FEMS Microbiology Letters 184 (2000) 199^206

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Eect of pretreatment of rubber material on its biodegradability by various rubber degrading bacteria
Mahmoud M. Berekaa a , Alexandros Linos a , Rudolf Reichelt b , Ulrike Keller b , Alexander Steinbu chel a ; *
b a Institut fu r Mikrobiologie der Westfa lischen Wilhelms-Universita t Mu nster, Corrensstrasse 3, D-48149 Mu nster, Germany Institut fu r Medizinische Physik und Biophysik der Westfa lischen Wilhelms-Universita t Mu nster, Robert-Koch-Strasse 31, D-48149 Mu nster, Germany

Received 24 November 1999; accepted 31 January 2000

Abstract
The effect of pretreatment of several cis-1,4-polyisoprene containing rubbers on their biodegradability was examined. Tests were carried out with six recently isolated and characterized rubber degrading bacteria belonging to the genera Gordonia (strains Kb2, Kd2 and VH2), Mycobacterium, Micromonospora and Pseudomonas. All strains were able to use natural rubber (NR) as well as NR latex gloves as sole carbon source. Extraction of NR latex gloves by organic solvents resulted in an enhancement of growth for three of the selected strains. On the other hand, growth of Gordonia sp. (strain Kb2 and Kd2), Mycobacterium fortuitum NF4 and Micromonospora aurantiaca W2b on synthetic cis-1,4-polyisoprene did only occur after removal of the antioxidants, that are usually added during manufacture to prevent aging of the materials. Detailed degradation studies performed with Gordonia sp. Kb2 revealed an enhanced mineralization of pretreated NR latex gloves and mineralization of purified natural rubber (NR), indicating the actual mineralization of cis-1,4-polyisoprene rubber constituent even after removal of non-rubber constituent that may act as co-metabolic substrate and support microbial growth. Further analysis by scanning electron microscopy (SEM) clearly demonstrated the enhanced colonization efficiency of these bacteria towards pretreated NR latex gloves. Colonization was additionally visualized by staining of overgrown NR latex gloves with Schiff's reagent, and the purple color produced in the area of degradation was an evidence for the accumulation of aldehydes containing oligomers. Further enhancement of latex gloves degradation could be achieved after successive replacement of mineral salts medium during cultivation. Thereby, a rapid disintegration of untreated NR latex gloves material was accomplished by Gordonia sp. strain VH2. 2000 Published by Elsevier Science B.V. All rights reserved.
Keywords : Biodegradation; Natural rubber; cis-1,4-Polyisoprene; Latex glove; Actinomycetes ; Scanning electron microscopy; Gordonia sp.; Gordonia polyisoprenivorans ; Micromonospora aurantiaca ; Mycobacterium fortuitum ; Pseudomonas aeruginosa

1. Introduction Raw natural rubber (NR) for technical applications is obtained from the latex of Hevea brasiliensis trees. Its dry weight consists more than 90% of cis-1,4-polyisoprene (Mw s 106 Da) and less than 10% of non-rubber constituents, like proteins, lipids, carbohydrate, resins and inorganic salts [1]. In contrast, raw synthetic cis-1,4-polyisoprene rubber (IR) does not contain any non-rubber constituents, except antioxidants, that are usually added during the manufacture to prevent ageing of the materials. For many commercial applications, raw rubber is subjected to a vulcanization process in which the cis-1,4-poly-

* Corresponding author. Tel. : +49 (251) 8339821; Fax: +49 (251) 8338388; E-mail : steinbu@uni-muenster.de

isoprene chains are crosslinked either by heating in the presence of sulfur, as in case of tires, or by irradiation and peroxidation, as in case of NR latex gloves. Therefore, further substances are added in these cases. Susceptibility of NR products towards a microbial attack is a well known phenomenon and has been suciently reviewed [2,3]. The isolation of a large number of rubber degrading bacteria has been reported so far [4^11]. Analytical data concerning biodegradation of the cis-1,4polyisoprene chain are available and indicated an oxidative attack at the double bonds of the polymeric chain to occur as the rst step in the degradation process [6,12,13]. During the past decades, investigations concerning biodegradation of rubbers focussed mainly on improving protection against biological damage [3]. The purpose of this study was to test degradation of rubbers by various rubber degrading bacteria after removal of antimicrobial substan-

0378-1097 / 00 / $20.00 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 4 8 - 3

