( N H 4 ) e S O j I of distilled water. The cells were harvested by centrifugation at 5000g for 15min. Under these culture conditions accumulation of polyester in the cells was not observed. To promote polyester synthesis about 4 g (dry weight) of aseptically centrifuged cells were transferred into a 2.6 litre jar fermenter (equipped with six conventional turbine impellers and three baffles) in 1.01itre of nitrogen-free mineral medium 27 containing carbon sources as ~-butyrolactone alone or together with either fructose or butyric acid. Temperature and pH were automatically controlled during the fermentation. Concentration of dissolved oxygen (DO) was controlled in the range of 4 - 6 p p m . The cells were cultivated in the nitrogen-free media for 48h at 30°C, harvested by centrifugation, and finally lyophilized. Polyesters were extracted from the lyophilized cells with hot chloroform in a Soxhlet apparatus, and purified by reprecipitation with hexane.

Analytical procedures The 1H-n.m.r. analysis of polyesters was carried out on a Jeol FX-100 spectrometer. The 1 0 0 M H z 1H-n.m.r. spectra were recorded at 27°C in a CDC13 solution of polyester (5mg/ml) with 45 ° pulse (15#s), 5s pulse repetition, 1000 Hz spectral width, 8 K data points, and 200 accumulations. The x3C-n.m.r. analysis of polyesters was carried out on a Jeol GX-500 spectrometer. The 125 MHz 13C-n.m.r. spectra of copolyesters were recorded at 27°C in a CDC13 solution with 45 ° pulse (5.3/as), 5 s pulse repetition, 25000 Hz spectral width, 64 K data points, and 15 000 accumulations. All molecular weight data were obtained at 40°C by using a Shimadzu 6 A g.p.c, system and a 6 A refractive index detector with a Shodex 80 M column. Chloroform was used as eluant at a flow rate of 0.5 ml/min, and a

sample concentration of 1.0mg/ml was used. Poly(styrene) standards with a low polydispersity were used to make a calibration curve. The films of polyester samples were prepared by conventional solvent casting techniques from chloroform solutions of polyesters using glass petri dishes as casting surfaces. The solution-cast films were kept for 3 weeks at room temperature to reach equilibrium crystallinity prior to analysis 26. Wide-angle X-ray diffraction measurements of polyester films were made on a Rigaku RAD-1VB system. CuKat radiation (2=0.1542 nm) was used as the source. The densities of polyester films were measured at 25°C using a density gradient column containing mixtures of toluene and carbon tetrachloride as the column liquid. The stress-strain curves of polyester films were obtained at 23°C with a strain rate of 20mm/min. The sample dimensions used were an initial length of 25 mm and width of 6.35 mm.

R e s u l t s and d i s c u s s i o n

Production and composition of P(3HB-co-4HB) Table I lists the results of P(3HB-co-4HB) production by A. eutrophus from ~-butyrolactone. The fermentation production of P(3HB-co-4HB) was carried out for 48 h at 30°C and pH 7.5 under aerobic conditions in the nitrogen-free culture solution containing ~-butyrolactone as the sole carbon source. The productivity and composition of copolyesters were influenced by the concentration of ~-butyrolactone in culture. The copolyester content in the dried cells decreased as the concentration of ~-butyrolactone increased. On the other hand, the 4HB fraction in copolyesters increased with the concentration of ~butyrolactone. Table 2 gives the results of P(3HB-co-4HB) production by A. eutrophus from substrate mixtures of ~-butyrolactone and fructose. Table 3 shows the results of P(3HBco-4HB) production from mixtures of ~-butyrolactone and butyric acid. The fermentation production was carried out for 48 h at 30°C in the pH range 6.5-8.5 of culture solution. When fructose or butyric acid was used as the sole carbon source, A. eutrophus produced the homopolyester of 3HB units. The productivity of copolyester was dependent upon the composition of carbon sources in the culture solution. The highest contents of copolyester in dried cells were observed in the culture solution o f p H 7.5 containing l0 g of ~-butyrolactone/l and l0 g of fructose/l (sample 6) or

Table 1 Fermentation production of P(3HB-co-4HB) from ),-butyrolactone by Alcaligenes eutrophus for 48 h at 30°C and pH 7.5 Polyester contenta

Polyester compositionb (moP/o)

Sample

y-butyrolactone

Cell dry wt

Molecular weight

no.

(g/i)

(g/l)

(wt % )

3H B

4H B

M n"10 - 3

M w/Mn

1 2 3 4

10 15 20 25

8.1 6.5 6.2 4.7

29 22 21 14

91 84 83 79

9 16 17 21

425 240 -

1.9 1.8 -

oPolyester content in dry cells bDetermined from ~H-n.m.r.spectra

Int. J. Biol. Macromol., 1990, Vol. 12, April

107

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate): Y.Doi et al. Table 2

Production of P(3HB-co-4HB) from 7-butyrolactone and fructose by A. eutrophus for 48 h at 30°C Carbon source (g/l)

Sample no.

