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        10
        Biodegradation of Natural
        and 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: linosa@uni-muenster.de
        2
          Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster,
          Corrensstraûe 3, 48149 Münster, Germany; Tel.: ‡ 49-251-8339821;
          Fax: ‡ 49-251-8338388; E-mail: steinbu@uni-muenster.de

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

2       Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . .                  .   .   .   .   .   .   .   .   .   323
2.1     General Considerations . . . . . . . . . . . . . . . . . . . . . .                      .   .   .   .   .   .   .   .   .   323
2.2     Early Investigations on the Biodegradation of Natural Rubber                            .   .   .   .   .   .   .   .   .   324
2.3     Biodegradation of Rubber Pipe Joint Rings . . . . . . . . . . .                         .   .   .   .   .   .   .   .   .   326
2.4     Degradation by Fungi . . . . . . . . . . . . . . . . . . . . . . . .                    .   .   .   .   .   .   .   .   .   328
2.5     Recent Developments . . . . . . . . . . . . . . . . . . . . . . . .                     .   .   .   .   .   .   .   .   .   329
2.6     Investigations in the Authors' Laboratory . . . . . . . . . . . .                       .   .   .   .   .   .   .   .   .   331
2.7     Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                   .   .   .   .   .   .   .   .   .   334

3       Microorganisms Capable of Rubber Biodegradation                 .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   335
3.1     Actinomycetes . . . . . . . . . . . . . . . . . . . . .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   335
3.1.1   Actinomycetes with Uncertain Classification . . .           .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   335
3.1.2   Actinomycetes with Reliable Classification . . . .          .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   338
3.2     Microorganisms Other than Actinomycetes . . . .             .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   340
3.2.1   Gram-Positive Bacteria . . . . . . . . . . . . . . . .      .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   341
3.2.2   Gram-Negative Bacteria . . . . . . . . . . . . . . .        .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   341
3.2.3   Fungi . . . . . . . . . . . . . . . . . . . . . . . . . .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   341

4       Optimization of Rubber Biodegradation . . . . . . . . . . . . . . . . . . . . . .                                           342
4.1     Previous Experiences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                      342
4.2     Recent Efforts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                    344

5       Enzymatic Mechanisms and Genetic Basis . . . . . . . . . . . . . . . . . . . . .                                            347
322   10 Biodegradation of Natural and Synthetic Rubbers

      5.1     Primary Degradation Reaction for cis-1,4-Polyisoprene .           .   .   .   .   .   .   .   .   .   .   .   .   347
      5.2     Analogous Degradation Known from Other Isoprenoids                .   .   .   .   .   .   .   .   .   .   .   .   348
      5.3     Catabolism of Rubber Degradation Products . . . . . . .           .   .   .   .   .   .   .   .   .   .   .   .   350
      5.4     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 demand
      BR             butadiene rubber
      CR             chloroprene rubber
      GC-MS          gas chromatography-mass spectrometry
      GPC            gel-permeation chromatography
      IIR            isobutylelene-isoprene rubber
      IR             isoprene rubber
      IR             infrared
      LSD            lignostilbene-a,b-dioxygenase
      NBR            nitrile-butadiene rubber
      NR             natural rubber
      RPE65          protein in the retinal pigment epithelium of mammals
      SBR            styrene-butadiene rubber
      SDS            sodium dodecylsulfate
      SDS±PAGE       sodium dodecylsulfate±polyacrylamide gel electrophoresis
      SEM            scanning electron microscopy
      SOD            superoxide dismutase
      TEM            transmission electron microscopy
      TLC            thin-layer chromatography

      1                                                    attention has been paid on the protective
      Introduction                                         aspect, because of its economic relevance
                                                           (Zyska, 1981, 1988). However, most reports
      The biodegradability of various rubbers and          regarding this topic did not make clear
      rubber products plays an important role from         whether the rubber hydrocarbon itself or
      the view of protecting rubber goods against          non-rubber constituents were biologically
      biological damage and deterioration and of           attacked.
      providing environmentally compatible solu-              In this report, the current knowledge about
      tions for the disposal and recycling of rubber       the biodegradation of rubber hydrocarbon is
      waste. During the past few decades, much             summarized. Thereby, special emphasis is
                                                                                2 Historical Outline   323

