Studies on copolyester synthesis by Rhodococcus ruber and factors influencing the molecular mass of polyhydroxybutyrate accumulated by Methylobacterium extorquens and Alcaligenes eutrophus

93

FEMS Microbiology Reviews 103 (1992) 93-102 © 1992 Federation of European Microbiological Societies 0168-6445/92/$15.00 Published by Elsevier

FEMSRE 00260

Studies on copolyester synthesis by Rhodococcus ruber and factors influencing the molecular mass of polyhydroxybutyrate accumulated by Methylobacterium extorquens and Alcaligenes eutrophus Alistair J. Anderson a, D. R o g e r Williams a, Behnam Taidi and David F. Ewing b

a, Edwin A. Dawes a

a Department of Applied Biology, School of Life Sciences and t, School of Chemistry, University of Hull, Hull, UK

Key words: Rhodococcus ruber ; Methylobacterium extorquens ; Alcaligenes eutrophus ; Copolyester biosynthesis; Polyhydroxyalkanoate; Polyhydroxybutyrate molecular mass 1. SUMMARY

Rhodococcus ruber NCIMB 40126 synthesizes copolyesters (polyhydroxyalkanoates) of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), containing primarily 3HV monomer units, from a variety of single, chemically unrelated substrates. The 3HV content of the polymer is dependent on the choice of substrate and succinic acid yields high values, e.g., 92-95 mol%. The 13C-NMR spectrum of copolymer produced from [2,3-t3C]succinate displayed similar enrichment of signals representing C-1,C-2, C-4 and C-5 of the 3HV component, whereas the C-3 signal showed negligible enrichment. Label from [1,4-13C]suc cinate was incorporated into the C-3 position of the 3HV monomer unit. The key enzymes of polyhydroxybutyrate (PHB) synthesis (3-ketothiolase, acetoacetyl-CoA reductase and PHB syn-

Correspondence to: A.J. Anderson, Department of Applied Biology, School of Life Sciences, University of Hull, Hull HU6 7RX, UK.

thase) are present, have been studied and their specificities shown to permit the synthesis of poly(3HB-co-3HV). Attempts to detect putative enzymes of succinate metabolism leading to propionyl-CoA formation have so far proved inconclusive. The organism can readily utilise as nitrogen source the amino acids valine and isoleucine, which yield propionyl-CoA in their metabolism. Under sulphate-limited conditions the copolymer accumulated from valine possesses an enhanced 3HV content relative to glucose as substrate. The weight-average molecular mass (M r) of PHB accumulated by Methylobacterium extorquens can be considerably higher (1.7 × 10 6) than previously reported and is dependent on the choice of carbon source, methanol yielding lower values (up to 6 >( 105) than succinate (up to 1.7 x 106). The M r of the PHB accumulated by both Alcaligenes eutrophus and M. extorquens is affected significantly by the concentration of the carbon source supplied, having lower values and higher polydispersities at higher substrate concentrations. During batch fermentation the M r of PHB in A. eutrophus decreases gradually with time whereas the converse is true for M. extorquens.

94 Polymercomposition(%(w/w)of driedcells) 0 I0 20 30 40

2. INTRODUCTION

I

A survey of bacteria capable of accumulating novel polyhydroxyalkanoates (PHAs) led to the discovery that certain Rhodococcus species synthesise copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), containing principally 3HV monomer units, from a range of single, chemically unrelated, carbon sources [1,2]. This behaviour contrasts with that of Alcaligenes eutrophus, the bacterium used by ICI plc for the commercial production of 'Biopor copolyester, which requires two substrates, glucose and propionic acid [3], and yields a lower proportion of 3HV in the copolymer than do Rhodococcus species. While polymers with a high 3HV content are not necessarily more useful individually, they do offer the opportunity for the production of blends. Accordingly, one of these organisms, Rhodococcus ruber NCIMB 40126, has been used for more detailed studies of the metabolic pathways and enzymology involved in copolymer synthesis. Recently Steinbiichel and Pieper [10] have reported a mutant of A. eutrophus which accumulates a copolymer of 3HB and 3HV, containing a low proportion (about 8 mol%) of 3HV, from various single, unrelated carbon sources. The weight-average molecular mass (M r) of polyhydroxybutyrate (PHB) is primarily speciesdependent [4] but it is also influenced by growth conditions [4,5]. The biochemical basis for the variation in M r of PHAs has not yet been reported and, in part, awaits detailed knowledge of the mechanism of PHA synthase action. We have studied the effect of the growth environment on the molecular mass of PHB produced by both A. eutrophus and M. extorquens with respect to the nature and concentration of the carbon source employed.