~-butyrolactone Fructose

pH a

Cell dry wt (g/I)

3 5 6 7 8 9 10 11

20 15 10 5 0 10 10 10

7.5 7.5 7.5 7.5 7.5 7.0 8.0 8.5

6.2 7.6 8.0 8.5 9.4 7.4 7.7 8.7

0 5 I0 15 20 10 10 10

Polyester composition (mol%)

Polyester content (wt%)

3HB

4HB

M ," 1O- 3

M w/Mn

21 37 48 46 37 39 38 32

83 88 92 96 100 91 92 90

17 12 8 4 0 9 8 10

240 164 453 479 104 162 313

1.8 2.9 1.7 1.9 2.5 2.6 2.2

Molecular weight m

"pH value in nitrogen-free culture media

Table 3

Production of P(3HB-co-4HB) from ~-butyrolactone and butyric acid by A. eutrophus for 48 h at 30°C Carbon source (g/l)

Sample no.

Butyric ),-butyrolactone acid

pH"

Cell dry wt (g/l)

3 12 13 14 15 16 17 18 19

20 15 10 5 0 10 10 10 10

7.5 7.5 7.5 7.5 7.5 6.5 7.0 8.0 8"5

6.2 8.7 8.9 6.9 8.7 5.1 7.5 8.5 9"0

0 5 10 15 20 10 10 10 10

Polyester composition (mol%)

Polyester content (wt%)

3HB

4HB

M," 10- 3

Mw/M"

21 38 65 59 58 13 42 43 55

83 81 76 89 100 86 73 85 90

17 19 24 11 0 14 27 15 10

240 264 289 281 146 226 339 374

1.8 2.4 2.0 2.1 2.7 2.3 2.4 2.1

Molecular weight m

a pH value in nitrogen-flee culture media m

3°l ~ 20 Mr

0

25

50

75

100

Fraction of y'-butyrol(:~tone (wt%) Figure I Relation between 4HB fraction in copolyester and the weight-fraction of 7-butyrolactone in carbon sources at pH 7.5. (O) Copolyesters from ),-butyrolactone and fructose (Table 2), and (O) copolyesters from v-butyrolactone and butyric acid

(Table 3)

butyric acid (sample 13). Optimal pH for the copolyester production was 7.5. The number-average molecular weights (M,) of copolyesters were in the range of 104000-479000, depending upon the fermentation conditions. As can be

108

Int. J. Biol. Macromol., 1990, Vol. 12, April

seen from Tables 2 and 3, the M, value increased with an increase in pH value of the culture solution. The molecular weight distributions of copolyester were unimodal and their polydispersities (Mw/M,) were in the range of 1.7-2.9. The 4HB fraction in copolyester was dependent upon both composition and pH of the culture solution. Figure I shows the relation between 4HB mole fraction and the weight fraction of 7-butyrolactone in the carbon sources. When fructose was used as the carbon source together with ~,-butyrolactone, the 4HB fraction in copolyester increased proportionally with the fraction of),-butyrolactone. In contrast, an unusual relation was recorded when butyric acid was used together with ~-butyrolactone. The highest value (24 mol%) of 4HB fraction was observed in the copolyester sample 13 produced from the mixture of butyric acid (10 g/l) and 7-butyrolactone (10 g/l). Figure 2 shows the relation between 4HB mole fraction and pH value of culture solution. The 4HB fraction in copolyester was not influenced by the pH value when fructose and 7-butyrolactone were used as the carbon sources. In contrast, the 4HB fraction was markedly influenced by the pH value when butyric acid was used with ),-butyrolactone, and a maximum value of 4HB fraction was observed at pH 7.0.

13C_n.m.r. analysis In order to investigate the mechanism of P(3HBco-4HB) biosynthesis in A. eutrophus, the microstructure of copolyesters was analysed by 13C.n.m.r. spectroscopy.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate): Y.Doi et ai. Fi#ure 3 shows the 125 MHz 13C-n.m.r. spectrum of sample 3 (P(3HB-co-17% 4HB)) produced from 7butyrolactone, together with the 13C chemical shift assignments25. The c~irbonyl resonances (~169-173) are resolved into four groups of peaks, arising from the different diad sequences of 3HB and 4HB units. As reported in a previous paper 25, the peaks at 169.1-169.3 are assigned to the carbonyl resonances in the 33(3HB3HB) sequence, and the other three groups of peaks at 169.9-170.1, 171.8-171.9, and 172.5-172.6 are assigned to 34(3HB-4HB), 43(4HB-3HB) and 44(4HB-4HB) sequences, respectively. The diad sequence distributions of 3HB and 4HB units in the copolyester samples 3, 11, and 13 were determined from the peak areas of carbonyl resonances. The results are given in Table 4. When the copolyester has a statistically random distribution of 3HB and 4HB units, the diad fractions, F33 , F34, F43 , and F44 can be expressed using the mole fraction of 3HB unit, 173, as follows.