given to the cis-1,4-polyisoprene chain, the        2
main constituent of natural rubber (NR ) and        Historical Outline
synthetic isoprene rubber (IR ). Due to the
chain's existence in nature, it is not surprising   2.1
to find most reports claiming its susceptibil-      General Considerations
ity, in contrast to reports about synthetic
rubbers of non-natural origin and differing         Summarizing examinations about the bio-
polymer composition, such as butadiene              degradation of rubbers implies the need to
rubber (BR ), styrene-butadiene rubber              pay attention only to those reports that
(SBR ), nitrile-butadiene rubber (NBR ), iso-       provided sufficient evidence for the catabo-
butylelene-isoprene rubber (IIR ), chloro-          lism of the rubber hydrocarbon chain. Ac-
prene rubber (CR ) and others, that mainly          cordingly, the following outline will include
reveal them as biologically recalcitrant mate-      only aspects dealing with the microbial, i.e.
rials (Seal, 1988).                                 bacterial and fungal, degradation of cis-1,4-
   One main approach will be to describe the        polyisoprene, the main constituent of natural
microorganisms that are capable of degrad-          rubber (NR ) and synthetic isoprene rubber
ing cis-1,4-polyisoprene, either in the raw         (IR ).
state or in rubber products, and of using the          In spite of numerous reports and reviews
rubber hydrocarbon as sole source for growth        concerning the biodeterioration of rubber
and biomass production. Different microbial         materials, which consisted of some other
strategies for an effective availability of this    diene hydrocarbon polymers, such as styr-
solid and hydrophobic substrate will be high-       ene-butadiene copolymerisate (SBR, Buna-
lighted. In this context, factors affecting the     S ), nitrile-butadiene copolymerisate (NBR,
rate of biodegradation, as well as known            Buna-N, nitrile rubber), isobutylene-iso-
approaches for an optimization of the degra-        prene copolymerisate (IIR, butyl rubber),
dation process with regard to a biotechno-          polybutadiene (BR ) and polychloroprene
logical application in rubber waste treatment,      (CR, neoprene) (ZoBell and Beckwith,
will be discussed. Analytical data concerning       1944; Blake et al., 1955; Snoke, 1957; Stein-
microbially caused surface modifications and        berg, 1961; Schwartz, 1963; Heap and Mor-
the production of degradation products will         rell, 1968; Cundell and Mulcock, 1972; Zyska,
be presented, especially from the view of           1981, 1988; Williams, 1985; Seal, 1988; Cain,
elucidating the primary cleavage mechanism          1992; Pommer, 1995; Tsuchii, 1995; Linos
of the polymer chain.                               and Steinbüchel, 1998), there was no con-
   Little is known so far about rubber-degrad-      vincing indication for the direct involvement
ing enzymes and their corresponding genes.          of microorganisms in rubber hydrocarbon
In this context, the first experimental ap-         metabolism. Moreover, it could be often
proaches in the authors' laboratory will be         shown, that microbial growth was promoted
introduced, and an attempt will be made to          at the expense of compounding ingredients
relate these data with results presented in the     typically used in rubber product manufac-
literature for the metabolism of isoprenoids        ture.
(e.g. carotenoids) and lignin model struc-             On the other hand, nonrubber substances
tures (e.g. lignostilbenes). It is generally        in commercial NR can amount to more than
intended to provide a stimulating basis for         5% of dry weight including proteins, carbo-
further investigations in this field.               hydrates, lipids, and inorganic salts (Subra-
                                                    maniam, 1995). Accordingly, the mere pres-
324   10 Biodegradation of Natural and Synthetic Rubbers

      ence and growth of microorganisms on NR              for metabolism of the polyisoprene chain
      does also not constitute proof for the utiliza-      must therefore exist. Unfortunately, to date
      tion of the rubber hydrocarbon as source of          no progress has been made in the elucidation
      carbon. However, the extent of microbial             of this phenomenon.
      growth and deterioration described in some
      communications was so high, that this could          2.2
      not be ascribed only to the utilization of           Early Investigations on the Biodegradation
      nonrubber constituents. Although final               of Natural Rubber
      proof for rubber hydrocarbon metabolism
      was missing, some of these reports were              As early as 1914, Söhngen and Fol (1914a, b)
      taken into consideration, due to their signifi-      reported about the decomposition of rubber
      cant contribution for further investigations in      by microorganisms. In order to prove assim-
      this field.                                          ilation of the hydrocarbon chain, highly
         Cis-1,4-polyisoprene represents a peculiar        purified NR was prepared and used as source
      polymer in all kind of laticiferous plants. A        of carbon. For purification, the authors dis-
      strong indication for the biodegradative po-         solved NR crepe sheet in benzene without
      tential of nature towards this molecule was          shaking, took only the clear upper phase of
      given by Taysum (1966), who showed that no           this solution, evaporated the solvent in glass
      accumulation of natural rubber occurred in           dishes and subsequently treated the NR films
      the soil of a 35-year-old plantation of rubber       obtained with a 2% trypsin solution for 8 h at
      trees (Hevea brasiliensis) in Malaysia, in spite     40 8C, for the effective removal of NR
      of the calculated annual deposition of               proteins. After washing for 16 h under
      ~1.7 g mÀ2 of cis-1,4-polyisoprene resulting         flowing water, the films were transferred to
      from the annual leaf fall of ~0.56 kg mÀ2 with       glass bowls containing water and inorganic
      a rubber content of 0.45% (w/w) in the dried         salts, sterilized at 1058C, inoculated with
      leaf material. No differences could be deter-        microorganisms, and cultivated (without
      mined when rubber tree soil was compared to          movement) for several weeks at different
      a 35-year-old Malayan grass soil, thereby            temperatures. No growth occurred with
      determining in both cases a concentration            different bacteria and fungi, which were first
      of 0.12% (w/w) of benzene-extractable mat-           enriched from soil and grew well on non-
      ter. Taysum concluded that ªconsiderable             purified NR, except in the case of two actino-
      quantities of rubber would have accumulated          mycetes, which formed colonies directly on
      if decomposition did not occurº.                     the purified NR material and were identified
         Assuming that cis-1,4-polyisoprene might          as Actinomyces elastica and Actinomyces fuscus.
      also serve as storage compound and energy            Additionally, the authors reported on the
      reservoir in rubber-containing herbs, also an        existence of holes in the rubber material,
      appropriate enzymatic equipment for its              which were formed beneath the adhering
      mobilization would be necessary. A first             actinomycete colonies ± both in liquid min-
      indication to this direction was obtained by         eral culture and on gelatin plates coated by
      Spence and McCallum (1935), who detected a           purified NR films ± and concluded that under
      seasonal fluctuation of rubber content in            these conditions increase in biomass could
      guayule plants (Parthenium argentatum).              only have taken place at the expense of the
      These authors determined a decrease in the           rubber hydrocarbon.
      latex amount at the beginning of each grow-             Encouraged by the results of Söhngen and
      ing season, and concluded that a mechanism           Fol, De Vries (1928) examined the possible
                                                                                 2 Historical Outline   325