3. RESULTS AND DISCUSSION

3.1. Studies with Rhodococcus ruber NCIMB 40126 3.1.1. Composition of copolyesters synthesised PHA biosynthesis in shake-flask batch cultures of R. ruber was achieved by the two-stage, growth

Glucose

I

I

I

I

- - 1

Pyruvate Lactate

- - 1 1

Acetate Citrate

2-Oxoglutarate Succinate

[ ] 3-Hydrowbut/rate

Fumare,te

[]

3-Hydroxyvalerate

~

3"Hydr°whexanoate

Malate

~ 4-Hydroxybut/rate

Oxaloacetate

[]

Propionate

5-Hydroxyvalerate

But/rate Valerate

__]

Hexanoate

Heptanoate Octanoate 4-Hydroxybutyrate 5-.Chlorovalerate 2-Hexenoate

Fig. 1. Composition of polymers produced by R. tuber from

various singlecarbon sources.

and accumulation phase technique described previously [2,6]. The composition of the PHAs extracted with chloroform from lyophilised bacteria by the methods previously reported [2] are recorded in Fig. 1. Noteworthy is the high proportion of 3HV monomer units relative to 3HB in the majority of the copolymers produced from carbohydrate substrates, while valerate yielded almost pure (99 mol%) polyhydroxyvalerate. The ratio of 3HV : 3HB monomer units in copolymers derived from different carbohydrate substrates is given in Table 1.

3.1.2. Pathways for poly(3HB-co-3HV) synthesis The biosynthetic pathway for PHB from acetyl-CoA has been known for almost 20 years (for recent reviews, see [7,8]). The specificities of the three key enzymes involved in PHB synthesis (3-ketothiolase, acetoacetyl-CoA reductase and PHB synthase) have been shown in A. eutrophus to permit the condensation of propionyl-CoA with

95

Table 1 Ratio of 3HV:3HB monomer units in copolyesters derived from carbohydrate substrates by R. ruber Substrate

3HV : 3HB (mol%)

Glucose Lactate Pyruvate Acetate Oxaloacetate Malate Fumarate Succinate 2-Oxoglutarate Citrate

3.8 5.1 3.6 2.5 3.6 1.3 1.5 12.0 7.0 3.0

acetyl-CoA and the subsequent reduction of the resulting 3-oxopentanoate to 3-hydroxypentanoate (3-hydroxyvalerate) and its incorporation into a random copolyester [6,9]. Clearly a similar route was feasible for copolyester formation in R. ruber although the possibility of the C 5 unit arising by route(s) independent of ketothiolase action could not be ruled out (Fig. 2).

3.1.3. Enzymology of PHA synthesis R. ruber was shown to contain the three key Routes to

propionyI-CoA

m

CH3.CH2.COSCoA

CH3COSCoA 3-Ketothiolase O

II

CH3.CH2.C.CH2.COSCoA AcetoacyI-CoAreduclase

Routes not involving 3-ketothiolase OH

I

CH3,CH2.CH.CH2.COSCoA PHA synthase

Fig. 2. Possible routes for biosynthesis of the 3-hydroxy-

valerate (3HV) monomer unit in poly(3HB-co-3HV).

