10 biodegradation of natural and synthetic rubbers · rubbers of non-natural origin and differing...

10

Biodegradation of Naturaland Synthetic Rubbers

Alexandros Linos1, Alexander Steinbüchel2

1 Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster,Corrensstraûe 3, 48149 Münster, Germany; Tel. :�49-251-8339821;Fax:�49-251-8338388; E-mail : [email protected]

2 Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster,Corrensstraûe 3, 48149 Münster, Germany; Tel. :�49-251-8339821;Fax:�49-251-8338388; E-mail : [email protected]

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3232.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3232.2 Early Investigations on the Biodegradation of Natural Rubber . . . . . . . . . 3242.3 Biodegradation of Rubber Pipe Joint Rings . . . . . . . . . . . . . . . . . . . . 3262.4 Degradation by Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3282.5 Recent Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3292.6 Investigations in the Authors' Laboratory . . . . . . . . . . . . . . . . . . . . . 3312.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

3 Microorganisms Capable of Rubber Biodegradation . . . . . . . . . . . . . . . 3353.1 Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3353.1.1 Actinomycetes with Uncertain Classification . . . . . . . . . . . . . . . . . . . 3353.1.2 Actinomycetes with Reliable Classification . . . . . . . . . . . . . . . . . . . . 3383.2 Microorganisms Other than Actinomycetes . . . . . . . . . . . . . . . . . . . . 3403.2.1 Gram-Positive Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3413.2.2 Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3413.2.3 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

4 Optimization of Rubber Biodegradation . . . . . . . . . . . . . . . . . . . . . . 3424.1 Previous Experiences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3424.2 Recent Efforts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

5 Enzymatic Mechanisms and Genetic Basis . . . . . . . . . . . . . . . . . . . . . 347

321

5.1 Primary Degradation Reaction for cis-1,4-Polyisoprene . . . . . . . . . . . . . 3475.2 Analogous Degradation Known from Other Isoprenoids . . . . . . . . . . . . 3485.3 Catabolism of Rubber Degradation Products . . . . . . . . . . . . . . . . . . . 3505.4 Recent Investigations in the Authors' Laboratory . . . . . . . . . . . . . . . . . 350

6 Biodegradation of Synthetic Rubbers . . . . . . . . . . . . . . . . . . . . . . . . 352

7 Biodegradation of trans-1,4-Polyisoprene . . . . . . . . . . . . . . . . . . . . . . 353

8 Anaerobic Biodegradation of cis-1,4-Polyisoprene . . . . . . . . . . . . . . . . . 354

9 Perspectives and Biotechnological Applications . . . . . . . . . . . . . . . . . . 354

10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

BOD biological oxygen demandBR butadiene rubberCR chloroprene rubberGC-MS gas chromatography-mass spectrometryGPC gel-permeation chromatographyIIR isobutylelene-isoprene rubberIR isoprene rubberIR infraredLSD lignostilbene-a,b-dioxygenaseNBR nitrile-butadiene rubberNR natural rubberRPE65 protein in the retinal pigment epithelium of mammalsSBR styrene-butadiene rubberSDS sodium dodecylsulfateSDS±PAGE sodium dodecylsulfate±polyacrylamide gel electrophoresisSEM scanning electron microscopySOD superoxide dismutaseTEM transmission electron microscopyTLC thin-layer chromatography

1

Introduction

The biodegradability of various rubbers andrubber products plays an important role fromthe view of protecting rubber goods againstbiological damage and deterioration and ofproviding environmentally compatible solu-tions for the disposal and recycling of rubberwaste. During the past few decades, much

attention has been paid on the protectiveaspect, because of its economic relevance(Zyska, 1981, 1988). However, most reportsregarding this topic did not make clearwhether the rubber hydrocarbon itself ornon-rubber constituents were biologicallyattacked.

In this report, the current knowledge aboutthe biodegradation of rubber hydrocarbon issummarized. Thereby, special emphasis is

10 Biodegradation of Natural and Synthetic Rubbers322

given to the cis-1,4-polyisoprene chain, themain constituent of natural rubber (NR) andsynthetic isoprene rubber (IR). Due to thechain's existence in nature, it is not surprisingto find most reports claiming its susceptibil-ity, in contrast to reports about syntheticrubbers of non-natural origin and differingpolymer composition, such as butadienerubber (BR), styrene-butadiene rubber(SBR), nitrile-butadiene rubber (NBR), iso-butylelene-isoprene rubber (IIR), chloro-prene rubber (CR) and others, that mainlyreveal them as biologically recalcitrant mate-rials (Seal, 1988).

One main approach will be to describe themicroorganisms that are capable of degrad-ing cis-1,4-polyisoprene, either in the rawstate or in rubber products, and of using therubber hydrocarbon as sole source for growthand biomass production. Different microbialstrategies for an effective availability of thissolid and hydrophobic substrate will be high-lighted. In this context, factors affecting therate of biodegradation, as well as knownapproaches for an optimization of the degra-dation process with regard to a biotechno-logical application in rubber waste treatment,will be discussed. Analytical data concerningmicrobially caused surface modifications andthe production of degradation products willbe presented, especially from the view ofelucidating the primary cleavage mechanismof the polymer chain.