decomposition of rubber hydrocarbon by             up to 59% of the material was present as a
fungi. In experiments with nonpurified NR          deteriorated latex clot, and up to 35% as water-
smoked sheet as sole carbon source (93%            soluble material. No distinction was made
rubber content), being inoculated with differ-     between the rubber and microorganisms.
ent Penicillium and Aspergillus species and        However, the authors also attempted to
cultivated stationary in 10% NaCl solution,        quantify the adhering biomass by dissolving
the author detected an increase in mold            the remaining brownish latex clot in benzene
biomass of up to 6% of the initial rubber          containing 1% trichloroacetic acid, in which
weight and a final weight loss of NR rising        bacteria and other nonrubber substances
from 15.5% after 19 months to 30.9% after 5        were insoluble. Analytical data showed that
years. By comparison, a noninoculated con-         the fraction of adhering cell material ranged
trol showed negligible weight loss. Examina-       from 12.5% to 28.7% of the weight of
tion of these data and those obtained after        analogously treated sterile controls, suggest-
chemical analysis of the remaining sub-            ing that growth was in fact promoted by the
strates resulting in a slight increase in nitro-   rubber hydrocarbon and not only by the
gen concentration in the 5-year-old sample         nonrubber constituents.
led the author to conclude that the rubber            Kalinenko (1938), using the latex overlay
hydrocarbon was being consumed preferen-           technique, reported the isolation of several
tially by the fungi, rather than the proteins.     actinomycetes and fungi, for example Asper-
   Spence and van Niel (1936) were the first to    gillus oryzae and Penicillium species, claiming
use latex overlay plates for the isolation of      that they all were able to consume large
rubber-degrading bacteria. Thereby, mineral        quantities of NR in diluted latex obtained
agar is overlayed by a thin layer of NR latex      from Taraxacum kok-saghyz. This is a rubber
that is dispersed in mineral agar. Rubber-         dandelion plant that was specially cultivated
degrading colonies developed on such plates        in the USSR during World War II to satisfy the
can be recognized by the production of             demand for NR. One of the actinomycetes
clearing zones (translucent halos) through         also produced holes in a thin film of pre-
the opaque agar layer. The authors reported        viously dialyzed kok-saghyz latex. However,
the (appropriate) isolation of four not closely    Shaposnikov et al. (1952a,b), who also tested
characterized actinomycetes, which they sub-       growth on natural rubber films placed on
sequently used as pure cultures for testing        mineral salts agar plates after evaporation of a
growth on NR. For this purpose, liquid             benzene solution, confirmed growth of acti-
cultures were prepared containing a mineral        nomycetes by weight-loss experiments (Sha-
salt medium with potassium nitrate instead         posnikov et al. 1952a), but noticed the ab-
of ammonium chloride as the nitrogen               sence of any fungal growth on NR (Shapos-
source in order to prevent latex coagulation       nikov et al. 1952b). In fact, the same was
after autoclaving. NR latex was used as            observed later by Nette et al. (1959), who
substrate; this was first dialyzed repeatedly      isolated several fungi and bacteria from
against refreshed phosphate buffer (pH 6.8 ±       pieces of contaminated rubber. These authors
7.2) in order to remove ammonia (present as a      reported weight losses of purified rubber
preservative) and water-soluble organic im-        films on mineral agar plates by three actino-
purities. Following incubation for 28 days at      mycetes (one Proactinomyces and two Actino-
30 8C, the weight of solids in the inoculated      myces strains) ranging between 25.8% and
samples was determined and compared with           43.2%, by a Bacillus sp. of 20.7%, by a
that in sterile controls. Results indicated that   Mycobacterium sp. of 17.2%, and by several
326   10 Biodegradation of Natural and Synthetic Rubbers