enzymes for P H A synthesis. A 3-ketothiolase was purified 35-fold by DEAE-Sepharose CL-6B and Sephacryl S-200 treatment and only one peak of activity was recovered from these columns. Assays were conducted for the thiolysis reaction. Activity was detected over the range (C4 to C 8) of substrates tested. Dithiothreitol (DTI', 1 mM) and 2-thioethanol (10 mM) both stabilised activity over 6 days with the C4 substrate but a differential effect was noted with these agents and the C 8 substrate, suggesting the presence of a second ketothiolase, a possibility which awaits confirmation. Activity in the thiolysis reaction was 80-fold higher with C4 than C 5 with the partially purified enzyme but there was a smaller difference with crude cell extracts (16-fold) and even less (3-fold) when crude extracts were supplemented with DTI'. The organism possesses two acetoacetyi-CoA reductases linked respectively to N A D H and NADPH. The N A D P H enzyme, which gives 1.5% activity with NADH, was purified some 50-fold by procedures similar to those used for 3-ketothiolase. The product was (R)3-hydroxybutyryl-CoA. The enzyme displayed an optimum pH of 6.0 and higher activity with C 5 than C 4 as substrate but did not react significantly with longer carbon chain substrates. Non-denaturing polyacrylamide gel electrophoresis (PAGE) yielded two major protein bands, only one of which was active, while SDS-PAGE gave only one major protein band, corresponding to a molecular mass of 30000. Western blots, kindly performed by Karen White (ICI Seeds Ltd) with antibody raised to the NADPH-linked acetoacetyl-CoA reductase of A. eutrophus, indicated considerable homology with the R, ruber enzyme. The N A D H enzyme, which was purified 87-fold by a similar protocol, displayed an optimum pH of 4.5 and yielded (S)3-hydroxybutyrate as product. It had a broad substrate specificity over the range (C4 to C8) tested and gave 5.5% activity with NADPH. SDS-PAGE yielded two major protein bands corresponding to molecular masses of 45000 and 31000; gel filtration chromatography on Sephacryl S-200 with protein standards indicated a molecular mass of 64000, suggesting the protein is a dimer.

96 PHA synthase activity of granules was measured in two different ways, first by incorporation of t4C substrates into polymer and second, by release of CoA in the synthase reaction. The latter method is valid only if deacylase activity is eliminated. This was accomplished by successive ultracentrifugation of crude granule preparations on glycerol (50% v / v ) and gradients of sucrose (0.1-2.0 M) or glycerol (10-70% v/v); the resulting granules were free of deacylase activity. The enzyme was active only towards C 4 and C 5 (R)3hydroxyacyl-CoAs, with activities of 0.54 and 0.18 U (rag p r o t e i n ) - t respectively. Interestingly, activity was also displayed with (S)3-hydroxybutyrylCoA (0.25 U (mg protein)- t ), which correlated with our previous observation that copolymer produced by R. ruber may not be optically pure [2]; these findings are now the subject of more intensive investigation.

3.1.4. Routes for propionyl-CoA formation The possible pathway(s) for propionyl-CoA formation in R. ruber were investigated, with particular emphasis on the metabolism of succi-

HOOC.CH2.CH2.CO(~H COASH insse HOOC,CH2.CH2.COSCoA I Methy~nak~yl-CoA mutase H (~ HOOC--t--CH 3 COSCoA $uccinyl-CoA

deca rboxylase

1

MethylmalonyI-CoAracemase

H (F~ HOOC--t--COSCoA CH 3

decarbole/laseMet "~1hylm1[Tr~an~ alH3"CO' °nytC' °Aco2 HOOC, COOH~ CH2-C~O.COOHIytalseCO2 [ CH3CH2"COSC°A I C02 Fig. 3. Possible routes for conversion of succinic acid to propionyl-CoA.

