Abstract

Using natural rubber latex as the sole source of carbon and energy 50 rubber-degrading bacteria were isolated. Out of those 50 isolates, 33 were identified as Streptomyces species and 8 as Micromonospora species. Screening of 1220 bacteria obtained from different culture collections revealed 46 additional rubber-degrading bacteria (Streptomyces 31 strains, Micromonospora 5, Actinoplanes 3, Nocardia 2, Dactylosporangium 1, Actinomadura 1, unidentified 3). All rubber-degrading isolates were identified as members of the actinomycetes, a large group of mycelium-forming Gram-positive bacteria. Interestingly no Gram-negative bacterium could be isolated. In most strains expression of extracellular rubber-degrading enzymes was repressed by glucose and/or succinate. The reduction of the average molecular mass of solution-cast films of natural rubber from 640.000 to 25.000 in liquid culture upon bacterial growth indicates the participation of an endo-cleavage mechanism of degradation.

Introduction

Natural rubber (NR) is a macromolecular isoprenoid [poly(cis-1,4-isoprene)] and is synthesized by more than 2000 plant species mostly belonging to the Euphorbiaceae and by some fungi. Despite the development of chemosynthetic rubbers NR is still produced in large amounts (∼107 tons/year) from the rubber tree Hevea brasiliensis and is used for production of tyres, latex gloves, condoms, etc.

Degradation of NR was first studied by Söhngen and Fol [1], who used solution-cast films of NR as the carbon source for isolation of NR-degrading microorganisms. Spence and van Niel [2] developed a more sensitive clear zone technique by emulsifying NR latex in mineral agar resulting in an opaque medium. Growing on those media NR-degrading microorganisms form translucent halos around the colonies. This technique was used to isolate a few NR-degrading fungi and bacteria ([3, 4] and references cited therein). Evidence for the presence of an extracellular polyisoprene oxygenase, which specifically cleaves NR, was shown for a Xanthomonas strain, and acetonyl-diprenyl-acetaldehyde was identified as a low molecular mass degradation product [5]. Besides NR, chemically cross-linked (e.g. vulcanized) rubber can be also biodegraded slowly by microorganisms [6–8].

Despite many studies on microbial rubber degradation during the last 8 decades only very little is known about the distribution of NR-degrading bacteria and the biochemical mechanisms of NR degradation. In order to analyze the biological mechanism of polyisoprene degradation in detail we screened various culture collections and isolated and characterized a large number of NR-degrading bacteria from various ecosystems.

Materials and methods

Growth conditions

Routinely, a mineral medium (liquid or solidified with 1.5% agar) consisting of (g/l) Na2HPO4·12H2O (9.0), KH2PO4 (1.5), NH4Cl (1.0), MgSO4·7H2O (0.2), CaCl2·2H2O (0.02), Fe(III)[NH4]citrate (0.0012), trace element solution (10.000×, 0.1 ml) was used with carbon sources as indicated. This medium was supplemented with 0.05% yeast extract for the cultivation of Xanthomonas strains. Sugars and organic acids were sterilized by filtration or autoclaving, respectively. Hexadecane, hexane/octane mixture, isoprene, citronellol, citronellolic acid, farnesol or squalene was poured (100 μl each) onto a filter paper and put into the top of a petri dish. Poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyoctanoate) (PHO) and poly(6-hydroxyhexanoate) (polycaprolactone, PCL) were applied as a top layer on a mineral bottom layer as described earlier [9]. For purification of Streptomyces colonies a selective medium consisting of (g/l) starch (10), vitamin-free casein (0.3), KNO3 (2), NaCl (2), KH2PO4 (2), MgSO4·7H2O (0.01), CaCO3 (0.02), FeSO4 (0.05), pH (HCl) 7.0 was used. Color and development of aerial mycelium was followed on soy-mannitol medium (soy meal and mannitol, each 2%) and glucose-yeast extract-malt extract medium (each 0.5%). Growth temperature was 30°C.

Preparation of rubber latex

Freshly tapped latex of Hevea brasiliensis was obtained from the Rubber Research Institute of Malaysia. Latex was purified by centrifugation and resuspension of the cream in water to give a 5% rubber dry weight latex. For the preparation of solid media mineral plates were overlayed with 7 ml of the same medium supplemented with heat-sterilized latex (0.2% rubber dry weight) resulting in an opaque overlay.

Identification and isolation of rubber-degrading bacteria

Samples from various ecosystems were diluted with sterile mineral medium and vortexed for 2 min. 0.1 ml of the dilutions was spread on mineral plates with NR as the sole carbon source and incubated at 30°C. Colonies with translucent halos were purified by alternating transfers to complex media and NR mineral medium plates.

