APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1992, p. 744-746
Vol. 58, No. 2
0099-2240/92/020744-03$02.00/0 Copyright 0 1992, American Society for Microbiology
Effect of Nitrogen Limitation on Long-Side-Chain Poly-3Hydroxyalkanoate Synthesis by Pseudomonas resinovorans BRUCE A. RAMSAY,'* ILIE SARACOVAN,2 JULIANA A. RAMSAY,' AND ROBERT H. MARCHESSAULT2 Department de Genie Chimique, tcole Polytechnique de Montreal, C.P. 6079, succursale "A," Montreal, Que'bec H3C 3A7,' and Chemistry Department, McGill University, Montreal, Quebec H3A 2A7,2 Canada Received 22 July 1991/Accepted 21 November 1991 Pseudomonas resinovorans produced poly-.(-hydroxyalkanoates (PHAs) when grown on hydrocarbons but not on glucose. In a chemostat culture, the PHA composition was l-hydroxybutyrate (C4)- -hydroxyhexanoate (C6--hydroxyoctanoate (C8--hydroxydecanoate (CIO) (1:15:75:9) on octanoate and C4-C6-C8-C10 (8:62: 23:7) on hexanoic acid. Contrary to the reported behavior of Pseudomonas oleovorans, the PHA accumulation rate increased under ammonium limitation on octanoate. the agitation rate at 900 rpm and the aeration rate at 1.5 liter h-'. The temperature and pH were 30°C and 7.0, respectively. The dissolved oxygen concentration was kept at 40% of saturation or higher. Shake-flask studies were performed in 500-ml Erlenmeyer flasks containing 100 ml of mineral salts medium with sodium octanoate or glucose as the carbon source at pH 7.0. The contents of the flasks were incubated on a New Brunswick G10 Gyrotory shaker at 250 rpm and 30°C for 48 h. The biomass dry weight was determined as previously described (10). The centrifuged biomass from 10 ml of culture broth was analyzed for protein content by the biuret reaction (13) using 1.0% (wt/vol) bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) as a standard. The ammonium ion concentration in the culture broth supernatant was quantified with a gas ammonia probe (Orion Research Laboratories, Cambridge, Mass.), by using (NH4)2SO4 solutions as standards. To determine the concentration of unassimilated octanoate, 2 ml of methanol containing 0.5% (vol/vol) pentanoic acid (as an internal standard) and 2% (vol/vol) concentrated H2SO4 was added to 1 ml of supernatant and heated for 1 h at 105°C in a screw-cap tube. After cooling the solution to room temperature, 2 ml of distilled water was added and the methyl esters were extracted into 2 ml of ether. Subsequent analysis was by gas chromatography (11). To quantify the intracellular PHA, biomass samples were harvested by centrifugation and heated in acidified methanol (2). Chloroform-extracted methyl esters (2 RIl) of the PHA monomers were quantified by gas chromatography (11). Shake-flask studies showed that P. resinovorans grew well on glucose but no PHA was produced. In the chemostat culture, the carbon-to-nitrogen (C/N) ratio was varied by increasing the amount of octanoate in the feed at a dilution rate of 0.25 h-'. Under nitrogen-limited conditions at C/N ratios greater than 7.33 mol mol-1 (i.e., greater than 4 g of octanoate liter-' in the feed), the PHA monomer composition remained virtually constant at 15:75:9 P-hydroxyhexanoate (C6)-3-hydroxyoctanoate (C8)-4-hydroxydecanoate (Clo), with traces of ,-hydroxybutyrate (C4). Under carbonlimited conditions, there was not enough polymer to accurately measure its composition. As with P. oleovorans, the predominant monomer reflected the chain length of the carbon source. When grown on hexanoic acid at a dilution rate of 0.125 h-', the monomer composition was 8:62:23:7 C4-C6-C8-C10. There was a dramatic increase in the amount
In the last decade, knowledge about the variety of poly,-hydroxyalkanoates (PHAs) which exist in nature or can be produced in the laboratory has been greatly expanded (3). Long-side-chain (LSC) PHAs are of interest since their physical properties are very different from those of the most common PHA, poly-p-hydroxybutyrate (PHB) (7, 8). However, the LSC PHAs have yet to find commercial application. One reason for this is their high cost of production. It was originally believed that they could only be made from hydrocarbons such as alkanes or alkanoic acids (C6 to C12) (5), but recently LSC PHAs, especially those containing P-hydroxydecanoate monomers, have