Little is known so far about rubber-degrad-ing enzymes and their corresponding genes.In this context, the first experimental ap-proaches in the authors' laboratory will beintroduced, and an attempt will be made torelate these data with results presented in theliterature for the metabolism of isoprenoids(e.g. carotenoids) and lignin model struc-tures (e.g. lignostilbenes). It is generallyintended to provide a stimulating basis forfurther investigations in this field.

2

Historical Outline

2.1

General Considerations

Summarizing examinations about the bio-degradation of rubbers implies the need topay attention only to those reports thatprovided sufficient evidence for the catabo-lism of the rubber hydrocarbon chain. Ac-cordingly, the following outline will includeonly aspects dealing with the microbial, i.e.bacterial and fungal, degradation of cis-1,4-polyisoprene, the main constituent of naturalrubber (NR) and synthetic isoprene rubber(IR).

In spite of numerous reports and reviewsconcerning the biodeterioration of rubbermaterials, which consisted of some otherdiene hydrocarbon polymers, such as styr-ene-butadiene copolymerisate (SBR, Buna-S), nitrile-butadiene copolymerisate (NBR,Buna-N, nitrile rubber), isobutylene-iso-prene copolymerisate (IIR, butyl rubber),polybutadiene (BR) and polychloroprene(CR, neoprene) (ZoBell and Beckwith,1944; Blake et al. , 1955; Snoke, 1957; Stein-berg, 1961; Schwartz, 1963; Heap and Mor-rell, 1968;Cundell andMulcock,1972;Zyska,1981, 1988; Williams, 1985; Seal, 1988; Cain,1992; Pommer, 1995; Tsuchii, 1995; Linosand Steinbüchel, 1998), there was no con-vincing indication for the direct involvementof microorganisms in rubber hydrocarbonmetabolism. Moreover, it could be oftenshown, that microbial growth was promotedat the expense of compounding ingredientstypically used in rubber product manufac-ture.

On the other hand, nonrubber substancesin commercial NR can amount to more than5% of dry weight including proteins, carbo-hydrates, lipids, and inorganic salts (Subra-maniam, 1995). Accordingly, the mere pres-

2 Historical Outline 323

ence and growth of microorganisms on NRdoes also not constitute proof for the utiliza-tion of the rubber hydrocarbon as source ofcarbon. However, the extent of microbialgrowth and deterioration described in somecommunications was so high, that this couldnot be ascribed only to the utilization ofnonrubber constituents. Although finalproof for rubber hydrocarbon metabolismwas missing, some of these reports weretaken into consideration, due to their signifi-cant contributionfor further investigations inthis field.

Cis-1,4-polyisoprene represents a peculiarpolymer in all kind of laticiferous plants. Astrong indication for the biodegradative po-tential of nature towards this molecule wasgiven by Taysum (1966), who showed that noaccumulation of natural rubber occurred inthe soil of a 35-year-old plantation of rubbertrees (Hevea brasiliensis) in Malaysia, in spiteof the calculated annual deposition of~1.7 g mÿ2 of cis-1,4-polyisoprene resultingfrom the annual leaf fall of~0.56 kg mÿ2 witha rubber content of 0.45% (w/w) in the driedleaf material. No differences could be deter-mined when rubber tree soil was compared toa 35-year-old Malayan grass soil, therebydetermining in both cases a concentrationof 0.12% (w/w) of benzene-extractable mat-ter. Taysum concluded that ªconsiderablequantities of rubber would have accumulatedif decomposition did not occurº.

Assuming that cis-1,4-polyisoprene mightalso serve as storage compound and energyreservoir in rubber-containing herbs, also anappropriate enzymatic equipment for itsmobilization would be necessary. A firstindication to this direction was obtained bySpence and McCallum (1935),who detected aseasonal fluctuation of rubber content inguayule plants (Parthenium argentatum).These authors determined a decrease in thelatex amount at the beginning of each grow-ing season, and concluded that a mechanism

for metabolism of the polyisoprene chainmust therefore exist. Unfortunately, to dateno progress has been made in the elucidationof this phenomenon.

2.2

Early Investigations on the Biodegradationof Natural Rubber

As early as 1914, Söhngen and Fol (1914a, b)reported about the decomposition of rubberby microorganisms. In order to prove assim-ilation of the hydrocarbon chain, highlypurified NR was prepared and used as sourceof carbon. For purification, the authors dis-solved NR crepe sheet in benzene withoutshaking, took only the clear upper phase ofthis solution, evaporated the solvent in glassdishes and subsequently treated the NR filmsobtained with a 2% trypsin solution for 8 h at40 8C, for the effective removal of NRproteins. After washing for 16 h underflowing water, the films were transferred toglass bowls containing water and inorganicsalts, sterilized at 1058C, inoculated withmicroorganisms, and cultivated (withoutmovement) for several weeks at differenttemperatures. No growth occurred withdifferent bacteria and fungi, which were firstenriched from soil and grew well on non-purified NR, except in the case of two actino-mycetes, which formed colonies directly onthe purified NR material and were identifiedas Actinomyces elastica and Actinomyces fuscus.Additionally, the authors reported on theexistence of holes in the rubber material,which were formed beneath the adheringactinomycete colonies ± both in liquid min-eral culture and on gelatin plates coated bypurified NR films ± and concluded that underthese conditions increase in biomass couldonly have taken place at the expense of therubber hydrocarbon.

