Polymer International Polym Int 48 :23–32 (1999)

Ring-opening homopolymerization andcopolymerization of lactones. Part 2. Enzymaticdegradability of poly(b-hydroxybutyrate)stereoisomers and copolymers ofb-butyrolactone with e-caprolactone andd-valerolactoneC Jaimes ,1 R Dobreva-Schue� ,1 O Giani-Beaune,1* F Schue� ,1 W Amas s 2and A Amas s 21 Laboratoire de Chimie Montpellier II , Sciences et Techniques du Languedoc, Place Bataillon,Macromole� culaire, Univers ite� Eugene

34095 Montpellier , Cedex 5, France

2 Department of Chemical Engineering and Applied Chemis try , As ton Univers ity , As ton Triangle, Birmingham,B4 7ET,UK

Abstract : In this study, we have prepared poly( [R,S ]-b-hydroxybutyrate) (P( [R,S ]-b-HB) or PHB)

from [R,S ]-b-butyrolactone ( [R,S ]-b-BL), using diþ erent aluminoxane catalyst systems

(triethylaluminium/water, triisobutylaluminium/water, trioctylaluminium/water and tetraiso-

butyldialuminoxane/water). By varying the ratio of catalyst to water and using a method of

fractionation of polymers, PHB with diþ erent isotactic diad fractions (i) (from 0.41 to 0.72) and crys-

tallinities were obtained. Copolymers poly(butyrolactone-co-caprolactone) (P(BL-co-CL)) and

poly(butyrolactone-co-valerolactone) (P(BL-co-VL)) have also been synthesized from the ring-

opening copolymerization of [R,S ]-b-BL with either e-caprolactone (CL) or d-valerolactone (VL)

using tetraisobutyldialuminoxane (TIBAO) catalyst. The enzymatic degradability of these polymers

was studied in aerobic and anaerobic media. The objective of this work was to determine the inýuence

of the tacticity and crystallinity of the polymers on their degree of biodegradation and on their initial

degradation rate. It was shown that the degradation rate measured for bacterial PHB 100% [R ] was

the highest and the degree of aerobic biodegradation reached after 36 days was around 94% . A 40–50%biodegradation was obtained for synthetic PHB, highly isotactic and predominantly syndiotactic. The

non-crystalline and atactic PHB synthesized from TIBAO catalyst had a very high degree of biodegra-

dation of around 88% . This result may suggest that not only are the [R ]-BL units hydrolysed but also

the [S ]-BL units. The inýuence of the crystallinity on the initial degradation rate was observed for the

copolymers P(BL-co-CL) and P(BL-co-VL) of various feed ratios. All these copolymers synthesized

from TIBAO catalyst, exhibit a high degree of biodegradation of around 85% except for copolymers

containing a very high portion of unsubstituted units, CL or VL. The anaerobic biodegradation of PHB

and copolymers P(BL-co-CL) is much lower than the aerobic biodegradation, as are the initial rates,

even for bacterial P( [R ]-HB).

1999 Society of Chemical Industry(

Keywords: polyhydroxybutyrate; copolymerization; e-caprolactone; d-valerolactone, enzymatic degradability

INTRODUCTION

Poly([R]-b-hydroxybutyrate) (P([R]-HB)) in an iso-tactic form isolated from bacteria, is a biodegradablethermoplastic with a melting temperature of around180¡C,1,2 which has recently attracted much interestas a biodegradable, non-toxic engineering plastic.3,4

Bacterial polyesters, including PHB, are especiallysusceptible to enzymatic degradation by a variety of

organisms in biological environments because bac-terial PHB contains only [R] units in its chain. Inthe case of synthetic polymers containing a mixtureof [R] and [S] stereoblocks, it seems that the [S]units do not undergo enzymatic degradation. PHBsynthetic analogues have been viewed as interestingmodel systems in order to investigate the eþects ofstructure and morphology on biodegradability.