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ces that were added for dierent purposes by the manufacturing companies and hence providing preliminary approaches for a future microbial treatment of rubber waste by combining chemical and biological methods. Furthermore, rst results concerning optimization of the degradation process by improving cultivation conditions are also presented. 2. Materials and methods 2.1. Rubbers Raw NR (SMR 10) and IR (SKI3) were obtained from Continental AG (Hannover, Germany). NR latex concentrate (Neotex Latz) was obtained from Weber and Schaer (Hamburg, Germany) and NR latex gloves (rotiprotect1) were purchased from Roth (Karlsruhe, Germany). 2.2. Microorganisms All rubber degrading bacteria used in this study were isolated in our laboratory [10,11,13,15]: Gordonia sp. VH2 (DSM 44266), Gordonia sp. Kb2 (DSM 44215), Gordonia polyisoprenivorans Kd2T (DSM 44302), Micromonospora aurantiaca W2b (DSM 44438), Mycobacterium fortuitum NF4 (DSM 44216) and Pseudomonas aeruginosa AL98 [15] were isolated and described as reported previously. In addition, Bacillus subtilis 168 (DSM 402) was used. 2.3. Cultivation experiments To examine the microbial degradation of various kinds of rubber, growth experiments with rubber as the sole carbon source were done. Cultivations were carried out in 500-ml Erlenmeyer asks containing 50 ml mineral salts medium (MSM) as described in [16], and the rubber materials were added at concentrations of 0.5% (w/v). Solid NR and latex gloves were cut into small pieces of 2^3 mm, while natural latex concentrate was added directly. Before inoculation the entire media were sterilized in an autoclave. During incubation at 30C, the cultures were agitated at 180 rpm on a rotary shaker. All cultures were inoculated with cells obtained from 3^6 days preculture in Luria^Bertani (LB) complex medium [17], which were washed twice with sterile saline before use. 2.4. Methods for purication of rubber materials Purication of solid NR was carried out by two dierent methods: (1) Extraction: NR was extracted in a Soxhlet apparatus with 250^300 cycles of a 90% (v/v) aqueous methanol solution followed by 500^600 cycles of acetone. (2) Precipitation: 3 g of NR were dissolved in 100 ml chloroform and subsequently precipitated by adding 100 ml acetone ; this step was repeated several times until the

acetone solution remained clear. After either purication, the rubber was left to dry, washed with distilled water and subsequently used as sole carbon source for the cultivation experiments. To extract antimicrobial substances from NR latex gloves, the material was treated with acetone or chloroform as follows : 1 g of latex gloves was extracted with 100 ml acetone or chloroform for 10^12 h. During this period the solvent was replaced one or two times with fresh acetone or chloroform. The treated material was left to dry and was subsequently used as carbon source. To extract antioxidants from IR, the material was treated with acetone as follows : 3 g of solid IR were extracted with 100 ml of acetone for 1^2 days. During this period acetone was repeatedly (approximately 3^5 times) replaced by fresh acetone until no colored substances were extracted from IR anymore. The puried material was left to dry and was subsequently used as carbon source. 2.5. Study of the growth inhibitory eect of antioxidants The eect of antioxidants on the growth of NR-degrading bacteria was tested as follows : 100 mg of the colored antioxidant substances extracted from IR (see above) were dissolved in 2 ml acetone. This colored substance was applied onto sterile paper discs, left to dry at air and subsequently placed on LB complex agar medium [17] plated with a cell suspension of Gordonia sp. strain Kb2. As a control, discs containing only acetone were left to dry and used in the same way. 2.6. Scanning electron microscopy (SEM) For SEM studies, 3 g of washed IR were dissolved in 100 ml chloroform. Rectangular aluminum pieces with an area of about 1 cm2 were repeatedly dipped into the solution and dried in a soft air stream. The coated pieces were sterilized with 95% ethanol and added to 30 ml of previously autoclaved MSM solution in 300-ml asks. Pieces of NR latex gloves and IR-coated aluminum overgrown by rubber-degrading bacteria were removed from liquid cultures at various cultivation periods, xed with 2.5% glutaraldehyde in 0.1 M phosphate buer (PBS ; pH 7.3) according to Srensen [18]. After washing with PBS, the cultures were postxed in 1% osmium tetroxide in 0.1 M PBS (pH 7.3) and dehydrated in graded ethanol (3%, 50%, 70%, 90%, 96% and absolute ethanol). The dehydrated samples were subjected to the critical point drying with liquid CO2 according to the standard procedure. Subsequently, the samples were mounted on aluminum specimen stubs using electrically conducting carbon (PLANO, Wetzlar, Germany) and sputter-coated with approximately 15 nm gold using argon gas as the ionizing plasma. Imaging was performed with a S-450 scanning electron microscope (Hitachi, Japan) with secondary electrons at 20 kV acceleration voltage and at room temperature. Micro-