      fungi of maximum 2.5% of the initial rubber          controls, it cannot be excluded that, in the
      film weight (with an experimental error of           inoculated samples an additional reaction of
      Æ 2%). Unfortunately, no further data were           iodine with released degradation products
      given beyond determination of weight loss.           might have occurred, resulting generally in
         ZoBell and Grant (1942) and ZoBell and            higher values of oxygen consumption. An-
      Beckwith (1944) isolated several strains de-         other weak point of this study was that the
      signated as Actinomyces, Proactinomyces, Mi-         organism utilized was not clearly identified.
      cromonospora, Mycobacterium, Bacillus, and              Considering biodegradation of vulcanized
      Pseudomonas. The authors tried to estimate           NR, in which the long hydrocarbon chains
      the extent of rubber oxidation by determining        have been linked together by sulfur bridges
      the microbial oxygen consumption in liquid           and several compounding ingredients have
      culture. For this purpose, highly purified NR        been added during manufacture, Blake and
      (99%, obtained from the Goodyear Compa-              Kitchin (1949) reported the severe pitting and
      ny) was dissolved in benzene, and the upper          disappearance of NR gum used for rubber
      part evaporated in glass-stoppered bottles           insulation of underground cables as a result
      thereby forming a thin rubber film at the            of microorganism action. The authors de-
      bottom. The bottles were filled with water           tected a complete failure of insulation resist-
      saturated with oxygen, inoculated, closed,           ance after 3 months in soil burial tests, and
      and cultivated for 10 days at ~25 8C. After          documented the extent of mass decomposi-
      incubation, the amount of oxygen remaining           tion, referring to actinomycetes and ªred
      in the liquid was determined and compared            colonies of Gram-positive micrococciº as
      with that in a noninoculated control. The            being the causative agents. It was claimed
      authors pointed out that, by considering             that the rubber hydrocarbon must also have
      rubber to be (C5H8)x, a consumption of               been consumed as substrate, though quanti-
      3.3 mg of oxygen would be required to                tative data were not at all presented.
      completely oxidize 1 mg of rubber. In other
      words, this relation corresponds to a com-
      plete mineralization of rubber into carbon           2.3
      dioxide and water according to the following         Biodegradation of Rubber Pipe Joint Rings
      equation:
                                                           Rook (1955) was the first to perform system-
      (C5H8) ‡ 7O2 3 5CO2 ‡ 4H2O
                                                           atic work on the biodegradation of vulcanized
        The data obtained revealed an oxidation of         NR by using pure cultures. Employing
      60 ± 75% when 1 mg was used as substrate             purified NR latex and the technique of Spence
      (ZoBell and Grant, 1942), and 78% when               and van Niel (1936), he isolated several
      2 mg was used (ZoBell and Beckwith, 1944).           clearing zone-forming actinomycetes from
      Considering these small amounts and the              corroded rubber rings that were used for
      additional observation of increasing biomass,        connecting asbestos cement pipes in water
      rubber hydrocarbon metabolism may be                 distribution pipelines in the Netherlands.
      obvious. However, the method to determine            Two of the strains, which were designated as
      the remaining oxygen was based on liberation         Streptomyces species, were tested for the
      of iodine in the presence of oxygen (modified        utilization of vulcanized rubber in liquid
      Winkler technique), and the authors re-              cultures containing pipe-joint ring material
      marked that iodine also reacts with rubber.          as sole carbon source immersed in a mineral
      In spite of the comparison with sterile              salts medium. After 8 months of stationary
                                                                                2 Historical Outline   327

cultivation at 25 ± 30 8C, small holes became     well as NR, was demonstrated for the first
visible on the rubber strip cultivated with one   time.
of the strains. These holes reached a diameter       Cundell and Mulcock (1973, 1975, 1976)
of 1.5 mm after 12 months, and actinomycete       performed further investigations on the
filaments could be detected microscopically       biodegradation of NR vulcanizates. They
in the cavities. From this observation, the       confirmed microbial susceptibility of sulfur-
author concluded that ªthe extent of the          cured NR pipe-joint rings after testing respi-
breakdown was such that the rubber hydro-         ratory activities, such as carbon dioxide
carbon must also have been involvedº. From        evolution and uptake of oxygen, as well as
today's point of view, this statement might be    loss of tensile strength (Cundell and Mul-
correct, because clearing zone-forming bac-       cock, 1973), or by applying scanning electron
teria are now known generally to use cis-1,4-     microscopy (SEM ) and infrared (IR ) spec-
polyisoprene for growth (Jendrossek et al.,       troscopy (Cundell and Mulcock, 1975). The
1997; Linos et al., 2000a), although at that      relative ability of microorganisms to release
time the mere observation of hole formation       carbon dioxide from the surface of rubber
did not constitute sufficient proof for bio-      strips when cultivated in a Leeflang test bath
logical polymer breakdown.                        was estimated for a period of 18 months after
   Leeflang (1963) extended Rook's studies        successive transfer of rubber strips every 2
and isolated two similar strains by smearing      months from the bath in sealed glass jars, and
tiny fragments of seemingly unattacked            determination of the evolved CO2 during the
rubber pipe-joint ring material directly onto     next 1-week period, thereby trapping the gas
glucose-peptone agar plates. The surface of       in an aqueous barium peroxide solution and
such materials was previously liberated from      measuring the amount of CO2 liberated (by
the slimy bacterial layer formed during           acidification of the BaCO3 formed). Results
biodeterioration and the underlying deterio-      showed increasing values for the NR sample,
rated rubber. Tests with obtained pure cul-       but decreasing values for other vulcanizates
tures revealed wrinkling of NR rubber vul-        containing synthetic rubbers, for example
canizates, formation of holes and loss in         SBR, BR, CR, IIR, and NBR. Oxygen uptake
elasticity after 2 years exposure at 25 8C in     was determined once after 12 months using
mineral salts. The same two types of Strepto-     Warburg respirometry, and this resulted in an
myces sp. were isolated together from more        increasing respiratory activity for the NR
than 50 samples of deteriorated gasket ma-        sample during a 12-h measurement. Addi-
terial taken from installations in the Nether-    tionally, a substantial loss in tensile strength
lands and Belgium. The author also devel-         of NR vulcanizate (up to two-thirds of the
oped a basin test for proving microbial           initial value) was observed after 2 years'
susceptibility of different rubbers. In this      exposure in the Leeflang bath. Following
so-called Leeflang test bath ± which was later    SEM analysis of deteriorated rubber pipe-
used by several investigators ± rubber strips     joint samples (Cundell and Mulcock, 1975,
were hung in a basin through which a slow         1976), the occurrence of microbial cells was
and constant flow of potable unchlorinated        demonstrated on the rubber surface, with
water was maintained at 20 ± 25 8C in the         both mycelia and hyphae that were typical of
dark. The basin was inoculated by placing a       actinomycetes being visualized. However, the
piece of deteriorated rubber in the bottom.       claim that these organisms belonged to the
Using this procedure, the microbial suscept-      genera Streptomyces and Nocardia, respective-
ibility of synthetic isoprene rubber (IR ), as    ly, was very vague due to the fact that non-pure
328   10 Biodegradation of Natural and Synthetic Rubbers