Table 2 Enzymes involved in the formation of propionyl-CoA from succinate in bacteria En~me (a) (S)-Methylmalonyl-CoAmutase succinyl-CoA~ (S) methylmalonyl-CoA (b) Methylmalonyl-CoAcarboxyltransferase (R)-methylmalonyl-CoA+ pyruvate propionyI-CoA+ oxaloacetate (c) PropionyI-CoAcarboxylase (R)-methylmalonyl-CoA propionyl-CoA+ CO 2

EC No. 5.4.99.2 2.1.3.1 4.1.1.41

nate in view of the high proportion of 3HV in the copolymer synthesised from this substrate. Fig. 3 shows some possible routes from succinate to propionyl-CoA which (apart from the hypothetical succinyl-CoA decarboxylase) are well established in the Propionibacteria, and Table 2 records some of the enzymes involved. However, efforts to detect activity of the methylmalonylCoA carboxyltransferase [12], succinyl-CoA decarboxylase and propionyl-CoA carboxylase [13] enzymes in cell extracts have so far proved inconclusive. Other possibilities include the acryloyI-CoA pathway observed in Clostridium propionicum and other rumen bacteria, wherein lactyl-CoA is dehydrated to acryloyl-CoA which is then reduced to propionyl-CoA. An assay was also devised to test for a hypothetical thiol condensation of methylmalonyl-CoA with acetyl-CoA, to yield 3oxopentanoyl-CoA, CO 2 and CoASH, but again without success.

3.1.5. Isotopic studies of succinate conversion to copolyester The high 3HV content of poly(3HB-co-3HV) accumulated by R. ruber from succinate as substrate indicates its efficient conversion to 3HV. 13C-NMR studies were therefore undertaken to identify the distribution of the succinate carbons in the copolymers. [1,4-13C] and [2,3-t3C]suc cinate were used in these experiments with the results shown in Fig. 4. In the 3HV monomer unit C-2 and C-3 of the substrate are located in C-l, C-2, C-4, and C-5, and C-1 and C-4 of succinate

97

are found in the C-3 position of 3HV. This labelling pattern is in accord with a ketothiolasemediated condensation of propionyl-CoA with acetyl-CoA. But it also accords with the incorporation of 3HV derived from valerate synthesised from succinate via propionyl-ACP and the fatty acid synthetase route, i.e., eliminating ketothiolase action, and thus cannot be used to distinguish between these two possible routes. Label from [2,3-13C]succinate was detected in all four carbon atoms of the 3HB monomer unit, which is the expected pattern for the known pathways leading from succinate to acetyI-CoA. To secure additional information concerning the association of the succinate carbon atoms in the 3HV monomer units, experiments were conducted with [1,2-13C]succinate, specifically to answer the question of whether the C-3 of 3HV is associated with C-1 and C-2, or with C-4 and C-5. The findings disclosed that the C-1 and C-2 of succinate cannot be incorporated into the 3HV monomer unit as an unseparated pair. The doublet/singlet ratio of the signals accords with a pathway that involves the conversion of succinylCoA to methylmalonyl-CoA, followed by its reaction with pyruvic acid, catalysed by methylmalonyl-CoA carboxyltransferase, to yield propionyl-CoA which reacts with acetyl-CoA to form 3-hydroxyvalerate mediated by ketothiolase.

3.1.6. Isotopic studies of acetate conversion to copolyester Poly(3HB-co-3HV) accumulated by R. ruber

HOOC.~H2.~H2.COOH

1

n

7.3 CH 2

+1

.

-- - O - - C m C H 2 - -

I

H

O

.N

C.

• CH 3

.I . •O--C~CH2m

O

.N

C

I

H

m

3HV

3HB

Fig. 4. Distribution of succinic acid carbon atoms in poly (3HB-co-3HV) accumulated by R. tuber.