* Corres pondence to : O Giani-Beaune, Laboratoire de ChimieMacromole� culaire, Univers ite� Montpellier II, Sciences etTechniques du Languedoc, Place Eugene Bataillon, 34095

Montpellier, Cedex 5, France

Contract/grant s pons or : European Commis s ion.Contract/grant number : Brite-Euram no BRE2 CT 92 0205.(Received 20 March 1998; revis ed vers ion received 23 July 1998;

accepted 10 Augus t 1998)

( 1999 Society of Chemical Industry. Polym Int 0959–8103/99/$17.50 23

C Jaimes et al

P([R]-HB) of natural origin is biodegradable invarious environments such as soil, anaerobic andaerobic activated sewage sludge and seawater. Themicro-organisms (bacteria, fungi, etc.) excrete extra-cellular PHB-depolymerase to degrade environ-mental P([R]-HB) and utilize the decomposedcompounds as nutrients. The extracellular PHB-depolymerases have been puriüed from some micro-organisms such as Pseudomonas lemoigne,5Alkaligenes faecalis,6 and Penicillium pinophilum.7

Recently, Kumagai et al8 reported that the rate ofenzymatic hydrolysis of P([R]-HB) ülm by thePHB-depolymerase from A. faecalis increased with adecrease in crystallinity. Marchessault and co-workers9,10 prepared PHB polymers by poly-merization of racemic b-butyrolactone ([R,S]-b-BL)in the presence of catalyst and separatedAlMe3/H2Othem into isotactic, atactic and syndiotactic fractions.The fraction with high isotactic diad tacticity contentshowed little or no degradation over a 50h incu-bation period, whereas the fraction of intermediateisotactic diad content degraded in a continuoussteady fashion at a rate that was less than that forbacterial PHB. The low isotactic diad fractionunderwent a rapid initial degradation, followed by nofurther mass loss.

Kemnitzer et al11 prepared PHB stereopolymersby the polymerization of [R]-b-BL with [S]-b-BL atvarious feed ratios in the presence of ZnEt2/H2Ocatalyst. The fraction of [R]-b-BL units varied from6 to 94% and the stereochemical eþects on the rate ofenzymatic degradation were studied with the PHB-depolymerase from P. funiculosum. They demon-strated for [R] stereocopolymer compositions of 95,90, 85 and 81%, that the initial surface degradationrates were slower than those measured for a 100%[R]-PHB sample. Therefore, the preference for [R]-BL repeat units by P. funiculosum esterase appears todominate over crystallinity eþects. However, theinitial surface degradation rates for the 67 and 77%[R]-PHB sample were larger than that measured forthe 100% [R]-PHB sample, which suggested that acritical degree of disruption of the crystalline orderoccurred at these stereochemical compositions, accel-erating the observed degradation rate.

In this paper, we have studied the enzymaticdegradabilities of PHB polymer samples of diþerenttacticities, and copolymers of b-butyrolactone with e-caprolactone P(BL-co-CL) and with d-valerolactoneP(BL-co-VL) at various feed ratios. These polymersand copolymers were prepared in the presence ofaluminoxane catalysts by synthesis proceduresreported in previous papers.12h14 In these previousstudies, the molecular weights, structure, thermalproperties and crystallization behaviour of the poly-mers were investigated using gel permeation chroma-tography (GPC), nuclear magnetic resonance (NMR)and diþerential scanning calorimetry (DSC).

The biodegradation of synthetic PHB is of interestin determining whether or not this material may be

used as a substitute for the naturally-occurringpolymer, especially in commercial applicationsrequiring biodegradability, such as medicine or pack-aging.

EXPERIMENTAL

Materials

(R,S)-b-BL, e-CL and d-VL were obtained com-mercially from Aldrich Chemical Co., dried over

under vacuum, fractionally distilled underCaH2vacuum and stored under a nitrogen atmosphere.Tetraisobutyldialuminoxane (TIBAO; (iBu)4Al2O)30% in cyclohexane was purchased from Schering ;trialkylaluminium compounds and(AlEt3 , AlOct3)were obtained from Aldrich Chemical Co. All cata-lysts were stored and manipulated under a nitrogenatmosphere and used as received. Toluene was dis-tilled from sodium–potassium alloy and stored over

under vacuum.CaH2

Preparation of polymers and copolymers

P(BL-co-CL) and P(BL-co-VL)

All reactants were transferred by syringe to ampoulesthrough serum caps under a nitrogen atmosphere.The ampoule was connected to a vacuum line(10~6 mmHg). The catalyst was ürst added, andsolvent (toluene or cyclohexane) was transferred bycold distillation. The reactor was cooled with anexternal bath at [ 78¡C and the monomer mixturewas added by syringe. The contents of the ampoulewere degassed during three freeze–thaw cycles andünally sealed under vacuum. The solution waswarmed to room temperature with magnetic stirring.Polymerization reactions were then carried out at60¡C for a period of 7 days, after which the ampouleswere opened, their contents were removed and dis-persed with a magnetic stirrer in diethyl ether(100ml g~1 of initial monomers mixture). The solidwas ültered and added to chloroform (100ml g~1 ofinitial monomers mixture), stirred for 12h at 25¡Cand then reýuxed for 1h. The clear chloroform solu-tion obtained was concentrated to 10ml g~1 of initialmonomer mixture and this solution was precipitatedin diethyl ether The polymer was ültered to(Et2O).give the crude product.

