Comonomer composition distribution of P(3HB-co-3HP)s produced by Alcaligenes latus at several pH conditions

Macromol. Chem. Phys.200,1047–1053 (1999) 1047

Comonomer composition distribution of P(3HB-co-3HP)sproduced byAlcaligenes latusat several pH conditions

Yi Wang, Masafumi Ichikawa, Amin Cao, Naoko Yoshie, Yoshio Inoue*

Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku,Yokohama 226-8501, [email protected]

(Received: August 31, 1998; revised: November 2, 1998)

SUMMARY: A series of poly(3-hydroxybutyric acid-co-3-hydroxypropionic acid) (P(3HB-co-3HP)) sampleswas biosynthesized by fermentation withAlcaligenes latususing sucrose/3-hydroxypropionic acid as carbonsource at different pH conditions from 6.0 to 8.0. The copolymers were compositionally fractionated usingthe mixed solvent chloroform/heptane. It was found thatA. latusproduces P(3HB-co-3HP)s with different3HP content under the same conditions except for the pH value of the medium. The higher the pH value ofthe fermentation medium is, the less 3HP units are introduced into the copolyester. The comonomer composi-tion distribution for the copolyesters also depends on the pH value of medium. The copolyester sample pro-duced at pH 6.0 has a much broader composition distribution than those produced at pH values between 6.5and 8.0. The copolyester with the narrowest distribution was obtained at pH 7.0. It was also found that thedistribution becomes broader with increasing fermentation time.

IntroductionPoly(hydroxyalkanoates) (PHAs) are intracellular carbonand energy reserve materials accumulated by a variety ofbacteria under certain unbalanced growth conditions1–3).PHAs show thermoplastic or elastic properties dependingon the chemical structures of the polymer chain constitu-ents, and have been considered as potential candidates forbiodegradable plastic materials. Bacterial poly(3-hydro-xybutyric acid) (P(3HB)) is a typical one which hasattracted much industrial attention as an environmentallydegradable material for a wide range of agricultural, mar-ine, and medical applications2). However, P(3HB) israther brittle, its processability window is narrow, and itsproduction cost is high, as compared to the common che-mosynthesized plastics produced from oil. In order tomodify such disadvantageous physical properties ofP(3HB), a series of biodegradable and biocompatiblecopolyesters, such as poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) (P(3HB-co-4HB))4–6), poly(3-hydro-xybutyric acid-co-3-hydroxyvaleric acid)[P(3HB-co-3HV)]7, 8), and poly(3-hydroxybutyric acid-co-3-hydroxy-propionic acid)[P(3HB-co-3HP)]9–14), have been bio-synthesized. In general, morphology and several physicalproperties of copolymers strongly depend on their como-nomer composition and their sequence structure15).Hence, a detailed characterization of the chain micro-structures of the copolymers is a requisite to establishtheir structure properties relationships.

Every copolymer is considered to be a kind of polymerblend by nature, that is, a copolymer material is com-posed of molecules with more or less a range of comono-

mer compositions. The range of compositon distributionin chemosynthesized copolymers is generally believed tobe very narrow. On the other hand, some bacterial copo-lyesters have been found to have a complex compositiondestribution, i.e., their composition distributions areextremely broad and the various porperties are related totheir composition distributions14, 16–18).

The apparent comonomer composition of bacterialcopolyesters can be controlled by using different carbonsources during biosynthesis, but the composition distribu-tions are broad and up to now uncontrollable.

Microbial P(3HB-co-3HP)s with a wide range of 3HPcontent have been synthesized byA. latus from mixedcarbon substrates of sucrose or 3-hydroxybutyric acid and3-hydroxypropionic acid. The sequence distributions ofthese copolymers were found to be statistically random,affording a biodegradable thermoplastic. The chemicalstructure is similar to that of P(3HB-co-3HV)18), with arandom sequence distribution which is well known tococrystallize. P(3HB-co-3HP) samples fractionated withthe mixed solvent chloroform/heptane have been revealedto show a broad and complex comonomer compositiondistributions14, 18). The morphology and physical proper-ties of the fractions with narrow comonomer distributionhave also been investigated14, 16). The thermal propertiesand crystallization behavior of the fractionated samplesvary significantly with 3HP content, and they are differ-ent from those of the original unfractionated ones.