Encouraged by the results of Söhngen andFol, De Vries (1928) examined the possible


decomposition of rubber hydrocarbon byfungi. In experiments with nonpurified NRsmoked sheet as sole carbon source (93%rubber content), being inoculated with differ-ent Penicillium and Aspergillus species andcultivated stationary in 10% NaCl solution,the author detected an increase in moldbiomass of up to 6% of the initial rubberweight and a final weight loss of NR risingfrom 15.5% after 19 months to 30.9% after 5years. By comparison, a noninoculated con-trol showed negligible weight loss. Examina-tion of these data and those obtained afterchemical analysis of the remaining sub-strates resulting in a slight increase in nitro-gen concentration in the 5-year-old sampleled the author to conclude that the rubberhydrocarbon was being consumed preferen-tially by the fungi, rather than the proteins.

Spence and van Niel (1936) were the first touse latex overlay plates for the isolation ofrubber-degrading bacteria. Thereby, mineralagar is overlayed by a thin layer of NR latexthat is dispersed in mineral agar. Rubber-degrading colonies developed on such platescan be recognized by the production ofclearing zones (translucent halos) throughthe opaque agar layer. The authors reportedthe (appropriate) isolation of four not closelycharacterized actinomycetes, which they sub-sequently used as pure cultures for testinggrowth on NR. For this purpose, liquidcultures were prepared containing a mineralsalt medium with potassium nitrate insteadof ammonium chloride as the nitrogensource in order to prevent latex coagulationafter autoclaving. NR latex was used assubstrate; this was first dialyzed repeatedlyagainst refreshed phosphate buffer (pH 6.8 ±7.2) inorder to removeammonia (presentasapreservative) and water-soluble organic im-purities. Following incubation for 28 days at30 8C, the weight of solids in the inoculatedsamples was determined and compared withthat in sterile controls. Results indicated that

up to 59% of the material was present as adeteriorated latexclot, and up to35% as water-soluble material. No distinction was madebetween the rubber and microorganisms.However, the authors also attempted toquantify the adhering biomass by dissolvingthe remaining brownish latex clot in benzenecontaining 1% trichloroacetic acid, in whichbacteria and other nonrubber substanceswere insoluble. Analytical data showed thatthe fraction of adhering cell material rangedfrom 12.5% to 28.7% of the weight ofanalogously treated sterile controls, suggest-ing that growth was in fact promoted by therubber hydrocarbon and not only by thenonrubber constituents.

Kalinenko (1938), using the latex overlaytechnique, reported the isolation of severalactinomycetes and fungi, for example Asper-gillus oryzae and Penicillium species, claimingthat they all were able to consume largequantities of NR in diluted latex obtainedfrom Taraxacum kok-saghyz. This is a rubberdandelion plant that was specially cultivatedintheUSSRduringWorldWarII tosatisfy thedemand for NR. One of the actinomycetesalso produced holes in a thin film of pre-viously dialyzed kok-saghyz latex. However,Shaposnikov et al. (1952a,b), who also testedgrowth on natural rubber films placed onmineral salts agar plates after evaporation of abenzene solution, confirmed growth of acti-nomycetes by weight-loss experiments (Sha-posnikov et al. 1952a), but noticed the ab-sence of any fungal growth on NR (Shapos-nikov et al. 1952b). In fact, the same wasobserved later by Nette et al. (1959), whoisolated several fungi and bacteria frompiecesofcontaminatedrubber.Theseauthorsreported weight losses of purified rubberfilms on mineral agar plates by three actino-mycetes (one Proactinomyces and two Actino-myces strains) ranging between 25.8% and43.2%, by a Bacillus sp. of 20.7%, by aMycobacterium sp. of 17.2%, and by several


fungi of maximum 2.5% of the initial rubberfilm weight (with an experimental error of�2%). Unfortunately, no further data weregiven beyond determination of weight loss.

ZoBell and Grant (1942) and ZoBell andBeckwith (1944) isolated several strains de-signated as Actinomyces, Proactinomyces, Mi-cromonospora, Mycobacterium, Bacillus, andPseudomonas. The authors tried to estimatethe extent of rubber oxidation by determiningthe microbial oxygen consumption in liquidculture. For this purpose, highly purified NR(99%, obtained from the Goodyear Compa-ny) was dissolved in benzene, and the upperpart evaporated in glass-stoppered bottlesthereby forming a thin rubber film at thebottom. The bottles were filled with watersaturated with oxygen, inoculated, closed,and cultivated for 10 days at ~25 8C. Afterincubation, the amount of oxygen remainingin the liquid was determined and comparedwith that in a noninoculated control. Theauthors pointed out that, by consideringrubber to be (C5H8)x, a consumption of3.3 mg of oxygen would be required tocompletely oxidize 1 mg of rubber. In otherwords, this relation corresponds to a com-plete mineralization of rubber into carbondioxide and water according to the followingequation:

(C5H8) �7O2!5CO2�4H2O

The data obtained revealed an oxidation of60 ± 75% when 1 mg was used as substrate(ZoBell and Grant, 1942), and 78% when2 mg was used (ZoBell and Beckwith, 1944).Considering these small amounts and theadditional observationof increasingbiomass,rubber hydrocarbon metabolism may beobvious. However, the method to determinetheremainingoxygenwasbasedonliberationof iodine in the presence of oxygen (modifiedWinkler technique), and the authors re-marked that iodine also reacts with rubber.In spite of the comparison with sterile

controls, it cannot be excluded that, in theinoculated samples an additional reaction ofiodine with released degradation productsmight have occurred, resulting generally inhigher values of oxygen consumption. An-other weak point of this study was that theorganism utilized was not clearly identified.