Polymer characterization

All molecular weight data reported in the tables wereobtained by gel permeation chromatography (GPC).The instruments used were a Knauer HPLC pumptype 364, a Knauer diþerential refractometer and aKnauer PL gel 10lm Mixted B column(600mm ] 7.5mm). Chloroform was used as eluantat a ýow rate of 1ml min~1. Samples were analysedas 2% w/v solutions and injection volumes of 20–30ll were used. Polystyrene standards with a lowdispersity were used to generate a calibration curve.

1H NMR measurements were recorded at250MHz on a Bruker AC 250 spectrometer. The

24 Polym Int 48 :23–32 (1999)

Enzymatic degradability of PHB stereoisomers and lactone copolymers

sample concentrations employed were typically 0.1%w/v. 1H NMR spectra of copolymers were recordedat 25–30¡C with as solvent. Tetra-CDCl3methylsilane (TMS) was used as internal reference.

13C NMR measurements of 1–1.5% w/v wererecorded at 50.3MHz on a Bruker WP 200 SXspectrometer with a total of 2000–4000 scans col-lected and a delay time of 0.5 s.

The heat of fusion melting temperature(*Hm),and glass transition temperature for all(Tm) (Tg)

polymer samples were determined by using a Mettlermodel DSC-30. Samples were heated at rate of10¡Cmin~1 from [ 150 to 200¡C, quickly cooledand then scanned a second time by using the sameheating rate and temperature range as the ürst scan.Data used for and were taken from the ürst*Hm Tmscan and those for from the second scan.Tg

Polymer fractionation

The crude product was treated with acetylacetone(AcAc) (10ml g~1 (PHB 1–2) or 0.5ml g~1 (PHB3–7) of crude polymer). The crude product suspen-sion was stirred for 24h at ambient temperature,after which time the AcAc treated polymer was pre-cipitated out by the slow addition of diethyl ether(10ml per ml of AcAc). The insoluble AcAc-treatedpolymer was recovered by ültration and washed withEt2O.

Enzymatic degradation

Biodegradation under aerobic conditions in waterAn automated electrolytic respirometer device(BSD-digi/Sapromat) was used to follow the oxygenconsumption as a function of time. The device was amanostatic closed system maintaining a constantpressure. The sample in powder form (50–200mg),was agitated in the test ýask with a magnetic stirrer,in order to maintain the initial concentration.O2The polymer, the sole source of carbon and energy inthe medium, was weighed into the test vessel (500mlýasks), as was the reference material (e.g. AVICEL,PHB). No test material was weighed into the blankvessels. 250ml of the prepared mineral salt solution(phosphate buþered mineral salt, pH 7) was added tothe reaction vessels ; then inoculum (an activatedsludge from research sewage treatment at a concen-tration of 1% v/v) and a magnetic stirrer were added.Incubation was allowed to take place in the dark atconstant temperature (20^ 1¡C). Each test wascarried out in duplicate.

During biodegradation the micro-organisms con-sumed oxygen and produced carbon dioxide. Biodeg-radation rates were determined by following the rateof oxygen consumption during aerobic degradationin water. The oxidation curves (oxygen consumptionrelative to time) gained from the BOD data (BOD isthe biochemical oxygen demand) gave informationabout the degradability of the test material, themaximum degradation rate and degradation levelafter a given number of days. Degradation is

expressed as percentage of the theoretical oxygendemand (% ThOD), calculated by the followingincineration equation (C

cH

hO

o\ polymer):

CcH

hO

o] (2c ] h/2[ o)O ] c CO2] (h/2)H2O

(1)

The theoretical oxygen demand (ThOD) can thenbe calculated with the molecular weight of oxygenM(O) and polymer M(P) as

ThOD \ (2c ] h/2[ o)M(O)/M(P) (2)

The actual oxygen consumption (BOD) of thepolymer can then be related to the ThOD.

Biodegradation under anaerobic conditions in waterPlastic material was exposed to sludge obtained froman anaerobic digester in a waste-treatment plant. Thevolume of gas, carbon dioxide and methane(percentage of and in the gas phase andCO2 CH4dissolved as dissolved inorganic carbon (DIC))CO2evolved was measured as a function of time. Theincrease in dissolved organic carbon (DOC), thebiomass content, and weight of residual polymer atthe termination of the test were also used in assessingthe degree of biodegradability. At any time theextent of biodegradability could be assessed from thepercentage of carbon present as gas, calculatedaccording the Buswell–Mueller equation.