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graphs were recorded from a high-resolution cathode-ray tube using negative lm (Agfapan, APX 100). 2.7. Staining of rubber-degrading colonies Staining of actively growing colonies on the NR latex glove surface with Schi's reagent [19] was performed in tightly stoppered bottles. 10 ml of Fuchsin reagent was added to latex samples, and a purple color developed after 10^30 min incubation at room temperature. Excess reagent was then discarded, and 10 ml of the sulte solution were added to suppress nonspecic reactions. Composition of Fuchsin reagent : 2 g Fuchsin dissolved in a solution consisting of 50 ml glacial acetic acid plus 10 g Na2 S2 O5 plus 100 ml 0.1 N HCl plus 50 ml H2 O. Composition of sulte solution: To 5 g Na2 S2 O5 , 5 ml concentrated HCl (37^38%) and water to 100 ml were added. 2.8. Mineralization of the rubber substrate Evidence for degradation and mineralization of the cis1,4-polyisoprene rubber hydrocarbon chain was obtained by determination of the amount of CO2 released during cultivation of cells on the rubber substrate which was provided as sole source for carbon. The released CO2 was trapped in a 0.25-M solution of Ba(OH)2 resulting in the precipitation of CO2 as BaCO3 . The decrease in alkalinity was determined by titration with 0.25 N HCl and compared to a non-inoculated control for dierent time intervals [10]. 3. Results 3.1. Enhanced mineralization of pretreated NR latex gloves The capability of various rubber degrading bacteria to utilize NR latex gloves and the eect of purication of this material by extraction with organic solvents was investigated by measurement of the amount of CO2 released during an incubation period of 40 days. Gordonia sp.

Fig. 1. Mineralization of NR latex gloves by Gordonia sp. strain Kb2. The degree of mineralization is shown as percent of carbon released as CO2 during the time course of the experiment using untreated (-a-), chloroform-treated (-b-) or acetone-treated (-F-) NR latex glove material as sole carbon source. Data represented are results of a single experiment.

strain Kb2 was cultivated in the presence of treated and untreated NR latex gloves. Results shown in Fig. 1 clearly demonstrated the enhancement of NR latex gloves degradation after treatment of the glove material with organic solvents such as acetone or chloroform. This treatment led to a more than 2^3-fold increase of the mineralization of rubber expressed by percentage CO2 released during growth. Results shown in Table 1 revealed similar eects also with other rubber degrading bacteria as indicated by enhanced growth following the increase of turbidity and biolm formation in liquid cultures and the degradation of NR latex gloves by M. aurantiaca strain W2b, and P. aeruginosa AL98 and G. polyisoprenivorans Kd2. In contrast, the growth of the potent rubber degrading bacteria Gordonia sp. strain VH2 and M. fortuitum strain NF4 on NR latex glove material was not positively aected by any of these treatment methods. 3.2. Mineralization of raw and puried natural rubber material The eect of NR purication on the growth of Gordonia sp. strain Kb2, and the mineralization of raw and puried NR was also followed by measurement of the amount of

Table 1 Eect of pretreatment of NR latex glove material as well as of cis-1,4-polyisoprene rubber (IR) material on growth of various bacterial isolates Bacterial strain Growth on NR latex gloves Untreated Gordonia sp. strain Kb2 G. polyisoprenivorans strain Kd2 Gordonia sp. strain VH2 M. fortuitum strain NF4 M. aurantiaca strain W2b P. aeruginosa strain AL98
a

Growth on IR
a

Treated +++ +++ +++ ++ +++ +++

Untreated 3 3 +++ 3 3 3

Treatedb ++ ++ +++ ++ ++ ++

+ + +++ ++ + ++

- Growth was qualitatively estimated as follows: (3) no growth was recorded; (+) very weak growth; (++) moderate growth and (+++) good growth. NR latex glove material was used as sole carbon source in the medium after removal of antimicrobial substances by treatment with chloroform or acetone as described in Section 2. b IR material was used as sole carbon source in the medium after removal of antioxidants (e.g. 6-PPD) by treatment with acetone as described in Section 2.