been produced from carbohydrates by a variety of bacteria (6). Knowledge of their production kinetics is required for the commercial exploitation of LSC PHAs. In a chemostat culture, LSC PHA synthesis by Pseudomonas oleovorans grown on octanoate was not significantly stimulated by nitrogen limitation (11). This was surprising since the limitation of nitrogen, phosphorous, and certain other elements greatly increases the polymer accumulation rate in most PHB-accumulating bacteria. P. oleovorans and other PHA-producing fluorescent pseudomonads cannot make homopolymeric PHB. Since their metabolic pathway for PHA synthesis is different from that of PHB producers such as Alcaligenes eutrophus (3), the regulation of LSC PHA accumulation may not be affected by nutrient limitation. This paper describes the effect of nitrogen limitation on the LSC PHA accumulation rate by another fluorescent pseudomonad, Pseudomonas resinovorans. P. resinovorans ATCC 14235 was grown on a mineral salts medium containing (per liter) 3.7 g of Na2HPO4 7H20, 0.83 g of KH2PO4, 2.0 g of (NH4)2SO4, 0.2 g of MgSO4 7H20, 60 mg of ferrous ammonium citrate, 10 mg of CaCl2 .2H20, and 1 ml of microelement solution. Each liter of microelement solution contained 0.3 g of H3BO3, 0.2 g CoCl2 6H20, 0.1 g of ZnSO4 *7H20, 30 mg of MnCl2 4H20, 30 mg of NaMoO4- 2H2O, 20 mg of NiCl2 6H20, and 10 mg of CuSO4 * 5H20. Sodium octanoate with 99% purity (Omega Chemical Co., Quebec, Canada) or hexanoic acid (Aldrich Chemical Co., Milwaukee, Wis.) was used as the carbon source. Continuous fermentations were performed in a Multigen F-2000 2-liter fermenter (New Brunswick Scientific, Edison, N.J.). The working volume was 1.5 liters with -
-
-
*
Corresponding author. 744
VOL. 58, 1992
NOTES
0.5
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16
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14at
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o i
8
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0
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.
8 10 12 14 OCTANOATE IN FEED (g L 1)
16
5
25
4
Iz
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0 0 0
0
o
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04
0
4
o a
2
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745
10
15
20
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0.
0
C/N RATIO FIG. 2. Effect of octanoate feed concentration on the percentage of PHA accumulated and the specific PHA productivity (qp) by P. resinovorans. Values were calculated from data in Fig. 1.
z
-j 1
0
4
OCTANOATE IN FEED (g L-1) 0
5
10
15
20
25
C/N RATIO FIG. 1. Effect of octanoate feed concentration on growth (bottom) and PHA production (top) by P. resinovorans at D = 0.25 h-1.
of PHA produced after the steady-state NH4+ concentration approached 0 with octanoate as the carbon source (Fig. 1). This effect of nitrogen limitation was similar to that found for PHB production on glucose in the chemostat culture of A. eutrophus (9) but unlike that of the most-studied LSC PHA producer, P. oleovorans (11). Unlike P. oleovorans, P. resinovorans responded to nitrogen limitation by greatly increasing the PHA production rate. At the point of maximum PHA accumulation, the specific PHA production rate (based on the cellular protein content) and the amount of PHA produced (expressed as the weight percent of dry biomass) were both about five times greater than that found during balanced growth (Fig. 2). This occurred at the ratio of 17 mol of octanoate mol-1 of NH4+ ion in the feed (i.e., at 9 g of octanoate liter-l in Fig. 1 and 2). Exceeding this C/N ratio resulted in decreased productivity probably due to inhibition by unmetabolized octanoate. At 9 g of octanoate liter-' in the feed, there was 2.2 g of unmetabolized octanoate liter-' in the reactor. Above 10 g of octanoate liter-1, the bacteria washed out because of substrate inhibition. The maximum specific productivity obtained at D = 0.25 h-1 was 50 mg of PHA g-1 of cellular protein h-1, compared with 74 mg of PHA g-1 of cellular protein h-1 for P. oleovorans at D = 0.24 h-1 (11). Thus P. oleovorans, although not very responsive to nitrogen limitation, is still more productive. It suggests that PHA synthesis in P. oleovorans is not highly regulated, whereas P. resinovorans, also a fluorescent pseudomonad, possesses a regulation mechanism similar to PHB-accumulating microorganisms such as A. eutrophus. Examination of the kinetic data for the production of poly(,-hydroxyoctanoate-co-4-hydroxydecanoate) by P. aeruginosa PAC1 grown on gluconate (14)