      cultures were seen, and thus insufficient            (as confirmed by transmission electron mi-
      taxonomic data were presented. This latter           croscopy, TEM ), as well as its entire tensile
      finding represents a weak point in most              strength. Isolation of pure cultures using
      reports dealing with the microbial degrada-          Spence and van Niel's latex overlay technique
      tion of rubbers. For IR spectroscopic analysis,      revealed several strains of the same species,
      deteriorated material of a 1-year-old sample         which was claimed to belong to the Nocardia
      was scraped from the rubber surface, ground          asteroides complex according to results from
      up with a pestle and mortar, mixed with              previous extensive taxonomic analysis of
      powdered potassium iodide, pressed into              analogous actinomycetes isolated from de-
      pellets and scanned in the spectrophotome-           teriorated pipe-joint rings (Hutchinson and
      ter. The absorbtion spectrum obtained was            Ridgway 1975, 1977; Hutchinson, 1977;
      compared with that corresponding to repeat-          Hookey et al., 1980; Orchard and Goodfel-
      ing isoprene units in NR, and revealed               low, 1980; Cross, 1981). Liquid culturing
      changes in the region of CH3-, CH2-, carbonyl-       performed with the pure cultures in mineral
      and hydroxyl-groups, as well as a decrease of        salts medium in fact revealed the same
      the C ˆ C double bond intensity. Unfortu-            deterioration and microbial pattern as after
      nately, there was no information about               inoculation with mixed cultures (confirmed
      removal of cell material from the deteriorated       by SEM ), and the authors concluded that
      fraction used for spectroscopy, so that inter-       N. asteroides was the main cause for the
      pretation of the spectra required great cau-         biodeterioration of such NR vulcanizates.
      tion. In any case, the authors noted that the        This hypothesis was additionally favored by
      ªprocess is probably initiated by a mono-            the fact that N. asteroides strains could not be
      oxygenase cleaving the rubber hydrocarbon,           found on resistant NR and synthetic vulcan-
      followed by a stepwise degradation of the            izate samples, while all of the other evidently
      polyisoprenoid chainº (Cundell and Mulcock,          nondeteriorating bacteria of the surface mi-
      1975). In a further report (Cundell and              croflora were present on either of these
      Mulcock, 1976), these authors also described         materials, in spite of the presence of antiox-
      the isolation of two types of a single `Strepto-     idants and other protecting agents.
      myces' strain by employing the latex overlay
      technique. In this study, pure cultures were         2.4
      cultivated with peroxide-cured NR strips for         Degradation by Fungi
      18 months at 24 8C on a rotary shaker; this led
      to weight losses of between 7.3% and 13.4% of        Kwiatkowska et al. (1980) performed soil
      the initial rubber weight determined after           burial tests of NR vulcanizate sheets of
      removal of the bacterial layer from the strips,      definite composition and detected substan-
      though no further data were provided.                tial weight losses reaching up to 40% of the
         For continuing studies on rubber pipe-joint       initial weight after 91 days. Further character-
      rings, Hanstveit et al. (1988) used a modified       ization also revealed changes in the network
      Leeflang test bath method to study the               chain density of the materials, determined as
      biodeterioration of susceptible and resistant        a function of the duration of soil burial and the
      NR-vulcanizates. After 2 years' exposure, the        carbon black loading rate in the vulcanizate,
      authors detected actinomycete growth only            as well as the main occurrence of the fungus
      on the susceptible sample, which has lost 3%         Fusarium solani on the rubber surface. These
      of its volume due to severe pitting and              authors claimed that this microorganism was
      penetration of one kind of microorganism             the cause of NR degradation, and referred
                                                                                 2 Historical Outline   329

also to appropriate degradation tests, which        weight of up to 20% and in intrinsic viscosity
were performed with the pure culture,               of up to 35%. Using GPC, a relative reduction
though specific data to prove this assumption       of the molecular weights of the rubber
were not presented.                                 polymers was also detected in the samples
   More detailed investigations on the bio-         inoculated with Fusarium solani, Cladospori-
degradation of cis-1,4-polyisoprene by fungi        um cladosporioides and Paecilomyces lilacinus.
were documented twice during 1982. In the           Interestingly, the attack on rubber stopped
first report ( Williams, 1982), the author          after 30 days, resulting in no further changes
introduced experiments with Penicillium va-         concerning biomass, weight loss and molec-
riabile, a fungal strain isolated from deterio-     ular weight distribution, but could be restored
rated NR and IR samples after soil burial           again after successive removal of the fungal
tests. Spore suspensions inoculated onto NR         protective layer and transfer of the rubber
smoked sheet in a humidity cabinet led to a         substrate to fresh mineral medium every 20
successive increase of biomass on the mate-         days. The appropriate experiment with
rial's surface, as shown by cell protein            C. cladosporioides thereby resulted in de-
determination every 14 days, and was accom-         creased values for average molecular weight
panied by a weight loss of rubber strips of up      after each treatment.
to 13% after 56 days. However, further
increase in biomass and in weight loss              2.5
beyond this time period could not be deter-         Recent Developments
mined. Using solution viscosity measure-
ment as analytical tool, the author estimated a     Further strong evidence for the microbial
15% reduction in the molecular weight of            degradation of rubber polymer was achieved
polyisoprene after 70 days. However, IR             by Tsuchii et al. (1985). These authors re-
spectroscopy performed on the deteriorated          ported isolation of the actinomycete Nocardia
rubber did not reveal any changes in chemical       sp. strain 835A from soil, which caused
structure, and examination of acetone ex-           considerable weight losses (up to 100% after
tracts by thin-layer chromatography (TLC ),         8 weeks) when being cultivated in liquid
gel-permeation chromatography (GPC ) and            culture with different cis-1,4-polyisoprene-
gas chromatography-mass spectrometry                containing materials (0.06 ± 0.07%, w/v), for
(GC-MS ) did not prove the existence of any         example NR pale crepe sheet, synthetic
degradation products. The author therefore          isoprene rubber (IR ) and different NR vul-
suggested that ªmicroorganisms attack such          canizates, including latex gloves, rubber
polymers from the chain ends, forming short         bands, rubber tubing, rubber stoppers and
chain intermediates, and growing at the             tire treads. Strips from latex gloves were
expense of these unitsº.                            degraded most rapidly, reaching a 90%
   In the second report (Borel et al., 1982),       weight loss after 17 days and showing a
several rubber-deteriorating fungi could be         continuous increase in biomass on the rubber
isolated from mineral agar plates containing        surface according to cell protein determina-
powdered NR as sole substrate and deterio-          tion. In order to obtain putative degradation
rated tire material or soil dispersed in the agar   products, chloroform extracts were made
as inoculum. Liquid cultivation performed           from the glove/cell mixture and analyzed by
with isolated pure cultures for 20 days             two-dimensional TLC and GPC, respectively.
revealed the formation of a mycelial layer          These analyses revealed the existence of two
on the rubber surface, as well as losses in         distinct degradation fractions representing
330   10 Biodegradation of Natural and Synthetic Rubbers

