Biodegradation of Aliphatic Homopolyesters and Aliphatic−Aromatic Copolyesters by Anaerobic Microorganisms

Biodegradation of Aliphatic Homopolyesters andAliphatic -Aromatic Copolyesters by Anaerobic Microorganisms

Dunja-Manal Abou-Zeid,† Rolf-Joachim Muller,* and Wolf-Dieter Deckwer

GBF, Gesellschaft fur Biotechnologische Forschung mbH,Mascheroder Weg, D 38124 Braunschweig, Germany

Received February 2, 2004; Revised Manuscript Received May 19, 2004

The anaerobic degradability of natural and synthetic polyesters is investigated applying microbial consortia(3 sludges, 1 sediment) as well as individual strains isolated for this purpose. In contrast to aerobic conditions,the natural homopolyester polyhydroxybutyrate (PHB) degrades faster than the copolyester poly-(hydroxybutyrate-co-hydroxyvalerate) (PHBV). For the synthetic polyester poly(ε-caroplacton) (PCL),microbial degradation in the absence of oxygen could be clearly demonstrated; however, the degradationrate is significantly lower than for PHB and PHBV. Other synthetic polyesters such as poly(trimethyleneadipate) (SP3/6), poly(tetramethylene adipate) (SP4/6), and aliphatic-aromatic copolyesters from 1,4-butanediol, terephthalic acid, and adipic acid (BTA-copolymers) exhibit only very low anaerobic microbialsusceptibility. A copolyester with high amount of terephthalic acid (BTA 40:60) resisted the anaerobicbreakdown even under thermophilic conditions and/or when blended with starch. For the synthetic polymers,a number of individual anaerobic strain could be isolated which are able to depolymerize the polymers andselected strains where identified as new species of the genusClostridiumor Propionispora. Their distinguisheddegradation patterns point to the involvement of different degrading enzymes which are specialized todepolymerize either the natural polyhydroxyalkanoates (e.g., PHB), the synthetic polyester PCL, or othersynthetic aliphatic polyesters such as SP3/6. It can be supposed that these enzymes exhibit comparablecharacteristics as those described to be responsible for aerobic polyester degradation (lipases, cutinases, andPHB-depolymerases).

Introduction

The past two decades have witnessed a growing publicand scientific concern regarding the use of biodegradableplastic material as a solution for the existing problem ofplastic waste. A number of biodegradable plastics have beensuccessfully developed over the past few years to meet thespecific demands, e.g., in agriculture and packaging indus-tries.1,2 The best understood and most extensively studiedplastics with regard to biodegradation are poly(hydroxyal-kanoates) (PHA), which are polymers naturally produced bybacteria.3,4 However, for practical applications biodegradablealiphatic synthetic polyesters such as poly(ε-caprolactone)(PCL), poly(ethylene succinate-co-butylene succinate) (tradename “Bionolle”), or polylactide (PLA) have predominantlybeen used up to now.5 Due to the limited material propertiesof aliphatic polyesters, new biodegradable aliphatic-aromaticcopolyesters have been developed and recently introducedinto the market, e.g., under the trade name Ecoflex (BASFAG, Germany) or Eastar Bio (Eastman Chemicals, U.S.A.).6,7

Most research on biodegradation processes is focused onaerobic environments such as surface water, soil, or compost.In contrast, only little is known about anaerobic biodegrada-tion of plastics, although anaerobic digestion of biowaste

becomes more and more established because of the additionalenergetic benefit of biogas recovery. Most studies publishedon anaerobic biodegradation of plastics focus on mixed andunspecified microbial communities such as diverse anaerobicsludges and/or sediments evaluating the anaerobic biodeg-radation of polyhydroxyalkanoates, PCL and PLA,8-18 orstarch- or cellulosesters.19-21 Nishida and Tokiwa22 charac-terized the distribution of PCL degrading aerobic andanaerobic microorganisms in different environments, but didnot isolate or identify any strain. Investigations usingindividual cultures were restricted to poly(hydroxybutyrate)(PHB) degradation by an organism described asIlyobacterdelafildii which was isolated and identified by Janssen andco-workers.23,24

Recently, we published systematic investigations on theanaerobic degradation of polyhydroxybutyrate (PHB) andpoly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and thesynthetic aliphatic polyester poly(ε-caprolactone) (PCL),using mixed microbial consortia and especially isolatedanaerobic individual strains (four strains of the genusClostridium).25,26 Although contrary findings had beenpublished about the anaerobic biodegradability of the syn-thetic aliphatic polyester PCL, we could unambiguouslydemonstrate the biodegradation of PCL by anaerobic mi-croorganisms. However, the observation of a faster degrada-tion of the PHB homopolyester compared with the copoly-ester PHBV under anaerobic conditions indicates that the

* To whom correspondence should be addressed. Tel:+49 531 6181619.Fax: +49 531 6181175. E-mail: [email protected].

† Present address: University of Alexandria, Alexandria, Egypt.

1687Biomacromolecules 2004,5, 1687-1697

10.1021/bm0499334 CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 07/10/2004

substrate specificity of the microorganisms and possibly alsodegradation mechanisms are different from that of the aerobicsituation, where usually PHBV was found to degrade fasterthan PHB.

Since it is obviously not possible to conclude from theaerobic degradation behavior of polyesters on their microbialsusceptibility in the absence of oxygen, it was of particularinterest to look if also other synthetic aliphatic polyestersand especially aliphatic-aromatic copolyesters, which areof high commercial relevance, can be degraded underanaerobic conditions. Therefore, we investigated the anaero-bic degradability of the polyesters poly(butylene succinate),poly(butylene adipate), and poly(butylene terephthalate-co-butylene adipate) and compared it with the results obtainedfor the natural poly(hydroxyalkanoates) and PCL tested in aprevious study.25 For the investigations presented here, weapplied microbial consortia as well as specifically isolatedindividual anaerobic strains.

Material and Methods

Polymers.The chemical structures of the different poly-esters chosen for the degradation studies are shown in Figure1. Table 1 summarizes the chemical components, thecomposition, and some important physical parameters.

Sample Preparation and Sterilisation.Polyester process-ing into thin films by compression molding and sterilizationby UV-radiation was performed as described by Abou-Zeidet al.25 PCL Tone 787 and PCL-S as well as BTA45:55 andBTA-S were available as films. Using punches with defineddiameters, test specimens of defined surface areas were cut.

For degradation tests in liquid cultures, the processed filmswere sterilized by H2O2 treatment. Films were inserted singlyin small Petri dishes (q ) 35 mm, Greiner, Frieckenhausen)and exposed to 10% (vol/vol) H2O2 for 1 h per each side,dried under a laminar air flow at room temperature overnight,and washed thereafter in three subsequent volumes of 500

mL sterile distilled water using sterile forceps. No significantchanges in the material properties due to the treatment couldbe observed.