shows that the rate of polymer accumulation by this bacterium is also greatly increased by nitrogen limitation. The selective advantage of PHA accumulation to microorganisms is as a carbon and energy reserve (4) and as an electron sink (12) when carbon sources are readily available but an absence of some other nutritional element makes growth impossible. A high, constant rate of PHA accumulation should be a competitive disadvantage since it diverts valuable carbon and energy from growth. This may explain why constitutive producers such as P. oleovorans and Alcaligenes latus (1) are rare. However, at least in the case of P. oleovorans, there is a compensating effect. As its specific growth rate increases, the ratio of PHA synthesis to growth rate declines, allowing the flow of carbon to be directed more to growth than to polymer synthesis (11). If LSC PHAs are to become available commercially, extensive screening must be conducted to select for higheryielding strains. Among such cultures, microorganisms like P. resinovorans, whose PHA accumulation is regulated by nutrient limitation, have greater economic potential than P. oleovorans. By separating growth and accumulation, less expensive substrates directed exclusively towards growth can be used. For example, P. resinovorans could be grown on an inexpensive carbohydrate such as glucose, and then octanoate could be added when the NH4+ concentration becomes limiting. Thus, the more expensive substrate should be dedicated to polymer accumulation. We acknowledge the financial support of the Natural Science and Engineering Research Council of Canada and Xerox Canada.
REFERENCES 1. Braunegg, G., and B. Bogensberger. 1985. Zur kinetik des wachstums und der speicherung von poly-D(-)-3-hydroxybuttersaure bei Alcaligenes latus. Acta Biotechnol. 5:339-345. 2. Braunegg, G., B. Sonnleitner, and R. M. Lafferty. 1978. A rapid method for the determination of poly-f3-hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol. 6:2937. 3. Dawes, A. J., and E. A. Anderson. 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54:450-472.
746
NOTES
4. Dawes, E. A., and P. J. Senior. 1973. The role and regulation of energy reserve polymers in micro-organisms. Adv. Microbiol. Physiol. 10:135-266. 5. Haywood, G. W., A. J. Anderson, and E. A. Dawes. 1989. A survey of the accumulation of novel polyhydroxyalkanoates by bacteria. Biotechnol. Lett. 11:471-476. 6. Haywood, G. W., A. J. Anderson, D. F. Ewing, and E. A. Dawes. 1990. Accumulation of a polyhydroxyalkanoate containing primarily 3-hydroxydecanoate from simple carbohydrate substrates by Pseudomonas sp. strain NCIMB 40135. Appl. Environ. Microbiol. 56:3354-3359. 7. Holmes, P. A. 1985. Applications of PHB-a microbially produced biodegradable thermoplastic. Phys. Technol. 16:32-36. 8. Marchessault, R. H., C. J. Monasterios, F. G. Morin, and P. R. Sundararajan. 1990. Chiral poly(P-hydroxyalkanoates): an adaptable helix influenced by the alkane side-chain. Int. J. Biol. Macromol. 12:158-165. 9. Ramsay, B. A., K. Lomaliza, C. Chavarie, B. Dube, P. Bataille, and J. A. Ramsay. 1990. Production of poly-(,-hydroxybutyric-
APPL. ENVIRON. MICROBIOL.
co-p-hydroxyvaleric) acids. Appl. Environ. Microbiol. 56:20932098. 10. Ramsay, B. A., J. A. Ramsay, and D. G. Cooper. 1989. Production of poly-p-hydroxyalkanoic acid by Pseudomonas cepacia. Appl. Environ. Microbiol. 55:584-589. 11. Ramsay, B. A., I. Saracovan, J. A. Ramsay, and R. H. Marchessault. 1991. Continuous production of long-side-chain poly-phydroxyalkanoates by Pseudomonas oleovorans. Appi. Environ. Microbiol. 57:625-629. 12. Senior, P. J., and E. A. Dawes. 1973. Poly-p-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem. J. 125:55-66. 13. Stickland, L. H. 1951. The determination of small quantities of bacteria by means of the biuret reaction. J. Gen. Microbiol. 43:159-271. 14. Timm, A., and A. Steinbuchel. 1990. Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl. Environ. Microbiol. 56:3360-3367.