Considering biodegradation of vulcanizedNR, in which the long hydrocarbon chainshave been linked together by sulfur bridgesand several compounding ingredients havebeen added during manufacture, Blake andKitchin (1949) reported the severe pitting anddisappearance of NR gum used for rubberinsulation of underground cables as a resultof microorganism action. The authors de-tected a complete failure of insulation resist-ance after 3 months in soil burial tests, anddocumented the extent of mass decomposi-tion, referring to actinomycetes and ªredcolonies of Gram-positive micrococciº asbeing the causative agents. It was claimedthat the rubber hydrocarbon must also havebeen consumed as substrate, though quanti-tative data were not at all presented.

2.3

Biodegradation of Rubber Pipe Joint Rings

Rook (1955) was the first to perform system-atic work on the biodegradation of vulcanizedNR by using pure cultures. EmployingpurifiedNRlatexandthe techniqueofSpenceand van Niel (1936), he isolated severalclearing zone-forming actinomycetes fromcorroded rubber rings that were used forconnecting asbestos cement pipes in waterdistribution pipelines in the Netherlands.Two of the strains, which were designated asStreptomyces species, were tested for theutilization of vulcanized rubber in liquidcultures containing pipe-joint ring materialas sole carbon source immersed in a mineralsalts medium. After 8 months of stationary


cultivation at 25 ± 30 8C, small holes becamevisible on the rubber strip cultivated with oneof the strains. These holes reached a diameterof 1.5 mm after 12 months, and actinomycetefilaments could be detected microscopicallyin the cavities. From this observation, theauthor concluded that ªthe extent of thebreakdown was such that the rubber hydro-carbon must also have been involvedº. Fromtoday's point of view, this statement might becorrect, because clearing zone-forming bac-teria are now known generally to use cis-1,4-polyisoprene for growth (Jendrossek et al.,1997; Linos et al. , 2000a), although at thattime the mere observation of hole formationdid not constitute sufficient proof for bio-logical polymer breakdown.

Leeflang (1963) extended Rook's studiesand isolated two similar strains by smearingtiny fragments of seemingly unattackedrubber pipe-joint ring material directly ontoglucose-peptone agar plates. The surface ofsuch materials was previously liberated fromthe slimy bacterial layer formed duringbiodeterioration and the underlying deterio-rated rubber. Tests with obtained pure cul-tures revealed wrinkling of NR rubber vul-canizates, formation of holes and loss inelasticity after 2 years exposure at 25 8C inmineral salts. The same two types of Strepto-myces sp. were isolated together from morethan 50 samples of deteriorated gasket ma-terial taken from installations in the Nether-lands and Belgium. The author also devel-oped a basin test for proving microbialsusceptibility of different rubbers. In thisso-called Leeflang test bath ± which was laterused by several investigators ± rubber stripswere hung in a basin through which a slowand constant flow of potable unchlorinatedwater was maintained at 20 ± 25 8C in thedark. The basin was inoculated by placing apiece of deteriorated rubber in the bottom.Using this procedure, the microbial suscept-ibility of synthetic isoprene rubber (IR), as

well as NR, was demonstrated for the firsttime.

Cundell and Mulcock (1973, 1975, 1976)performed further investigations on thebiodegradation of NR vulcanizates. Theyconfirmed microbial susceptibility of sulfur-cured NR pipe-joint rings after testing respi-ratory activities, such as carbon dioxideevolution and uptake of oxygen, as well asloss of tensile strength (Cundell and Mul-cock, 1973), or by applying scanning electronmicroscopy (SEM) and infrared (IR) spec-troscopy (Cundell and Mulcock, 1975). Therelative ability of microorganisms to releasecarbon dioxide from the surface of rubberstrips when cultivated in a Leeflang test bathwas estimated for a period of 18 months aftersuccessive transfer of rubber strips every 2months from the bath in sealed glass jars, anddetermination of the evolved CO2 during thenext 1-week period, thereby trapping the gasin an aqueous barium peroxide solution andmeasuring the amount of CO2 liberated (byacidification of the BaCO3 formed). Resultsshowed increasing values for the NR sample,but decreasing values for other vulcanizatescontaining synthetic rubbers, for exampleSBR, BR, CR, IIR, and NBR. Oxygen uptakewas determined once after 12 months usingWarburg respirometry, and this resulted in anincreasing respiratory activity for the NRsample during a 12-h measurement. Addi-tionally, a substantial loss in tensile strengthof NR vulcanizate (up to two-thirds of theinitial value) was observed after 2 years'exposure in the Leeflang bath. FollowingSEM analysis of deteriorated rubber pipe-joint samples (Cundell and Mulcock, 1975,1976), the occurrence of microbial cells wasdemonstrated on the rubber surface, withboth mycelia and hyphae that were typical ofactinomycetesbeingvisualized.However, theclaim that these organisms belonged to thegenera Streptomyces and Nocardia, respective-ly,wasvery vaguedue to the fact thatnon-pure