12"

V-~ Vol glc NH~ Isoleu, glc ~ll-~--_I~ J ' ' 8-.-.,-- ==

10

Isoleu, glc

-

I

C

ff O"Vol,glc

i

1

If

I

2

3

/

I

I

I

4 5 6 Time (aoys)

I

/~'N

7 i

I

I

12

13

Fig. 5. G r o w t h o f R. tuber with branched-chain amino acids,

valine and isoleucine, as sources of nitrogen or carbon. Nutrients were supplied at the following concentrations: glucose, 55.5 mM; ammonium, 53 mM; valine and isoleucine, 20 mM. Valine, A; valine+NH~, n ; valine+glucose, <3; valine+ glucose+NH~-, v ; isoleucine, z~; isoleucine+NH~, II; isoleucine + glucose, e; isoleucine + glucose + NH ~-, v .

from [1,2-13C]acetate and containing 3HV as the major monomer unit was subjected to 13C-NMR analysis. In this experiment [1,2-13C]acetate (99% enrichment) and unlabelled acetate were used in the ratio of 1 : 3 as substrate. Preliminary findings suggest that the 3HB monomer is synthesised, as would be expected, from two intact acetate moieties. In contrast, only C-1 and C-2 of the 3HV monomer units are synthesised from an intact acetate molecule; the carbon incorporated into C-3, C-4 and C-5 is derived from acetate molecules which have been cleaved.

3.1.7. Growth and copolymer accumulation with valine and isoleucine The metabolism of the branched-chain amino acids valine and isoleucine involves the formation of propionyl-CoA. It was of interest, therefore, to discover whether, as with A. eutrophus (Linda Naylor, personal communication), R. ruber can utilise these amino acids and produce from them a copolymer with an enhanced 3HV content. Growth experiments with a glucose basal salts medium showed that both valine and isoleucine could serve effectively as sources of nitrogen and eventually support as much bacterial mass as did an NH~- addition. However, they were much less

98 Table 3

Table 4

Composition of copolyesters accumulated by R. ruber w h e n supplied with branched-chain amino acids

Composition of copolyesters accumulated by R. ntber under conditions of sulphate limitation when supplied with glucose and valine

Accumulation substrate(s)

PHA content (% w / w of dried cells)

Composition of p o l y m e r (mol %) 3HB 3HV

Valine Valine + Glucose Isoleucine lsoleucine + Glucose

11.4

30

70

12.6 2.6

3! 38

69 62

12.3

31

69

Bacteria w e r e g r o w n in glucose-ammonium salts medium and the basal salts polymer accumulation medium contained 20 mM L-valine or L-isoleucine and 55.5 mM glucose, as appropriate.

effective as the sole carbon source, whether supplemented with NH~- or not (Fig. 5); both rate and extent of growth were seriously diminished. The composition of the polymer produced by R. ruber after growth on glucose-ammonium salts medium followed by an accumulation phase with amino acid alone or plus glucose was investigated (Table 3). It' is apparent that under these conditions the amino acids do not enhance the 3HV content of the copolymer accumulated and, indeed, yield a rather lower content than does glucose alone (75 mol%, Fig. 1). However, in these accumulation experiments, the amino acid 60.0

_ _

30

Accumulation substrate(s)

PHA content (Composition of p o l y m e r (% w / w (mol %) of dried cells) 3HB 3HV

(a) Glucose (b) Valine (c) Glucose + Valine

16.2 26.2

30.3-+1.3(4) 18.0-+0.2(3)

69.7-+1.3(4) 82.0+0.2(3)

27.7

21.9_+0.4(3)

78.1+0.4(3)

Bacteria for (a) w e r e g r o w n in glucose-ammonium salts medium containing 6.5 I~M sulphate. Bacteria for (b) and (c) w e r e g r o w n in glucose-valine salts medium containing 6.5 #M sulphate. T h e accumulation media were devoid of added sulphate and contained, as appropriate, 20 mM c-valine and 55.5 mM glucose.

could serve as a nitrogen source in a medium otherwise incapable of supporting growth. Accordingly the experiments with valine and glucose were repeated under conditions of sulphate limitation. The organism was grown on a medium containing 6.5/x M SO4z-. Under these conditions (Table 4) valine gave rise to a higher 3HV content of copolymer than did glucose or glucose plus valine. The possible role of valine metabolism in copolymer formation [10] thus warrants further investigation. 6.0 q 6.0

9.0

30.0-

6.0

20.0 --

4.0

3.0

I0.0

2.0

Mr •

o

!