Source of Inocula.Three different sources of technicallymanaged and controlled disposal systems were used as asource of anaerobic bacteria for all degradation tests and theisolation of microbial strains:

(I) Anaerobic sludge (wastewater sludge: WWS) from ananaerobic digester of a municipal wastewater treatment plant(Gifhorn, Germany)

(II) Anaerobic methane producing sludge collected froman anaerobic laboratory reactor of the Institute for Technol-ogy of Carbohydrates (Technical University, Braunschweig,Germany) fed with wastewater from the sugar industry(laboratory sludge: LS)

(III) Thermally treated biowaste (TBW) from the anaerobicbiowaste treatment plant in Braunschweig-Watenbu¨ttel,Germany

(IV) Additionally, a sample from a natural environmentwas taken, namely an anaerobic river sediment (AS) fromSpittelwasser, a sidearm of the Elbe river, Germany. Sedi-ments of the Spittelwasser are highly loaded with organiccontaminants. It is known that for such sediments only theupper few millimeter exhibit aerobic conditions, while themicrobial activity in this layer consumes the entire oxygendiffusing from the surface into the sediment.

The sludges were used directly after sampling for pre-liminary degradation tests and the preparation of polyesterdegrading enrichment cultures. Sludge samples were ad-ditionally stored under nitrogen at 4°C for further experi-ments.

Media for Cultivation and Degradation Experiments.The composition of the mineral salt medium used in thiswork was according to Abou-Zeid.25,26

MSV Medium (Components per 1 L Medium).K2HPO4,0.35 g; KH2PO4, 0.27 g; NH4Cl, 0.5 g; CaCl2‚2H2O, 0.075g; FeCl2‚4H2O, 0.02 g; MgCl2‚6H2O, 0.1 g; trace elementsolution, 1 mL; vitamin solution, 1 mL; selenite/tungstatesolution; resazurin (0.1%), cystein-HCl (0.025%), and Na2S(0.025%) were added to ensure anaerobic conditions.

Trace Element Solution. MnCl2‚4H2O, 0.5 g; H3BO3, 0.05g; ZnCl2, 0.05 g; CuCl2‚2H2O, 0.03 g; CoCl2‚6H2O, 0.5 g;NiCl2‚6H2O, 0.05 g; Na2MoO4‚2H2O, 0.01 g. Demineralizedwater was added to complete 1 liter.

Vitamin Solution. Biotine, 2 mg; folic acid, 2 mg;pyridoxalhydrochloride, 10 mg; thiamindichloride, 5 mg;riboflavine, 5 mg; nicotinic acid, 5 mg; DL-calciumpantho-tenate, 5 mg; vitamin B 12, 0.1 mg;p-aminobenzoate, 5 mg;lipoic acid, 5 mg;. Components were dissolved in 1 l ofdemineralized water. The solution was membrane filtered(pore size 0.2µm) and stored at 4°C.

Selenite/Tungstate Solution.NaOH, 0.5 g; Na2SeO3‚5H2O,3 mg; Na2WO4‚2H2O, 4 mg. The salts were added to 1 ldemineralized water and stored at 4°C.

For preparing agar plates, 20 g of agar/L was added tothe MSV medium; The preparation of the polyester supple-mented agar plates for clear zone experiments is describedelsewhere.26

Figure 1. Chemical formula of the polyesters used in this study. (Theindex n represents the degree of polymerization, and the indices land m represent the composition of the random copolyesters.)

1688 Biomacromolecules, Vol. 5, No. 5, 2004 Abou-Zeid et al.

Mass Loss Determination.For mass loss measurementsof films after incubation the polyester samples were washedtwice with distilled water and dried to constant mass undervacuum. The mean mass difference of the films (at leasttriplicates) was expressed as mass loss (∆m in mg) oroptionally expressed as∆m A-1 in mg cm-2 (A ) totalsurface area of both sides of the polyester strip in cm2) sincepolymer depolymerization is a surface process.

Determination of the Biogas Formation.Monitoring ofbiogas production for the quantitative comparison of anaero-bic degradation with mixed cultures was done as describedby Abou-Zeid et al.25 according to ASTM D 5210-91.Biodegradation is expressed as percentage of the maximumtheoretical biogas formation as calculated from the so-calledBuswell equation:27

Determination of the Starch Content of Polyester-Starch Blends.The relative starch content of the polyester-starch blends was determined to follow up changes in thecomposition during degradation, either by a gravimetricmethod or by applying GPC. For the gravimetric determi-nation of the starch content, a preweighed circular polyesterfilm (q 38 mm) was dissolved in 1 mL dichloromethane in

a preweighed 1 mL test vial (Eppendorf). After centrifugation(15 min, 15 000 rpm), the supernatant was carefully removed.The remaining pellet was dispersed again in 1 mL ofdichloromethane and centrifuged. This washing procedurewas performed three times for each sample. Finally, the cupswere dried under vacuum at 37°C for 36 h and re-weighed.The content of insoluble starch was calculated from theweight differences. To estimate possible mass losses due tothe dissolution of the cup-material caused by the dichlo-romethane treatment, the cups were treated the same way asmentioned above without polyester samples. An increase inmass only up to 0.2% was noticed. The determination ofthe starch content by GPC was performed by comparing thepolyester content of pure polyester films to that of the starchcontaining blends. The blends were dissolved in chloroformto a known concentration. The residual solid starch wasthereafter separated by filtration (0.8 and 0.45µm filterpaper), and the solution containing the polyesters wasanalyzed by GPC. The polymer concentrations were calcu-lated from the GPC peak areas. GPC calibration curves ofpure PCL and BTA were established in a concentration rangeof 0.25-1.5 mg‚ml-1 in chloroform (R2 for PCL: 0.9997;R2 for BTA: 0.9996).

Enrichment Cultures. Enrichment cultures were preparedby adding to the three mesophilic sludges (sludge I, WWS;sludge II, LS; sludge IV, AS) strips of one of the six

Table 1. Polyesters Used for Anaerobic Degradation Experiments

polymer component(s) Tma Mw

b source

PHB 3-hydroxybutyrate 174 540 000 ICI, BillinghamUnited Kingdom(as Biopol BX G08)

PHBV 3-hydroxybutyrate/ 150 397 000 ICI, Billingham3-hydroxyvalerate (11,6 mol %) United Kingdom

(as Biopol BX PO270)PCL ε-caprolactone 60 50 000 Polyscience Inc.

Warrington, USAPCL Tone 787 ε-caprolactone 63 200 000 Novamont S.p.A.

Novara, ItalyPCL-S ε-caprolactone and starch (40% w/w) 63 187 000c Novamont S.p.A.