The polymer (50–200mg), anaerobic sludge (10%v/v from the anaerobic digester unit, research sewagetreatment plant), and inorganic test medium(phosphate-buþered mineral medium for anaerobes,pH 7, shaken) were incubated in a gas-tight vesselwith a normal volume of 250ml, at room tem-perature. The vessels were prepared at least in tripli-cate for the test compound, in duplicate for theblank, one vessel each for both positive referencesubstances (e.g. cellulose or PHB) and negative ref-erence substance (e.g. polyethylene). During the testthe temperature was maintained at 30¡C.

Degradation of a polymer was expressedCcH

hO

oas a percentage of the theoretical values for biogas(ThBiogas), methane and carbon dioxide(ThCH4)

which are calculated by the Buswell(ThCO2),(disproportionation) equation.

CcHhO

o] (c [ h/4[ o/2)H2O ]

(c/2[ h/8] o/4)CO2] (c/2[ h/8] o/4)CH4 (3)

The theoretical and production canCH4 CO2therefore be calculated as (mol g~1)\ThCH4(c/2[ h/8] o/4)/M(P), (mol g~1)\(c/2[ThCO2h/8] o/4)/M(P), and ThBiogas (mol g~1)\ThCH4] ThCO2 .

The actual and biogas production ofCH4 , CO2the polymer can then be related to the ThCH4 ,

and ThBiogas values. The amount of carbonThCO2

Polym Int 48 :23–32 (1999) 25

C Jaimes et al

dioxide dissolved in the medium cannot be followedby daily reading of the biogas production during thetest. It can only be determined at the end of the testas dissolved inorganic carbon (DIC). Thereforemethane values, followed by gas chromatographyduring the test, are often higher and more reliable.

RESULTS AND DISCUSSION

Studies on enzymatic degradation of polymersinvolve an understanding of a number of fundamen-tal aspects of both enzyme systems and the nature ofthe substrate. In terms of the enzyme, factors such asthe identity and location of the active sites, and alsooptimal conditions for physiological activity, such astemperature and pH, are key parameters to consider.As for the substrate, the morphology, accessibilityand mobility of chain segments of the appropriateconüguration for enzymatic attack are importantfactors governing the extent to which degradationwill occur. In order to develop the relationshipbetween polymer properties (stereoregularity,crystallinity) and susceptibility towards biodegrada-tion, it is necessary to compare the behaviour of syn-thetic and natural PHB. The biodegradation of somesynthetic copolymer by both aerobic and anaerobicsystems has been also tested.

Aerobic biodegradation of synthetic P( [R,S ] -b-BL)Table 1 summarizes a number of important charac-teristics of some of the synthetic PHBs used in thisstudy. The details for the synthesis and character-ization of these polymers are the subject of separatepublications.12,13

All the polymers were prepared by ring-openingpolymerization of [R,S]-b-BL but with various cata-lysts. The catalysts used in the synthesis of PHB-1and -2 came from the system TheH2O/AlEt3\ 1.crude polymers obtained were fractionated withAcAc and the high crystallinity fractions (i \ 0.7 and0.72) were isolated. Polymers PHB-3 and -4 wereprepared in the presence of andH2O/AlOct3\ 0.5

catalyst systems respectively.H2O/AliBu3\ 0.5

These were puriüed with a minimal quantity ofAcAc (0.5ml) to eliminate any residual aluminiumand to preserve the atactic character of the crudepolymer. PHB-5, -6 and -7 were synthesized fromracemic b-BL with an oligomeric aluminoxane cata-lyst obtained by reaction of TIBAO with water usingthree diþerent ratios. These polymersH2O/TIBAOwere fractionated with a minimal quantity of AcAc togive an isotactic diad content of 0.41, 0.48 and 0.51,respectively. The last polymer was prepared withTIBAO without water, and was not fractionated.PHB-8 is totally atactic with 0.50 isotactic diadcontent. Only PHB-1 and -2 exhibit a melting point,but their crystallinity level is lower than for bacterialPHB. All the other samples of synthetic PHB have aglass transition temperature but no melting tem-perature, so they are amorphous. Some of them areslightly syndiotactic (i \ 0.50) and so have more(RS ] SR) diads than (RR ] SS). We note that asthe percentage of isotactic diad content increases, theweight-average molecular weight increases.