cultures were seen, and thus insufficienttaxonomic data were presented. This latterfinding represents a weak point in mostreports dealing with the microbial degrada-tion of rubbers. For IR spectroscopic analysis,deteriorated material of a 1-year-old samplewas scraped from the rubber surface, groundup with a pestle and mortar, mixed withpowdered potassium iodide, pressed intopellets and scanned in the spectrophotome-ter. The absorbtion spectrum obtained wascompared with that corresponding to repeat-ing isoprene units in NR, and revealedchanges intheregionofCH3-,CH2-, carbonyl-and hydroxyl-groups, as well as a decrease ofthe C�C double bond intensity. Unfortu-nately, there was no information aboutremoval of cell material from the deterioratedfraction used for spectroscopy, so that inter-pretation of the spectra required great cau-tion. In any case, the authors noted that theªprocess is probably initiated by a mono-oxygenase cleaving the rubber hydrocarbon,followed by a stepwise degradation of thepolyisoprenoid chainº (Cundell and Mulcock,1975). In a further report (Cundell andMulcock, 1976), these authors also describedthe isolation of two types of a single `Strepto-myces' strain by employing the latex overlaytechnique. In this study, pure cultures werecultivated with peroxide-cured NR strips for18 months at 24 8C on a rotary shaker; this ledtoweight lossesofbetween7.3%and13.4%ofthe initial rubber weight determined afterremoval of the bacterial layer from the strips,though no further data were provided.

Forcontinuingstudiesonrubberpipe-jointrings, Hanstveit et al. (1988) used a modifiedLeeflang test bath method to study thebiodeterioration of susceptible and resistantNR-vulcanizates. After 2 years' exposure, theauthors detected actinomycete growth onlyon the susceptible sample, which has lost 3%of its volume due to severe pitting andpenetration of one kind of microorganism

(as confirmed by transmission electron mi-croscopy, TEM), as well as its entire tensilestrength. Isolation of pure cultures usingSpence and van Niel's latex overlay techniquerevealed several strains of the same species,which was claimed to belong to the Nocardiaasteroides complex according to results fromprevious extensive taxonomic analysis ofanalogous actinomycetes isolated from de-teriorated pipe-joint rings (Hutchinson andRidgway 1975, 1977; Hutchinson, 1977;Hookey et al. , 1980; Orchard and Goodfel-low, 1980; Cross, 1981). Liquid culturingperformed with the pure cultures in mineralsalts medium in fact revealed the samedeterioration and microbial pattern as afterinoculation with mixed cultures (confirmedby SEM), and the authors concluded thatN. asteroides was the main cause for thebiodeterioration of such NR vulcanizates.This hypothesis was additionally favored bythe fact that N. asteroides strains could not befound on resistant NR and synthetic vulcan-izate samples, while all of the other evidentlynondeteriorating bacteria of the surface mi-croflora were present on either of thesematerials, in spite of the presence of antiox-idants and other protecting agents.

2.4

Degradation by Fungi

Kwiatkowska et al. (1980) performed soilburial tests of NR vulcanizate sheets ofdefinite composition and detected substan-tial weight losses reaching up to 40% of theinitial weight after 91 days. Further character-ization also revealed changes in the networkchain density of the materials, determined asafunctionof thedurationofsoilburialandthecarbon black loading rate in the vulcanizate,as well as the main occurrence of the fungusFusarium solani on the rubber surface. Theseauthors claimed that this microorganism wasthe cause of NR degradation, and referred


also to appropriate degradation tests, whichwere performed with the pure culture,though specific data to prove this assumptionwere not presented.

More detailed investigations on the bio-degradation of cis-1,4-polyisoprene by fungiwere documented twice during 1982. In thefirst report (Williams, 1982), the authorintroduced experiments with Penicillium va-riabile, a fungal strain isolated from deterio-rated NR and IR samples after soil burialtests. Spore suspensions inoculated onto NRsmoked sheet in a humidity cabinet led to asuccessive increase of biomass on the mate-rial's surface, as shown by cell proteindetermination every 14 days, and was accom-panied by a weight loss of rubber strips of upto 13% after 56 days. However, furtherincrease in biomass and in weight lossbeyond this time period could not be deter-mined. Using solution viscosity measure-ment as analytical tool, the author estimated a15% reduction in the molecular weight ofpolyisoprene after 70 days. However, IRspectroscopy performed on the deterioratedrubber did not reveal any changes in chemicalstructure, and examination of acetone ex-tracts by thin-layer chromatography (TLC),gel-permeation chromatography (GPC) andgas chromatography-mass spectrometry(GC-MS) did not prove the existence of anydegradation products. The author thereforesuggested that ªmicroorganisms attack suchpolymers from the chain ends, forming shortchain intermediates, and growing at theexpense of these unitsº.

In the second report (Borel et al. , 1982),several rubber-deteriorating fungi could beisolated from mineral agar plates containingpowdered NR as sole substrate and deterio-rated tire material or soil dispersed in the agaras inoculum. Liquid cultivation performedwith isolated pure cultures for 20 daysrevealed the formation of a mycelial layeron the rubber surface, as well as losses in

weight of up to 20% and in intrinsic viscosityof up to 35%. Using GPC, a relative reductionof the molecular weights of the rubberpolymers was also detected in the samplesinoculated with Fusarium solani, Cladospori-um cladosporioides and Paecilomyces lilacinus.Interestingly, the attack on rubber stoppedafter 30 days, resulting in no further changesconcerning biomass, weight loss and molec-ularweightdistribution,butcouldberestoredagain after successive removal of the fungalprotective layer and transfer of the rubbersubstrate to fresh mineral medium every 20days. The appropriate experiment withC. cladosporioides thereby resulted in de-creased values for average molecular weightafter each treatment.