Novara, ItalySP 3/6 1,3-propanediol/adipic acid 44 38 000 GBF

Braunschweig, GermanySP 4/6 1,4-butanediol/adipic acid 62 40 000 GBF

Braunschweig GermanyBTA 10:90 1,4-butanediol (50 mol %) 56 25 000 GBF

adipic acid (45 mol %)/ Braunschweig Germanyterephthalic acid (5 mol %)

BTA 20:80 1,4-butanediol (50 mol %) 52 36 000 GBFadipic acid (40 mol %)/ Braunschweig Germanyterephthalic acid (10 mol %)

BTA 40:60 1,4-butanediol (50 mol %) 99 47 600 Huls AGadipic acid (30 mol %)/ Marl, Germanyterephthalic acid (20 mol %)

BTA 45:55 1,4-butanediol (50 mol %) 120 66 500 BASF AGadipic acid (27.5 mol %)/ Ludwigshafen, Germanyterephthalic acid (22.5 mol %)

BTA-S 1,4-butanediol (50 mol %) 92 145 000c Novamont S.p.A.adipic acid(27.5 mol %)/ Novara, Italyterephthalic acid (22.5 mol %)and starch (32% w/w)

a Tm: melting temperature; temperature at the maximum of melting peak (DSC). b Mw: weight average molar mass (determined by GPC with polystyrenecalibration). c Mw of the polyester component.

CnHaOb + (n - a/4 - b/2)H2O f (n/2 - a/8 + b/4)CO2 +(n/2 + a/8 - b/4)CH4

Biodegradation of Polyesters Biomacromolecules, Vol. 5, No. 5, 2004 1689

polyesters (see Figure 1) and optionally of all six polyesterstogether. Screening for polyester degrading microorganismswas then performed after 14 weeks and optionally after 18month incubation using the so-called “clear-zone method”with polyester containing agar plates and roll tubes asdescribed elsewhere.25,26 The isolates were preserved inairtight vials containing 50% (v/v) glycerol (87%) flushedand headspace filled with oxygen free N2 gas. The anaerobicvials were additionally put in airtight bags containing ananaerobic catalyst (Anaerocult A, Merck, Darmstadt, Ger-many) and stored at-20 °C.

Identification and Biochemical Characterization of theIsolated Strains.The identification of the isolated polyesterdegrading strains through partial 16S rDNA sequenceanalysis was carried out by the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Braunschweig,Germany). Selected isolates were characterized according tothe standard methods described by Holdemann et al.28 aswell as Krieg.29 These tests were also partially performedby the DSMZ, Germany. More information are givenelsewhere.49

Results

Evaluation of Anaerobic Biodegradablity by Mass LossMeasurements.The incubation of the polyester films in theanaerobic sludges served as a first indication to what extentthe different materials are susceptible to an anaerobicmicrobial attack. In our previous publication,25 we found asignificant mass loss of PHB and PHBV films in anaerobicmethane sludge (LS) after 10 weeks at 37°C. Under thesame conditions, the synthetic aliphatic polyester PCL alsoexhibited a clear microbial attack but mass losses were onlyabout 30% of those of the poly(hydroxyalkanoates). In Figure2, mass losses of these polymers and additionally of thesynthetic aliphatic polyester SP 4/6 and the aliphatic-aromatic copolyester BTA 40:60 in cultures with inoculafrom three different anaerobic environments after 14 weeksincubation at 35°C are shown.

As previously observed, PHB and PHBV disintegratedrapidly while PCL exhibited a significant slower degradation.In contrast, for SP4/6 and BTA 40:60 even after anincubation time of more than 3 month, only minor masslosses (less than 2 mg) of the samples could be observed,mainly in the laboratory sludge (LS). Obviously anaerobicdegradability of synthetic polyesters is strongly dependingon the structure or properties of the material. From the massloss data shown here, it is not possible to decide reliably ifa microbial attack in principle took place or not for the SP4/6 and BTA 40:60.

The tendency of the degradation behavior of the polyestersin the different sludges used here is generally comparable.However, no general rule of the degradation potential of thedifferent sludges can be derived from the data presented here.

Evaluation of Anaerobic Biodegradability by BiogasDetermination. Although mass loss measurements only givean indication if microorganisms principally can attack amaterial, the determination of the biogas (predominantly CO2

and CH4) which is formed during degradation reflects directlythe metabolic transformation of the material in the microbialcells.

In Figure 3, the biogas formation from the polyestersamples in the laboratory sludge (LS) and the wastewatersludge (WWS) at 37°C is shown as a function of theexposure time.

The successively decreasing anaerobic degradability PHB> PHBV > PCL, which was already previously observedin the laboratory sludge25 (Figure 3a), is confirmed by thedata obtained in the wastewater sludge (Figure 3b). For thesynthetic polyesters SP4/6 and BTA40:60 only very lowbiogas formation could be observed in LS (<5.5%). InWWS, a biogas formation of about 11% was calculated after42 days for both polymers, but also these results are not areliable prove for an anaerobic degradation since the biogasevolution from the sludges itself (background) was in thesame order of magnitude as the gas formation from the SP4/6and BTA40:60 samples and thus, the final data (correctedfor the background gas evolution) exhibit probably a higherror. Mass loss measurements of the polyester samples afterthe biogasification experiment indicate that nearly no bio-logical attack took place on both samples (Table 2).

For PCL and PHBV, an at least temporary accumulationof intermediates can be observed. With both anaerobicmicrobial consortia, the mass loss of the samples is muchhigher than the metabolization (biogas formation) for bothpolyesters which is an indirect indication of the presence ofwater soluble intermediates. This supports preliminaryobservations made in a previous paper.25

To reduce the problems of high background biogasevolution, a synthetic mineral medium inoculated with only10% v/v of anaerobic laboratory sludge (LS) was used inanother gas evolution measurement (Figure 4). The polyestersSP4/6 and BTA40:60 exhibiting up to this point a question-able biodegradability were omitted in this test and SP3/6known to be more easily biodegradable under aerobicconditions30,31 was used instead.

The diluted system ascertained a higher degree of accuracyof mineralization data due to low background gas evolution

Figure 2. Biological hydrolysis of the polyesters in different anaerobicenvironments after 14 weeks at 35 °C. (Polyester films, q ) 25 mm;initial mass of films, 39-49 mg; average of three films per test.) Thenumbers above the bars indicate the absolute mass of the residualpolymer.


(3-5% of the determined biogas due to mineralization ofthe polyesters in the diluted system, compared to over 30%in the native sludges). In contrast to Figure 3, PHBVmineralizes faster in the diluted system and exhibits almostthe same degradation behavior as PHB. The very low biogasdevelopment observed for SP 3/6 (negative values originatefrom the correction for the biogas formation in the blanktests) still leaves open if this kind of polyesters is reallysignificantly attacked by anaerobic microorganisms, althoughSP 3/6 is reported to be more rapidly degraded than SP4/6under aerobic conditions. The clear lag phase of about 5 daysand the pronounced stepwise degradation of the polyesterspoints to community dynamics through adaptation phasesof the different bacterial trophic groups. A similar behavior,however less pronounced, was also observed in the tests withthe undiluted sludges (Figure 3).

Biodegradation of Polyesters under Accelerated Deg-radation Conditions. Since the synthetic polyesters (exceptof PCL) were not or only very slowly attacked by anaerobicmicroorganisms, it was aimed to check if generally amicrobial susceptibility could be demonstrated applyingaccelerated degradation conditions.

