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1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149, Germany
2 Institut für Botanik und Botanischer Garten, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, D-48149, Germany
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences reported in this paper are DQ840025 (N. takedensis WE30), DQ840027 (N. jiangxiensis SM1), DQ840026 (N. nova L1b), DQ840028 (N. nova SH22a), DQ840029 (N. nova SEI2b) and DQ840030 (N. nova SEII5a).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and is an industrially important polymer used for many technical applications such as fabrication of automobile tyres. The trans isomer (gutta percha, GP) is synthesized by much fewer plants and occurs, for example, in South-east Asian trees Palaquium gutta and Eucommia ulmoides, the European shrub Euonymus europaeus and the South American tree Couma macrocarpa. GP is used for several applications due to its resistance to biological degradation. This polymer has been used since the last century as an insulation material for transatlantic telegraph cables, and it is still used for the production of conveyers, golf balls, decoration objects, chewing gum and in dentistry to fill root canals.
First investigations on the biodegradation of NR were carried out in the early 20th century (Söhngen & Fol, 1914; for a review, see Rose & Steinbüchel, 2005), but only recently have two genes encoding enzymes capable of poly(cis-1,4-isoprene) cleavage been isolated, independently from different bacteria. In the NR-degrading bacterium Xanthomonas sp. 35Y, RoxA was identified (Braaz et al., 2004) which acts as a dioxygenase on poly(cis-1,4-isoprene), resulting in the formation of short-chain isoprenoid intermediates. Lcp (Latex-clearing protein) was identified in Streptomyces sp. K30, and the lcp gene restored the wild-type phenotype in a chemically induced rubber-negative mutant of this bacterium lacking the ability to degrade NR as indicated by the absence of haloes if grown on NR latex-containing agar plates (Rose et al., 2005). Intermediates resulting from cleavage of poly(cis-1,4-isoprene) by actinomycetes harbouring Lcp have been identified as isoprenoids with approximately 20 isoprene units (Ibrahim et al., 2006). Apart from the number of isoprene moieties of the cleavage products, intermediates of poly(cis-1,4-isoprene) degradation produced by RoxA and Lcp exhibit a similar chemical structure, possessing one terminal ketone and one terminal aldehyde functional group. However, RoxA and Lcp share no sequence homologies, indicating two individually evolved enzyme systems for rubber cleavage. So far no proteins homologous to RoxA have been identified in bacteria other than Xanthomonas sp. 35Y, whereas the presence of genes encoding Lcp homologues has been shown in the genomes of several actinomycetes capable of rubber degradation. In Streptomyces sp. K30, two ORFs (oxiA and oxiB), putatively encoding a heterodimeric molybdenum-dependent aldehyde dehydrogenase, are located downstream of lcp. Recombinant strains of Streptomyces lividans TK23 expressing lcp and oxiBA do not accumulate aldehydes around the colonies if grown on NR latex as was observed for strains expressing only lcp. As revealed by transposon-induced rubber-negative mutants of Gordonia polyisoprenivorans, degradation intermediates of poly(cis-1,4-isoprene) enter the central metabolism via 
-oxidation with the involvement of a specific 
-methylacyl-coenzyme A racemase (Banh et al., 2005).