2.5

Recent Developments

Further strong evidence for the microbialdegradation of rubber polymer was achievedby Tsuchii et al. (1985). These authors re-ported isolation of the actinomycete Nocardiasp. strain 835A from soil, which causedconsiderable weight losses (up to 100% after8 weeks) when being cultivated in liquidculture with different cis-1,4-polyisoprene-containing materials (0.06 ± 0.07%, w/v), forexample NR pale crepe sheet, syntheticisoprene rubber (IR) and different NR vul-canizates, including latex gloves, rubberbands, rubber tubing, rubber stoppers andtire treads. Strips from latex gloves weredegraded most rapidly, reaching a 90%weight loss after 17 days and showing acontinuous increase inbiomassontherubbersurface according to cell protein determina-tion. In order to obtain putative degradationproducts, chloroform extracts were madefrom the glove/cell mixture and analyzed bytwo-dimensional TLC and GPC, respectively.These analyses revealed the existence of twodistinct degradation fractions representing


oligomerswithmolecular weights from103 to104 Da. Staining with Schiff's reagent andanalysis by IR spectroscopy indicated thepresence of carbonyl groups in the oligomers,while analysis by 1H- and 13C-NMR finallyestablished the occurrence of two degrada-tion products consisting of 19 and 114isoprene units, respectively, each carryingan aldehyde on one end of the molecule and aketone on the other end. On the basis of thesedata, the authors proposed an enzymaticcleavage mechanism proceeding by oxidativescission of the cis-1,4-double bond as theprimary step in biodegradation of the cis-1,4-polysoprene chain.

Further investigations performed in thesame laboratory (Tsuchii and Takeda, 1990)revealed an analogous degradation mecha-nism when latex of NR or IR was treated withthe extracellular crude enzyme of an isolatedGram-negative bacterium, designated asXanthomonas sp. strain 35Y. For the prepara-tion of crude enzyme, latex was purified bythe method of Spence and van Niel (1936),suspended in a mineral salts medium at0.05% (w/v) containing also a small amountof a surface-active agent, and cultivated for 5days at 30 8C after inoculation with the strain.Subsequently, the culture was centrifugedand the clear middle fraction of the centrifugetube was collected and filter-sterilized. Theenzymatic reaction was performed at 30 8Cfor different time intervals by incubatingsterile NR latex in phosphate buffer togetherwith the crude extract. After acidification andether extraction, the fraction obtained wasused for carbonyl content determination andanalysis by TLC, GPC and gas-liquid chro-matography (GLC). Enzyme activity ± whichcould be destroyed by heating and concen-trated by ultrafiltration ± was expressed as thequantity of carbonyl compounds produced inrelation to the amount of crude enzyme used.Chromatographic analysis of the enzymereaction again revealed the existence of two

fractions consisting of low- and high-molec-ular weight oligomers. Further analysis of themolecular weights by GC-MS, and of thestructure by 1H- and 13C-NMR, confirmed theexistence of two main degradation productsof two and 113 isoprene units, respectively,and with characteristic carbonyl end-groupsreferred to above. On the basis of successfulincorporation of 18O into the degradationdimer under an 18O2 atmosphere, the authorsconcluded that the cleavage reaction waspartly oxygenase-catalyzed.

However, according to the author's ownstatements, the ability of the Xanthomonasstrain to degrade rubber was rather poor(Tsuchii, 1999), and in subsequent reportsTsuchii and coworkers concentrated mainlyon the Nocardia sp. strain 835A. This was dueto the organism's remarkable rubber-degrad-ing potential and thus its putative use in thebiological disposal of rubber waste (Tsuchiiet al. , 1990, 1996, 1997; Kajikawa et al. , 1991;Tsuchii and Tokiwa, 1999). Appropriate re-sults are summarized in Section 4, in whichthe optimization of microbial rubber degra-dation is discussed.

Heisey and Papadatos (1995) describedseveral actinomycetes that were able tometabolize highly purified NR as sole carbonsource. Thin films of pale crepe rubber werefirst extensively extracted with different or-ganicsolventsandused toproduceNR-coatedglass slides which were subsequently used inthe isolation experiments. The pure culturesobtained were further cultivated with vulcan-ized NR, which corresponded to strips ofanalogously purified latex glove materialcontaining finally only 0.1% of proteinaceousimpurities. Besides colonization of the rub-ber surface, penetration of the material andgeneral alterationof itsphysical structure wasalso observed by SEM. Increasing weightlosses that reached >10% after 6 weekscorrelated well with an increase in theamount of cell protein, both in the culture


broth and on the rubber strips. Taxonomicdata based on fatty acid profiles and cell wallcharacteristics revealed seven Streptomyces,two Amycolatopsis and one Nocardia strain,and the authors concluded that actinomy-cetes were the main organisms involved inthe biodegradation of natural rubber hydro-carbon.