Anaerobic Degradation under Thermophilic Conditions.For aerobic conditions, it had been observed that thedegradation rate of specific polyesters strongly depends onthe temperature at which degradation occurs. This waspreviously documented, for example, by Kleeberg32 andinterpreted in detail by Marten31 and Marten et al..33 Theincreased degradability at higher temperatures is not onlycaused by a different microbial community at thermophilicconditions, but is also attributed to a better susceptibility ofthe polyester chains for the depolymerizing enzymes due toan increased chain mobility. It was of interest, particularlyfor the BTA-copolyester, for which no unambiguous anaero-bic biodegradability at mesophilic conditions could beobserved, if the temperature effect under anaerobic conditionsis as pronounced as it is observed in the presence of oxygen.Thermophilic anaerobic conditions are present in practice,

Figure 3. Time dependent mineralization expressed as percentage of the theoretical biogas volume evolved from anaerobic laboratory andwastewater sludge at 37 °C over a period of 42 days; biogas evolution is corrected for the biogas formation from the blank tests (polyester films,q ) 19 mm; initial mass of films, 35-40 mg; average of two films per test).

Table 2. Comparison of Mass Loss and Biogas Formation ofPolyester Samples in Anaerobic Sludges after 42 Days ofIncubation at 37 °Ca

laboratory sludge (SL) wastewater sludge (WWS)

polyester % biogasb % mass loss % biogasb % mass loss

PHB 101 100 101 100PHBV 29 57 31 63PCL 16 30 17 30SP 4/6 1.1 1.2 11 2.1BTA 40:60 5.5 0.5 11 1.0

a Polyester films with 19 mm diameter; total surface area per film 22.7cm2, weight of films 35-40 mg; two films per test. b Percentage of formedbiogas compared to the theoretical possible biogas evolution which wascalculated from the Buswell equation (see Material and Methods). Thetotal biogas formation of the samples was corrected by the biogas formedfrom the sludge (negative control).

Figure 4. Time dependent mineralization expressed as percentageof the theoretical biogas volume evolved from a MSV-mediuminoculated with a laboratory sludge enrichment culture inoculum (10%v/v) at 37 °C over a test period of 56 days; biogas evolution iscorrected for the biogas formation from the blank tests (polyester films,q ) 19 mm; initial mass of films, 35-40 mg; average of two films pertest).


for example, in thermophilic anaerobic digestion plants (highsolid sludges at, e.g., 50°C).

In Figure 5, the degradation rates of different polyestersin a anaerobically treated biowaste at 50°C are comparedwith those in a mesophilic wastewater sludge at 37°C(WSS). Degradation rates were calculated from mass lossmeasurements at different incubation times (3 and 6 weeks),and the maximum degradation rates are used to describe thedegradation behavior.

Under thermophilic conditions, generally higher maximumdegradation rates were obtained as compared to mesophilicconditions. Even higher degradation rates must be anticipatedfor PHB and PHBV, since at the first sampling the materialwas already completely disintegrated. A 28-fold increase ofthe weight loss after 6 weeks was determined for PCL (massloss caused by abiotic hydrolysis of this material did notexceed 1%) and for SP4/6 even a 420-fold higher mass losscould be observed at 50°C (abiotic hydrolysis: mass loss) 0.2 mg; 0.2%).

In contrast to the aliphatic polyesters, the mass loss ofthe aliphatic-aromatic copolyester BTA remained low, evenat thermophilic conditions. The somewhat higher loss ofmaterial at 50°C may be attributed mainly to the influenceof an abiotic hydrolysis.

Anaerobic Degradation of Polyester Starch Blends.Inpractice, many biodegradable polyesters are blended withstarch, once to adjust properties but also with the aim toincrease biodegradability.34 So it was questioned, if blendingthe polyesters PCL and BTA with starch, which is a readilymetabolizable polymeric substrate for many anaerobic mi-croorganisms, would increase the degradation rates of theblend as a whole.

For films of two polyester starch blends (PCL-S, 40 mass%starch and BTA-S, 32 mass% starch) the mass loss wasdetermined after incubation in wastewater sludge (WWS)and thermally treated biowaste (TBW) for 3 and 6 weeksincubation time (Table 3).

For the PCL sample (the small increase in weight for thePCL blind test is caused by a water uptake of the material,which was not removed during the drying process), blendingwith starch resulted in a significant increase of the degrada-tion rate, under mesophilic and thermophilic conditions aswell. However, the effect seems to be higher at mesophilicconditions. Here the degradation of the polyester componentis slower than at 50°C, and the addition of easily degradablestarch gives a higher benefit to the overall degradation asfor thermophilic conditions, where PCL degradation isanyhow much faster.

In contrast to PCL, only a slight increase in mass losseswas observed for the aliphatic-aromatic copolyester BTA.When the results of the blind samples are taken into account(Table 3), the increased degradation rates can be attributedentirely to abiotic hydrolysis.

The relative starch content of the polyester-starch blendsdecreased after incubation for 3 weeks at 37°C (at 50°Cno analysis was possible due to the complete disintegrationof the materials) from 39% to 8% for PCL-S and from 31%to 29% for BTA-S. This reveals that there is a predominantremoval of starch from the sample, increasing the relativepolyester content in the degraded samples.

Isolation of Polyester Degrading Anaerobic SingleStrains. Previously, the isolation of a number of anaerobicmicroorganisms able to depolymerize PHB and PCL has beenreported,25 and selected strains were taxonomically identifiedas new species belonging to the genusClostridium.26,35

The screening for polyester degrading anaerobic strainswas extended in this work (some of the PHB degradingstrains reported earlier lost their capability of PHB-degrada-tion when maintained on complex media) with the aim tofind: if also degrading microorganisms for the syntheticpolyesters SP3/6, SP 4/6 and the BTA-copolyesters can befound, the substrate selectivity of the isolates obtained, andwhat the distribution of the various degrading microorgan-isms in the different environments is.

From the anaerobic sludges and the sediment, a total of55 morphologically different anaerobic polyester degradingbacterial isolates (using the clear zone method as indicationfor polyester degradation) were obtained from enrichmentcultures using the different polyesters as enrichment sub-strates. For the natural polyesters (PHB, PHBV) and themoderately biodegradable PCL, degrading organisms couldbe found already after 14 weeks of enrichment culture.However, a preincubation period of 18 month was necessarywith the synthetic polyesters SP 3/6, SP 4/6, BTA 10/90,and BTA 20/80 to find polyester degrading strains. Noorganisms were found, even after such a long enrichmentphase, which could depolymerize the aliphatic-aromaticBTA-copolyesters with a content of the aromatic acidcomponent higher than 20 mol %.