In order to establish the relationships between fermen-tation conditions and comonomer composition distribu-tion of the resulting copolymers, it is important to clarify

Macromol. Chem. Phys.200, No. 5 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 1999 1022-1352/99/0505–1047$17.50+.50/0

1048 Y. Wang,M. Ichikawa, A. Cao,N.Yoshie,Y. Inoue

thefactorswhich canaffect thecomonomercompositionsandtheir distributions.In this study, we report the effectof thepH valueof thefermentationmedium on thecomo-nomercompositions and their distributionsof the result-ing P(3HB-co-3HP)s.

Experimental part

Materials

The strain Alcaligeneslatus (ATCC 29713) was used tosynthesizepolyesters.It wasstoredin 20%(w/v) glycerinat–808C in a deepfreezer. The monomerR-3-hydroxypropio-nic acid (3HPA) was purchasedfrom TOKYO KASEIKOGYO CO.,LTD.

Biosynthesesof P(3HB-co-3HP)s

The biosynthesesof original unfractionated samples ofP(3HB-co-3HP) were carried out by a one-stagefermenta-tion with A. latusat 308C. First, theinoculafor thefermenta-tions were grown on a shakerin a 500ml Sakaguchiflaskwith 100ml mediausingsucrose(5 g/l) ascarbonsourcefor15 h underaerobicconditions.Thesemediaas well as thefermentationmedia include the sameinorganic compoundsas described in other papers12–14,16–18): 8.6g ofNa2HPO4 N 12H2O, 1.5g of KH2PO4, 1.0g of (NH4)2SO4,0.2g of MgSO4 N 7H2O, 0.06g of ammonium iron(III)citrate, and 0.01g of CaCl2 N 2H2O per liter of distilledwater. 1 ml of microelementsolutionwasaddedto onelitermedium,including: 0.3g of H3BO3, 0.2g of CoCl2 N 6H2O,0.1g of ZnSO4 N 7H2O, 0.03g of MnCl2 N 2H2O, 0.03g ofNaMoO4 N 2H2O, 0.02g of NiCl2 N 6H2O, 0.01g ofCuSO4 N 5H2O perliter of 1.0N HCl. Then,thebacteriaweregrown in 2-l or 8-l fermenter(EYELA JAR FERMENTERMBF, TOKYO RIKAKIKAI CO., LTD.) using sucrose(15g/l) and3HPA (3 g/l) asmixed carbonsourcesfor 30 h.The pH wasregulatedwith 2 M NaOH and2 M H2SO4. Theaerationrateamountedto 0.5:1 (aerationrate(l/min): med-ium volume (l)), and the stirrerswere adjustedto 500rpm.Bacteriawereharvestedby centrifugation,washedwith dis-tilled water, andfollowed by lyophilization.

Copolyesterswere extracted from the lyophilized cellswith hot chloroformusinga Soxhletapparatus,andpurifiedby reprecipitationwith ethanol.

Fractionationof theoriginal P(3HB-co-3HP)s

The original samplesobtainedwere fractionatedwith themixed solvent chloroform/heptane.The details have beenreportedelsewhere14,16). Theoutline of the fractionationpro-cedurewas as follows: heptanewas slowly addedinto thecopolyester/chloroformsolution(startingpolymerconcentra-tion was 1 g/100ml) by a constantHC of heptanestepbystep,whereHC meanstheincrementalvolumeconcentrationof heptanein the mixed solvent betweentwo continuoussteps.The precipitatedmasswas isolatedby centrifugation(8000rpm, 30 min). This procedurewasrepeateduntil add-ing any amountof heptanecould not causeappreciablepre-

cipitation.Eachprecipitatewasdriedundervacuumat 508Cfor oneweek.

1H NMR1H NMR spectrawere measuredat 308C and 270MHz inCDCl3 solution on a JELO GSX-270spectrometerwith 4.5ls pulse width (458 pulse), 5s pulse repetition time, 2500Hz spectralwidth, 32k datapointsand32 freeinductiondecayaccumulations.3HPmole fractionsof the fractionatedsamplesalong with the original products were estimatedfrom therelativeintegratedintensitiesof CH2 (7) andCH (3)resonances.

Gel permeationchromatography(GPC)

Molecular weightsof the polyestersampleswere measuredby a TosohHLC-8020 GPC systemwith a TosohSC-8010controller and a refractometer. Chloroform was usedas aneluentat a flowing rateof 1.0ml/min andat 408C. Polystyr-enestandardsof low polydispersitywere usedto constructthe calibrationcurve.The number-average(M

—n) andweight-

averagemolecularweight (M—

w) andthe polydispersityindex(M

—w/M

—n) werecalculatedthrougha SC-8010dataprocessor.