In contrast to NR degradation, no micro-organisms capable of degrading poly(trans-1,4-isoprene) have been described to date. In addition, only very few studies have been published on microbial degradation of GP. Kupletskaya et al. (1960) tried to demonstrate biodegradability of GP by fermentation studies of Eucommia leaves; however, the micro-organisms isolated were incapable of degrading chemically pure poly(trans-1,4-isoprene). The weight losses of blends of poly(trans-1,4-isoprene) and starch observed by Arvanitoyannis et al. (1998) were solely due to degradation of the starch. So far, cleavage of either isomer of polyisoprene has only been demonstrated in vitro employing enzyme-mediator systems like lipoxygenase/linoleic acid or laccase/1-hydroxybenzotriazole/linoleic acid as radical producers (Enoki et al., 2003). This study aimed to isolate and characterize axenic cultures of poly(trans-1,4-isoprene)-degrading bacteria and to compare microbial degradation of GP and NR.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Cells of G. polyisoprenivorans VH2, Nocardia sp. and Rhodococcus opacus PD630 were cultivated at 30 °C in Standard I nutrient broth (St-I; Merck), Caso medium (Merck) or mineral salts medium (MSM) as described by Schlegel et al. (1961). The following polyisoprenoides were added as carbon sources to liquid MSM: 0.6 % (w/v) cryomilled synthetic poly(trans-1,4-isoprene) with a mean molecular mass of approximately 410 kDa (Aldrich) or 0.6 % (w/v) milled synthetic poly(cis-1,4-isoprene) with a mean molecular mass of 800 kDa (Aldrich). Synthetic poly(cis-1,4-isoprene) was milled in the presence of inert talcum (3MgO.4SiO2.H2O) in the ratio 1 : 1 to prevent coagulation of the polymer particles and was added before autoclaving, whereas poly(trans-1,4-isoprene) was frozen in liquid nitrogen before cryomilling in a ZM200 rotation mill (Retsch), yielding particles with grain sizes between 62 µm and 1.4 mm. GP was sterilized with 70 % (v/v) ethanol, added after autoclaving and subsequently evaporated in a vacuum. For mineralization experiments, poly(trans-1,4-isoprene) and poly(cis-1,4-isoprene) with a defined range of grain size was obtained by sieving in an AS200 sieve shaker (Retsch).
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); GP latex from the South American tree Couma utilis was applied accordingly.
Enrichment and isolation of GP-degrading bacteria.
Soil and plant samples were taken from different habitats of autochthonous Euonymus europaeus populations in Germany and also from different areas with Couma macrocarpa populations in the Amazon rainforest in Brazil. Samples were incubated in 50 ml MSM medium containing 0.6 % (w/v) cryomilled poly(trans-1,4-isoprene). If growth was macroscopically detected by an increase in turbidity, several strains of the respective cultures were brought to axenic cultures on solid St-I medium. The strains obtained were then individually tested in liquid culture for their capability to degrade synthetic poly(trans-1,4-isoprene). Purity of cultures capable of GP degradation was verified both by smearing on St-I medium and by direct determination of 16S rRNA gene sequences (see below).
Taxonomic studies.
Gram-staining was performed according to Gerhardt et al. (1994). Bactident oxidase test strips were applied for oxidase testing (Merck). Catalase activity was determined by adding 3 % (v/v) H2O2 to a freshly grown colony. Cleavage of L-alanyl-nitroanilid was tested by applying Bactident LAAP test strips (Merck).
Isolation of total DNA.
A sample (1.5 ml) of a well grown culture in MSM containing poly(trans-1,4-isoprene) was harvested by centrifugation and resuspended in 500 µl SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris/HCl, pH 7.5) containing 3 mg lysozyme ml–1. After 2 h incubation at 37 °C, 50 µl of an SDS solution (100 g l–1) and 20 µl of a proteinase K solution [20 g (l TE)–1; TE, 1 mM EDTA and 10 mM Tris/HCl, pH 8.0] were added and mixed gently, followed by another incubation for 2 h at 55 °C. Subsequently, 0.33 vols of a 5 M NaCl solution and 1 vol. chloroform were added and mixed gently. After 30 min incubation at room temperature, the phases were separated by centrifugation and the aqueous phase was transferred to a fresh tube. DNA was then precipitated by the addition of 0.7 vols 2-propanol and the precipitate was washed twice with 70 % (v/v) ethanol. After drying it was dissolved in an appropriate volume of TE buffer containing 0.01 % (v/v) RNase A solution (10 mg ml–1).
Determination of 16S rRNA gene sequence.