It should be mentioned that the eþect of molecularweights of samples may not be important in deter-mining susceptibility toward biodegradation exceptfor samples with low molecular weights. In fact, arecent study15 conducted on two samples of bacterialPHB having number-average molecular weights of768000 and 22000 and very comparable crystallinityshowed essentially the same rate constant towardenzymatic degradation. For a sufficiently low molec-ular weight sample, the higher solubility in the reac-tion medium and hence the greater surface exposureto the enzyme active sites, should result in a higherdegradation rate compared to a similar sample ofhigh molecular weight. In our study, the molecularweights of all samples listed in Table 1 are sufficient-ly high, so that solubility does not account for thediþerence in degradation rate. The results of aerobicbiodegradation of all the samples and bacterial PHBare summarized in Table 2.

Two diþerent aspects are to be considered: (i) theinýuence of tacticity and crystallinity of the sampleon the initial biodegradation rate; (ii) the ünal bio-

Sample Catalys t Diad Tga (¡C) T

mb (¡C) *H

mc (J gÉ1) X

cd(%) M1

we

fraction (i )

1 H2O/AlEt

3\ 1 0.72 È 139 33 23 310 500

2 H2O/AlEt

3\ 1 0.70 È 166 25 17 241 300

3 H2O/AlOct

3\ 0.5 0.43 1.0 È È È 11 200

4 H2O/AliBu

3\ 0.5 0.43 1.0 È È È 14 400

5 TIBAO 0.41 2.2 È È È 17 200

6 H2O/TIBAO\ 0.5 0.48 [2.0 È È È 19 000

7 H2O/TIBAO\ 1 0.51 [2.0 È È È 31 700

8 TIBAO 0.50 3.5 È È È 18 900

PHB Bacterial 1.0 È 179 97 67^ 5 800 000

(Zeneca)

aGlas s trans ition temperature determined from DSC.

bMelting temperature determined from DSC.

cMelting enthalpy.

dCrys tallinity degree calculated from the known crys tallinity of bacterial P([R]-HB) Zeneca.eWeight-average molecular weight calculated from GPC.

Table 1. Characterization of

s ynthetic poly(hydroxybutyrate)

P([R,S]-b-BL) and bacterialP([R]-HB)

26 Polym Int 48 :23–32 (1999)

Enzymatic degradability of PHB stereoisomers and lactone copolymers

Sample Diad Biodegradation Incubation Initial rate of

fraction (i ) in % ThODa time (days ) degradation

(% ThOD dayÉ1)b

1 0.72 41 36 2.7

2 0.70 37 36 1.5

3 0.43 42 42 2.3

4 0.43 28 42 1.6

5 0.41 50 42 2.9

6 0.48 63 42 4.6

7 0.51 62 42 3.9

8 0.50 88 48 11.6

PHB (Zeneca) 1.00 94 36 42.2

a % ThOD, theoretical percentage oxygen demand of the s ample giving the final degree of degradation. Eachs ample has been analys ed twice and we have taken into account the average of both res ults .

b Initial rate of degradation is the s lope at the beginning of the curve repres enting % ThOD as a function of

incubation time.

Table 2. Res ults of aerobic

biodegradation in s ynthetic

P([R,S]-b-BL) and bacterialP([R]-HB)

degradation which is determined by the biochemicaloxygen demand (% ThOD) of the sample and whichis directly related to the [R] content of the polymer.

The behaviour of % ThOD versus time is given inFig 1, where the complete degradation of bacterialPHB is observed after 40–50 days. This total degra-dation is directly related to its structure; indeed thespeciücity of the enzyme used for [R] linkages canbe established. Moreover the bacterial PHB has aninitial rate of degradation higher than those observedfor synthetic samples. These results show that thedegradation of [R] units is a more important factorthan the degree of crystallinity.

The initial rate of degradation of synthetic PHB,depicted in Table 2, is much lower than that of bac-terial PHB. The highest initial degradation rate forsynthetic PHB is observed for atactic polymers(i \ 0.50). The lowest initial rate is observed for thepredominantly isotactic synthetic PHB with i \ 0.70and for the syndiotactic synthetic PHB with i \ 0.43.

In order to explain this behaviour and take intoaccount the ünal degree of biodegradation, polymerproperties such as crystallinity, stereochemical com-position and accessibility16 to the enzyme need to beconsidered. The synthetic crystalline PHB with ahigh isotactic diad content (i \ 0.70) has the lowestinitial degradation rate. It has been shown that enzy-matic attack proceeds on the surface of polymerleaving the inner polymer matrix relativelyuntouched. The inability of the enzyme to act on thehighly crystalline polymer can be viewed as beingdue to the presence of a largely ordered compactpolymer structure. Hence the low efficiency of theenzyme could stem from the limited number ofexposed accessible linkages and also long [S] stereo-blocks which are not enzymate substrates.