Isolates from PHB and PHBV Enrichment Cultures.Asexpected from the mass loss experiments described above,in all three different microbial sources, PHB depolymerizingorganisms could be found (Table 4). However, sludges fromtechnically controlled processes (LS and WWS) harbor abroader spectrum of PHB-degraders (total of 26 morphologi-cally different isolates) compared to the natural habitat (AS),

Figure 5. Comparison of the calculated maximum degradation ratesof different polyesters under mesophilic (WWS; 37 °C) and thermo-philic conditions (TBW; 50 °C). The numbers represent absolute massloss percentages. (Polyester films, q ) 19 mm; initial mass of films,PHB, PHBV, BTA40:60, 60-76 mg; PCL, SP4/6, 80-100 mg; ratescalculated from weight losses after 3 and 6 weeks; average of threefilms per test.)


from which only a total of 4 different PHB degrading isolateswere obtained. Interesting was the finding that more strainswere isolated from the PHB enrichments (19 isolates) thanfrom the PHBV enrichment cultures (11 isolates) althoughthe microbial sources were identical. This fact correlates withthe differences in the biodegradability of PHB and PHBVpreviously observed. It additionally points to the impact ofpolyester characteristics on their biodegradability rather thanthe microbial population. For many isolates, supplementationof the medium with acetate, crotonate, and citrate, respec-tively, was necessary to obtain clear zone formation, whereasthe presence of glucose seems to repress the polymerdegradation.

Isolates from PCL Enrichment Cultures.As shown inTable 5, a total of 16 morphologically different isolates wereobtained capable of depolymerizing PCL. In contrast to thePHB and PHBV-degraders, co-substrate addition had nosignificant impact on PCL depolymerization with the excep-tion of acetate. Ten isolates formed clear zones on PCL-

MSV mineral salt agar without supplementation with co-substrates, the remaining 6 isolates depolymerized PCL onlyin the presence of acetate. As expected from the observationof PCL-mass loss in the sludges, the three different microbialsources are inhabited by PCL depolymerizing organisms.However, again more isolates were obtained from thetechnical sludges (LS and WWS) (6 and 9 isolates, respec-tively) compared to the natural habitat (AS) harboring onlyone isolate.

Isolates from SP3/6 Enrichment Cultures.Out of a totalof 15 morphologically different anaerobes isolated from 18month old enrichment cultures with SP 3/6, nine isolates werecapable of SP 3/6 depolymerization (Table 6). Comparableto PHB disintegration, the isolates exhibited different co-substrate requirements for clear zone formation. Especially,yeast extract enhanced polyester hydrolysis. Interestingly,no isolates were obtained from the natural environment(anaerobic river sediment) enrichment cultures, and mostisolates originated from WWS, exhibit a higher degradation

Table 3. Degradation Rates of PCL, BTA, and the Corresponding Starch Blends in Dependence on the Degradation Temperature after 3Weeks of Incubation in Different Sludges

wastewater sludge at 37 °C blind-test 37 °C1) thermally treated biowaste at 50 °C blind-test 50 °Ca

sampleincubation

timedegradation rate(mg/(week cm2)b mass loss (%)

degradation rate(mg/(week cm2)b mass loss (%)

PCL 3 weeks 0.01 ( 0.00 0.4 ( 0.1 0.26 ( 0.11 20.2 ( 8.8 1.66 weeks 1.8 ( 0.2 -0.32

PCL-S 3 weeks 0.10 ( 0.05 19.7 ( 9.5 0.40 ( 0.03 80.2 ( 8.0 156 weeks 23.7 ( 0.4 8.58

BTA 3 weeks 0.01 ( 0.003 0.9 ( 0.2 0.01 ( 0.01 0.9 ( 0.6 2.36 weeks 2.6 ( 0.2 2.23

BTA-S 3 weeks 0.06 ( 0.001 8.4 ( 0.2 0.05 ( 0.04 7.3 ( 6.3 7.86 weeks 9.5 ( 1.9 5.19

a Samples incubated in distilled water at 37 °C and 50 °C, respectively. b Calculation performed by calculating both surfaces of the polyester films.

Table 4. Screening of Anaerobic PHB and PHV-Degrading Organisms from Different Enrichment Cultures with PHB or PHBV as anEnrichment Substrate (14 Weeks Preincubation)

PHB as Enrichment Substrate

no. of organisms forming clear zones on PHB-agara

PHB-agar supplemented with the following:

microbial source total number of isolates no suppl. + acetate + crotonate + citrate + glucose

laboratory sludge (LS) 13 6 11 8 6 1(5-11 mm) (5-18 mm) (5-26 mm) (17-32 mm) (5 mm)

wastewater sludge (WWS) 3 2 1 3 0 0(5-22 mm) (12 mm) (5-18 mm)

anaerobic river sediment (AS) 3 0 1 2 0 0(6 mm) (4-12 mm)

total number of isolates 19 8 14 13 6 1

PHBV as Enrichment Substrate

no. of organisms forming clear zones on PHBV-agara

PHBV-agar supplemented with the following:

microbial source total number of isolates no suppl. + acetate + crotonate + citrate + glucose

laboratory sludge (LS) 4 1 4 1 1 0(5 mm) (4-20 mm) (20 mm) (12 mm)

wastewater sludge (WWS) 6 3 6 3 4 0(2-8 mm) (8-24 mm) (18-24 mm) (12-30 mm)

anaerobic river sediment (AS) 1 1 0 0 0 0(4 mm)

total number of isolates 11 5 10 4 5 0

a Numbers in brackets: diameter of clear zone formed on PHB or PHBV supplemented mineral salt agar plates (MSV-medium) after incubation forfour weeks at 35 °C.


potential and seemed to be more versatile concerning thecosubstrates supporting clear zone formation.

Substrate Specificity of the Isolates.The question arose ifthese 55 anaerobic bacterial isolates also degrade otherpolyesters than those of the corresponding enrichment cultureand if the strains can be divided into separate groupsdepending on their substrate (polyester) specificity.

To investigate the anaerobic biodegradability of differentBTA-polymers, all of the polyester degrading isolates weretested additionally for their capability to depolymerize BTApolyesters with lower molar ratios of aromatic to aliphaticconstituents (BTA 10/90; BTA 20/80; BTA 32/68; BTA 34/66; BTA 36/64; BTA 38/62) (Table 7).

As expected from the missing microbial susceptibility ofBTA 40:60 in the anaerobic sludges, only BTA 10/90; BTA20/80 were attacked by individual strains.

Interestingly, the total number of isolates capable ofdepolymerizing the different polyesters or polyester groups(Table 7) coincided with the degradation rates obtained withthe mixed microbial populations in the sludges. The generalsuccession of the depolymerization rate as well as the number

of depolymerizing organisms is as follows:

The polyester degrading isolates from Table 7 can bedivided into three main groups:

(1) The PHB and PHBV degrading isolates are specializedto depolymerize only the natural polyhydroxyalkanoates andcannot attack synthetic polyesters and vice versa.

