Cultivation engineering of microbial bioplastics production

FEMS Microbiology Reviews 103 (1992) 257-264 © 1992 Federation of European Microbiological Societies 0168-6445/92/$15.00 Published by Elsevier

257

FEMSRE 00242

Cultivation engineering of microbial bioplastics production Tsuneo Y a m a n e Laboratory of Bioreaction Engineering, Department of Food Science and Technology, Faculty of Agriculture, Nagoya Unicersity, Nagoya, Japan

Key words: PoIy-D( -- )-3-hydroxybutyrate; Theoretical yield; Overall yield; Productivity

1. SUMMARY Theoretical yields of poly-o(-)-3-hydroxybutyrate (PHB) from several carbon sources have been estimated from biochemical pathways leading to PHB. In estimating the yields, a special emphasis is made on recycling (or regeneration) of NADP + which is the co-substrate of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADP+-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. Theoretical and observed yields have been compared when polyhydroxyalkanoates (PHA) were synthesized from methanol and from n-amyl alcohol by a methylotroph, Paracoccus

rate on the basis of the residual biomass (total biomass-PHB contained) was proposed. The specific PHB formation rate decreased according to a mono-molecular decay model whose decay constant depended solely on the C / N ratio of the feed solution. From this model, an equation has been derived to calculate the volumetric productivity of PHB on the assumption that the total amount of the residual biomass is unchanged in the nitrogen-deficient PHB formation phase. Also, a graphical procedure has been shown to calculate the volumetric productivity of PHB. A comparison has been made between several data of PHB productivities that have been calculated from the literature.

denitrificans. An equation, which predicts the overall yield of PHB when allowance is made for non-PHB biomass formation in actual bacterial PHB production, has been derived as a function of both theoretical yields and PHB content of the total dry cell mass. The ratio of the overall (yield) to be theoretical yield is roughly proportional to the PHB content. A novel specific PHB formation

Correspondence to: T. Yamane, Laboratory of Bioreaction Engineering, Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Nagoya 464-01, Japan.

2. INTRODUCTION One of the obstacles which hamper large-scale commercialization of PHA is the considerabe high cost of its production. Factors determining the economics of PHA production on an industrial scale are the raw-material cost(s), the complexity of the technology and hence the capital and running costs of the production plant and the cost of product separation and purification. When all other items of cost, such as fixed costs (including depreciation), indirect labor cost, and cost of downstream processing are put aside,

258

the direct cost of production, ¥, for one batch run or fed-batch comprises mainly of the direct raw-material cost(s), the running cost (mostly utility costs such as steam, process water, coolingtower water, sterilized air, electricity, and natural gas or other fuel) and direct labor cost. Thus, ¥ for PHB production is given by the following equation (1). ks i

kt

kin

where ksi = cost of the ith substrate (medium component) (yen/g); Yp/si = PHB yield from the ith substrate (g P H B / g substrate); k t = runningcost constant (yen/h); 4, = volumetric productivity of PHB [g 1- h - l l; Vb = volume of culture broth (1); k I = constant of labor-cost term (yen/year); n = number of repetitions of culture per year; and P --- total PHB amount produced in a bioreactor [g]. The first term on of the right side of eqn. (1) is related to the PHB yield from the substrate, the second term to the productivity of PHB, and the third term to the direct labor cost. Since the direct labor'cost is quite complicated because it depends on the economic and social situations, only yield and productivity terms are discussed theoretically and experimentally in this article.

3. YIELD Among the substrates required, the carbon source is primarily significant in the case of PHA production since PHA is composed only of C, H and O atoms. Therefore the yield of PHA from the carbon source, Yp/c, is exclusively discussed in this paper. Yp/c is a very important factor for the industrial-scale PHA production. In this article, Yp/c for PHB formation under non-growing conditions is theoretically discussed, focusing on the mass balances of coenzymes (or more precisely co-substrates) such as NADP +, NAD +, ATP and Coenzyme A, which are involved in the metabolic pathway of PHB biosynthesis. Several papers have appeared concerning Yp/c estimation [2,3], but they have neglected the regeneration of the coenzymes, especially the involvement of NADP + regeneration in the PHB biosynthesis.