Taxonomic characterization of isolates

Fatty acid methyl esters were obtained from wet biomass (ca. 40 mg) by saponification, methylation and extraction and were separated by gas chromatography. Identity and composition of fatty acids were determined by the Microbial Identification System Library Generation Software (Microbial ID, Newark, DE, USA).

Results

Isolation of natural rubber-degrading bacteria

Thirty-three samples from different ecosystems in East Asia and Europe were screened for NR-degrading bacteria. In 30 samples NR-degrading bacteria were identified as indicated by (i) size of the colonies developing on solid media with purified NR latex as the sole source of carbon and energy in comparison to a control plate without NR and (ii) the appearance of translucent halos around the colonies. We could not isolate any NR-degrading bacteria from the sediment of an Asian river, from one of 15 different soil samples, and from a commercial compost. In most other cases we found NR-degrading bacteria even after 100- or 1000-fold dilution of soil suspensions. Screening of 1220 bacteria of culture collections led to the identification of 46 additional rubber-degrading bacteria (Streptomyces 31 strains, Micromonospora 5, Actinoplanes 3, Nocardia 2, Dactylosporangium 1, Actinomadura 1, unidentified 3) (Table 1).

1

Screening of culture collections for NR-degrading bacteria

Genus Number of strains tested Number of positives NR-degrading species DSM or Tüa 
Actinomadura 12 A. libanotica 43554 
Actinoplanes A. missouriensis 43046 
   A. italicus 43146 
   A. utahensis 43147 
Amycolatopsis   
Arthrobacter   
Bacillus   
Cellulomonas   
Corynebacterium   
Couchioplanes   
Dactylosporangium D. thailandense 43158 
Geodermatophilus   
Gordona 16   
Microbispora   
Micromonospora 38 Micromonospora sp. 43126 
   Micromonospora sp. 43170 
   Micromonospora sp. 43351 
   Micromonospora sp. 43426 
   Micromonospora sp. 43713 
Microtetraspora 12   
Mycobacterium 21   
Nocardia 32 N. brasiliensis 43112(Tü-69) 
   Nocardia sp. 43191 
Nocardiopsis   
Planomonospora   
Promicromonospora   
Pseudonocardia 37   
Rhodococcus 60   
Streptomyces 621 31 Streptomyces sp. 40296 
   Streptomyces sp. 40416 
   Streptomyces sp. 40441 
   Streptomyces sp. 40533 
   Streptomyces sp. 40566 
   Streptomyces sp. Tü-97 
   Streptomyces sp. Tü-1028 
   Streptomyces sp. Tü-1963 
   Streptomyces sp. Tü-2200 
   S. acrimycini Tü-42 
   S. albogriseus 40003 
   S. albadunctus 40478 
   S. antibioticus Tü-2 
   S. atroolivaceus 40137 
   S. aureocirculatus Tü-1471 
   S. daghestanicus 40149 
   S. flavoviridis 40153 
   S. fradiae Tü-39 
   S. griseus Tü 
   S. griseobrunneus 40066 
   S. griseoflavus 40456 
   S. griseoflavus (4 x) Tü-9, 15, 37, Tü-2043 
   S. griseoviridis 40229, Tü-430 
   S. nitrosporeus 40023 
   S. olivaceus Tü-1379 
   S. olivoviridis 40211 
   S. tauricus 40560 
Streptosporangium   
Sacharomonospora   
Saccharopolyspora   
Saccharothrix   
Thermomonospora   
Tsukamurella   
     
unclassified 83   
     
Acinetobacter   
Acetobacter   
Agrobacterium   
Azotobacter   
Azorhizobium   
Azoarcus   
Aquaspirillum   
Chromobacterium   
Comamonas   
Escherichia coli K12   
Methylobacterium   
Paracoccus   
Proteus vulgaris   
Pseudomonas 115   
Ralstonia/Alcaligenes 16   
Rhizobium   
Serratia marcescens   
Thiobacillus   
Xanthobacter 10   
Xanthomonas 45   
Zoogloea   
     