      oligomers with molecular weights from 103 to         fractions consisting of low- and high-molec-
      104 Da. Staining with Schiff's reagent and           ular weight oligomers. Further analysis of the
      analysis by IR spectroscopy indicated the            molecular weights by GC-MS, and of the
      presence of carbonyl groups in the oligomers,        structure by 1H- and 13C-NMR, confirmed the
      while analysis by 1H- and 13C-NMR finally            existence of two main degradation products
      established the occurrence of two degrada-           of two and 113 isoprene units, respectively,
      tion products consisting of 19 and 114               and with characteristic carbonyl end-groups
      isoprene units, respectively, each carrying          referred to above. On the basis of successful
      an aldehyde on one end of the molecule and a         incorporation of 18O into the degradation
      ketone on the other end. On the basis of these       dimer under an 18O2 atmosphere, the authors
      data, the authors proposed an enzymatic              concluded that the cleavage reaction was
      cleavage mechanism proceeding by oxidative           partly oxygenase-catalyzed.
      scission of the cis-1,4-double bond as the              However, according to the author's own
      primary step in biodegradation of the cis-1,4-       statements, the ability of the Xanthomonas
      polysoprene chain.                                   strain to degrade rubber was rather poor
         Further investigations performed in the           (Tsuchii, 1999), and in subsequent reports
      same laboratory (Tsuchii and Takeda, 1990)           Tsuchii and coworkers concentrated mainly
      revealed an analogous degradation mecha-             on the Nocardia sp. strain 835A. This was due
      nism when latex of NR or IR was treated with         to the organism's remarkable rubber-degrad-
      the extracellular crude enzyme of an isolated        ing potential and thus its putative use in the
      Gram-negative bacterium, designated as               biological disposal of rubber waste (Tsuchii
      Xanthomonas sp. strain 35Y. For the prepara-         et al., 1990, 1996, 1997; Kajikawa et al., 1991;
      tion of crude enzyme, latex was purified by          Tsuchii and Tokiwa, 1999). Appropriate re-
      the method of Spence and van Niel (1936),            sults are summarized in Section 4, in which
      suspended in a mineral salts medium at               the optimization of microbial rubber degra-
      0.05% (w/v) containing also a small amount           dation is discussed.
      of a surface-active agent, and cultivated for 5         Heisey and Papadatos (1995) described
      days at 30 8C after inoculation with the strain.     several actinomycetes that were able to
      Subsequently, the culture was centrifuged            metabolize highly purified NR as sole carbon
      and the clear middle fraction of the centrifuge      source. Thin films of pale crepe rubber were
      tube was collected and filter-sterilized. The        first extensively extracted with different or-
      enzymatic reaction was performed at 30 8C            ganic solvents and used to produce NR-coated
      for different time intervals by incubating           glass slides which were subsequently used in
      sterile NR latex in phosphate buffer together        the isolation experiments. The pure cultures
      with the crude extract. After acidification and      obtained were further cultivated with vulcan-
      ether extraction, the fraction obtained was          ized NR, which corresponded to strips of
      used for carbonyl content determination and          analogously purified latex glove material
      analysis by TLC, GPC and gas-liquid chro-            containing finally only 0.1% of proteinaceous
      matography (GLC ). Enzyme activity ± which           impurities. Besides colonization of the rub-
      could be destroyed by heating and concen-            ber surface, penetration of the material and
      trated by ultrafiltration ± was expressed as the     general alteration of its physical structure was
      quantity of carbonyl compounds produced in           also observed by SEM. Increasing weight
      relation to the amount of crude enzyme used.         losses that reached >10% after 6 weeks
      Chromatographic analysis of the enzyme               correlated well with an increase in the
      reaction again revealed the existence of two         amount of cell protein, both in the culture
                                                                                  2 Historical Outline   331