3.1. Theoretical and overall yields It is easily deduced from a simple mathematical consideration that Yp/c is greatest when nonPHB residual biomass is assumed to be absent [5]. In this article, the maximum value of Yp/c, i.e. the maximum PHB yield from the carbon source during the absence of non-PHB biomass formation, is designated by the theoretical yield, v(theor) "p/c ,

Table 1 Theoretical yield, v(the°r) • p/c , of PHB from various carbon sources based on biochemical stoichiometry and their market prices Carbon source

Assimilation pathway

N A D P ÷ regeneration

y(theor) (kg/kg)

Market price (¥/kg)

Glucose

Entner-Doudoroff Pentose-phosphate cycle Glycolysis + T C A Entner-Doudoroff Serine pathway Serine pathway A. eutrophus type R. rubrum type A. eutrophus type R. rubrum type

Glucose-6-phosphate Glucose-6-phosphate + 6-phosphogluconate Isocitrate Glucose-6-phosphate Isocitrate Isocitrate Isocitrate Isocitrate lsocitrate No need Isocitrate No need

0.48 0.44

120

Sucrose Methanol Methane Acetic acid Ethanol Butyric acid Butanol

a International market price of raw sugar

0.32 0.50 0.54 0.54 0.48 0.62 0.65 0.98 0.77 1.16

28 a 55 200 110 650 160

259 Acetoacetyl-CoA + N A D P H + H + ~ D ( - )-3hydroxybutyryl-CoA + N A D P ÷.

and the actual overall yield when allowance is made for preceding or simultaneous non-PHB biomass formation is designated by the overall yield, V(°verall) "p/c •

In some microorganisms, such as Zoogloea ramigera and Alcaligenes entrophus, another re-

3.2. Theoretical yield on biochemical stoichiometry

ductase which is N A D H - l i n k e d has been detected, but the product is L(+)-3-hydroxyacetylCoA, which cannot be the substrate for PHB polymerase. Only the N A D P H - l i n k e d reductase was found to participate in P H B synthesis from acetyl-CoA in a reconstituted system containing purified 3ketothiolase, acetoacetyl-CoA reductase and PHB synthase [7-9]. Thus, N A D P + generated from the reaction must be regenerated by another biochemical reaction(s) in order for P H B biosynthesis to continue. This fact forces us to look for a N A D P ÷ - r e generating reaction in addition to a PHB biosyn-

Two different approaches are possible to estimate the value of vtthe°r) i.e. on chemical stoip/c ' chiometry and on biochemical stoichiometry [6]. In the latter approach, both the metabolic pathway involved and the recycling of coenzymes must be carefully taken into consideration. PHB is synthesized from acetyl-CoA by three sequential reactions catalysed by 3-ketothiolase, acetoacetyl-CoA reductase and PHB synthase in most of the bacteria investigated so far [6]. It must be noted that acetoacetyl-CoA reductase is NADPH-linked, i.e. this enzyme catalyses only the following reaction:

(Entner-Doudoroff Pathway)

glucose ATP ADP

6-phosphogluconate-~

6-phosphoglucono-

i ....

!

<

~

~.

glucose-6-(~)

f

f .......

/ /

.

.

.

.

.

.

.

.

.

glyceroaldehyde- 3 ~ 2pyruvate t ~ / l t. . . . . . . . . . t- - --(~-- - -,4". . . . . . . t . . . . . -I - NAD' +Pi ' ~,~ r NADPH+H J NADP ~ i NADH+H'< 1~/ 2 C o A S H ' ~ ~--2NAD'

1,3-bisphosphoglycerate

2CO~ <-'J "->2NADH+2H'

A0P

2acet' ,I-CoA

ATP 3-phosphoglycerate

acetoacet yl-CoA 2-phosphoglycerate

J

> D(-)- 3-hydroxybutyryl-CoA

"-> ATP ~ADP

C o A S H ~'~

phosphoenolpyru~

Fig. 1. Biosynthesis of PHB from glucosevia the Entner-Doudoroff pathway.