As reported in earlier studies on bacterial PHBand model oligomeric compounds,6c hydrolytic chaincleavage may begin in an ‘exo’ way from the freehydroxyl terminus of the polymer chains and con-tinue in an ‘endo’ manner as the chains becomeshorter and more free surface area is exposed. In thecase of synthetic polymers, the presence of [S]stereoblocks would halt the degradation until another

accessible [R] stereoblock is exposed at the surface.The enzymatic activity will stop when all availablesurface [R] stereoblocks are removed, leaving animpenetrable ‘wall’ of [S] stereoblocks. This inter-pretation shows the inýuence of crystallinity onenzyme accessibility. Therefore, the highest initialdegradation rates were obtained for atactic syntheticPHB.

Another aspect of our work was to study the ünalbiodegradation of the polymers (Fig 2), which is notrelated to the tacticity or crystallinity of the poly-mers, but only to the [R] unit content. The ünal bio-degradation versus time proüles have been studiedfor polymers with variable isotactic diad fractions :i \ 1.0 for bacterial PHB, i \ 0.70 for PHB-2,i \ 0.50 for PHB-8, i \ 0.51 for PHB-7 and i \ 0.41for PHB-5. The choice of catalyst for the ring-opening polymerization of [R,S]-b-BL dictates the[R] and [S] unit length in the polymer. Moreover,the speciücity of the enzyme for the [R] linkages hasbeen previously established. Therefore, we may onlyobtain a ünal biodegradation of 50% for syntheticPHB, whereas ünal biodegradations of 41% and 37%for predominantly isotactic PHBs, and 2% and 28%for predominantly syndiotactic PHBs are observed.These four polymers were synthesized in the pres-ence of trialkylaluminium/water or(AlEt3 , AliBu3

catalyst systems. A study6b has shown thatAlOct3)the [RS] and [SR] units are less accessible to theenzyme than the [RR] units. Moreover, it has beenshown11,17 that the ability of the enzyme to attack[RRR] trimer sequences is higher than [RRS] and[SRR] sequences. The triad distributions, calculatedfrom the 13C NMR spectra of synthetic polymers arepresented in Table 3. For instance, PHB-1 and -2have an isotactic triad (ii) content of 0.60. If we con-sider that the (ii) triad is constituted equally of[RRR] and [SSS] sequences, it is apparent thatthese polymers contain about 30% of [RRR] trimersequences. In the same way, the content of [RRS]and [SRR] sequences in these polymers is about13%, because the [RRR] trimer and [RR] dimersequences are the most accessible, the ünal biodegra-dation of PHB-1 and -2 should be around 40%. This

Polym Int 48 :23–32 (1999) 27

C Jaimes et al

Figure 1. Curves repres enting the theoretical percentage oxygen demand (% ThOD) vers us incubation time for : (a) bacterial PHB; (b)PHB-1 ; (c) PHB-2 ; (d) PHB-3 ; (e) PHB-4 ; (f) PHB-5 ; (g) PHB-6 ; (h) PHB-7 ; (i) PHB-8. Each s ample (except (a)) has been analys ed twice:…,firs t analys is ; s econd analys is .L,

Figure 2. Biodegradation of bacterial P([R]-HB) and s yntheticP([R,S]-b-BL) as a function of the is otactic diad content.

Table 3. Triad s equence dis tributiona for s ynthetic P([R,S]-b-BL)

Sample (ii ) Triad (is ) Triad (s i ) Triad (s s ) Triad

RRR]SSS RRS]SSR RSS]SRR RSR]SRS

1 0.60 0.15 0.12 0.13

2 0.60 0.13 0.12 0.15

3 0.27 0.22 0.28 0.23

4 0.19 0.28 0.35 0.28

5 0.25 0.18 0.35 0.22

6 0.25 0.20 0.31 0.24

7 0.31 0.18 0.27 0.24

8 0.22 0.28 0.28 0.22

aThe triad fractions were determined from the methylene carbon

s ignals in the 13C NMR s pectra at 100.3MHz.