broth and on the rubber strips. Taxonomic           sample for 6 months on tire crumb material
data based on fatty acid profiles and cell wall     as sole carbon and energy source; additional
characteristics revealed seven Streptomyces,        investigations included the subsequent en-
two Amycolatopsis and one Nocardia strain,          richment of rubber-degrading bacteria on NR
and the authors concluded that actinomy-            or synthetic IR. Taxonomic analysis of the
cetes were the main organisms involved in           obtained pure cultures revealed strains be-
the biodegradation of natural rubber hydro-         longing to the nocardioform actinomycete
carbon.                                             genus Gordonia (formerly known as Gordo-
   A strong indication for this was also given      na). One of the strains was recently classified
by Jendrossek et al. (1997), who tested a large     as the novel species Gordonia polyisoprenivo-
number of Gram-positive and Gram-negative           rans Kd2T (Linos et al., 1999). Biodegradation
bacteria, which were either isolated from           of the cis-1,4-polyisoprene in NR (raw and
different natural habitats by using centri-         vulcanized) as well as in IR was confirmed
fuged fresh NR latex as carbon source, or were      after determining the extent of mineraliza-
obtained from different culture collections.        tion, which was expressed as percentage of
Using Spence and van Niel's latex overlay           carbon released from the rubber substrate as
technique, it was not possible to identify clear    CO2 during the time course of the cultivation
zone-forming bacteria other than actinomy-          experiments, thereby reaching values of
cetes. Screening of bacteria from culture           >50% for NR and >20% for IR after 52 days.
collections and fatty acid profile analysis of      For evaluation, it was assumed that the rubber
isolated strains revealed members of the            substrates consisted totally of carbon. Con-
genera Actinomadura, Actinoplanes, Dactylo-         sidering this, and the fact that the biomass
sporangium, Micromonospora, Nocardia and            also increased during cultivation, real bio-
Streptomyces, with Micromonospora and Strep-        degradation values were actually much high-
tomyces representing the most prominent             er. At the same time, solubilization, disinte-
genera. With most of the strains, clear zone        gration and continuous visual disappearance
formation was repressed, when readily uti-          of the solid rubber substrate could be ob-
lized substrates such as glucose and/or             served (Figure 1)
succinate were added to the latex-agar me-             Attempts to isolate solubilized rubber from
dium as co-substrates. The authors also noted       the turbid supernatant of liquid Gordonia
a reduction in the average molecular mass of        cultures by organic solvent extraction yielded
residual NR polymer from solution cast films,       solid material after 5 days, which was only
from 6.4”105 to 2.5”104 Da, implying the            soluble in chloroform (suggesting a higher-
participation of an endo-cleavage mechanism         molecular weight fraction), the amount of
in the biodegradation process; analytical data      which diminished during the time course of
were not presented, however.                        the cultivation experiment (Figure 2A ). An-
                                                    other indication for the solubilizing proper-
2.6                                                 ties of the strains was shown by preventing
Investigations in the Authors' Laboratory           latex coagulation, when NR latex was treated
                                                    with the filter-sterilized extract of a 5-day-old
Linos and Steinbüchel (1998) reported on the        Gordonia rubber culture. This effect was
isolation of several rubber-degrading bacteria      similar to that seen with detergents such as
from fouling water inside of deteriorated car       sodium dodecyl sulfate (SDS ) or Triton X-
tires, from soil of a rubber tree plantation, and   100, which were also tested for the prevention
from sewage sludge after adaptation of each         of latex coagulation (Figure 2B ).
332   10 Biodegradation of Natural and Synthetic Rubbers




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



         In a series of screening experiments for          before the start of the disintegration process.
      isolating various rubber degrading bacteria,         In contrast, actinomycetes ± which were able
      Linos et al. (2000a) specified that there were       to grow well on latex overlay plates by forming
      two different microbial strategies to make           characteristic clear zones ± were unable to
      rubber available as substrate. In the first,         grow appreciably on latex film plates. These
      bacteria grow only in direct contact with the        clear zone-formers, the isolation of which has
      rubber surface, and this leads to considerable       been described frequently in the literature,
      disintegration of the material during culti-         had a different strategy. As an example of such
      vation. Strains like the above-mentioned             a strain, the authors isolated and character-
      Gordonia isolates, as well as a newly intro-         ized a Micromonospora aurantiaca and dem-
      duced Mycobaterium fortuitum, were assigned          onstrated growth of this isolate, especially on
      to this category. These bacteria were barely         synthetic IR. SEM analysis revealed the
      able to grow on Spence and van Niel's latex          formation of mycelial corridors on the mate-
      overlay plates, as a consequence of the              rial's surface and its penetration by hyphae
      absence of direct contact with the rubber,           (Figure 3B ). Neither embedding of cells into
      which is distributed in the agar medium. By          the rubber matrix nor the formation of a
      contrast, good growth occurred when the              classical biofilm were observed. The authors
      latex was spread as a thin film on mineral agar      concluded that, as cells were unlikely to
      plates (latex film plates), thereby allowing         transport solid rubber to their interior before
      direct exposure of the bacteria to the rubber.       cleaving it into smaller molecules, the rubber-
      Detailed analysis of the growth behavior on IR       degrading activity of adhesive growers would
      and NR latex gloves by means of SEM revealed         most likely be bound to the cell surface, while
      that, during colonization, the strains were          the clear zone-forming bacteria excrete this
      tightly adhered and embedded into the                activity into the medium.
      rubber matrix, thereby forming characteristic           Chemical changes that arose directly on the
      penetration craters on the material's surface        rubber surface as result of the biodegradation
      (Figure 3A ). Another characteristic feature         process were determined using Fourier trans-
      was the formation of a biofilm (bacterial            form infrared (FTIR ) spectroscopy. This
      surface layer) over the entire rubber surface        comprised the Attenuated Total Reflectance
                                                                                               2 Historical Outline    333