Resultsand discussion

Biosynthesesof copolyesters

Tab.1 lists theresults of thebiosynthesesof copolyestersby A. latusat 308C usingsucrose(15 g/l) and3-hydroxy-propionicacid(3HPA) (3 g/l) asthemixedcarbonsource.The copolyestersamples were codedas C6.0, C6.5, C7.0,C7.5, C8.0, O6.5, O7.0-A, andO7.0-B according to thepH condi-tions of the fermentationmedia. The samplescodedasC6.0, C6.5, C7.0, C7.5, andC8.0 were producedat constantpHvalues,while thosecodedas O6.5, O7.0-A, and O7.0-B wereproducedat initial pH valuesof 6.5 and 7.0, set at thestarting point of fermentation but not controlled duringthe fermentation. The samplescoded as O7.0-A and O7.0-B

were obtainedfrom the samefermentation batch but atdifferentfermentationtimesof 30 and50h, respectively.

At first, we examinedthe change in pH value of themedium during fermentation, whenthe fermentationwascarried out without pH control. Fig. 1 indicatesthe timecourseof the pH valuesduring the fermentations startedat initial pH values of 6.5 and 7.0. When the initialpH was6.5, the pH value of the mediumwasfirstly con-stant at 6.5 for about 15 h, then it gradually lowered

Comonomercompositiondistributionof P(3HB-co-3HP)sproducedby... 1049

down to 5.7 in the next 9 h, and it remained almostunchanged duringthelast24 h. Whentheinitial pH valuewas 7.0, the features of the pH change were different,best describedas “V”-s haped. The pH becamelower,reached 6.2 in the first 24 h, thencamebackto 6.8 in thelast24 h.

The content of the 3HP units are rangedfrom 33.4mol-% to 12.8 mol-% depending on the pH value of thefermentation medium. The higher the pH value of themediumwas, the lessthe 3HP unit was introducedintothe copolyester, except in the caseof C7.5 (Tab.1). At a

constantpH 6.5, the highestdried cell weight, polyesterweight, and polyestercontentwere obtained. When thefermentation was started at an initial pH value of themedium of 6.0 or 6.25 in the flask culture withoutpH control, thepH value became 4.9or 5.2 after 42 h fer-mentation and the dried cell weight was only about0.4 g/l; further no polyestercould be harvested(data isnot listed). The observed pH dependence can beexplained as follows. The bacteria preferablyutilize themolecular form of 3HPA, ratherthan its ion form. Fromequilibrium Eq. (1), whenthe pH valueof the solution islarger, the equationwill move to the left side.For exam-ple, in themediumwith controlled pH 6.0 during fermen-tation, thereis moremolecularform of 3HPA thanin themedium with pH 8.0 with the same3HPA concentration,thus the 3HP content of the produced copolyesterin themedium with pH 6.0 becomeslarger (33.4mol-%) whilethatat pH 8.0 is lower (13.2mol-%).

CH2OHCH2COO– + H+ K CH2OHCH2COOH (1)

The fermentation medium with a pH value lower than6.0 or higherthan8.0 shouldnot be suitablefor the bac-teria to grow andaccumulate thepolyester. Consequently,from the fermentation under the condition of an initialpH value 6.0 or 6.25 and without control, only litt lepolyester can be obtained. And when the fermentationwent onat aconstant pH 6.0and8.0,thedriedcell weightandpolyesterweight weremuchlessthanthoseobtainedat theother pH values from 6.5to 7.5 (Tab.1).

Fig. 2 shows the time courseof the content of 3HPunits included in P(3HB-co-3HP) during fermentation,indicating clearly that the 3HP content in the polymersamplesincreaseswith time.The3HPcontentof thesam-

Tab.1. Biosynthesesof P(3HB-co-3HP)sby Alcaligenes latus from sucrose (15 g/l) and3-hydroxypropionicacid(3 g/l) at 308C

Sample pH range Batch timeh

Cell dry weightg=l

Polyester weightg=l

Polyestercontentwt:-%

3HP contentmol-%

Molecularweight

(10–5 N M—

w)

C6.0 6.0 28 1.3 0.4 30.1 33.4 7.4C6.5 6.5 29 7.8 4.2 45.2 23.0 8.0C7.0 7.0 29 3.8 1.5 40.0 16.1 8.6C7.5 7.5 30 3.2 1.2 38.5 20.9 6.5C8.0 8.0 29 0.9 0.3 34.2 13.2 10.5O6.5 6.5–5.7a) 48 3.7 1.4 36.8 29.8 5.8

O7.0-Ab) 7.0–6.2a) 30 2.9 1.3 46.2 12.8 2.8

O7.0-Bb) 7.0–6.2a) 50 4.6 2.1 45.2 18.8 3.3

a) pH valuesof themediumwerenot controlledduringfermentation. ThestartingpH valueswere6.5(O6.5) and7.0(O7.0-A, O7.0-B).b) Obtained from thesamefermentationbatchbut at differentfermentation time of 30 (O7.0-A) and50 (O7.0-B) h.