Total 1220 46   
Genus Number of strains tested Number of positives NR-degrading species DSM or Tüa 
Actinomadura 12 A. libanotica 43554 
Actinoplanes A. missouriensis 43046 
   A. italicus 43146 
   A. utahensis 43147 
Amycolatopsis   
Arthrobacter   
Bacillus   
Cellulomonas   
Corynebacterium   
Couchioplanes   
Dactylosporangium D. thailandense 43158 
Geodermatophilus   
Gordona 16   
Microbispora   
Micromonospora 38 Micromonospora sp. 43126 
   Micromonospora sp. 43170 
   Micromonospora sp. 43351 
   Micromonospora sp. 43426 
   Micromonospora sp. 43713 
Microtetraspora 12   
Mycobacterium 21   
Nocardia 32 N. brasiliensis 43112(Tü-69) 
   Nocardia sp. 43191 
Nocardiopsis   
Planomonospora   
Promicromonospora   
Pseudonocardia 37   
Rhodococcus 60   
Streptomyces 621 31 Streptomyces sp. 40296 
   Streptomyces sp. 40416 
   Streptomyces sp. 40441 
   Streptomyces sp. 40533 
   Streptomyces sp. 40566 
   Streptomyces sp. Tü-97 
   Streptomyces sp. Tü-1028 
   Streptomyces sp. Tü-1963 
   Streptomyces sp. Tü-2200 
   S. acrimycini Tü-42 
   S. albogriseus 40003 
   S. albadunctus 40478 
   S. antibioticus Tü-2 
   S. atroolivaceus 40137 
   S. aureocirculatus Tü-1471 
   S. daghestanicus 40149 
   S. flavoviridis 40153 
   S. fradiae Tü-39 
   S. griseus Tü 
   S. griseobrunneus 40066 
   S. griseoflavus 40456 
   S. griseoflavus (4 x) Tü-9, 15, 37, Tü-2043 
   S. griseoviridis 40229, Tü-430 
   S. nitrosporeus 40023 
   S. olivaceus Tü-1379 
   S. olivoviridis 40211 
   S. tauricus 40560 
Streptosporangium   
Sacharomonospora   
Saccharopolyspora   
Saccharothrix   
Thermomonospora   
Tsukamurella   
     
unclassified 83   
     
Acinetobacter   
Acetobacter   
Agrobacterium   
Azotobacter   
Azorhizobium   
Azoarcus   
Aquaspirillum   
Chromobacterium   
Comamonas   
Escherichia coli K12   
Methylobacterium   
Paracoccus   
Proteus vulgaris   
Pseudomonas 115   
Ralstonia/Alcaligenes 16   
Rhizobium   
Serratia marcescens   
Thiobacillus   
Xanthobacter 10   
Xanthomonas 45   
Zoogloea   
     
Total 1220 46   

aBacteria were obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (DSM) or from the culture collection of the corresponding author. ‘Tü’ refers to a culture collection of W. Wohlleben from the university of Tübingen (Germany).

Characterization of NR-degrading bacteria

The ability of the NR-degrading bacteria to use several low molecular mass monomers and high molecular mass polymers as carbon sources was tested (Table 2). Besides NR, all strains were able to utilize complex media such as nutrient broth (NB), Luria-Bertani broth (LB), soy mannitol as well as the polymers starch, gelatine, and chitin. Most strains were also able to hydrolyze poly-hydroxyalkanoates such as PHB and poly(6-hydroxyhexanoate) but not poly(3-hydroxyoctanoate). No growth was found for the NR-degrading bacteria with valerate, isoprene, farnesol, squalene, hexane, hexanoate, octane, crystalline cellulose (Avicel), or polybutadiene (poly(80%cis-/20%trans 1,4-butadiene)).

2

Phenotypic properties of NR-degrading bacteria

Strain Sourcea Species Natural rubber Hexadecan Citronellol PHB PCL Succinate Lactate Octanoate Glucose Fructose Arabinose Sucrose Xylose Inositol Mannitol Rhamnose Raffinose Mycelium Spore chain 
                    aerial substrate  
Identified bacterial strains: 
3831-24A M24WW Micromonospora sp. ± − − ± − − − ±R ± − − − − − − orange monosporic 
3833−24C M24WW Micromonospora sp. − − ± − − − +R ± ± ± − − − − orange monosporic 
3836−27B M27W Micromonospora sp. ± − − ± − − − +R − ± − − − − orange monosporic 
3849−37A G37LW Micromonospora sp. − − − ± − − − ±R ± − − − − − − orange monosporic 
3879−2A S2HB Micromonospora sp. − − − − − − ±R ± − − − − − − orange−black monosporic 
3881−24D M24WW Micromonospora sp. − − ± − − − ±R ± − − − − − − orange−black monosporic 
3882−25C M25WW Micromonospora sp. − − ± ± − − − ±R − ± − − − − orange−black monosporic 
3884−40A G40WW Micromonospora sp. − − − − −R − +R − ± − − − − orange−black monosporic 
                       