Vol. 58, No. 2
0099-2240/92/020744-03$02.00/0 Copyright 0 1992, American Society for Microbiology
Effect of Nitrogen Limitation on Long-Side-Chain Poly-3Hydroxyalkanoate Synthesis by Pseudomonas resinovorans BRUCE A. RAMSAY,'* ILIE SARACOVAN,2 JULIANA A. RAMSAY,' AND ROBERT H. MARCHESSAULT2 Department de Genie Chimique, tcole Polytechnique de Montreal, C.P. 6079, succursale "A," Montreal, Que'bec H3C 3A7,' and Chemistry Department, McGill University, Montreal, Quebec H3A 2A7,2 Canada Received 22 July 1991/Accepted 21 November 1991 Pseudomonas resinovorans produced poly-.(-hydroxyalkanoates (PHAs) when grown on hydrocarbons but not on glucose. In a chemostat culture, the PHA composition was l-hydroxybutyrate (C4)- -hydroxyhexanoate (C6--hydroxyoctanoate (C8--hydroxydecanoate (CIO) (1:15:75:9) on octanoate and C4-C6-C8-C10 (8:62: 23:7) on hexanoic acid. Contrary to the reported behavior of Pseudomonas oleovorans, the PHA accumulation rate increased under ammonium limitation on octanoate. the agitation rate at 900 rpm and the aeration rate at 1.5 liter h-'. The temperature and pH were 30°C and 7.0, respectively. The dissolved oxygen concentration was kept at 40% of saturation or higher. Shake-flask studies were performed in 500-ml Erlenmeyer flasks containing 100 ml of mineral salts medium with sodium octanoate or glucose as the carbon source at pH 7.0. The contents of the flasks were incubated on a New Brunswick G10 Gyrotory shaker at 250 rpm and 30°C for 48 h. The biomass dry weight was determined as previously described (10). The centrifuged biomass from 10 ml of culture broth was analyzed for protein content by the biuret reaction (13) using 1.0% (wt/vol) bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) as a standard. The ammonium ion concentration in the culture broth supernatant was quantified with a gas ammonia probe (Orion Research Laboratories, Cambridge, Mass.), by using (NH4)2SO4 solutions as standards. To determine the concentration of unassimilated octanoate, 2 ml of methanol containing 0.5% (vol/vol) pentanoic acid (as an internal standard) and 2% (vol/vol) concentrated H2SO4 was added to 1 ml of supernatant and heated for 1 h at 105°C in a screw-cap tube. After cooling the solution to room temperature, 2 ml of distilled water was added and the methyl esters were extracted into 2 ml of ether. Subsequent analysis was by gas chromatography (11). To quantify the intracellular PHA, biomass samples were harvested by centrifugation and heated in acidified methanol (2). Chloroform-extracted methyl esters (2 RIl) of the PHA monomers were quantified by gas chromatography (11). Shake-flask studies showed that P. resinovorans grew well on glucose but no PHA was produced. In the chemostat culture, the carbon-to-nitrogen (C/N) ratio was varied by increasing the amount of octanoate in the feed at a dilution rate of 0.25 h-'. Under nitrogen-limited conditions at C/N ratios greater than 7.33 mol mol-1 (i.e., greater than 4 g of octanoate liter-' in the feed), the PHA monomer composition remained virtually constant at 15:75:9 P-hydroxyhexanoate (C6)-3-hydroxyoctanoate (C8)-4-hydroxydecanoate (Clo), with traces of ,-hydroxybutyrate (C4). Under carbonlimited conditions, there was not enough polymer to accurately measure its composition. As with P. oleovorans, the predominant monomer reflected the chain length of the carbon source. When grown on hexanoic acid at a dilution rate of 0.125 h-', the monomer composition was 8:62:23:7 C4-C6-C8-C10. There was a dramatic increase in the amount
In the last decade, knowledge about the variety of poly,-hydroxyalkanoates (PHAs) which exist in nature or can be produced in the laboratory has been greatly expanded (3). Long-side-chain (LSC) PHAs are of interest since their physical properties are very different from those of the most common PHA, poly-p-hydroxybutyrate (PHB) (7, 8). However, the LSC PHAs have yet to find commercial application. One reason for this is their high cost of production. It was originally believed that they could only be made from hydrocarbons such as alkanes or alkanoic acids (C6 to C12) (5), but recently LSC PHAs, especially those containing P-hydroxydecanoate monomers, have been produced from carbohydrates by a