(2) PCL degrading strains showed no depolymerizationactivity toward other polymers. Hence, no organisms orig-inally screened on PCL degrade SP 3/6, SP 4/6 or BTA-copolymers.

(3) In contrast, strains isolated from SP 3/6-enrichmentcultures (total of 9) show a wide substrate spectrum withinthe synthetic polyesters but do not hydrolyze the polyhy-droxyalkanoates.

Discussion

Biological degradation of polyesters under anaerobicconditions differ from that in the presence of oxygen with

Table 5. Screening of Anaerobic PCL-Degrading Organisms from Different Enrichment Cultures with PCL as an Enrichment Substrate (14Weeks Preincubation)

no. of organisms forming clear zones on PCL-agara

PCL-agar supplemented with the following:

microbial source total no. of isolates no supplement + acetate

laboratory sludge (LS) 6 4 2(4-5 mm) (4-6 mm)

wastewater sludge (WWS) 9 5 4(3-8 mm) (2-8 mm)

anaerobic river sediment (AS) 1 1 0(2-4 mm)

total number of isolates 16 10 6

a Numbers in brackets: diameter of clear zone formed on PHB or PHBV supplemented mineral salt agar plates (MSV-medium) after incubation for twoweeks at 35 °C.

Table 6. Screening of Anaerobic SP3/6-degrading Organisms from Different Enrichment Cultures with SP 3/6 as an Enrichment Substrate(18 month preincubation)

no. of organisms forming clear zones on SP 3/6-agara

SP 3/6 agar supplemented with the following:

microbial source total number of isolates no suppl. + acetate + crotonate + yeast extract + LLSb

laboratory sludge (LS) 3 1 1 0 1 0(1-5 mm) (15-20 mm) (12 mm)

wastewater sludge (WWS) 6 6 4 2 6 2(5-22 mm) (1-9 mm) (1-15 mm) (12-30 mm) (1-4 mm)

anaerobic river sediment (AS) 0 0 0 0 0 0total number of isolates 9 7 5 2 7 2

a Numbers in brackets: diameter of clear zone formed on PHB or PHBV supplemented mineral salt agar plates (MSV-medium) after incubation forfour weeks at 35 °C. b sterile laboratory sludge supernatant instead of MSV-medium used; to obtain the supernatant, methane sludge was centrifuged at11000 rpm for 30 min, filtered twice through paper filters and autoclaved twice for 20 min at 121 °C.

Table 7. Substrate Spectrum and Number of Depolymersing Isolates Evaluated by Clear Zone Formation on Mineral Salt Agar Plates(MSV-Medium) Supplemented with Different Polyesters

no. of strains capable of degrading

isolates from enrichmentwith the following:

no. of testedisolates SP 3/6 SP4/6 PCL

BTA10:90

BTA20:80

BTA40:60 PHB PHBV

SP 3/6 9 9 7 4 2 2 0 0 0PCL 16 0 0 16 0 0 0 0 0PHB 30 0 0 0 0 0 0 30 27total number of isolates 55 9 7 20 2 2 0 30 27

PHB > PHBV > PCL . SP 3/6- SP 4/6> BTA 10:90-BTA 20:80 (. BTA 40:60)


respect to the absolute degradation rates and the influenceof the polymer structure on the biodegradation. A largenumber of publications are available focusing on aerobicbiodegradation of natural and also synthetic polymers, butonly few papers are dealing with the behavior of biodegrad-able plastics under anaerobic conditions. Furthermore mostof these papers focused on biodegradation in complexmicrobial environments such as anaerobic sewage sludge orsediments, from which only few reliable facts about mecha-nistic aspects can be drawn, since anaerobic microbialcommunities are complex systems.

In a previous paper,25 we demonstrated that it is alsopossible to use individual strains besides anaerobic microbialconsortia (four new species of the genusClostridia wereidentified25,26) to investigate enzymatically catalyzed depo-lymerization of poly(hydroxyalkanoates) and PCL, althoughanaerobic digestion is recognized as a complex processinvolving the coordinated activity of a number of differentbacterial trophic groups.36 It could clearly be shown thatPHB, PHBV, and also PCL, which are all well degradableunder aerobic conditions, are also microbially attacked inthe absence of oxygen. However, differences in aerobicconditions were detected, since the copolyester PHBVexhibited clearly no faster degradation as the hompolyesterPHB. In addition to the polyesters mentioned above, onlyvery few other polymers have been evaluated with respectto their anaerobic biodegradability up to now.

From the investigations presented here, it can be suspectedthat synthetic polyesters others than PCL (such as SP 3/6,SP 4/6 and BTA-copolyesters) degraded very slowly underanaerobic conditions, although their ready aerobic degrada-tion has been demonstrated.31,37-39 The long time period ofenrichment (18 month), necessary to isolate strains capableto depolymerize these synthetic polyesters, point to the factthat only few special anaerobic organisms are able to attackthe chain structures of these polymers. This supposition issupported by the number of degrading strains isolated fromthe enrichment cultures, decreasing from 30 strains capableto attack the natural poly(hydroxyalkanoates) and 16 strainsdegrading PCL to only 9 isolates forming clear zones withSP 3/6.

The PHB degrading anaerobic isolates seem to be special-ized on PHB degradation and metabolization, only. Theenzyme, presumably a specific PHB depolymerase, is ap-parently induced by PHB. Generally, the addition of a secondcarbon substrate (additional energy source) such as acetate,crotonate or citrate (Table 4) obviously enhanced biomassformation and hence increased indirectly polyester degrada-tion (clear zone formation). PHB depolymerization is mainlystimulated by acetate (80% of the total number of isolates)and crotonate (57% of the 30 isolates), a fact which can beexplained by acetate being an intermediary metabolite ofmany anaerobes40 and crotonate is a key metabolite in3-hydroxybutyrate metabolism.41 Glucose on the other handsupported growth of the isolates but not degradation (clearzone formation) with one exception. Obviously, glucosesuppresses the PHB depolymerizing enzyme secretion,probably by catabolite repression.40,42 It is known forclostridia that easily degradable substrates might mask

abilities for biosynthesis and biodegradation and henceextracellular enzymes of polymeric substrates.40,42 It isassumed that the presence of glucose as a growth substrateresults in what is known as inducer (3-hydroxybutyrate)expulsion as has been observed for other clostridia.42,43

Accordingly, the PHB depolymerizing enzyme is not inducedand PHB is not degraded.

In contrast, catabolite repression of the polymer degrada-tion was not observed for the organisms depolymerizingsynthetic polyester and rich medium addition enhancedpolyester depolymerization. Thus, here no problems withpreservation of organisms and/or instability of degradationcharacter occurred.