Amplification of 16S rRNA genes was done by PCR using Taq DNA-polymerase (Invitrogen) and the primers 27f (5'-GAGTTTGATCCTGGCTCAG-3') and 1525r (5'-AGAAAGGAGGTGATCCAGCC-3'). Purification of the PCR product was carried out as described previously (Rainey et al., 1996). The purified 16S rRNA gene was then directly sequenced using the following primers: 27f (5'-GAGTTTGATCCTGGCTCAG-3'), 343r (5'-CTGCTGCCTCCCGTA-3'), 357f (5'-TACGGGAGGCAGCAG-3'), 519r [5'-G(T/A)ATTACCGCGGC(T/G)GCTG-3'], 536f [5'-CAGC(C/A)GCCGCGGTAAT(T/A)C-3'], 803f (5'-ATTAGATACCCTGGTAG-3'), 907r (5'-CCGTCAATTCATTTGAGTTT-3'), 1114f (5'-GCAACGAGCGCAACCC-3'), 1385r [5'-CGGTGTGT(A/G)CAAGGCCC-3'] and 1525r (5'-AGAAAGGAGGTGATCCAGCC-3') applying the Big Dye 3.1 kit and a 3730 DNA capillary sequence analyser (Applied Biosystems). Sequences were aligned manually with published sequences from representative actinomycetes obtained from EMBL. BLASTN was used to determine the percentage of identical nucleotides to 16S rRNA gene sequences in the GenBank database. Phylogenetic trees were constructed by applying CLUSTAL X software (www-igbmc.u-strasbg.fr/BioInfo/; Thompson et al., 1997) using the neighbour-joining method (Saitou & Nei, 1987) and were visualized by NJPLOT (ftp://pbil.univ-lyon1.fr/pub/mol_phylogeny/njplot; Perrière & Gouy, 1996).
Determination of percentage mineralization.
Evidence for biodegradation of the poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) hydrocarbon chains to CO2 was obtained by determination of respiratory CO2 released during cultivation of cells in the presence of polyisoprene as sole carbon source. Determination was carried out in tightly closed Erlenmeyer flasks by using the property of Ba(OH)2 to precipitate CO2 as BaCO3. The flasks, containing 100 ml MSM medium, 0.6 % milled poly(cis-1,4-isoprene) or poly(trans-1,4-isoprene), and a test tube, containing 15 ml of a 0.2 M Ba(OH)2 solution, were inoculated with 25 or 40 mg cells (fresh wt) of a culture grown for 48 h. At each measuring point, the flasks were aerated, and the test tubes were replaced by new tubes containing fresh Ba(OH)2 solution. Consumption of HCl for precipitation of 
as BaCO3 was determined for each period by titration with 0.1 M HCl according to the following equation, yielding the percentage mineralization.
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As indicator, phenolphthalein was added (20 µl of a 1 %, w/v, solution in 2-propanol) and the end point of titration was determined by alteration of the colour from magenta to uncoloured. A non-inoculated Erlenmeyer flask was handled in the same way as control.
Scanning electron microscopy (SEM).
Deterioration of samples of poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) by selected strains was verified by SEM. A sample (40 ml) of a 1 % solution of poly(trans-1,4-isoprene) in chloroform was placed into a glass Petri dish to obtain a thin polymer film after evaporation of the solvent. The film was then cut into pieces. Poly(cis-1,4-isoprene) was applied as cubic pieces with a side length of about 1 cm. MSM cultures (200 ml) containing 1 g milled GP plus thin GP squares or 1 g milled poly(cis-1,4-isoprene) plus the cubes of poly(cis-1,4-isoprene) were inoculated with 70 mg cell fresh weight of the respective strain grown for 48 h in St-I medium. The polymer pieces were withdrawn from their culture vessel after the incubation periods indicated in the text and were then washed with distilled water or used directly for SEM. Samples were gold-sputtered with an Emitech vacuum sputter device K550x (Ashford) and examined with a Hitachi S-3000N scanning electron microscope at 20 kV under high vacuum conditions. Micrographs were recorded digitally.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Numerous enrichment cultures yielded only 13 poly(trans-1,4-isoprene)-degrading strains in total. Some strains, which were isolated from different enrichment cultures but inoculated with the same environmental sample, turned out to be identical, as revealed by 16S rRNA gene sequencing. Thus, only six strains, differing in their 16S rRNA gene sequences, were obtained and subsequently characterized. Five of these different strains were isolated from cultures inoculated with samples received from C. utilis populations in the Amazon rainforest in Brazil, and only one strain was isolated from a culture inoculated with a soil sample from an autochthonous E. europaeus population in Germany (for details, see Table 1
). No bacterial strains capable of GP degradation could be isolated from samples taken from autochthonous E. europaeus populations located near the villages Wustrow, Trochel, Freude and Bleckede (Germany), from a wastewater treatment plant (Münster, Germany), from a composting plant (Münster, Germany) and from more than 30 samples taken from unassigned locations in the Amazon rainforest in Brazil. All newly isolated bacterial strains colonized the surface of the GP grains in liquid cultures (data not shown). Later it was shown that the isolates grew adhesively on poly(cis-1,4-isoprene) particles as has been described for species of Gordonia and Nocardia (Arenskötter et al., 2004; Ibrahim et al., 2006). These strains exhibited no clear zone formation when cultivated on overlay agar plates prepared from GP or NR latex.