variety of bacteria (6). Knowledge of their production kinetics is required for the commercial exploitation of LSC PHAs. In a chemostat culture, LSC PHA synthesis by Pseudomonas oleovorans grown on octanoate was not significantly stimulated by nitrogen limitation (11). This was surprising since the limitation of nitrogen, phosphorous, and certain other elements greatly increases the polymer accumulation rate in most PHB-accumulating bacteria. P. oleovorans and other PHA-producing fluorescent pseudomonads cannot make homopolymeric PHB. Since their metabolic pathway for PHA synthesis is different from that of PHB producers such as Alcaligenes eutrophus (3), the regulation of LSC PHA accumulation may not be affected by nutrient limitation. This paper describes the effect of nitrogen limitation on the LSC PHA accumulation rate by another fluorescent pseudomonad, Pseudomonas resinovorans. P. resinovorans ATCC 14235 was grown on a mineral salts medium containing (per liter) 3.7 g of Na2HPO4 7H20, 0.83 g of KH2PO4, 2.0 g of (NH4)2SO4, 0.2 g of MgSO4 7H20, 60 mg of ferrous ammonium citrate, 10 mg of CaCl2 .2H20, and 1 ml of microelement solution. Each liter of microelement solution contained 0.3 g of H3BO3, 0.2 g CoCl2 6H20, 0.1 g of ZnSO4 *7H20, 30 mg of MnCl2 4H20, 30 mg of NaMoO4- 2H2O, 20 mg of NiCl2 6H20, and 10 mg of CuSO4 * 5H20. Sodium octanoate with 99% purity (Omega Chemical Co., Quebec, Canada) or hexanoic acid (Aldrich Chemical Co., Milwaukee, Wis.) was used as the carbon source. Continuous fermentations were performed in a Multigen F-2000 2-liter fermenter (New Brunswick Scientific, Edison, N.J.). The working volume was 1.5 liters with -
-
-
*
Corresponding author. 744
VOL. 58, 1992
NOTES
0.5
0--
16
I
14at
In IV
o i
8
12
0
-J) 0S
*P I
10 8
I
m
t0
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'
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4
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.
6
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2
500 -
*
l
400
E
I
300
°
mn
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200
O)
z
100
+sd
0 °
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0 0
0
2
.
6
1
0
.
8 10 12 14 OCTANOATE IN FEED (g L 1)
16
5
25
4
Iz
.3 00-:
0 0 0
0
o
Z o
iO
04
0
4
o a
2
I
745
10
15
20
0
Q
0.
0
C/N RATIO FIG. 2. Effect of octanoate feed concentration on the percentage of PHA accumulated and the specific PHA productivity (qp) by P. resinovorans. Values were calculated from data in Fig. 1.
z
-j 1
0
4
OCTANOATE IN FEED (g L-1) 0
5
10
15
20
25
C/N RATIO FIG. 1. Effect of octanoate feed concentration on growth (bottom) and PHA production (top) by P. resinovorans at D = 0.25 h-1.
of PHA produced after the steady-state NH4+ concentration approached 0 with octanoate as the carbon source (Fig. 1). This effect of nitrogen limitation was similar to that found for PHB production on glucose in the chemostat culture of A. eutrophus (9) but unlike that of the most-studied LSC PHA producer, P. oleovorans (11). Unlike P. oleovorans, P. resinovorans responded to nitrogen limitation by greatly increasing the PHA production rate. At the point of maximum PHA accumulation, the specific PHA production rate (based on the cellular protein content) and the amount of PHA produced (expressed as the weight percent of dry biomass) were both about five times greater than that found during balanced growth (Fig. 2). This occurred at the ratio of 17 mol of octanoate mol-1 of NH4+ ion in the feed (i.e., at 9 g of octanoate liter-l in Fig. 1 and 2). Exceeding this C/N ratio resulted in decreased productivity probably due to inhibition by unmetabolized octanoate. At 9 g of octanoate liter-' in the feed, there was 2.2 g of unmetabolized octanoate liter-' in the reactor. Above 10 g of octanoate liter-1, the bacteria washed out because of substrate inhibition. The maximum specific productivity obtained at D = 0.25 h-1 was 50 mg of PHA g-1 of cellular protein h-1, compared with 74 mg of PHA g-1 of cellular protein h-1 for P. oleovorans at D = 0.24 h-1 (11). Thus P. oleovorans, although not very responsive to nitrogen limitation, is still more productive. It suggests that PHA synthesis in P. oleovorans is not highly regulated, whereas P. resinovorans, also a fluorescent pseudomonad, possesses a regulation mechanism similar to PHB-accumulating microorganisms such as A. eutrophus. Examination of the kinetic data for the production of poly(,-hydroxyoctanoate-co-4-hydroxydecanoate) by P. aeruginosa PAC1 grown on gluconate (14)