28 Polym Int 48 :23–32 (1999)

Enzymatic degradability of PHB stereoisomers and lactone copolymers

Sample Copolymer compos itiona Tg(¡C) T

m(¡C) (*H

m) M1

w(J gÉ1)

P(BL-co-CL)

9 92 BL : 8 CL [3 È È 17 580

10 67 BL : 33 CL [17 È È 29 310

11 45 BL : 55 CL [37 È È 26 470

12 16 BL : 84 CL [50 49 25 40 560

13 5 BL : 95 CL [54 55 72 55 230

14 100 CL [62 60 105

P(BL-co-VL)

15 90 BL : 10 VL [4.5 È È 7750

16 69 BL : 31 VL [17 È È 8300

17 48 BL : 52 VL [30 È È 11 430

18 26 BL : 74 VL [43 È È 13 800

19 11 BL : 89 VL [54 36 34 17 190

20 100 VL [63 52 65 13 100

aCopolymer compos ition has been calculated from the methylene hydrogens s ignal for VL and

CL units and from the methine hydrogene for BL units in the 1H NMR s pectra at 250MHz.

Table 4. Characteris tics of

s ynthetic copolymers

P(BL-co-CL) and P(BL-co-VL)

hypothesis is in good agreement with the resultsobtained: 40% for PHB-1 and 37% for PHB-2. ForPHB-5 and -8 (synthesized in the presence ofTIBAO with or without water) however, a very highünal biodegradation of 88% is exhibited, althoughaccording to the previous argument, the ünal biodeg-radation should be located around 40%. This latterresult suggest that [R] units as well as a proportionof [S] units are hydrolysed together.

Aerobic biodegradation of synthetic copolymers

P(BL-co-CL) and P(BL-co-VL)

In this part, we aim to relate the physical and struc-tural properties of synthetic copolymers P(BL-co-CL) and P(BL-co-VL) to their biodegradability inaerobic and anaerobic medium. For this study, wecompare the initial biodegradation rate of diþerentsynthesized copolymers with the correspondinghomopolymers such as synthetic and bacterial PHB,polycaprolactone (PCL) and polyvalerolactone

(PVL). Table 4 summarizes a number of importantcharacteristics of the copolymers studied. All thecopolymers were synthesized by ring-opening poly-merization of [R,S]-b-BL with e-CL and d-VL atvarious feed ratios in the presence of TIBAO cata-lyst. The details for the synthesis and character-ization of these copolymers are the subject of aseparate paper.14

The glass transition temperature melting tem-Tg ,perature enthalpy of fusion and molecularTm , *Hmweights of copolymers were determined from DSCand GPC measurements, respectively. CopolymersP(BL-co-CL) 9–11 and copolymers P(BL-co-VL)15–18 are amorphous because they exhibit nomelting temperature. Copolymers P(BL-co-VL) 12and 13 and P(BL-co-VL) 19 are semi-crystallinebecause they present both glass transition andmelting temperatures ; the crystallinity increases withan increase of value. A previous study14 showed*Hmthat these copolymers behave as a compatible

Sample Biodegradation Incubation time Initial

in % ThOD (days ) biodegradation rate

(% ThOD dayÉ1)

P(BL-co-CL)

9 81 48 8.3

10 81 48 9.5

11 74 48 3.6

12 73 48 3.8

13 77 48 7.3

14 43 36 2.1

P(BL-co-VL)

15 84 35 13.8

16 85 35 10.8

17 85 35 10.8

18 83 35 6.1

19 25 35 1.8

20 25 35 8.7

Table 5. Res ults of aerobic

biodegradation of s ynthetic

P(BL-co-CL) and P(BL-co-VL)

copolymers

Polym Int 48 :23–32 (1999) 29

C Jaimes et al

Figure 3. Curves repres enting the theoretical percentage oxygen demand (% ThOD) vers us incubation time for s ynthetic copolymersP(BL-co-CL) and PCL. Each s ample has been analys ed twice: firs t analys is ; s econd analys is .…, L,

mixture of polymers, whereas PHB is not misciblewith PCL and PVL. In fact, we have suggested thatthey would consist of homopolymers and copolymerswith various lengths of comonomer sequences.

The biodegradation in an aerobic medium is givenin Table 5 and the curves representing the percent-age of ThOD as a function of incubation time areshown in Figs 3 and 4. The initial rate and degree ofbiodegradation of copolymers are compared to valuesfor the corresponding homopolymers PHB, PCL andPVL.

PCL homopolymer degraded to about 43% after36 days, but biodegradation is not complete at this

Figure 4. Curves repres enting the theoretical percentage oxygen

demand (% ThOD) vers us incubation time for s yntheticcopolymers P(BL-co-VL) and PVL: P(90BL-co-10VL) ;],…,P(48BL-co-52VL) ; P(26BL-co-74VL) ; P(11BL-co-89VL) ;L, =, K,PVL.

stage. In fact, it is known that PCL is fully biode-gradable but at a lower rate than crystalline PHB.