3880−19B M19S Microtetraspora sp. − ± − ± ± − +R − − ± − white (sparse)  
                       
3813−1A S1HB S. coelicolor − − ± +R +R ± +R − − − − − yellow dark brown RF 
3815−1F S1HB S. coelicolor − ± ± +R +R ± +R − − − − − beige dark brown RF 
3817−3B S3S S. coelicolor − − − +R +R − +R ± − − − − yellow dark brown RF 
3826−17A M17R S. coelicolor − ± − +R +R ± +R − − − − − yellow dark brown RF 
3832−24B M24WW S. coelicolor − ± − +R +R ± +R − − − − − yellow dark brown RF 
                       
3814−1D S1HB S. griseus − ± ± − − − ± − − − yellow light brown RF 
3816−1L S1HB S. griseus − ± +R ± − +R − ± ± − − − yellow light brown RF 
3819−14B M14SHB S. griseus − ± +R ± − +R − − ± − − − yellow light brown RF 
3820−14C M14SHB S. griseus − ± − − − +R − − ± − − − yellow light brown RF 
3821−14D M14SHB S. griseus − ± ± − ± ± +R − − ± − − − yellow light brown RF 
3822−14F M14SHB S. griseus − ± +R ± − +R − − ± − − − yellow light brown RF 
3823−14H M14SHB S. griseus − ± ± − +R − − ± − − − yellow dark brown RF 
3824−15A M15S S. griseus − ± ± − +R − − ± − − − yellow light brown RF 
3827−17B M17R S. griseus − ± ± +R ± +R − − ± − − − yellow light brown RF 
                       
3825−16B M16R S. halstedii − ± ± +R ± − +R − ± ± − − grey dark brown SP 1−3 turns 
3837−30A G30S S. halstedii − − ± +R − +R ± − − grey dark brown RF 
3839−30C G30S S. halstedii − − +R +R − +R − − − grey dark brown RF 
3847−34C G34C S. halstedii − − +R +R − +R − − − grey dark brown RF 
                       
3818−5E S5S S. rochei − ± ± +R ± − +R − − grey dark brown SP 5−10 turns 
3829−17G M17R S. rochei − ± +R − − +R − ± − − grey dark brown SP 1−2 turns 
3840−31A G31C S. rochei ± ± +R ± − +R − − grey dark brown SP 5−10 turns 
3843−33B I33S S. rochei − ± − +R − − +R − − grey dark brown SP 5−10 turns 
3846−34B G34C S. rochei − ± +R − − +R ± − grey dark brown SP 5−10 turns 
3848−35A G35C S. rochei ± − ± − +R − − grey dark brown SP 5−10 turns 
                       
3844−33C I33S S. violaceoruber − ± − − +R ±R − +R ± grey brownish SP 1−3 
                       
3838−30B G30S Streptomyces sp. − − − ± ± − − +R − − grey dark brown SP 5−8 turns 
3828−17C M17R Streptomyces sp. − ± − − +R − − +R − − − grey dark yellow SP 5−10 turns 
3842−32D I32S Streptomyces sp. − ± ± +R ± − +R − − grey dark yellow SP 2−5 turns 
3850−38A G38S Streptomyces sp. − ± − ± ± − +R − ± − grey dark yellow SP 2−5 turns 
3834−25A M25WW Streptomyces sp. − ± ± +R − − +R − − grey dark yellow SP 2−5 turns 
3841−31B G31C Streptomyces sp. − ± +R − − +R − − grey dark yellow SP 5−10 turns 
3835−26A M26W Streptomyces sp. ± − ±R +R − +R − − − grey orange SP 1−3 open 
3883−32C I32S Streptomyces sp. − ± ± +R ± − +R − − grey yellow brown  
 
Bacterial isolates not yet identified: 
3845−33D I33S  − − +R ± − +R ± −    
4A S4S  ± − − − − − − ± − − − − − − −    
8A S8S  ± − − − − − +R − −    
18A M18WW  − − ± − − − ±R ± − − − − −    
20A M20S  − ± ± +R − − +R ± − − −    
21B M21S  − ± − − − − − +R − − − −    
33E I33S  − − − − − +R − − − − ± −    
39B G39S  − − ± − − − ±R − − − −    
 