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Fig. 2. Mineralization of dierent types of untreated and puried NR by Gordonia sp. strain Kb2. The degree of mineralization is shown as percent of carbon released as CO2 during the time course of the experiment on NR latex concentrate (-E-), untreated solid NR (-a-), extracted NR (-b-) and precipitated NR (-F-). Data represented are results of a single experiment.

products containing aldehyde groups during the degradation. Further investigations showed that the colonization of rubber-degrading bacteria on the surface of NR latex gloves increased during the time course of the experiment. Whereas during the rst week spots representing small colonies appeared, these spots had increased in size after 3 weeks and nally covered the entire surface of the glove material that was subsequently disintegrated in liquid medium after 5 weeks. Although the rubber surface was completely covered with dense growth of the rubber-degrading cells after the rst week of cultivation, there were nearly no suspended cells in the culture medium. Only after 3^4 weeks an increasing number of cells were released from the latex gloves surface into the medium. After 8 weeks holes could be seen on the surface, and the number of suspended cells had increased signicantly. 3.4. Eect of pretreatment on synthetic rubber degradation Several rubber degrading bacteria were also tested for growth on synthetic cis-1,4-polyisoprene and to use it as sole source for carbon. For this purpose, IR was provided as thin lm covering both sides of aluminum pieces. SEM analysis of the overgrown IR material of each strain was done at dierent time intervals. It became evident that the response of bacterial strains towards IR degradation was clearly dierent before and after removal of the antioxidants. Results in Table 1 revealed that Gordonia sp. strain Kb2, G. polyisoprenivorans Kd2, M. aurantiaca W2b, M. fortuitum NF4 and P. aeruginosa AL98 grew only after the extraction of the antioxidants from IR, whereas growth of Gordonia sp. strain VH2 was not positively aected by the extraction process. The inhibitory eect of antioxidants on the growth of NR-degrading bacteria was investigated. For this purpose, Gordonia sp. strain Kb2 was cultivated on LB complex agar medium [17] in the presence of a sterile disc containing the antioxidants extracted from synthetic cis-1,4-polyisoprene. Fig. 5 showed that the growth of Gordonia sp. strain Kb2 was obviously inhibited by antioxidants as indicated by the occurrence of clear zone formation. Whereas, no clear zones were observed around discs containing no antioxidants. A similar inhibitory eect was also observed with other Gram-positive bacteria such as B. subtilis strain 168. 3.5. Optimization of the cultivation condition Beside enhanced degradation of NR latex gloves by pretreatment processes, further optimization of NR latex gloves lm degradation was achieved after cultivation of Gordonia sp. strain VH2 in MSM with latex gloves as sole source of carbon. Two dierent approaches were established to enhance the rate of degradation. In the rst approach, stationary incubation of the culture after 6 weeks of shaking led to complete disintegration of the rubber

CO2 released during an incubation period of 40 days (Fig. 2). In contrast to NR latex gloves, during mineralization of both, puried and raw NR, a similar degradation behavior was observed for either substrates. This was revealed by severe adherence of the cells to the hydrophobic rubber substrate and with more than 30% of the carbon released as CO2 from untreated raw NR as well as from NR latex concentrate. On the other hand, the amount of CO2 released during cultivation on precipitated- and extracted-NR rubber were clearly lower. 3.3. SEM and other analysis The eect of acetone treatment of NR latex gloves on colonization and enhanced degradation of NR latex gloves was also clearly demonstrated by SEM studies. For this purpose, Gordonia sp. strain Kb2 and M. aurantiaca strain W2b were cultivated with non-treated and acetone-treated NR latex gloves as sole source of carbon. SEM's showed that the colonization of Gordonia sp. strain Kb2 on untreated latex gloves was very weak after 2 weeks of incubation (Fig. 3A). On the other hand, the surface of acetone-treated latex gloves was completely colonized by a biolm of cells of strain Kb2 after the same incubation period (Fig. 3B). Similar observations were recorded during growth of M. aurantiaca strain W2b on latex gloves. SEM micrographs demonstrated weak colonization of Micromonospora cells on untreated material only after 6 weeks of incubation (Fig. 3C), whereas, 2 weeks of incubation were sucient to obtain dense mycelia masses on the surface of acetone-treated latex gloves (Fig. 3D). Furthermore, colonization behavior of rubber degrading Gordonia sp. strain Kb2 on the surface of pretreated NR latex glove was examined by Schi's reagent. Fig. 4 shows that actively growing colonies on the rubber surface were clearly visualized by staining with this reagent and gave a clear purple color according to a previous study [14] thus providing evidence for the occurrence of degradation