Concerning the anaerobic PCL-degrading strains isolated(Tables 5 and 6), to our knowledge, no reports on character-ized anaerobic single strains exist in the literature up to now.These strains are also specialists since they only showeddepolymerization activity toward PCL. No organism origi-nally screened on PCL, degrades the other synthetic poly-esters SP 3/6, SP 4/6, BTA 40:60 or the natural PHAs. Twostrains, identified asClostridium sp. noV., are lipase negative,although the described aerobic PCL depolymerizing enzymesare reported to be also lipases besides cutinases.44,45,46

Catabolite repression was not observed for PCL degradation,and the strains did not metabolize the depolymerizationproducts, i.e., the monomers of PCL. The hydrolyzingenzyme depolymerizes PCL probably due to structuralsimilarities between PCL and another natural polymer, suchas cutin, possessing structurally similar elements.22 AlsoMurphy et al.47 presented genetic, regulatory, and enzymaticevidence for the involvement of a cutinase in aerobic PCLdegradation. In addition, they showed that PCL dimers andtrimers are structurally similar to natural inducers of cutinase.It is therefore possible that anaerobic PCL degradationfollows the same principle as aerobic PCL degradation.

The nine strains (Table 6) able to degrade the othersynthetic polyesters (none of them did attack the naturalpolyhydroxyalkanoates) showed a wide substrate spectrumwithin the synthetic polyesters. One isolate was taxonomi-cally characterized asPropionispora sp. noV.48 (since thegenusPropionisporaVibroides noV. gen., noV sp. has justrecently been established by Biebl et al.,49 only a littleinformation is available about these organisms). To ourknowledge, this is the first report describing the anaerobicdepolymerization of these synthetic polyesters by an indi-vidual culture. From the observation that the strain onlyexhibited a very limited growth of the polyesters, it can besupposed that the isolated strain did not metabolize thedepolymerization products of the polyesters. The involveddepolymerizing enzyme seems to be quite unspecific andrepresents probably lipase-like enzyme induced by thepresence of gratuitous inducers, i.e., the synthetic aliphaticpolyesters. Kleeberg32 documented a similar situation whereaerobic BTA depolymerizing strains ofThermobifida fusca(former name:Thermomonospora fusca) secreted an extra-cellular hydrolase which unspecifically depolymerized thecopolyester and several other synthetic aliphatic polyesters,too. The resulting depolymerization products were also notmetabolized by the strain.39 In the present case, probably a


nonspecific lipase producing organism had to adapt itsenzyme regulation mechanisms to the synthetic and unusualpolyester substrate. Similarly, several strains of clostridiasuch asC. thermocellumsynthesize xylanases, for instance,but grow only poorly on xylan owing to an inability tometabolize the degradation products.50-53

The degradation rate of the aliphatic synthetic polyestersby Propionispora sp. noV. decreases with increasing meltingpoint of the materials (SP 3/6 (44°C) > PCL (60°C) > SP4/6 (62°C)). This is congruent to observations reported foraerobic degradation of polyesters.31,33,55 Lipases attack thepolyester chains quite nonspecifically. Degradation is notcontrolled predominantly by the chemical structure of theester bonds but by the ability of the polymer chains to fitinto the active site of the lipases, which is located withinthe enzyme interior. Thus, the degradation is stronglydepending on the mobility of the chains, which can becharacterized by the difference between the melting tem-perature of the given polyester and the incubation temper-ature.33

In contrast to aerobic conditions, the aliphatic-aromaticcopolyesters were anaerobically only attacked at low contentsof aromatic component (up to 20 mol % terephthalic acid ofthe acid component). Here, two reasons for this differentbehavior can be discussed. Generally, the degradation ratesin the anaerobic environment are slower than in the presenceof oxygen, and thus, it may be that the absolute degradationrates of the BTA-copolyesters with higher amounts ofterephthalic acid (exhibiting also high melting points) are tolow to be detected with the experimental tools used in thiswork. However, it cannot be excluded that the anaerobicenzymes (lipases) are not able to cleave any ester bond invicinity of an aromatic group and, thus, can only attack thecopolyesters when extended aliphatic sequences exist.

Obviously, under anaerobic as well as under aerobicconditions, at least three different enzyme systems aresupposed to be involved in the anaerobic degradation of thedifferent polyesters: PHB depolymerases, lipases, andenzymes described as cutinases. None of the PHA depoly-merases shows significant lipase activity or attacks syntheticpolyesters.54 However, several lipases hydrolyze polyestersof ω-hydroxyalkanoic acids such as PCL. Cutinases, on theother hand, are serine hydrolases for primary alcoholesters55,56which depolymerize cutin and synthetic polyesterssuch as PCL.

Acknowledgment. We gratefully acknowledge the sup-port of the scholarship of Mrs. D.-M. Abou-Zeid by theDAAD (Deutscher Akademischer Austauschdienst e.V.). Wethank Dr. I. Wagner-Do¨bler for supplying the anaerobic riversediment, BASF, and Novamont for providing polyestersamples.

References and Notes

(1) Gross, R. A.; Kalra, B.Science2002, 297, 803.(2) Amass, W.; Amass, A.; Tighe, B.Polym. Int.1998, 47, 89.(3) Braunegg, G.; Lefebvre, G.; Genser, K. F.J. Biotechnol. 1998, 65,

127.(4) Doi, Y. Microbial polyesters; VCH Publishers Inc.: New York, 1990.(5) Okada, M.Prog. Polym. Sci. 2002, 27 (1), 87.

(6) Yamamoto, M.; Witt, U.; Skupin, G.; Beimborn, D.; R.-J. Mu¨ller,R.-J. In Biopolymers; Doi, Y., Steinbuchel, A., Eds.; Wiley-VCH:Weinheim, Germany, 2002; Vol. 4, Chapter 3, pp 299-314.

(7) Muller, R.-J.; Kleeberg, I.; Deckwer, W.-D.J. Biotechnol. 2001, 86(2), 87.

(8) Federle, T. W.; Barlaz, M. A.; Pettigrew, C. A.; Kerr, K. M.; Kemper,J. J.; Nuck, B. A.; Schechtmann, L. A.Biomacromolecules2002, 3(4), 813.

(9) Itavaara, M.; Karjomaa, S.; Selin, J.-F.Chemosphere2002, 46 (6),879.

(10) Eldsater, C.; Erlandsson, B.; Renstad, R.; Albertsson, A.-C.; Karlsson,S. Polymer1999, 41 (4), 1297.

(11) Nishide, H.; Toyota, K.; Kimura, M.Soil Sci. Plant Nutr.1999, 45(4), 963.

(12) Albertsson, A.-C.; Renstad, R.; Erlandsson, B.; Eldsa¨ter, C.; Karlsson,S. J. Appl. Polym. Sci.1998, 20, 61.

(13) Gartiser, S.; Wallrabenstein, M.; Stiene, G.J. EnViron. Polym.Degrad.1998, 6 (3), 159.

(14) Reischwitz, A.; Stoppok, E.; Buchholz, K.Biodegradation1998, 8,313.

(15) Shin, P. K.; Kim, M. H.; Kim, J. M.J. EnViron. Polym. Degrad.1997, 5 (1), 33.

(16) Budwill, K.; Fedorak, P. M.; Page, W. J.J. EnViron. Polym. Degrad.1996, 4 (2), 91.

