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A Gram-negative bacterium, identified as Pseudomonas aeruginosa AL98, is a potent degrader of natural rubber and synthetic cis-1,4-polyisoprene

Alexandros Linos , Rudolf Reichelt , Ulrike Keller , Alexander Steinbüchel
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb08890.x 155-161 First published online: 1 January 2000

Abstract

A Gram-negative bacterium, strain AL98, was isolated from foul water inside of a deteriorated car tire on a farmer's field in Münster, Germany. The strain was able to considerably disintegrate natural rubber (NR), either in the raw state as NR latex concentrate or in the vulcanized state as NR latex glove, as well as raw synthetic cis-1,4-polyisoprene (IR). Determination of carbon dioxide evolution and living cell number during batch cultivation with each of the materials as sole source of carbon, revealed mineralization of the rubber polymer during biomass increase. Surface investigation by scanning electron microscopy gave evidence for an adhesive growth behavior of the strain proceeding by colonizing the rubber surface, merging into the rubber and forming a biofilm prior to disintegration of the material. Schiff's reagent staining performed with NR latex gloves indicated production and accumulation of aldehyde groups during colonization. The solid glove substrate disappeared completely after a prolonged cultivation period as a result of continuous degradation. Taxonomic analyses of the strain, which were also based on similarity examination of the complete 16S rRNA gene, revealed classification of strain AL98 as a strain of Pseudomonas aeruginosa. This is the first report about the isolation of a Gram-negative bacterium exhibiting strong rubber decomposing properties.

Keywords
  • Biodegradation
  • Natural rubber
  • cis-1,4-polyisoprene
  • Latex gloves
  • Biofilm
  • Scanning electron microscopy
  • Pseudomonas aeruginosa

1 Introduction

Raw natural rubber (NR) for technical applications is obtained as latex from the rubber tree Hevea brasiliensis. More than 90% of the dry weight of latex consists of cis-1,4-polyisoprene (IR) (Mw >106 Da). Prior to conversion into rubber products it undergoes further processing, like concentration, mastication and vulcanization, i.e. crosslinking of the polymer chains. IR can also be synthesized chemically thereby improving, but not replacing, all properties of the natural product. Both kind of polyisoprene rubbers are known to be susceptible towards microbial action [13]. Most NR degrading bacteria were identified as members of the group of actinomycetes [410]. There is only one report about treatment of NR latex with the extracellular crude extract of a Gram-negative bacterium, a Xanthomonas sp., leading to the formation of oligomers with Mw's between 103 and 104 Da [11]. However, the ability of this strain to colonize and to decompose solid rubber was rather poor [12].

In this communication, the isolation of the rubber degrading strain AL98, which was identified as a representative of the species Pseudomonas aeruginosa, is reported, indicating that not only actinomycetes are potent degraders of NR and IR. Since members of this Gram-negative species are physiologically and genetically well characterized, this strain may offer advantages for the elucidation of the physiological and genetic causes of rubber degradation.

2 Materials and methods

2.1 Rubbers

NR latex concentrate (Neotex Latz) was obtained from Weber and Schaer (Hamburg, Germany) and IR (SKI3) from Continental AG (Hannover, Germany). NR latex gloves (rotiprotect™) were purchased from Roth (Karlsruhe, Germany).

2.2 Cultures

Liquid cultures were carried out in 300-ml Erlenmeyer-flasks containing a mineral salts medium (MSM), as previously described [13]. NR latex concentrate was added at 0.8% (v/v) to 30 ml MSM, which corresponds to 0.5% dry matter (w/v) considering the specifications of the supplier. NR latex gloves were cut into small pieces of 0.15 g, and each one was added without further treatment to 30 ml MSM. IR was cut into small pieces of about 2–3 mm in diameter, extracted with acetone (1–2 days), and 0.15 g were added to 30 ml MSM. Sterilization by heat prior to inoculation was done in an autoclave.

For scanning electron microscopy (SEM), IR-cultures were prepared as follows: 3 g of acetone extracted IR rubber were dissolved in 100 ml chloroform to give a 3% IR solution. Thin rectangular pieces of aluminum with a surface area of about 1 cm2 were dipped into this solution and dried in a stream of air. This procedure was repeated until both sides of the aluminum pieces were coated completely with IR material. The coated pieces were sterilized with 96% ethanol and added to 30 ml of already autoclaved MSM solution in 300-ml flasks. Cells were precultivated over night at 30°C in 10 ml Luria-Bertani (LB) complex medium, washed twice with saline solution, resuspended in 1 ml saline, and 10 μl were inoculated to the rubber containing cultures. The inoculated flasks were incubated at 30°C and shaken at 180 rpm.