40.0

~ 0 ~

~

_ 2.0

e~

20.0

M

~

-- 1.0

2.0 I

I

0 PHB 0.0

i

I

I

I

I

I

5

10

15

20

25

Sodiumsuccinate(g 1"1)

oo ...10.0

0.0

I

I

I

0.5

1.0

15

0.0 ..I 00

Methanol (% v/v)

Fig. 6. Effect of nature and concentration of carbon source on accumulation and molecular mass (M r) of PHB by M. extorquens in shake-flask culture. (a) Succinate; (b) methanol.

99

60.0 --

3.2. Studies with Methylobacterium extorquens

1.5 - -

- - 30.0

PHB

O~ 0 3.2.1. Effect of the nature and concentration of the carbon source on the M~ of PHB accumulated 3.2.1.1. Shake-flask culture experiments. Comparisons were made of succinic acid and methanol as substrates for PHB formation by M. extorquens in shake-flask culture. M~ was higher on succinate (up to 1.7 x 10 6) than on methanol (up to 6 x 105), a value considerably greater than previously reported for the related organism Protomonas extorquens [5], namely 8 x 10 ~, for growth on methanol. Increasing the initial medium concentration of succinate decreased the specific growth rate, increased the amount of PHB synthesised in the accumulation phase but resulted in a decrease in Mr and an increase in polydispersity (Fig. 6a). Similar experiments with methanol also revealed a decrease in specific

40.0 --

|.0

--

_

o

Z

L

m

=

-6

e~

20.0 --

0.5

_

_

Mr/M n

ZXLb-Z~1

0.0_

I

0.0

%~

i,

I

10

10.0

I

20

30

I

0.0

40

Glucose (g I"1) Fig. 8, Effect of initial concentration of glucose on accumula-

tion and molecular mass ( M r) of PHB by A. eutrophus in shake-flask culture.

_..1

/ 5C

~, ~ - - 1

3.0

1 / Mr

I /

2,0°_

=

/

~.

10

PH8 o~-

.~-AA" A /

• II

i

-

AA [ n

/ * ,'

oo~

/\

"

-o5~ ~

A'A'AA'A - A A-A--AA'A ~ ~ i "\ n B/

n~n

/NH~):SO4

,,o_~.oo -n~ ,' %0\ o , \_\/

t

t

, D/

c|

+

/o~

+ / o

~

II

'

GO 2.0

____o.__.._o~_e o

/-

./"

I

.20

,'"-.

I

\

-oMetb°ool

,

\

,2 ~z

5e

"

~

z

,=

<

20.0

_

-i5~

I/

'

~

/

.~o.

/

"

-20/

I 0

4

II 14

0/

\

\

,

\ ,,:~

\ 0

t

l

I

t

18

22

26

30

Time

V

o

\ o..~ :

j ~,~

A I 34

'

~,

" 0

i 38

z

\

\ ~

,

~

,

42

46

50

iJ

.k

9.0

- 3.0

64

(h)

Fig. 7. Growth and accumulation of PHB by M. extorquens on methanol in a batch fermenter. Arrows and broken lines indicate additions of methanol except at A where ammonium was also added. Growth (log A62o) , e; m e t h a n o l , o ; ammonium, zx ; PHB, • ; M r, I .

100 60

~"

1.6

40

AIA •

~

2o

o 1.o

PHB

n~n

~

•

(NH4)2SO4 l-l) ~A (g i

I

0

12

A/

I

I

,,.. A

I-4 '~,

I

0

I

%

°$

2.0

20

Z

0.5

=

(NH4)2SO 4 (g l-l) ~ A

~ . O/°-o....

0-0

/~

ulucose

r,.) 0.5

.~..