Taxonomic classification
In liquid MSM cultures containing GP as sole source of carbon and energy, all strains exhibited coccoid growth, and strains WE30, L1b and SH22a also formed mycelia on solid complex St-I or Caso medium. Growth was clearly reduced at temperatures above 30 °C and no growth was observed at temperatures higher than 37 °C. All strains were Gram-positive and non-motile; no spores could be detected. Oxidase and LAAP tests were negative for all strains, but the catalase test demonstrated enzyme activity.
The sequences of complete 16S rRNA genes were determined as described in Methods to characterize the taxonomic position of the novel isolates in more detail. The new sequences were aligned with sequences deposited in GenBank to determine closely related species. The 16S rRNA gene sequence of N. takedensis WE30 shared highest similarity (99.9 %) with that of Nocardia takedensis DSM 44801T which was previously isolated from moat sediment and activated sludge (Yamamura et al., 2005). Isolate WE30 is therefore referred to as N. takedensis WE30. The 16S rRNA gene sequences of the isolates L1b, SH22a, SEI2b and SEII5a exhibited highest similarities (99.8, 98.9, 98.7 and 99.5 %, respectively) to that of Nocardia nova DSM 44481T (Wallace et al., 1991). These strains are therefore referred to as N. nova strains L1b, SH22a, SEI2b and SEII5a, respectively. The 16S rRNA gene sequence of strain SM1 shared highest similarities (99.3 %) with that of Nocardia jiangxiensis DSM 17684T (Cui et al., 2005). This isolate is therefore referred to as N. jiangxiensis SM1. The phylogenetic tree shown in Fig. 1 illustrates the relationship between the novel isolates to all validly described type strains of the genus Nocardia. Since 16S rRNA analysis identified N. takedensis DSM 44801T as the type strain with the closest relationship to N. takedensis WE30, this strain was also investigated for its ability to degrade GP.
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(a) clearly illustrates a correlation between the size of the supplied granules and the extent of GP degradation by N. takedensis WE30. If smaller particles were provided, mineralization proceeded more rapidly when compared to cultures with larger granules. In cultures with GP grains of 125–250 µm, 40 % of the substrate was metabolized to CO2 within 78 days, while only 25 % of the poly(trans-1,4-isoprene) granules of 1250–1400 µm was mineralized in the same period by N. takedensis WE30. This indicated that only the surface of the material is accessible to microbial attack, as is usually the case with solid biodegradable materials.
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). The type strain of N. takedensis mineralized only about 20 % of the GP during the same period, and the slowest rate of degradation was observed with N. jiangxiensis SM1 which released only about 3 % of the poly(trans-1,4-isoprene) as CO2 (Fig. 2b). Mineralization of poly(trans-1,4-isoprene) by cultures of G. polyisoprenivorans VH2 and R. opacus PD630 was also measured as controls. G. polyisoprenivorans VH2 has been characterized as a potent degrader of poly(cis-1,4-isoprene) by Linos & Steinbüchel (1998), but was incapable of degrading poly(trans-1,4-isoprene) (Fig. 2b). Also, R. opacus PD630 did not degrade poly(trans-1,4-isoprene) as indicated by mineralization studies (Fig. 2b). In addition, no growth on poly(trans-1,4-isoprene) could be detected macroscopically for these two bacteria.
In the past, numerous bacterial strains have been described that degrade both synthetic poly(cis-1,4-isoprene) and NR (Jendrossek et al., 1997; for review, see Rose & Steinbüchel, 2005); however, these strains do not degrade the trans isomer GP as revealed from a laboratory screening of several of them (unpublished data). In this study we decided to clarify if the novel GP-degrading strains and N. takedensis DSM 44801T also are capable of degrading poly(cis-1,4-isoprene). Therefore, all six poly(trans-1,4-isoprene)-degrading strains and N. takedensis DSM 44801T were tested for their ability to utilize poly(cis-1,4-isoprene) as a source for carbon and energy, and degradation was compared by mineralization experiments to that of G. polyisoprenivorans VH2 (Fig. 2c). All the novel Nocardia strains and N. takedensis DSM 44801T were indeed able to utilize poly(cis-1,4-isoprene) very efficiently and metabolized this polymer for the most part even more rapidly than G. polyisoprenivorans VH2. The highest degree of mineralization after 78 days was obtained with N. nova SH22a (43 %). N. nova L1b metabolized only 34 % of the poly(cis-1,4-isoprene) to CO2, but was also more efficient than G. polyisoprenivorans VH2 (25 %). However, the values for mineralization of poly(cis-1,4-isoprene) by the novel GP-degrading strains diverged to a lesser extent than observed for their mineralization of the trans isomer.