shows that the rate of polymer accumulation by this bacterium is also greatly increased by nitrogen limitation. The selective advantage of PHA accumulation to microorganisms is as a carbon and energy reserve (4) and as an electron sink (12) when carbon sources are readily available but an absence of some other nutritional element makes growth impossible. A high, constant rate of PHA accumulation should be a competitive disadvantage since it diverts valuable carbon and energy from growth. This may explain why constitutive producers such as P. oleovorans and Alcaligenes latus (1) are rare. However, at least in the case of P. oleovorans, there is a compensating effect. As its specific growth rate increases, the ratio of PHA synthesis to growth rate declines, allowing the flow of carbon to be directed more to growth than to polymer synthesis (11). If LSC PHAs are to become available commercially, extensive screening must be conducted to select for higheryielding strains. Among such cultures, microorganisms like P. resinovorans, whose PHA accumulation is regulated by nutrient limitation, have greater economic potential than P. oleovorans. By separating growth and accumulation, less expensive substrates directed exclusively towards growth can be used. For example, P. resinovorans could be grown on an inexpensive carbohydrate such as glucose, and then octanoate could be added when the NH4+ concentration becomes limiting. Thus, the more expensive substrate should be dedicated to polymer accumulation. We acknowledge the financial support of the Natural Science and Engineering Research Council of Canada and Xerox Canada.
REFERENCES 1. Braunegg, G., and B. Bogensberger. 1985. Zur kinetik des wachstums und der speicherung von poly-D(-)-3-hydroxybuttersaure bei Alcaligenes latus. Acta Biotechnol. 5:339-345. 2. Braunegg, G., B. Sonnleitner, and R. M. Lafferty. 1978. A rapid method for the determination of poly-f3-hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol. 6:2937. 3. Dawes, A. J., and E. A. Anderson. 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54:450-472.
746
NOTES
4. Dawes, E. A., and P. J. Senior. 1973. The role and regulation of energy reserve polymers in micro-organisms. Adv. Microbiol. Physiol. 10:135-266. 5. Haywood, G. W., A. J. Anderson, and E. A. Dawes. 1989. A survey of the accumulation of novel polyhydroxyalkanoates by bacteria. Biotechnol. Lett. 11:471-476. 6. Haywood, G. W., A. J. Anderson, D. F. Ewing, and E. A. Dawes. 1990. Accumulation of a polyhydroxyalkanoate containing primarily 3-hydroxydecanoate from simple carbohydrate substrates by Pseudomonas sp. strain NCIMB 40135. Appl. Environ. Microbiol. 56:3354-3359. 7. Holmes, P. A. 1985. Applications of PHB-a microbially produced biodegradable thermoplastic. Phys. Technol. 16:32-36. 8. Marchessault, R. H., C. J. Monasterios, F. G. Morin, and P. R. Sundararajan. 1990. Chiral poly(P-hydroxyalkanoates): an adaptable helix influenced by the alkane side-chain. Int. J. Biol. Macromol. 12:158-165. 9. Ramsay, B. A., K. Lomaliza, C. Chavarie, B. Dube, P. Bataille, and J. A. Ramsay. 1990. Production of poly-(,-hydroxybutyric-
APPL. ENVIRON. MICROBIOL.
co-p-hydroxyvaleric) acids. Appl. Environ. Microbiol. 56:20932098. 10. Ramsay, B. A., J. A. Ramsay, and D. G. Cooper. 1989. Production of poly-p-hydroxyalkanoic acid by Pseudomonas cepacia. Appl. Environ. Microbiol. 55:584-589. 11. Ramsay, B. A., I. Saracovan, J. A. Ramsay, and R. H. Marchessault. 1991. Continuous production of long-side-chain poly-phydroxyalkanoates by Pseudomonas oleovorans. Appi. Environ. Microbiol. 57:625-629. 12. Senior, P. J., and E. A. Dawes. 1973. Poly-p-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem. J. 125:55-66. 13. Stickland, L. H. 1951. The determination of small quantities of bacteria by means of the biuret reaction. J. Gen. Microbiol. 43:159-271. 14. Timm, A., and A. Steinbuchel. 1990. Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl. Environ. Microbiol. 56:3360-3367.