"" "/'...o--~,-~'-o-o~-~

1.5 0

~g

/ o

:\ ~ "'6\

0

/

co:/

(%

o

~

O

L ~

',

O-o_ o

~

'j['-~-;.-;.-,~O

~

"-''0.~

1.0

_

=

XA

xXka

I

I

4

8

T_ 12

0---0--0.~

:

C'-,., "'-.

o/ 1.0

O~o~

-~ 16

12

4

I

I

I

16

20

24

0

0

28

Time (h) Fig. 9. Growth and accumulation of PHB by A. eutrophus on glucose in a batch fermenter. Arrows and broken lines indicate additions of glucose. Growth was monitored by CO 2 output. Growth (log % CO2) , o; glucose, ©; ammonium, A ; PHB, • ; Mr, II.

growth rate, decrease in M r and increase in polydispersity with increasing substrate concentration but, conversely, the PHB content declined (Fig. 6b). 3.2.1.2. Fed-batch culture experiments. PHB accumulation commenced immediately after nitrogen exhaustion and continued for about 16 h. The M r of the polymer increased gradually during the accumulation phase (Fig. 7). In similar experiments under prolonged nitrogen and carbon exhaustion, M. extorquens utilised its accumulated PHB, and the M r decreased (data not shown).

3.3. Studies with Alcaligenes eutrophus 3.3.1. Effect of glucose concentration on the M, of PHB accumulated 3.3.1.1. Shake-flask culture experiments. Over the range of glucose concentrations studied (1040 g / l ; 55.5-222 mM) the PHB content decreased slightly although glucose consumption doubled. The M r decreased with increasing glu-

cose concentration while the polydispersity increased (Fig. 8). 3.3.1.Z Fed-batch culture experiments. PHB production started immediately after nitrogen exhaustion occurred and proceeded rapidly for about 8 h. The M r of the polymer synthesised decreased gradually from the first measurement (4 h after nitrogen exhaustion), and the polydispersity increased until the cessation of PHB synthesis (Fig. 9). In similar experiments (data not shown) evidence for an increase in M r during the initial stages of PHB synthesis was obtained but this was followed by the pattern of Fig. 7. Prolonged starvation for nitrogen and carbon led to mobilisation of PHB accompanied by a gradual fall in M r and increase in polydispersity. The variation of M r of PHB during batch fermentation of A. eutrophus could be explained in terms of simultaneous synthesis and degradation of the polymer, some evidence for which exists [14], or the synthesis of PHB of lower M r during the later stages of polymer accumulation.

101 A decrease in M r of P H B d u r i n g its a c c u m u l a tion by A. eutrophus was observed previously u n d e r c o n d i t i o n s of p h o s p h a t e exhaustion [4]. A small a m o u n t ( 1 - 5 % w / w ) of P H B is present in both A. eutrophus a n d M. extorquens d u r i n g e x p o n e n t i a l growth. It has b e e n f o u n d to be of relatively high m o l e c u l a r mass (1.4 × 10 6 for A. eutrophus a n d 1.6 x 10 5 for M. extorquens).

ACKNOWLEDGEMENTS W e are grateful for financial s u p p o r t from the E u r o p e a n C o m m i s s i o n u n d e r their E C L A I R prog r a m m e , from ICI Bio P r o d u c t s a n d F i n e C h e m i cals, a n d the Science a n d E n g i n e e r i n g R e s e a r c h C o u n c i l for the award of a C A S E S t u d e n t s h i p to B.T. W e also t h a n k D r David Byrom a n d Mrs L i n d a Naylor of ICI plc a n d D r A l e x a n d e r Steinbiichel, G 6 t t i n g e n , for their valuable assistance a n d discussions.

REFERENCES [t] Haywood, G.W., Anderson, A.J. and Dawes, E.A. (1989) A survey of the accumulation of novel polyhydroxyalkanoates by bacteria. Biotechnol. Lett. 11,471-476. [2] Haywood, G.W., Anderson, A.J., Williams, D.R., Dawes, E.A. and Ewing, D.F. (1991) Accumulation of poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int. J. Biol. Macromol. 13, 83-88. [3] Byrom, D. (1990) Industrial production of copolymer from Alcaligenes eutrophus. In: Novel Biodegradable Microbial Polymers (Dawes, E.A., Ed.), pp. 113-117. Kluwer, Dordrecht.

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