Fig. 1. Time courseof pH valuesof the fermentation mediumproducingP(3HB-co-3HP) by A. latus using 15g/l sucroseand3 g/l 3-hydroxypropionicacidasmixedcarbonsourceat 308C


ple after50 h fermentation wastwo timeslarger thanthatafter15 h.

All P(3HB-co-3HP) samples shown in Tab.1 havealmost the sameaverage molecular weight (M

—w). From

Fig. 3, which showsthe time coursesof the cell growthandthe production of P(3HB-co-3HP), it wasfound thatafter48 h some of thepolyesterwasdegradedby thebac-teria.

Fractionation of P(3HB-co-3HP)s

Theeightsampleslistedin Tab.1 werefractionatedby thechloroform/heptane solvent/nonsolvent method14,16).Resultsare listed in Tab.2 and3. Fig. 4 and5 showthe

weight percentage(wt.-%) of fractionsvs. the3HPmono-mercontent (mol-%)of samplesC6.5, O6.5, O7.0-A andO7.0-B.

TheC6.0 sample(0.5g), whichwasobtainedby fermen-tation at constant pH 6.0, wasfractionatedinto five frac-tionsby using 42.0–61.0vol.-% of heptane(Tab.2). The3HP contentsof the fractions decreasedwith increasingheptaneconcentration.The 3HP contentsof the first andthe last fractionswere62.3and20.5mol-%, respectively.The valuesof the averagemolecular weight (M

—w) of all

fractions were as high as 105, but show a little decreasewith increasing heptane content up to 48.0 vol.-%. Theresults indicatethat theC6.0 samplewasfractionatedwiththe chloroform/heptane systemmainly by the differencein the 3HP content.The polydispersity of the third frac-tion is muchlarger thanthe others.At presentthe reasonis not known.

Fig. 2. Time courseof 3HP content included in P(3HB-co-3HP)producedby fermentationof A. latus at 308C in the med-ium with an initial pH 7.0 without control during fermentation,using15g/l sucrose and3 g/l 3-hydroxypropionic acidasmixedcarbonsource

Fig. 3. Time coursesof the productionof P(3HB-co-3HP) ataninitial pH 7.0without controlandat 308C

Fig. 4. Weight percentof fractions vs. 3HP mol-% for P(3HB-co-3HP)samples,(a) C6.5 producedat a constantpH 6.5 and (b) O6.5 procuced at aninitial pH 6.5without pH control


Tab.2. Resultsof thefractionation of C6.0, C6.5, C7.0, C7.5 andC8.0 producedby A. latus from sucrose (15g/l) and3-hydroxypropionicacid(3 g/l) at 308C anda constantpH in batchculture

Samplefraction

Conc: of heptanevol:-%

Amount of sample in fractionwt:-%

3HP contentmol-%

10–5 N M—

w M—

w/M—

n

C6.0 Originala) – 100.0 33.4 7.4 6.01 42.0 4.0 62.3 9.1 9.92 45.0 18.8 45.3 9.4 3.13 48.0 39.0 32.3 8.3 15.44 52.0 26.4 26.9 5.7 7.25 61.0 5.8 20.5 2.3 4.4

C6.5 Originala) – 100.0 23.0 8.0 1.81 45.0 1.5 31.4 8.4 1.62 46.0 5.9 28.8 11.2 1.73 47.0 36.5 22.5 10.4 1.54 48.0 22.9 18.0 5.2 2.25 49.0 15.2 15.1 3.8 1.56 50.0 3.4 16.9 2.5 2.17 56.0 1.2 17.6 0.6 3.1

C7.0 Originala) – 100.0 16.1 8.6 2.21 56.0 6.9 17.7 4.5 4.82 57.0 44.4 16.7 9.4 2.23 58.0 30.7 12.8 8.6 2.64 59.0 3.2 11.6 6.5 1.75 62.0 6.2 14.5 0.01 1.8