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Fig. 3. Microbial degradation of NR latex gloves. SEM of latex gloves showed very weak growth of Gordonia sp. strain Kb2 on untreated NR latex gloves (A) and complete cover of acetone-treated NR latex gloves with Gordonia cells (B) after 2 weeks of incubation. SEM micrographs demonstrated also the start of growth of M. aurantiaca strain W2b on untreated NR latex gloves after 6 weeks (C) and condensed mycelial mass on acetone-treated NR latex gloves after 2 weeks (D).

material within the next 12 weeks. In the second approach, a semicontinuous method of cultivation was tested in which MSM was replaced by fresh medium after 6 weeks of cultivation. This replacement of the medium led to a complete disintegration of the rubber material within the following week. Another replacement of the medium led to complete visual loss of the rubber material during the following 2 weeks.

4. Discussion The eect of pretreatment of dierent rubber materials on its biodegradability by several bacteria was investigated. Manufacture of products from synthetic and NR is usually accompanied by the addition of compounding ingredients. Many of these substances are known to prevent microbial growth [20]. In case of NR latex gloves, no

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Fig. 4. Colonization of Gordonia sp. strain Kb2 on treated NR latex gloves. After dierent time intervals, growth was visualized by staining with Schi's reagent. a: Start of colonization after 1 week as very small colonies. b: During the third week, colonies increased in surface area and penetrated deeper into the material. c: During the fth week the material was completely covered with a dense biolm and disintegration of the material started. d: Control experiment showing non-inoculated NR latex gloves after 5 weeks of incubation (magnication : 18.75U).

information about these ingredients was available from the manufacturer. Interestingly, organic extraction of NR latex gloves revealed one possibility to improve the biodegradation process with respect to potential biotechnological treatment of rubber waste. This was especially demonstrated for Gordonia sp. Kb2 and M. aurantiaca W2b that showed an increased substrate mineralization of the rubber substrate (Fig. 1) and also enhanced growth supported by SEM analysis (Fig. 3) and an increased colonization eciency (Fig. 4) as indicated by Schi's reagent test with the pretreated glove substrate. However, removal of non-rubber constituents from raw NR could also result in a decreased mineralization rate (Fig. 2), indicating the possible importance of these compounds as co-substrates supporting biodegradation of the polymer. For IR, the addition of the antioxidant 6PPD (N-(1,3dimethyl-butyl)-NP-phenyl-p-phenylene diamine) was reported from the supplier. Removal of this antimicrobial substance by organic solvent extraction led to an increased

growth of several microorganisms on this substrate. It was remarkable that growth of almost all selected NR-degrading strains on IR was only possible after acetone-extraction of the material (Table 1). This nding suggests also to use treated IR as enrichment substrate to isolate additional rubber degrading bacteria. Direct screening of cis-1,4polyisoprene degraders by this way would also make subsequent discussion about metabolism of rubber hydrocarbon or non-rubber constituents, as in case of NR, unnecessary. However, NR is also known to contain naturally occurring antioxidants, e.g. phenolic compounds, proteins etc. [21]. Therefore, it would be interesting to see, if strains enriched on pretreated IR were also able to grow on NR. On the other hand, growth of Gordonia sp. VH2 on untreated IR indicated the possible occurrence of a suitable resistance mechanism towards the used antioxidant. This assumption is supported by the fact that the strain was originally enriched on this IR material [10].

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Fig. 5. The inhibitory eect of antioxidants. A bacterial suspension of Gordonia sp. strain Kb2 cells was plated on LB-agar and incubated at 30C for 48 h in the presence of a disc containing the extracted antioxidants (a) or lacking the antioxidant (b).

Beside the use of pretreated rubber material, improving of biodegradation of rubber could be further enhanced by optimization of the cultivation conditions. The employed bacterium, Gordonia sp. VH2, led to a considerable increased disintegration of untreated NR latex glove material, especially after successive replacement of the MSM during cultivation. Therefore, semicontinuous or continuous processes may be promising to establish biotechnological processes to the disposal of products made from synthetic or natural rubbers [22,23]. Acknowledgements This work was supported in part by a grant from the Egyptian Government Fellowship Program. Furthermore, the authors appreciate the expert photographic work of Mrs. G. Kiefermann from the Institut fu r Medizinische Physik und Biophysik. References
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