The value of D is equal to 1.0 for a statistically random copolymer. The D value for a blocky copolymer should be larger than 1.0. The D values of samples 3, 11, and 13 are also given in Table 4. The D values of samples 3 and 11 are close to 1.0. whereas the D value of sample 13 is as large as 66. Sample 11 is produced from the mixture of 7-butyrolactone and fructose (Table 2). Therefore, it may be concluded that A. eutrophus cells produce a random copolyester of 3HB and 4HB units from 7-butyrolactone and fructose. The sample 13 is produced from the mixture of y-butyrolactone and butyric acid (Table 3). The sample 13 with the D value of 66 is either a block copolymer or a mixture of 3HB- and 4HB-rich random copolymers. We carried out solvent extraction of the sample 13 with boiling acetone for 3 h in a Soxhlet apparatus, and obtained an acetone-soluble fraction (24wt%) and an insoluble fraction (76wt%). The 4HB content of the acetone-soluble fraction (sample 13-S) was as high as 86 mol%, determined from the 1H n.m.r, spectrum. On

F33=F32 F34. = F43 = F 3 ( 1 - F 3 )

F44 = (l-F3) 2

'cH

(1)

o,

o

Here, we define the parameter D by equation (2).

(2)

D = (F33 F44)/(F34 F,r3 )

3HB

4HB f3 4

0

O

E

m

2

20-

169

m -1Mr

1

10-

0

I

I

I

I

6

7

8

9

J

180 I~0 140 120 ~

pH

Figure 2 Relationbetween4HB fraction in copolyesterand pH

60

2b

ppm

Figure 3 125MHz ~aC-n.m.r. spectrum of P(3HB-co-

value of culture solution. (O) Copolyestersfrom y-butyrolactone (10g/l) and fructose (10g/l) (Table 2), and (o) copolyestersfrom 7-butyrolactone (lOg/I) and butyric acid (lOg/l) (Table 3)

Table 4

~) 6 in

17%4HB) sample 3 in CDCI3 at 27°C. Chemical shifts are in ppm from tetramethyl-silane

Diad sequence distributions of 3HB and 4HB units in copolyester samples

Samples

F33

F34

F43

F44

Da

3, P(3HB-co-17%4HB) 11, P(3HB-co-10%4HB) 13, P(3HB-co-24%4HB) 13-Sb, P(3HB-co-86%4HB) 13-P, P(3HB-co-7%4HB)

0.66 0.81 0.73 0.02 0.885

0.17 0.09 0.04 0.12 0.06

0.15 0.09 0.05 0.12 0.05

0.02 0.01 0.18 0.74 0.005

0.52 1.0 66 1.0 1.5

° D = F33F44/F34F43 ~Samples 13-S and 13-I are respectively the acetone-soluble fraction (24 wt%) and the acetone-insoluble fraction (76 wt%) of sample 13

Int. J. Biol. Macromol., 1990, Vol. 12, April

109

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate): Y.Doi et al. CoA-SH 9CH2CH2CH2C I I

~

AMP

~ HOCH2CH2CH2COH

HOCH2CHaCH2C-SCoA

~/~

~H~ ~ (OCHCH2C-)x

( OCH2CH2CH2C)y

3"B

4.B

TCA cycle

I

Physical properties of P(3HB-co-4HB)

COA-SH

CH3CO-SCoA~ v ; ~ CH3COCH2CO-SCoA

$

NADH ~ NAD+

?

NADPH

(D)CH3 CH2CO-SCoA

l

NADP+

.?H

(L)CHHCHCH2CO-SCoA

Fructose

CH3CH=CHCO-SCoA.