C7.5 Originala) – 100.0 20.9 6.5 2.01 46.0 2.3 27.1 7.2 1.62 48.0 10.4 23.3 6.8 1.73 50.0 33.1 19.5 6.4 1.54 52.0 15.7 17.0 4.5 2.35 54.0 23.4 13.3 4.0 2.26 62.0 5.5 16.8 0.9 1.4

C8.0Originala) – 100.0 13.2 10.5 2.11 54.0 17.5 18.7 12.9 1.72 56.0 37.5 12.5 9.4 2.33 58.0 22.6 11.3 8.1 2.74 61.0 11.7 16.8 1.3 1.4

a) Unfractionatedsample.

Fig. 5. Weight percentof fractionsvs. 3HP mol-% for P(3HB-co-3HP)samplesobtained from the samefermentation batchat an initial pH 7.0without pH control for (a) 30h and(b) 50h


When the C6.5 sample(4.0g) was fractionated,sevenfractions with 3HP contents of 31.4–15.1 mol-% wereobtained(Tab.2). The 3HP contentsof the last two frac-tions were a litt le larger than the fifth fraction and themolecular weight of the last one was much lower com-paredto thoseof the other fractions.For thesefractions,they were alsofractionatedby a synergic effect of differ-encesin comonomer compositionandmolecularweight.The distribution of the 3HPcontent becomesnarrowto avalueof 16.3mol-% (Fig. 4(a)).

The C7.0 sample(1.5g) wasfractionatedinto five frac-tions (Tab.2). The last fraction still showed a larger3HPcontentthanthethird andtheforth fractionsbut its mole-cular weight was much smaller than the others. Thecomonomerdistribution of C7.0 wasquitenarrow, just 6.1mol-%.

The C7.5 sample (1.5g) was fractionatedinto six frac-tions (Tab.2). The 3HP contentdecreasedfrom the firstto the fifth fraction. The distribution of it was broaderthan that of C7.0 and reached 13.8 mol-%. The last frac-tion hasa larger3HPcontentandamuch lower molecularweightthanthefifth one.

As the C8.0 sample (0.3g) was fractionated, four frac-tions were obtained; the decreasingtendencyof the 3HPcontent wassimillar to that of C7.5 andthe distribution ofthe3HPcomposition was7.4 mol-% (Tab.2).

Tab.3 and Fig. 4(b) showthe results of the fractiona-tion of the O6.5 (5.0g). Twelve fractions were obtainedusing 42.0–64.0 vol.-% of heptane.The thirteenthfrac-tion was obtainedas the residueafter an evaporation ofthe solvent chloroform/heptane. The 3HP contentdecreasedsignificantly from thefirst to theninth fraction,i. e., changedfrom 77.2 mol-% to 20.0 mol-%, and thenshowed rathersimillar valuesor evenslightly increasingvaluesfrom thetenthto thelastfraction.The3HPcontentof the fifth fraction with the largestweight percent wasalmostthesameasthatof theoriginal copolyester. Beforethis experiment, we found that the last fraction of everysamplealwayshasa large3HPcontentanda small mole-cular weight. We supposedthata litt le part of thecopoly-mer degradedwhen being fractionatedfor such a longtime. While themolecular weight of thedegraded portionbecame small, it was difficult to collect the fraction bycentrifugation eventhough it wasprecipitated. Whenwe

Tab.3. Resultsof the fractionation of O6.5, O7.0-A andO7.0-B producedby A. latus from sucrose(15g/l) and3-hydroxypropionic acid(3 g/l) at 308C andaninitial pH 6.5or 7.0without pH control in batchculture

Samplefraction

Conc: of heptanevol:-%

Amount of sample in fractionwt:-%

3HP contentmol-%

10–5 N M—

w M—

w/M—

n

O6.5 Originala) – 100.0 29.8 5.8 2.51 42.0 1.2 77.2 3.6 2.02 44.0 0.8 56.5 3.8 3.73 46.0 1.8 45.0 3.7 2.54 48.0 7.8 37.1 4.3 2.85 50.0 33.9 30.2 6.1 1.76 52.0 32.5 23.8 6.2 2.17 54.0 12.8 22.5 4.5 1.98 56.0 4.3 21.9 2.6 2.29 58.0 2.1 20.0 1.8 1.8

10 60.0 1.3 22.3 4.1 2.811 62.0 0.8 22.2 3.6 2.012 64.0 0.5 23.6 3.9 2.213 b) 0.3 24.2 1.6 2.8