(17) Urmeneta, J.; Mas-Castella, J.; Guerrero, R.J. Appl. EnViron.Microbiol. 1995, 61 (5), 2046.

(18) Budwill, K.; Fedorak, P. M.; Page, W. J.Appl. EnViron. Microbiol.1992, 58 (4), 1398.

(19) Rivard, C.; Moens, L.; Roberts, K.; Brigham, J.; Kelley, S.EnzymeMicrob. Technol. 199517, 848.

(20) Gu, J.-D.; Eberiel, D.; McCarthy, S. P.; Gross, R. A.J. EnViron.Polym. Degrad.1993, 1, 143.

(21) Gu, J.-D.; McCarthy, S. P.; Smith, G. P.; Eberiel, D.; Gross, R. A.Polym. Mater. Sci. Eng.1992, 67, 230.

(22) Nishida, H.; Tokiwa, Y.Chem. Lett.1994, 8, 1547.(23) Janssen, P. H.; Schink, B.Biodegradation1993, 4, 179.(24) Janssen, P. H.; Hartfoot, C. G.Arch. Microbiol. 1990, 154, 253.(25) Abou-Zeid, D.-M.; Muller, R.-J.; Deckwer, W.-D.J. Biotechnol.2001,

86 (2), 113.(26) Abou-Zeid, D.-M.Anaerobic Biodegradation of Natural and Synthetic

Polyesters. Dissertation 2001, TU-Braunschweig, Germany; Inter-net: http://opus.tu-bs.de/opus/volltexte/2001/246.

(27) Buswell, A. M.; Muller, H. F. Ind. Eng. Chem.1952, 44, 550.(28) Holdemann, L. V.; Moore, M. E. C.Anaerobe laboratory manual,

4th edition; Virginia Polytechnic Institute and State University:Blacksburg, VA, 1978.

(29) Krieg, N. R. Systematics. InManual of methods of general bacteriol-ogy; Gerhardt, P., Murray, R. G. E., Costilow, R. N., Nester, R. N.,Wood, E. W., Krieg, N. R., Phillips, G. B., Eds.; American Societyof Microbiology: Washington, DC, 1981.

(30) Witt, U.; Muller, R.-J.; Augusta, J.; Widdecke, H.; Deckwer, W.-D.Makromol. Chem.1994, 195, 793.

(31) Marten, E.Korrelation zwischen der Struktur und der enzymatischenHydrolyze Von Polyestern. Dissertation 2000, TU-Braunschweig,Germany; Internet: http://opus.tu-bs.de/opus/volltexte/2000/136.

(32) Kleeberg, I.Untersuchungen zum mikrobiellen AbbauVon aliphatisch-aromatischen Copolyestern sowie Isolierung und Charakterisierungeines polyesterspaltenden Enzyms. Dissertation 1999, TU-Braunsch-weig, Germany; Internet: http://opus.tu-bs.de/opus/volltexte/2000/90.

(33) Marten, E.; Mu¨ller R.-J.; Deckwer W.-D.Polym. Degrad. Stab.2003,80 (3), 485.

(34) Bastioli, C. Starch-polymer composites. InDegradable Polymers,Principles and Applications; Scott, G., Gilead, D., Eds., Chapman& Hall: London, 1995; pp 112-133.

(35) Abou-Zeid, D.-M.; Biebl, H.; Sproër, C.; Muller. Int J. Syst. EVol.Microbiol. In press

(36) Gujer, W.; Zehnder, A. J. B.Wat. Sci. Technol.1983, 15 (8/9), 127.(37) Witt, U.; Muller, R.-J.; Deckwer, W.-D.J. EnViron. Polym. Degrad.

1996, 4 (1), 9.(38) Witt, U.; Muller, R.-J.; Deckwer, W.-D.J. EnViron. Polym. Degrad.

1997, 5 (2), 81.(39) Witt, U.; Einig, T.; Yamamoto, M.; Kleeberg, I.; Deckwer, W.-D.;

Muller, R.-J.Chemosphere2001, 44 (2), 289.(40) Andreesen, J. R.; Bahl, H.; Gottschalk, G. Introduction to the

physiology and biochemistry of the genus Clostridium. InBiotech-nology handbooks; Minton, N. P., Clarke, D. J., Eds.; Plenum Press:New York, 1989; Vol. 3Clostridia, pp 27-62


(41) Bader, J.; Gu¨nther, H.; Schleicher, E.; Simon, H.; Pohl, S.; Mannheim,W. Arch. Microbiol. 1980, 125, 159.

(42) Mitchell, W. J.AdV. Microbiol. Physiol.1998, 39, 31.(43) Diez-Gonzalez, F.; Russel, J. B.FEMS Microbiol. Lett.1969, 136,

123.(44) Tokiwa, Y.; Suzuki, T.; Takeda, K.Agric. Biol. Chem.1988, 52 (8),

1937.(45) Oda, Y.; Oida, N.; Urakami, T.; Tonomura, K.FEMS Microbiol.

Lett. 1997, 152, 339.(46) Murphy, C. A.; Cameron, J. A.; Huang, S. J.; Vinopal, R. T.Appl.

Microbiol. Biotechnol.1998, 50, 692.(47) Murphy, C. A.; Cameron, J. A.; Huang, S. J.; Vinopal, R. T.Appl.

EnVironm. Microbiol. 1996, 62 (2), 456.(48) Abou-Zeid, D.-M.; Biebl, H.; Sproër, C., Muller, R.-J.IJSEM2004,

in press.(49) Biebl, H.; Schwab-Hanisch, H.; Sproër, C. and Lu¨nsdorf, H.Arch.

Microbiol. 2000, 174, 239.

(50) Garcia-Martinez, D. V.; Shinmyo, A.; Madia, A.; Demain, A. L.Eur.J. Appl. Microbiol. Biochem.1980, 9, 189.

(51) Morag, E.; Bayer, E. A.; Lamed, R.J. Bacteriol.1990, 172, 6098.(52) Bronnenmeier, K.; Staudenbauer, W. L.Enzyme Microbiol. Technol.

1993, 12, 431-436.(53) Hazlewood, G. P.; Davidson, K.; Clarke, J. H.; Durrant, A. J.; Hall,

J.; Gilbert, H. J.Enzyme Microbiol. Technol.1990, 12, 656.(54) Jager, K.-E.; Steinbu¨chel, A.; Jendrossek, D.Appl. EnViron. Micro-

biol. 1995, 61 (8), 3113.(55) Kazlauskas, R. J.Trends Biotechnol.1994, 12, 464.(56) Svendsen, A. Sequence comparisons within the lipase family. In

Lipases: their structure, biochemistry and application; Woolley, P.,Petersen, S. B., Eds.; Cambridge University Press: New York, 1994;pp 3-6.

BM0499334


Documents

Biodegradation of Aliphatic Homopolyesters and Aliphatic−Aromatic Copolyesters by Anaerobic Microorganisms