PHB

260

thetic route: (1) glucose-6-phosphate dehydrogenase, (2) 6-phosphogluconate dehydrogenase (decarboxylating), (3) isocitrate dehydrogenase (NADP +-linked, cytoplasmic). The first enzyme is active in the EnterDoudoroff pathway and both the first and second are enzymes from the pentose-phosphate cycle. Enzyme(s) (1) or (1) plus (2) is the most frequently encountered when the carbon source is carbohydrate, but microorganisms seem to resort to enzyme (3) when they are provided with alcohol, organic acids, alkanes, or other substrates. Coenzyme A, another co-substrate involved in the three sequential reactions, is self-recycled on the balance within the three reactions. Although neither NAD + nor ATP are required in the three sequential reactions, one must also consider recycling of NAD+/NADH and A T P / A D P in all pathways including PHB biosynthesis. Deficits in these co-substrates may not allow smooth flow of metabolites. Under aerobic conditions, recycling of N A D * / N A D H is mainly achieved by the respiratory chain and is accompanied by formation of several mol ATP. Sometimes, a surplus of ATP merely dissipates biochemical energy by ATPase, or polyphosphate accumulates under conditions of excess phosphate [10]. Following the above-mentioned principle, ypttheor) is estimated when each of C 1, C, and C 4 /c compounds is supplied separately, taking the balance of NADP+/NADPH, NAD÷/NADH and A T P / A D P into consideration. For example, when glucose is converted to acetyl-CoA via the Entner-Doudoroff pathway, which is employed by Pseudomonas sp., Azotobacter sp. and Alcaligenes eutrophus, the biosynthetic pathway of PHB from glucose including ATP and NAD + formations and NADP+/NADPH recycling is as depicted in Fig. 1, where NADP + is regenerated by glucose-6phosphate dehydrogenase. The net reaction is glucose + (3p + 1)ADP + (3p + 1)P i + ( 3 / 2 ) 0 2 -~ ( 1 / n ) P H B + 2CO 2 + (3p + 1)ATP + 3H20 where p is the P / O ratio. From the net reaction ypttheor) = 86/180 = 0.48. /c

The estimated values of v(the°r) "p/c are summarized in Table 1, together with market prices of the carbon sources, seen as both yield and price determine the raw-material cost (see eqn. (1)). It is noteworthy that there are two different metabolic pathways leading to PHB in the cases of butanol and butyric acid. /3-Oxidation of butyric acid gives rise to crotonyl-CoA. In Alcaligenes eutrophus and many other bacteria, crotonyl-CoA is converted to L(+)-3-hydroxybutyryl-CoA followed by the D(--)-form via acetoacetyl-CoA by both L(+)-3-hydroxyacyl-CoA dehydrogenase and acetoacyl-CoA reductase [69]; while in Rhodospirillum rubrum, crotonyl-CoA is directly converted to D(-)-3-hydroxybutyrylCoA by crotonyl-CoA hydrotase [11]. In the A. eutrophus-type pathway, NADP + must be regenerated by another enzyme, i.e. isocitrate dehydrogenase in the TCA cycle; but in a R. rubrum-type pathway, no such co-substrate regeneration is necessary, which results in a considerably higher value of "v(the°r) i.e. the value calculated by "o/c , chemical stoichiometry.

3.3. Comparison of theoretical and obserced yields We have recently found that Paracoccus denitrificans (Mierococcus denitrificans, an autotrophic methylotroph, can produce not only homopolymers (PHB from methanol and poly-3hydroxyvaleric acid from n-amyi alcohol) but also a copolymer of P(3HB)-co-P(3HV) [12,13]. P. denitrificans follows the ribuiose bisphosphate pathway of methanol assimilation [14,15], whereby methanol is first completely oxidized to CO z which is followed by CO 2 assimilation in the ribulose bisphosphate cycle. The ribulose bisphosphate cycle consumes a considerably amount of ATP to assimilate CO 2 (8 moi ATP consumed/5 tool CO 2 assimilated). If 1 tool of ATP is assumed to be formed from ADP through 1 tool of methanol oxidation to CO 2, a PHB yield as low as 0.15 (kg/kg) is predicted from the ATP balance, which is comparable with the observed yield of 0.11 (kg/kg) (obtained recently in a batch culture [12] (Table 2). Although all metabolic steps relevant to the PHB biosynthesis of methanol, i.e. methanol oxidation, ribulose bisphosphate pathway, TCA cy-