O7.0-A Originala) – 100.0 12.8 7.4 2.81 52.0 51.6 15.9 9.5 1.92 54.0 39.6 11.6 5.9 1.83 56.0 5.0 12.8 2.5 2.34 64.0 2.3 12.2 c) c)

O7.0-B Originala) – 100.0 18.8 3.9 3.31 52.0 37.9 24.9 5.2 2.22 54.0 39.0 15.6 4.3 2.13 56.0 10.0 15.1 1.9 2.04 64.0 9.7 18.6 0.6 1.4

a) Unfractionatedsample.b) Recoveredfrom thechloroform/heptanesolutionof fractionno.12.c) Not determined.


fractionatedthis sample at 08C, the fraction with a smallmolecular weightwasnot obtained, but the last four frac-tions still have a larger 3HP content than the ninth frac-tion. Thus, the increasing 3HP contentof the last frac-tionsis alsoinfluencedby other, unknownfactors.

Both samplesO7.0-A (0.5g) and O7.0-B (1.5 g) obtainedfrom thesamefermentationbatchbut at differentfermen-tation times of 30 and 50 h, respectively, were fractio-natedto four fractionsusing52.0–64.0vol.-% of heptane(Tab.3). However, the3HPcontentsof fractionswith thesamefraction number were quite different. In caseofO7.0-A, the 3HP contents of the fractions were between15.9mol-% and11.6 mol-%, while thoseof the fractionsof O7.0-B werebetween24.9 and15.1 mol-%. The como-nomercompositiondistribution of O7.0-B wasasabouttwotimesbroaderthanthatof O7.0-A (Fig. 5).

The comonomer compositiondistributionsof the sam-ples C6.0 and O6.5 were much broader than the others,reaching41.8 and 57.2 mol-%, respectively. At pH 7.0,thedistribution became thenarrowestwith a valueof 6.1mol-%. The distributions of the other sampleswere lessthan17.1mol-%. In caseof O6.5, thepH of the fermenta-tion changedfrom an initial value of 6.5 to 5.7 (Fig. 1).At about25 h, the pH was lower than 6.0. Thus it wasconcludedthat whenthe pH of the medium is lower than6.0, the comonomercomposition distribution of P(3HB-co-3HP) produced by A. latus becomesvery broad.Onthe other hand,7.0 and6.5 may be the bestpH valuestoobtainP(3HB-co-3HP)with a narrow distribution andthehighestcopolyester yield, respectively.

Thecomparisonof thefractionationsof O7.0-A andO7.0-B

leadsto the conclusionthat the comonomercompositiondistribution becomesbroader with ongoing fermentation.

ConclusionIn conclusion, the pH value of the medium during thebiosynthesis of the copolyester P(3HB-co-3HP) by A.latus influencessignificantly the comonomer composi-tion andits distribution. The 3HP content decreases withincreasingpH, andthe comonomer compositiondistribu-tion of P(3HB-co-3HP) will becomenarrowerwhen thepH value of the medium is controlled to be constant,compared to that without a pH control. The comparisonof the fractionation resultsfor the samples C6.5 and O6.5

supports this conclusion(Fig. 4).The comonomer distribution of the copolyester is not

just influenced by the pH because even at a constantpH value the product is composed of copolyesterswithdifferent comonomercompositions.WhenthepH valueis6.0, the distribution of the copolyestercomonomercom-

position is muchbroader thanthoseof samplesproducedat otherpH valuesfrom 6.5 to 8.0.Among the pH valuesused, 7.0 is thebestpH to producea copolyester with nar-row comonomercompositiondistribution.

Comparing the resultsfor the samples O7.0-A andO7.0-B

leads to the conclusionthat the time of fermentation alsoinfluences the comonomer composition distribution(Fig. 5). This resultmay arisefrom the difference in therateof utilizationof carbonsources(sucroseand3-hydro-xypropionicacid)by microorganisms.Themixing weightratio of the carbon sourcesin the culture mediumshouldchangeduringthefermentation, if microorganismsutilizepreferentially one component of the mixed carbonsources. The studyof this point is now in progressin ourlaboratory.

Acknowledgement: Part of this work was supportedby aGrant-in-Aid for InternationalJointResearchin theAreaof Glo-bal Environment from NEDO/RITE(1998).

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Documents

Comonomer composition distribution of P(3HB-co-3HP)s produced by Alcaligenes latus at several pH conditions