Solid cultures were performed in Petri dishes containing either LB agar or MSM latex agar. Thereby two kind of latex agar plates were used: (1) latex film plates, prepared by spreading the latex concentrate as a thin film directly on MSM agar, and (2) latex overlay plates, prepared by pouring a thin layer of MSM agar containing dispersed NR latex concentrate at a concentration of 0.02% (w/v) above the MSM agar containing no carbon source. Incubation of the inoculated plates was done at 30°C.

Substrate utilization pattern was carried out by using the BBL Oxi/Ferm Tube II bacterial identification system (Becton Dickinson, Cockeysville, MD, USA) as specified by the manufacturers.

2.3 Mineralization

Measurement of mineralization was performed in airtight 500-ml Erlenmeyer-flasks by determining the CO2 release after different cultivation periods, as described recently [9]. The amount of rubber substrate provided comprised in all cases 0.25 g in 50 ml MSM.

2.4 Staining with Schiff's reagent

Staining of NR latex gloves with Schiff's reagent was recently shown [14]. The analogous procedure applied here was as follows: In a tightly stoppered bottle, 10 ml of the fuchsin reagent was added to a sample, and the purple color was developed for 10–30 min at room temperature. Excess amount of the reagent was then discarded, and 10 ml of the sulfite solution was added in order to suppress the non-specific color reaction of the blank sample. The composition of the fuchsin reagent [15] was the following: 2 g fuchsin dissolved in 50 ml glacial acetic acid plus 10 g Na2S2O5 plus 100 ml 0.1 N HCl plus 50 ml H2O. The composition of the sulfite solution was: 5 g Na2S2O5 plus 5 ml concentrated HCl (37–38%), ad 100 ml with H2O.

2.5 Scanning electron microscopy

NR-latex gloves and IR-coated thin aluminum pieces were taken from liquid cultures at varying cultivation periods, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PBS; pH 7.3) according to Sørensen [16]. After washing with PBS, the cultures were postfixed in 1% osmium tetroxide in 0.1 M PBS (pH 7.3) and dehydrated in graded ethanol (30%, 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 (SEM; Hitachi Ltd., Japan) with secondary electrons at 20 kV acceleration voltage and at room temperature. Micrographs were recorded from a high-resolution cathode-ray tube using negative film (Agfapan, APX 100).

2.6 Analysis of 16S rDNA

Extraction of genomic DNA was carried out as described previously [17]. Amplification of the 16S rRNA gene was performed using oligonucleotides as primers as described in [18]. Nucleotide sequences of the purified PCR product were determined with a 4000L DNA sequencer (LI-COR Inc., Biotechnology Division, London, NE, USA) and a Thermo Sequenase fluorescence-labelled primer cycle-sequencing kit (Amersham Life Science, Little Chalfont, UK) as specified by the manufacturers employing the primers described in [18]. The 16S rDNA sequences were aligned manually with published sequences from representatives of pseudomonads obtained from EMBL.

3 Results

3.1 Isolation of strain AL98

Environmental samples from foul water inside of deteriorated car tires on a farmer's field in Münster, Germany, were collected and subsequently screened for the occurrence of rubber degrading bacteria. The isolation of two novel actinomycetes belonging to the genus Gordonia (former Gordona) after enrichment on granular tire material was previously reported [9]. One of the samples was enriched on NR latex concentrate in liquid culture that showed a substantial increase in turbidity and a considerable disintegration of the solid latex material that was initially coagulated completely to one clump under the chosen cultivation conditions (shaking at 180 rpm in MSM). This enrichment culture was subsequently tested for growth on latex film plates. Overnight incubation revealed greenish areas on the latex film. Further enrichment from such areas on LB plates revealed single colonies of a bacterial isolate, designated as strain AL98, that formed greenish colonies on LB plates after 1 day and also led to a dark greenish staining of the LB agar after 2 days. Growth on latex film plates occurred as a biofilm without classical colony formation causing greenish staining of both, the latex material that was in direct contact to the cells and also the MSM agar. This greenish color turned to a faint reddish color during prolonged incubation (>1 week); this was accompanied by a clearing of the latex film in the biofilm area. No growth of the strain could be determined on latex overlay plates.