261 Table 2 Comparison

of theoretical

and observed

yields

of PHA

by

overall yield of PHB with the theoretical described above [4] as follows:

yield

Paracoccus denitrificans Carbon

PHA

source

Methanol n-Amy1 alcohol

Theoretical

(g/g)

Observed

ycovrralr, P/C -= y(theor)

Chemical

Biochemical

(g/g)

P(3HB)

0.67 a

0.15 h

0.11

P(3HV)

1.14 c

1.14 d

0.97

CP

yuheor)

P/C

a Chemical stoichiometry: 8nCH ,OH + 3n0, -+ 2(C,H,0Z ),, + 10n HzO. h (1 mol ATP formed)/(l mol MeOH oxidized) is assumed. ’ Chemical stoichiometry: 2nC5H,,0H + 3n02 + 2(C,H,O,), +4n H,O. d No involvement of NADP+/(NADPH + H+ ) recycling is assumed.

cle and PHB biosynthesis including all the cosubstrate regeneration, have not yet been well established in P. denitrifcans, it is clear that the low PHB yield was due to dissipation of carbon atoms as CO, gas. In other words, a large amount of CO, derived from methanol oxidation was not assimilated but was wasted as CO, from the bioreactor. On the other hand, when we incubated the strain in nitrogen-free medium with n-amyl alcohol, the strain accumulated a polyester composed of 92% P (3HV) and 8% P (3HB) in amounts of up to 45% of total cell dry weight. The yield of polyester from n-amyl alcohol was 0.97 (kg/kg), which is very close to the theoretical value (1.14) calculated by chemical stoichiometry (Table 2). This result seems to indicate that the biosynthesis of P (3HV) from n-amyl alcohol does not require NADP+/(NADPH + H+) regeneration.

3.4. Ocerall yield The overall yield is the yield observed which is calculated from the actual microbial reaction where PHB accumulates in the starved stage succeeding the cell-growth stage or where PHB accumulated partially in parallel with cell growth. Making simple assumptions, it is possible to derive an equation which correlates the actual

P/C

y /

x/c +

(1 -

Y$-/

Yx,JCp

(2) where Yd/trera”’is the overall yield of PHB from the carbon source (kg/kg); YX+ is the cell-growth yield from the carbon source under balanced growth conditions (kg/kg) and C, is the content of PHB in the total biomass. Figure 2 is a diagram resulting from eqn. (2) as a function of C,, using several values of Y$p)/YX,c as parameter. From the observed value of growth yield under balanced growth, the value of Yr$p)/YX,c seems to lie within a narrow range of approx. l.l- 1.3. This indicates that the dependence of the value of y@verall, yd;hceor) on the variations in / y~.+;,,~ is not significant, and YdFra”)/ yPtL) . IS roughly proportional to C,. Eqn. (2) or P/C Fig. 2 will be useful to estimate Y$Tra”). For example, when PHB production from sucrose is attempted using a microorganism that employs the Entner-Doudoroff pathway, and YX,c= 0.40 and C, = 0.70, then YdFra”’ is calculated as 0.33

C, Cg PHBlg dry cell3 Fig.

2. Diagram

(WcrJll) from to estimate Yp,c and C,.

y;;p,,

y,,,

262

(kg P H B / k g sucrose), which means that 3.1 kg sucrose is required to produce 1 kg of PHB.

(a)

60

4. PRODUCTIVITY

~0

Productivity, or space-time yield, which is • in eqn. (1), is directly concerned with the running costs of a process. • is a factor related to the rate of the process. For bacterial PHA production, increases by attaining (1) the greatest amounts of cells, (2) having the highest content of PHA, (3) in the shortest operating time. (b)

In any microbial reaction, the cells' activity to synthesize the metabolite concerned is estimated by their specific formation rate (g metabolite (g dry cell mass) -t h-L). When the microorganism produces and accumulates PHB intracellularly, the whole dry cell mass, Xtotal, consists of two parts, namely PHB, XpH B, and residual biomass, Xresidua., where Xresidual is calculated by subtracting the PHB concentration ( S r e s i d u a l = S t o t a l XeHB). We considered that only Sresidual would represent anabolically active biomass composed of nucleic acids and proteins. Therefore we proposed a novel specific PHB formation rate, qr'HS, based on the residual biomass, as a more rational specific rate than the one based on the total biomass [16]: 1