Visualization of GP and rubber degradation by SEM
Films of GP and cubes of synthetic poly(cis-1,4-isoprene) were incubated for 1, 3 and 5 weeks with cells of N. takedensis WE30 and N. nova L1b, and were then examined by SEM to observe the degradation of poly(trans-1,4-isoprene) and poly(cis-1,4-isoprene) visually in a chronological sequence (Figs 3 and 4). GP films incubated with cells of N. takedensis WE30 exhibited a few cavities with diameters of approximately 10 µm after only 1 week (Fig. 3a). After 3 weeks incubation, approximately 20 cavities mm–2 with diameters of about 30 µm were observed (Fig. 3b). After 5 weeks incubation, the surfaces of the GP films had almost totally deteriorated; cavities were confluent and separated cavities could hardly be distinguished (Fig. 3c); only a few holes with diameters of about 60 µm permeating the entire GP film were visible. This progression was also observed in cultures of N. nova L1b. In the samples withdrawn after 1 week incubation, signs of degradation were hardly visible by SEM (Fig. 3e), whereas after 3 weeks incubation, the material exhibited approximately 15 cavities mm–2 with diameters of about 20 µm (Fig. 3f). The numbers and diameters of the cavities continued to increase, and after 5 weeks incubation with N. nova L1b approximately 50 holes mm–2 with diameters of about 40 µm were observed (Fig. 3g). In contrast, GP films of a non-inoculated control exhibited no evidence of deterioration (Fig. 3d). Consequently, the appearance of cavities was clearly due to microbial attack of poly(trans-1,4-isoprene) by N. takedensis WE30 and N. nova L1b. In addition, the GP films exhibited no cavities or other signs of degradation when incubated with cells of G. polyisoprenivorans VH2 (Fig. 3h), thus confirming the inability of this poly(cis-1,4-isoprene)-degrading bacterium to degrade the trans isomer.
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. In the absence of bacterial cells (uninoculated control), the surface was not modified (Fig. 4a). In contrast, cells of G. polyisoprenivorans VH2 caused remarkable deterioration as indicated by the appearance of large cavities in the rubber (Fig. 4b), as already shown for this strain (Linos et al., 2000). The surfaces of the rubber particles were attacked to a similar extent by N. takedensis WE30 (Fig. 4c) and N. nova L1b (Fig. 4d), thereby confirming that these strains are also potent NR-degrading bacteria. However, despite the adhesive growth of all the isolates on GP and poly(cis-1,4-isoprene), cells were never visible in the scanning electron micrographs. This is probably due to the procedure of sample preparation and due to only very weak adhesion of the cells to the polymer materials.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Obviously, the growth of the micro-organisms in this case was only due to other compounds present in the leaves. Additionally, several bacterial strains well known as poly(cis-1,4-isoprene)-degraders cannot utilize the trans isomer of polyisoprene (unpublished data), and even purified RoxA, which is responsible for poly(cis-1,4-isoprene) cleavage by Xanthomonas sp. 35Y, was shown to be incapable of cleaving poly(trans-1,4-isoprene) in vitro (Braaz et al., 2004). However, GP as a natural compound ought to be biodegradable (Steinbüchel, 2005). This assumption led us to investigate if poly(trans-1,4-isoprene)-degrading bacterial strains could be isolated from the habitats of plants synthesizing GP. This is the first report of micro-organisms which are unequivocally capable of degrading GP. Although more than 100 environmental samples were investigated, bacterial growth could only be detected macroscopically in 6 enrichment cultures, each yielding one strain in axenic culture metabolizing poly(trans-1,4-isoprene). It is possible that cultivatable GP-degrading bacteria are endemic and occur only in habitats where poly(trans-1,4-isoprene) is present due to decaying material from plants synthesizing GP. GP-utilizing bacteria are obviously not ubiquitously distributed in nature, otherwise previous investigations should have succeeded in isolating poly(trans-1,4-isoprene)-degrading strains.