A strong indication for this was also givenby Jendrossek et al. (1997), who tested a largenumber of Gram-positive and Gram-negativebacteria, which were either isolated fromdifferent natural habitats by using centri-fugedfresh NRlatexascarbonsource, orwereobtained from different culture collections.Using Spence and van Niel's latex overlaytechnique, it was not possible to identify clearzone-forming bacteria other than actinomy-cetes. Screening of bacteria from culturecollections and fatty acid profile analysis ofisolated strains revealed members of thegenera Actinomadura, Actinoplanes, Dactylo-sporangium, Micromonospora, Nocardia andStreptomyces, with Micromonospora and Strep-tomyces representing the most prominentgenera. With most of the strains, clear zoneformation was repressed, when readily uti-lized substrates such as glucose and/orsuccinate were added to the latex-agar me-dium as co-substrates. The authors also noteda reduction in the average molecular mass ofresidualNRpolymerfromsolutioncast films,from 6.4�105 to 2.5�104 Da, implying theparticipation of an endo-cleavage mechanismin the biodegradation process; analytical datawere not presented, however.

2.6

Investigations in the Authors' Laboratory

Linos and Steinbüchel (1998) reported on theisolation of several rubber-degrading bacteriafrom fouling water inside of deteriorated cartires, from soil of a rubber treeplantation, andfrom sewage sludge after adaptation of each

sample for 6 months on tire crumb materialas sole carbon and energy source; additionalinvestigations included the subsequent en-richment of rubber-degrading bacteria on NRor synthetic IR. Taxonomic analysis of theobtained pure cultures revealed strains be-longing to the nocardioform actinomycetegenus Gordonia (formerly known as Gordo-na). One of the strains was recently classifiedas the novel species Gordonia polyisoprenivo-rans Kd2T (Linos et al., 1999).Biodegradationof the cis-1,4-polyisoprene in NR (raw andvulcanized) as well as in IR was confirmedafter determining the extent of mineraliza-tion, which was expressed as percentage ofcarbon released from the rubber substrate asCO2 during the time course of the cultivationexperiments, thereby reaching values of>50% for NR and >20% for IR after 52 days.Forevaluation, itwasassumedthat therubbersubstrates consisted totally of carbon. Con-sidering this, and the fact that the biomassalso increased during cultivation, real bio-degradation values were actually much high-er. At the same time, solubilization, disinte-gration and continuous visual disappearanceof the solid rubber substrate could be ob-served (Figure 1)

Attempts to isolate solubilized rubber fromthe turbid supernatant of liquid Gordoniacultures by organic solvent extraction yieldedsolid material after 5 days, which was onlysoluble in chloroform (suggesting a higher-molecular weight fraction), the amount ofwhich diminished during the time course ofthe cultivation experiment (Figure 2A). An-other indication for the solubilizing proper-ties of the strains was shown by preventinglatex coagulation, when NR latex was treatedwith the filter-sterilized extract of a 5-day-oldGordonia rubber culture. This effect wassimilar to that seen with detergents such assodium dodecyl sulfate (SDS) or Triton X-100, which were also tested for the preventionof latex coagulation (Figure 2B).


In a series of screening experiments forisolating various rubber degrading bacteria,Linos et al. (2000a) specified that there weretwo different microbial strategies to makerubber available as substrate. In the first,bacteria grow only in direct contact with therubber surface, and this leads to considerabledisintegration of the material during culti-vation. Strains like the above-mentionedGordonia isolates, as well as a newly intro-ducedMycobateriumfortuitum,wereassignedto this category. These bacteria were barelyable to grow on Spence and van Niel's latexoverlay plates, as a consequence of theabsence of direct contact with the rubber,which is distributed in the agar medium. Bycontrast, good growth occurred when thelatex was spreadas a thin film onmineral agarplates (latex film plates), thereby allowingdirect exposure of the bacteria to the rubber.Detailedanalysisof thegrowthbehavioronIRandNRlatexglovesbymeansofSEMrevealedthat, during colonization, the strains weretightly adhered and embedded into therubber matrix, thereby formingcharacteristicpenetration craters on the material's surface(Figure 3A). Another characteristic featurewas the formation of a biofilm (bacterialsurface layer) over the entire rubber surface

before the start of the disintegration process.In contrast, actinomycetes ± which were ableto grow well on latex overlay plates by formingcharacteristic clear zones ± were unable togrow appreciably on latex film plates. Theseclear zone-formers, the isolation of which hasbeen described frequently in the literature,hadadifferentstrategy.Asanexampleofsucha strain, the authors isolated and character-ized a Micromonospora aurantiaca and dem-onstrated growth of this isolate, especially onsynthetic IR. SEM analysis revealed theformation of mycelial corridors on the mate-rial's surface and its penetration by hyphae(Figure 3B). Neither embedding of cells intothe rubber matrix nor the formation of aclassical biofilm were observed. The authorsconcluded that, as cells were unlikely totransport solid rubber to their interior beforecleaving it intosmaller molecules, the rubber-degrading activity of adhesive growers wouldmost likely be bound to the cell surface, whilethe clear zone-forming bacteria excrete thisactivity into the medium.

Chemical changes thatarosedirectlyon therubber surface as result of the biodegradationprocessweredeterminedusingFourier trans-form infrared (FTIR) spectroscopy. Thiscomprised the Attenuated Total Reflectance


Fig. 1 Degradation of NR (SMR 10) by Gordonia polyisoprenivorans VH2. Left: Noninoculated control. Right:Complete disintegration of the inoculated sample after 6 weeks.