Fig. 2   A: Solubilized material recovered from a 5-
day-old NR latex culture of G. polyisoprenivorans VH2
after ultracentrifugation and chloroform extraction of      Fig. 3 Secondary electron micrographs demonstrat-
the solid-free supernatant. B: Demonstration of the         ing two microbial strategies at the surface of synthetic
solubilizing properties of the supernatant of a 5-day-      cis-1,4-polyisoprene (IR ). A: Growth behavior of G.
old NR latex culture of G. polyisoprenivorans VH2. Left:    polyisoprenivorans VH2 after 4 days, showing adher-
Latex coagulate. Middle: Prevention of latex coagu-         ance and embedding of the cells into the rubber
lation after addition of 3 mL of filter-sterilized super-   matrix, thereby forming characteristic penetration
natant. Right: Total inhibition of latex coagulation        craters. B: Growth behavior of Micromonospora
after addition of sodium dodecylsulfate (SDS) as            aurantiaca W2b after 7 days, showing formation of
detergent.                                                  mycelial corridors on the material's surface and its
                                                            penetration by hyphae. Scale bars, 5 mm. Micrographs
                                                            from Linos et al. (2000a.).

(ATR ) technique (Linos et al., 2000a), a
method which allows nondestructive, in-situ                 sample surface, the spectra detected revealed
analysis of surfaces coated by microbial                    signals corresponding to those known from
biofilms (Schmitt and Flemming, 1998).                      the literature for cis-1,4-polyisoprene in He-
Latex glove material overgrown by the Gordo-                vea-NR. These spectra exhibited the following
nia cells was analyzed, and the spectra                     changes when compared with non-inoculat-
confirmed the presence of a bacterial surface               ed controls: (1) a decrease in the number of
layer by detecting characteristic marker                    cis-1,4 double bonds; (2) the appearance of
bands corresponding to bacterial proteins,                  ketone and aldehyde groups; and (3) the
fatty acids and polysaccharides. After proving              formation of two different bonding environ-
mechanical removal of the biofilm from the                  ments. These observations were finally in-
334   10 Biodegradation of Natural and Synthetic Rubbers

      terpreted as the consequence of an oxidative         distinguish between these two bacteria, when
      reduction of the polymer chain, in similar           complex or readily utilizable carbon sources
      manner to that shown for the above-men-              were used for spreading the AL98 culture on
      tioned Nocardia sp. strain 835A (Tsuchii             agar plates, because of the inability of the
      et al., 1985).                                       Gordonia strain to grow in the presence of the
         In further screening experiments, Linos           Pseudomonas strain, which was shown to
      et al. (2000b) reported about the isolation of a     produce an inhibitory substance towards
      Gram-negative bacterium from foul tire               Gordonia. Due to the fact that biomass of
      water, which was taxonomically character-            Pseudomonas also increased during cultiva-
      ized as Pseudomonas aeruginosa AL98. The             tion with rubber, it was assumed that growth
      culture was first enriched on NR latex               of the Pseudomonas strain occurred at the
      concentrate, and exhibited the ability to            expense of the degradation products of cis-1,4-
      produce greenish areas when cultivated on            polyisoprene being produced primarily by the
      latex film plates. Successive transfer of cell       action of the Gordonia strain. This possibility
      material from such areas on LB plates                was favored by the observation that Pseudo-
      revealed single colonies of a greenish bacte-        monas also totally lost its ability to grow on
      rium, that was used for testing growth on            rubbers after numerous transfer passages on
      different substrates such as latex concentrate,      LB plates, in contrast to the Gordonia strain,
      latex glove and synthetic IR. The determina-         whose ability remained unaffected.
      tion of growth parameters such as CO2-                  Further investigations in the authors' labo-
      release and living cell number resulted in           ratory described the effect of pretreatment of
      enhanced mineralization values for all sub-          rubber material on its biodegradability by
      strates (up to 36% after 6 weeks), as well as        these bacteria (Berekaa et al., 2000); the
      increased values for suspended biomass (up           results of these studies are summarized in
      to 35-fold). SEM analysis also revealed a            Section 4.
      property of the strain as the effective colo-
      nization of the rubber surface, and formation        2.7
      of a biofilm during cultivation. However,            Conclusions
      visible disintegration of the rubber started
      only after 2 ± 3 weeks, when cells from LB           Accumulation of knowledge on the biodegra-
      culture were used as inoculum, in contrast to        dation of cis-1,4-polyisoprene-containing
      cells from rubber cultures, where disintegra-        rubbers during the past century was mainly
      tion had already started after 2 days. The           accompanied by progress of the analytical
      reason for this behavior remained unknown            equipment needed for these investigation.
      until recent experiments in the authors'             The simple visual and microscopic observa-
      laboratory (Linos et al., 2001) showed that          tions used during the early stages were in turn
      the AL98 culture was a mixed culture, due to         replaced by electron microscopy and spec-
      the appearance of an additional strain, when         troscopy approaches, enabling detailed illus-
      acetonylacetone was used as carbon source,           tration to be made of microbial rubber
      and which the Pseudomonas strain could not           colonization and the chemical structure of
      utilize. This new isolate turned out to express      rubber degradation products to be elucidated.
      good rubber-decomposing activities and to            Moreover, developments in molecular biol-
      represent another strain of Gordonia polyiso-        ogy opened the way for the performance of
      prenivorans, according to 16S rDNA analysis.         reliable taxonomic classification of the iso-
      The authors stressed that it was not possible to     lated rubber-degrading microorganisms, and

				
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