×

d(XpH B × VB) dt

QPHB =" Sresidua I X V b

1 ( Xtota

I -

XpHB)

× X

V b

d( Xp. × Vb) dt

(3) When we analysed our experimental data of PHB production from methanol by fed-batch cultures according to eqn. (3), we found that the change in qPHB after nitrogen limitation could be expressed by the following equation, analogous to a monomolecular decay model: qPHB = ( q P H B ) 0 X

exp( --kdt )

(4)

In eqn. (4), t is the time elapsed after the nitrogen source has been exhaustively consumed

¢

~

~

~

~--~--

i

t

I

2

0

4.1. Specific formation rate of PHB

~

1

i

i

i

I

t

t

t

I

0.1

"-~0.01

\

0.001

. . . .

0

'

\

\

\

. . . . .

50

100 t

(h)

Fig. 3. Effect of ammonia feeding on PHB production by fed-batch cultivation of Protomonas extorquens with a constant ratio of methanol/ammonia [17]. (a) Time course of the PHB content of the cells. (b) Time course of the specific PHB production rate. The dotted line in (b) shows the time course of qPHB by stopping ammonia feeding, i.e. C / N =o0. From the initial stages of the fed-batch cultures, C / N ratios of feeding solution were present at 0, 0.75; e, 10.0; ®, 12.5; ¢, 16.3; zx, 21.3; v , 25.0 (mol/mol).

and (qeHa)0 is the value of qPHB at time 0. k d was proposed as the specific reduction rate of PHB. The fact that qPHa could be expressed by eqn. (4) verifies the soundness of our definition of qPHB by eqn. (3). Interestingly, we found that k a depended solely on the ratio of the amount of methanol fed to the amount of ammonia fed, that is, fed C / N ratio, as shown in Fig. 3 in the

263

fed-batch cultures with a constant ratio of methanol/ammonia [17]. The following empirical equations were obtained from Fig. 3: XpHB/Xtota

I =

--

(-CellGrowthPhase~

PHB Production Phase

1

tg

tp

',

~- td --~

:

77 + 42 × I n ( C / N )

k d = 5.9 × 10 -4 × ( C / N ) " 4

/

o

] Fig. 4. Graphical determinationof PHB productivityin batch or fed-batchculture.

( qPHB)O X p H B X V b ~-- ( X p H B X Vb) 0 "{- - -

kd

kd

(8)

It needs to be noted, however, that Alcaligenes latus is quite unique since this strain synthesizes

{1 - e x p ( --kdt)}

PHB in a growth-associated manner, i.e. tg = 0 [17]. This enables us to expect a higher 4) for A. latus than by other bacteria. From eqns. (6a) and (7) one can calculate q~ by

4.2. (Volumetric) productivity of PHB Since batch or fed-batch cultures have a down time necessary for cleaning, refilling, sterilizing and emptying, the volumetric productivity, q~, of PHB is given by: td + i f )

Time ['h

tf = tg + tp

(65)

qD = X p t l B / t p - - X p H B / (

Culture

production phase under nutrient-deficient conditions (to), which is often applied to the bacterial PHB production, tf is given by:

- exp( --kdt)} (6a)

When Vb is constant, as in special cases, eqn. (6a) is reduced to: (qPHB)O X p . B = ( X p n B ) 0 -f- - X

'

Ld

(5)

It is reasonable to assume that Xr¢sidua,X Vb is unchanged in the nitrogen-deficient phase, so that XpH B X Vu can be expressed as a function of t by substituting eqn. (3) into eqn. (4), which after integration gives:

)< ( Xresidua, )< V b ) { 1

7

=

( t d "t- tg +

t p ) × (Xprm)tg>

(Vb)tg (Vb)t'-------~o

+ (qPnB)tg XSresidual

ka

(7)

where t d is the down time and tf is the actual time of the microbial reaction. In a two-stage process consisting of the first rapid microbial growth phase (tg), followed by the second PHB