(ATR) technique (Linos et al. , 2000a), amethod which allows nondestructive, in-situanalysis of surfaces coated by microbialbiofilms (Schmitt and Flemming, 1998).Latex glove material overgrown by the Gordo-nia cells was analyzed, and the spectraconfirmed the presence of a bacterial surfacelayer by detecting characteristic markerbands corresponding to bacterial proteins,fatty acids and polysaccharides. After provingmechanical removal of the biofilm from the

sample surface, the spectra detected revealedsignals corresponding to those known fromthe literature for cis-1,4-polyisoprene in He-vea-NR. These spectra exhibited the followingchanges when compared with non-inoculat-ed controls: (1) a decrease in the number ofcis-1,4 double bonds; (2) the appearance ofketone and aldehyde groups; and (3) theformation of two different bonding environ-ments. These observations were finally in-


Fig. 2 A: Solubilized material recovered from a 5-day-old NR latex culture of G. polyisoprenivorans VH2after ultracentrifugation and chloroform extraction ofthe solid-free supernatant. B: Demonstration of thesolubilizing properties of the supernatant of a 5-day-old NR latexculture of G.polyisoprenivorans VH2.Left:Latex coagulate. Middle: Prevention of latex coagu-lation after addition of 3 mL of filter-sterilized super-natant. Right: Total inhibition of latex coagulationafter addition of sodium dodecylsulfate (SDS) asdetergent.

Fig. 3 Secondaryelectronmicrographsdemonstrat-ing two microbialstrategiesat the surfaceof syntheticcis-1,4-polyisoprene (IR). A: Growth behavior of G.polyisoprenivorans VH2 after 4 days, showing adher-ance and embedding of the cells into the rubbermatrix, thereby forming characteristic penetrationcraters. B: Growth behavior of Micromonosporaaurantiaca W2b after 7 days, showing formation ofmycelial corridors on the material's surface and itspenetrationbyhyphae.Scalebars,5 mm.Micrographsfrom Linos et al. (2000a.).

terpreted as the consequence of an oxidativereduction of the polymer chain, in similarmanner to that shown for the above-men-tioned Nocardia sp. strain 835A (Tsuchiiet al. , 1985).

In further screening experiments, Linoset al. (2000b) reported about the isolation of aGram-negative bacterium from foul tirewater, which was taxonomically character-ized as Pseudomonas aeruginosa AL98. Theculture was first enriched on NR latexconcentrate, and exhibited the ability toproduce greenish areas when cultivated onlatex film plates. Successive transfer of cellmaterial from such areas on LB platesrevealed single colonies of a greenish bacte-rium, that was used for testing growth ondifferent substrates such as latex concentrate,latex glove and synthetic IR. The determina-tion of growth parameters such as CO2-release and living cell number resulted inenhanced mineralization values for all sub-strates (up to 36% after 6 weeks), as well asincreased values for suspended biomass (upto 35-fold). SEM analysis also revealed aproperty of the strain as the effective colo-nization of the rubber surface, and formationof a biofilm during cultivation. However,visible disintegration of the rubber startedonly after 2 ± 3 weeks, when cells from LBculture were used as inoculum, in contrast tocells from rubber cultures, where disintegra-tion had already started after 2 days. Thereason for this behavior remained unknownuntil recent experiments in the authors'laboratory (Linos et al., 2001) showed thatthe AL98 culture was a mixed culture, due tothe appearance of an additional strain, whenacetonylacetone was used as carbon source,and which the Pseudomonas strain could notutilize. This new isolate turned out to expressgood rubber-decomposing activities and torepresent another strain of Gordonia polyiso-prenivorans, according to 16S rDNA analysis.Theauthorsstressedthat itwasnotpossible to

distinguish between these two bacteria, whencomplex or readily utilizable carbon sourceswere used for spreading the AL98 culture onagar plates, because of the inability of theGordonia strain to grow in the presence of thePseudomonas strain, which was shown toproduce an inhibitory substance towardsGordonia. Due to the fact that biomass ofPseudomonas also increased during cultiva-tion with rubber, it was assumed that growthof the Pseudomonas strain occurred at theexpenseof thedegradationproductsof cis-1,4-polyisoprenebeingproducedprimarilyby theaction of the Gordonia strain. This possibilitywas favored by the observation that Pseudo-monas also totally lost its ability to grow onrubbers after numerous transfer passages onLB plates, in contrast to the Gordonia strain,whose ability remained unaffected.

Further investigations in the authors' labo-ratory described the effect of pretreatment ofrubber material on its biodegradability bythese bacteria (Berekaa et al. , 2000); theresults of these studies are summarized inSection 4.

2.7

Conclusions

Accumulation of knowledge on the biodegra-dation of cis-1,4-polyisoprene-containingrubbers during the past century was mainlyaccompanied by progress of the analyticalequipment needed for these investigation.The simple visual and microscopic observa-tionsusedduringtheearlystageswere inturnreplaced by electron microscopy and spec-troscopy approaches, enabling detailed illus-tration to be made of microbial rubbercolonization and the chemical structure ofrubber degradation products to be elucidated.Moreover, developments in molecular biol-ogy opened the way for the performance ofreliable taxonomic classification of the iso-lated rubber-degrading microorganisms, and


10 biodegradation of natural and synthetic rubbers · rubbers of non-natural origin and differing...

Documents