3.2 Taxonomic classification

Strain AL98 was a motile, Gram-negative, oxidase-positive and catalase-positive rod-shaped bacterium (0.5 μm by 2 to 3 μm). Due to these characteristics, identification analysis by the BBL Oxi/Ferm Tube II system was performed. Thereafter, substrate utilization pattern revealed the following results: growth on arginine, N2, xylose, glucose (aerobic), urea and citrate; no growth on glucose (anaerobic), lactose, sucrose, indole, maltose, mannitol and phenylalanine; indistinct reaction for lysine. The obtained number code resulted in a classification to P. aeruginosa, irrespective of the result of the lysine reaction.

Further taxonomic characterization of strain AL98 was carried out by analysis of the 16S rRNA gene. The almost complete gene was sequenced consisting of 1530 nucleotides. According to the results of the EMBL data base searching, the sequence revealed highest similarities ranging from 99.9% to 100% to several P. aeruginosa strains listed. The next highest similarities were 98.7% to Pseudomonas resinovorans and 97.7% to Pseudomonas alcaligenes. According to Moore et al. [19]P. aeruginosa strains possess a 32-bp region in their 16S rDNA, corresponding to E. coli positions 66–103, which is hypervariable among different species of the genus Pseudomonas, but 100% conserved among P. aeruginosa strains. Exactly the same sequence, which is typical to P. aeruginosa, was also determined for strain AL98 at the positions 58–89. The 16S rRNA gene sequence data have been submitted to the EMBL nucleotide sequence data base and are listed under the accession no. AJ249451. Strain AL98 was deposited in the culture collection of the Institut für Mikrobiologie, Münster, Germany, as P. aeruginosa AL98.

3.3 Disintegration of natural rubber

Pure cultures of P. aeruginosa AL98 were obtained from LB plates after several passages on this medium and tested for growth on NR latex concentrate. Visible disintegration of the coagulated rubber started only after 2–3 weeks of incubation. In contrast, when AL98 cells from latex cultures were used as inoculum, disintegration of the material already started after 2 days. If these ‘adapted cells’ were subsequently subjected to several passages on LB plates, again a delayed start of the rubber disintegration process was observed. It was generally observed that growth was even more delayed the more passages on LB medium were performed. Simultaneous observation by light microscopy revealed in any case only one type of motile rods. However, washed cells from adapted cultures tended generally to adsorb on the walls of Eppendorf cups or glass tubes used for washing in contrast to cells from LB cultures, which were microscopically identical to cells from latex culture, but did not show any adhesive properties. Growth tests performed with several other strains of P. aeruginosa from different culture collections, such as PAO1, DSM 50071, DSM 939 and ATCC 27853, revealed no growth on natural rubber and exhibited even a decrease in living cell number during incubation on media containing rubber (data not shown).

3.4 Growth on cis-1,4-polyisoprene

Several IR containing materials were used as sole carbon sources for determining the ability and the extent of growth of P. aeruginosa AL98 on rubbers: NR-latex concentrate, which contains >90% dry weight of IR, NR latex gloves, which correspond to vulcanized NR, i.e. with crosslinks between the polymer chains, as well as raw synthetic IR. In all cases growth was determined with adapted cells. Fig. 1A refers to the time course of mineralization of the rubber substrates. For evaluation of the CO2-release, it was assumed that the employed rubbers consisted totally of carbon. After 6 weeks, best mineralization was detected with NR latex concentrate (36%), followed by NR latex gloves (26%) and IR (21%). In addition, also increases of the biomass of the same cultures were detected for all three substrates as shown in Fig. 1B showing the time course of living cell count during incubation on these substrates. In spite of the preferentially adhesive growth behavior, an increase in the number of cells suspended in the medium during cultivation with NR latex concentrate (35-fold), NR latex glove (11-fold) as well as IR (seven-fold) occurred.

1

Cultivation of P. aeruginosa AL98 on the IR containing rubbers NR latex concentrate (●), NR latex glove (○) and IR (▾). A: Time course of mineralization expressed as % CO2 released from total carbon. B: Increase of the number of living suspended cells during cultivation. Values are means of triple measurements.

3.5 SEM and other analysis of rubber surfaces

The colonization behavior and biofilm formation of P. aeruginosa AL98 on NR latex glove material were investigated by SEM (Fig. 2). Compared to a non-inoculated control (Fig. 2A), the rubber surface was completely covered by a biofilm after a cultivation period of 6 weeks (Fig. 2B). Fig. 2C shows cells merging into the rubber material, which occurred after 2 weeks, whereas after 6 weeks incubation additional disintegration of the material became visible (Fig. 2D).

2

Secondary electron micrographs showing growth of P. aeruginosa AL98 on NR latex gloves. A: Non-inoculated control showing rubber surface. B: Biofilm formed at the rubber surface after 6 weeks. C: Details from growth after 2 weeks. D: Details from growth after 6 weeks. Bars correspond to 5 μm.