Interestingly, all strains isolated in this study were taxonomically assigned to the genus Nocardia based on their 16S rRNA gene sequences. Based on these data the isolates L1b, SH22a, SEI2b and SEII5a were assigned as strains of N. nova, isolate WE30 as a strain of N. takedensis and isolate SM1 as a strain of N. jiangxiensis. These GP-degrading bacteria belong therefore to the mycolic-acid-containing Actinobacteria [Corynebacterium, Mycobacterium, Nocardia (CMN) group]. All GP-degrading bacteria were also able to degrade NR. In this regard they behaved like most rubber-degrading bacteria representing the so-called ‘adhesively growing group’ of rubber degraders (Linos et al., 2000). In contrast to several NR-degrading streptomycetes, these GP-degrading bacteria do not produce translucent haloes on agar plates containing emulsified NR latex of Hevea brasiliensis or latex from the South American tree Couma utilis that contains both poly(trans-1,4-isoprene) and poly(cis-1,4-isoprene). Instead, they grew adhesively on both isomers if incubated in liquid cultures like all rubber-degrading species of the genus Gordonia do on NR (Arenskötter et al., 2004).
Mineralization experiments clearly revealed that the GP-degrading isolates, as well as N. takedensis DSM 44801T, metabolized synthetic poly(trans-1,4-isoprene), although they exhibited marked differences regarding the percentage of mineralization. Mineralization studies and SEM clearly revealed that the poly(cis-1,4-isoprene)-degrading bacterium G. polyisoprenivorans VH2 did not exhibit any detectable degradation of poly(trans-1,4-isoprene), and nor did the negative control R. opacus PD630. This confirmed previous observations that bacteria capable of degrading poly(cis-1,4-isoprene) do not degrade the trans isomer. In contrast, all poly(trans-1,4-isoprene)-degrading bacteria isolated described in this study and N. takedensis DSM 44801T were also able to degrade poly(cis-1,4-isoprene). Therefore, the ability to degrade GP always seems to occur in combination with the capability to degrade NR. In general, biodegradation of both the cis and trans isomers is a similarly slow process. It will be interesting to determine if GP-degrading bacteria possess one unspecific enzyme system for degradation of both isomers of polyisoprenoides, or if two separate enzyme systems exist in these bacteria: one responsible for degradation of the trans isomer and the other for the cis isomer. In the latter case, it should be possible to isolate bacteria capable of utilizing only poly(trans-1,4-isoprene) but not poly(cis-1,4-isoprene). Previous observations that biodegradation of poly(trans-1,4-isoprene) is a rare process are supported by the fact that none of the many poly(cis-1,4-isoprene)-degrading bacteria isolated so far is able to degrade poly(trans-1,4-isoprene).
All newly isolated strains capable of degrading poly(trans-1,4-isoprene) colonized the surface of the water-insoluble polymer during growth in liquid cultures, as has been described for taxonomically related bacteria that degrade the cis isomer. Therefore, the surface area of GP particles or the surface to volume ratio affect the percentage of mineralization since only the exposed surface is supposed to be accessible to the bacteria and their enzymes. This was clearly demonstrated with granule fractions of GP varying in grain size; the highest percentage of mineralization was observed with particles of the smallest grain size (125–250 µm). Therefore, to obtain reliable and comparable quantitative data on the degradation of poly(trans-1,4-isoprene) or on degradation by different micro-organisms, the granule size should be kept within a defined and narrow range.
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ACKNOWLEDGEMENTS |
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Edited by: J. D. Semrau
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Received 14 July 2006;
revised 20 September 2006;
accepted 31 October 2006.
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, 125–250; 
, 250–500; 
, 500–710; 
, 710–1000; 
, 1250–1400 µm) were inoculated with 40 mg cell fresh weight of isolate WE30. (b) Mineralization of GP by novel GP-degrading strains. Cultures (100 ml) in mineral salts medium containing 0.6 % (w/v) synthetic poly(trans-1,4-isoprene) (granule size, 250–500 µm) were inoculated with 25 mg fresh cell weight of the respective strains (
, L1b; 
, N. takedensis; 
, SH22a; 
, SM1), or of G. polyisoprenivorans VH2 (