For all P(BL-co-VL) copolymers, a ünal degree ofbiodegradation of around 75–80% is observed. Incontrast, the initial degradation rate decreases as theBL content in the copolymer increases except in thecase of P(5BL-co-95CL) copolymer. For copolymersin which the BL-unit content varies from 45 to 92%,the kinetics of biodegradation are similar to that ofsynthetic PHB-8 prepared with TIBAO as catalyst.

The crystallinity seems to be a determinant factorinýuencing the initial degradation rate. For instance,the initial degradation rate is strongly aþected forPCL because the polymer exhibits a high crys-tallinity compared to the copolymers P(BL-co-CL).BL units aþect the polymer morphology and allowthe presence of more accessible linkages.

We observe that the initial degradation rate ofP(BL-co-VL) copolymers decreases progressivelywhen the VL-unit content increases. For amorphouscopolymers 15–18, the degree of biodegradation isvery high (around 85%) and similar to that of syn-thetic PHB-8, but when the VL content is higher (upto 75% for copolymers 19 and 20), the degree of bio-degradation strongly decreases because of theincrease of crystallinity.

Anaerobic biodegradation of synthetic

P( [R,S ] -b-BL) and copolymers P(BL-co-CL)The anaerobic biodegradation results of syntheticP([R,S]-b-BL), bacterial P([R]-HB), and copoly-mers P(BL-co-CL) are given in Table 6. The curves

30 Polym Int 48 :23–32 (1999)

Enzymatic degradability of PHB stereoisomers and lactone copolymers

Sample ThCH4a ThCO

2b ThBiogas c Incubation Initial

(%) (%) (%) time (days ) biodegradation

rate

(% ThBiogas dayÉ1)

PHB

8 62 13 47 53 5.5

Bacterial 86 36 68 53 3.8

P(BL-co-CL)

10 69 14 49 53 3.6

11 60 17 48 53 2.0

12 40 6 28 53 0.8

13 7 4 6 53 0

14 7 1 6 53 0.15

aTheoretical percentage of converted methane CH4.

bTheoretical percentage of converted CO2.

c Theoretical percentage of biogas produced and(CH4

CO2).

Table 6. Res ults of anaerobic

biodegradation of s ynthetic PHB

and copolymers P(BL-co-CL)

showing the theoretical percentage of biogas produc-ed (% ThBiogas including and versus theCO2 CH4)incubation time, are presented in Fig 5. Firstly, itcan be seen that the biodegradation rates in anaerobic medium are lower than the rates of aerobicbiodegradation. The same observation can be madeconcerning the degrees of biodegradation.

The degree of anaerobic biodegradation (%ThBiogas) for synthetic PHB-8 of around 48% is ingood agreement with the expected theoretical valuecorresponding to the [R] unit content of the polymerwhich is around 50%.

In the same way as in aerobic conditions, theinitial biodegradation rates decrease uniformly when

the crystallinity of the material increases. Hence,whatever the degradation medium (aerobic oranaerobic), the inýuence of crystallinity is similar.

CONCLUSIONS

The results discussed above suggest that polymerssynthesized from TIBAO catalyst exhibit very goodbehaviour in aerobic biodegradation. Because of themethod and inoculum used for the tests, it is possibleto degrade synthetic polymers by about 85–90% after50 days. This result may mean that the TIBAO cata-lyst permits the synthesis of polymers having anideal relative distribution of [R] and [S] units in

Figure 5. Curves repres enting the theoretical percentage of biogas produced (% ThBiogas ) in anaerobic conditions vers us incubationtime for s ynthetic PHB and copolymers P(BL-co-CL). Each s ample has been analys ed twice: firs t analys is ; s econd analys is .…, L,

Polym Int 48 :23–32 (1999) 31

C Jaimes et al

their chain. In the same way, copolymers synthesizedfrom TIBAO catalyst exhibit a high ünal biodegra-dation of around 85%. As we have shown in a pre-vious paper, these copolymers comprise a compatiblemixture of homopolymers and block copolymers, sothat when an atactic homopolymer such as P([R,S]-b-BL) is mixed with a semi-crystalline copolymerP(BL-co-CL), for instance, a more biodegradablepolymer is obtained.

ACKNOWLEDGEMENTS

We gratefully acknowledge the ünancial supportobtained from the European Commission, Brite-Euram no BRE2 CT 95 0205.

The authors acknowledge the assistance of Dr WMu� ller, Dr P Pu� chner and Dr A Boley (University ofStuttgart) in the measurement of the biodegradationrates.

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