Biofilm formation was also demonstrated after 6 weeks by staining the overgrown latex gloves with Schiff's reagent. The purple color produced by the reagent provided evidence that degradation products containing aldehyde groups were produced and accumulated during the microbial degradation, as was recently reported by Tsuchii [14].

Complete disintegration of the glove material was also observed after prolonged cultivation periods of more than 3 months. First experiments to optimize this disintegration process resulted in a much accelerated degradation of gloves, when MSM was replaced from time to time by fresh MSM during liquid batch cultivation, indicating that semi-continuous culturing is a possible strategy for enhancing degradation rate, as previously discovered for a rubber degrading Nocardia strain [20].

Growth of P. aeruginosa AL98 on IR is shown in the SEM micrographs of Fig. 3. Biofilm formation was already visible after 1 week of cultivation. Fig. 3B demonstrates AL98 rods colonizing the rubber surface, and Fig. 3C shows cells merging into the rubber and the strong contacts between the cells by means of pilli. Finally, Fig. 3D illustrates a region of the biofilm formed after 4 weeks on the IR surface.

3

Secondary electron micrographs showing growth of P. aeruginosa AL98 on synthetic IR. A: Non-inoculated control showing rubber surface. B: Colonization of the rubber surface after 1 week. C: Details from colonization after 1 week. D: Biofilm formed at the rubber surface after 4 weeks. Bars correspond to 50 μm (D), 5 μm (A,B), and 0.5 μm (C), respectively.

4 Discussion

Screening procedures for the isolation of rubber degrading bacteria led to the pure culture of the Gram-negative bacterium AL98 with enhanced capability to disintegrate raw and vulcanized natural rubber as well as synthetic IR. Taxonomic studies including 16S rRNA analysis revealed this bacterium as a member of the species P. aeruginosa. These findings provide evidence that beside actinomycetes also Gram-negative bacteria are potent degraders of NR and IR. Taxonomic classification of strain AL98 to P. aeruginosa, which is one of the best studied bacterial species, offers advantages with respect to elucidate the physiological and genetic basis of rubber degradation, since rubber degrading bacteria belonging to the group of actinomycetes are almost not accessible to genetic methods.

P. aeruginosa AL98 exhibited adhesive growth towards solid rubbers in an analogous manner like recently shown for some actinomycetes [9,14]. Similar to these bacteria, strain AL98 expressed strong rubber decomposing properties and did not produce any clearing zones (translucent halos) on latex overlay plates like other weaker rubber decomposers isolated previously [4,5,8].

The observed decrease of rubber degrading property after successive transfer on LB nutrient medium reveals that this ability of P. aeruginosa AL98 is not stable. On the other hand, rubber degrading activity could be restored again after long time adaptation on rubber substrate. The reason for this is still unknown and has to be examined in detail. It is possible that AL98 harbors plasmids encoding the genetic information for rubber degradation, which is lost after successive cultivation on complex medium. However, first trials to isolate putative degradation plasmids from adapted AL98 cultures by the alkaline lysis method according to [21] gave no indication for this. On the other hand, SEM studies of IR colonization revealed that there was a strong contact between cells by pili (Fig. 3C), so that also a transfer of genetic information from one cell to another could theoretically occur.

Staining with Schiff's reagent revealed that aldehyde groups were formed at the rubber surface during degradation. This finding corresponds well to the results obtained by an analogous examination of a previously described Nocardia strain [14]. Earlier studies with this isolate showed that oligomers containing carbonyl groups at their ends were formed during degradation indicating oxidative scission of the IR chain at the double bonds [6]. Further examination must clarify whether this scheme is also suitable for P. aeruginosa AL98. During preliminary studies, in which growth of adapted AL98 cells on low-rank coal as sole carbon source consisting of lignin like structures [22] was tested, an almost eight-fold increase of the living cell number of suspended cells was observed after a cultivation period of 10 weeks (data not shown). Production of biomass from this substrate also implies oxidative degradation of the lignin-like polymer.

Acknowledgments

We would like to thank Mr. Mahmoud M. Berekaa for his help during preparation of IR-substrate for SEM. Provision of the description of the method for the staining with Schiff's reagent by Dr. Akio Tsuchii from the National Institute of Bioscience and Human-Technology, Higashi, Tsukuba, Ibaraki, Japan, is gratefully acknowledged. Furthermore, the authors appreciate the expert photographic work of Mrs. G. Kiefermann from the Institut für Medizinische Physik und Biophysik.

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