× {1 - e x p ( - k a t o ) } ]

It is also possible to calculate qb graphical as shown in Fig. 4 even though q~ cannot be ex-

Table 3 Comparison of P(3HB) productivities by various bacteria Bacterium

Carbon source

Final conc. (g l - t )

Max. content (%)

Time required (h)

Productivity ~ (g 1-1 h - I )

Reference

Protomonas extorquens Alcaligenes latus Alcaligenes eutrophus Alcaligenes eutrophus

Methanol Sucrose CO 2 Fructose

136 8.5 35.6 3.7

64 77 58 58

121 17 60 47

1.12 0.50 0.59 0.079

17 18 19 20

a Calculated on the assumption t o = 0.

(9)

264 p r e s s e d m a t h e m a t i c a l l y , q~ is t h e s l o p e o f a s t r a i g h t line w h i c h c r o s s e s o r t a n g e n t s t h e c u r v e o f XpH B VS. (t d + tf). Table 3 summarizes comparisons of PHB productivities calculated from the literature. Our data a r e t h e h i g h e s t so far b u t e v e n so t h e y a r e as low as t h e o r d e r o f 1 g P H B × I - ~ × h -~. F u r t h e r i n v e s t i g a t i o n is r e q u i r e d to f u r t h e r i n c r e a s e P H B productivity.

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in polyhydroxyalkanoate synthesis by Alcaligenes eutrophus. FEMS Microbiol. Lett. 57, 1-6. [10] Doi, Y., Kawaguchi, Y., Nakamura, Y. and Kunioka, M. (1989) Nuclear magnetic resonance studies of poly(3-hydroxybutyrate) and polyphosphate metabolism in Alcaligenes eutrophus. Appl. Environ. Microbiol. 55, 2932-2938. [11] Moskowitz, G.I. and Merrick, J.M. (1969) Metabolism of poly-/3-hydroxybutyrate, II. Enzymatic synthesis of o( - )/3-hydroxybutyryl Coenzyme A by an enoyl hydrase from Rhodospirillum rubrum. Biochemistry 8, 2748-2755. [12] Ueda, S., Matsumoto, S., Takagi, A. and Yamane, T. (1993) Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from methanol and n-amyl alcohol by methylotrophic bacteria, Paracoccus denitrificans and Methylobacterium extorquens. Appl. Environ. Microbiol. 58, in press. [13] Ueda, S., Matsumoto, S., Takagi, A. and Yamane, T. (1992) Application of n-amyl alcohol as a substrate for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacteria. FEMS Microbiol. Len. 98, 57-60. [14] Cox, R.B. and Quayle, J.R. (1975)The autotrophic growth of Micrococcus denitrificans on methanol. Biochem. J. 150, 569-571. [15] Shively, J.M., Saluja, A. and McFadden, B.A. (1978) Ribulose bisphosphate carboxylase from methanol-grown Paracoccus denitrificans. J. Bacteriol. 134, 1123-1132. [16] Suzuki, T., Yamane, T. and Shimizu, S. (1986) Kinetics and effect of nitrogen source feeding on production of poly-fl-hydroxybutyric acid by fed-batch culture. Appl. Microbiol. Biotechnol. 24, 366-369, [17] Suzuki, T., Yamane, T. and Shimizu, S. (1986) Mass production of poly-/3-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding. Appl. Microbiol. Biotechnol. 24, 370-373, [18] Braunegg, G. and Bogensberger, B. (1985) Zur Kinetik des Wachstums und der Speicherung von Poly-o(-)-3hydroxybuttersiiure bei Alcaligenes latus. Acta Biotechnol. 5, 339-345. [19] Ishizaki, A. and Tanaka, K. (1991) Production of poly-flhydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697 T. J. Ferment. Bioeng. 71, 254257. [20] Mulchandani, A., Luong, J.H.T. and Groom, G. (1989) Substrate inhibition kinetics for microbial growth and synthesis of poly-fl-hydroxybutyric acid by AIcaligenes eutrophus ATCC 17697. Appl. Microbiol. Biotechnol. 30, 11-17.