FADFADH2~

CH2=CHCH2CO-SCoA¢~--~

H20

CH3CH2CH2CO-SCoA

AMP+PPI ATP

CoA-SH CH3CH2CH2COOH

Figure 4 Schematicpathway of P(3HB-co-4HB) biosynthesis the other hand, the 4HB content of the acetone-insoluble fraction (sample 13-1) was 7 mol%. The diad sequence distributions of samples 13-S and 13-1 were determined from the 13C-n.m.r. spectra. The result is given in Table 4. The D values of both samples 13-S and 13-1 are close to 1.0. These results indicate that the mixture of random copolymers with two different 4HB fractions (7 and 86 mol%) is produced in A. eutrophus cells when butyric acid and 7-butyrolactone are used as the carbon sources. Figure 4 shows a hypothetical pathway of P(3HBco-4HB) biosynthesis in A. eutrophus. When 7-butyrolactone is used as the sole carbon source, 4-hydroxybutyryl-coenzyme A(CoA) is first formed in the cells. A portion of 4-hydroxybutyryl-CoA is then metabolized into D-3-hydroxybutyryl-CoA via a complex metabolic pathway which has been suggested to occur in Clostridium kluyveri 2s, as shown in Figure 4. A random copolyester of 3HB and 4HB units is produced by the copolymerization of o-3-hydroxybutyryl-CoA with 4-hydroxybutyryl-CoA under the action of polyhydroxybutyrylpolymerase. The 4HB fraction in the random copolyester increased with the concentration of ?-butyrolactone in the culture solution (Table 1). This result suggests that the mole fraction of 4-hydroxybutyryl-CoA to D-3-hydroxybutyryl-CoA in cells is influenced by the concentration of 7-butyrolactone in the culture solution. When fructose is used as the carbon source for A. eutrophus, D-3-hydroxybutyryl-CoA is produced from acetyl-CoA via acetoacetyl-CoA29 31, as shown in Figure 4. Therefore, D-3-hydroxybutyryl-CoA is formed from fructose and ?-butyrolactone when both the carbon sources are present in culture solution, resulting in a decrease in the 4HB fraction of a random copolyester P(3HB-co-4HB). When butyric acid was used with 7-butyrolactone, a mixture of random copolyesters with two different 4HB fractions was synthesized in A. eutrophus cells (Table 4). Butyric acid is known to be metabolized to D-3-hydroxy-

!10

butyryl-CoA via acetoacetyl-CoA in the fl-oxidation cycle 1°. An intermediate produced in the fl-oxidation cycle from butyric acid in cells may inhibit the transformation of 4-hydroxybutyryl-CoA into o-3-hydroxybutyryl-CoA, resulting in the formation of a random copolyester with a high 4HB fraction. A detailed study on the effect of butyric acid on P(3HB-co-4HB) production is in progress.

Int. J. Biol. Macromol., 1990, Vol. 12, April

The physical properties of random copolyester films were studied in the range of 0-16mo1% 4HB. The copolyester films were prepared by conventional solvent casting techniques from chloroform solutions of polyesters, as described in the experimental section. Table 5 gives the X-ray crystallinities and densities of P(3HBco-4HB) films. The crystallinity and density decreased with an increase in the 4HB fraction. Table 6 shows the result of stress-strain test of P(3HB-co-4HB) films at 23°C. The stresses are calculated on the unstained cross-section. The sample became soft with an increase in the 4HB fraction, and the elongation to break increased from 5 to 444% as the 4HB fraction increased from 0 to 16 mol%. The true tensile strength of P(3HB-co-16% 4HB) film is expected to be ca. 100 MPa, if the cross-section is corrected.

Biodegradation of P (3H B-co-4 H B ) Microbial P(3HB) is biodegradable in soil and activated sludge 7. An extracellular P(3HB) depolymerase has been isolated from Pseudomonas lemoignei 2° and Alcaligenes faecalis 21. In this study we prepared two samples of P(3HB) and P(3HB-co-10% 4HB) films (0.7 mm thickness) and measured the rate of biodegradation in an activated sludge, obtained from the sewagetreatment plant at Tokyo Institute of Technology. The two samples of polyester films were kept at 30°C under aeration in the activated sludge. Figure 5 shows the films Table 5 Crystallinity and density of P(3HB-co-4HB) films Polyester composition (mol%) 3HB

4HB

Crystallinit/ (%)

Density b (g/ml)

100 97 90 84

0 3 10 16

60±5 55±5 45±5 45± 5

1.250 ND 1.232 1.234

Determined by X-ray diffraction b Determined at 25°C

a

Table 6 Stress-strain test results of P(3HB-co-4HB) films at 23°C 4HB fraction (mol%) Properties Stress at yield (MPa) Elongation at yield (%) Tensile strength (MPa) Elongation to break (%)

0

3

10

16

43 5

34 4 28 45

28 5 24 242

19 7 26 444

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate):

6 Time (Weeks)

P (3HB)

P (3HB-co-10%4HB)

7 8 9 10 11 12 13 14

Figure 5 Microbial polyester films of P(3HB) and P(3HBco-10%4HB) placed for 2 and 5 weeks at 30°C in an activated sludge under aeration. Initial film dimensions; 3 cm diameter and 0.07 mm thickness

after 2 a n d 5 weeks in the activated sludge. The (3HB-co-10% 4HB) film was almost decomposed after 2 weeks, a n d disappeared after 5 weeks. Thus, the rate of b i o d e g r a d a t i o n of P ( 3 H B - c o - 1 0 % 4HB) film was faster t h a n that of P ( 3 H B ) film.

15 16 17 18 19 20 21 22

Acknowledgements

23

This work is supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture in Japan.

24 25 26

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