Bacteria from culture collections: 
DSM40003  S. albogriseolus − − +R − − +R − −     
DSM40023  S. nitrosporeus − − − − ±R +R − +R − − ± − − − −     
DSM40066  S. griseobrunneus ± − − − +R − − +R − − − − −     
DSM40137  S. atroolivaceus − − − − +R − − − − −     
DSM40149  S. daghestanicus − − − − − − +R ± − ± − − −     
DSM40153  S. flavoviridis − − − − +R − − +R − −     
DSM40211  S. olivoviridis − − − − − +R − − − − −     
DSM40229  S. griseoviridis − − − − +R − − +R − − −     
DSM40456  S. griseoflavus − − +R − − +R ± − −     
DSM40478  S. albaduncus − − − − +R − − +R − −     
DSM40560  S. tauricus  − ± − − +R +R − +R −    
DSM40533  Streptomyces sp. − − − − ± − +R ± − − −     
DSM40441  Streptomyces sp. − − − − +R − − +R − −     
DSM40566  Streptomyces sp. − − − +R − − +R              
DSM43554  Actinomadura libanotica ± ± − − − − − +R − − − − −     
DSM43146  Actinoplanes italicus − − − +R − − +R − − −     
DSM43147  Actinoplanes utahensis − − − − ±R − − +R − − − −     
DSM43170  Micromonospora sp. − − − ± − − +R ± − − − −     
DSM43713  Micromonospora sp. − − − − − +R ± ± − − −     
DSM43423  Micromonospora sp. ± − − − ± − − +? − − − − −     
DSM43191  Nocardia sp. − − − +R − − +R − − − − −     
DSM43158  Dactylosporangiumthailandense − − − − − − +R − − ±     
Strain Sourcea Species Natural rubber Hexadecan Citronellol PHB PCL Succinate Lactate Octanoate Glucose Fructose Arabinose Sucrose Xylose Inositol Mannitol Rhamnose Raffinose Mycelium Spore chain 
                    aerial substrate  
Identified bacterial strains: 
3831-24A M24WW Micromonospora sp. ± − − ± − − − ±R ± − − − − − − orange monosporic 
3833−24C M24WW Micromonospora sp. − − ± − − − +R ± ± ± − − − − orange monosporic 
3836−27B M27W Micromonospora sp. ± − − ± − − − +R − ± − − − − orange monosporic 
3849−37A G37LW Micromonospora sp. − − − ± − − − ±R ± − − − − − − orange monosporic 
3879−2A S2HB Micromonospora sp. − − − − − − ±R ± − − − − − − orange−black monosporic 
3881−24D M24WW Micromonospora sp. − − ± − − − ±R ± − − − − − − orange−black monosporic 
3882−25C M25WW Micromonospora sp. − − ± ± − − − ±R − ± − − − − orange−black monosporic 
3884−40A G40WW Micromonospora sp. − − − − −R − +R − ± − − − − orange−black monosporic 
                       
3880−19B M19S Microtetraspora sp. − ± − ± ± − +R − − ± − white (sparse)  
                       
3813−1A S1HB S. coelicolor − − ± +R +R ± +R − − − − − yellow dark brown RF 
3815−1F S1HB S. coelicolor − ± ± +R +R ± +R − − − − − beige dark brown RF 
3817−3B S3S S. coelicolor − − − +R +R − +R ± − − − − yellow dark brown RF 
3826−17A M17R S. coelicolor − ± − +R +R ± +R − − − − − yellow dark brown RF 
3832−24B M24WW S. coelicolor − ± − +R +R ± +R − − − − − yellow dark brown RF 
                       
3814−1D S1HB S. griseus − ± ± − − − ± − − − yellow light brown RF 
3816−1L S1HB S. griseus − ± +R ± − +R − ± ± − − − yellow light brown RF 
3819−14B M14SHB S. griseus − ± +R ± − +R − − ± − − − yellow light brown RF 
3820−14C M14SHB S. griseus − ± − − − +R − − ± − − − yellow light brown RF 
3821−14D M14SHB S. griseus − ± ± − ± ± +R − − ± − − − yellow light brown RF 
3822−14F M14SHB S. griseus − ± +R ± − +R − − ± − − − yellow light brown RF 
3823−14H M14SHB S. griseus − ± ± − +R − − ± − − − yellow dark brown RF 
3824−15A M15S S. griseus − ± ± − +R − − ± − − − yellow light brown RF 
3827−17B M17R S. griseus − ± ± +R ± +R − − ± − − − yellow light brown RF 
                       
3825−16B M16R S. halstedii − ± ± +R ± − +R − ± ± − − grey dark brown SP 1−3 turns 
3837−30A G30S S. halstedii − − ± +R − +R ± − − grey dark brown RF 
3839−30C G30S S. halstedii − − +R +R − +R − − − grey dark brown RF 
3847−34C G34C S. halstedii − − +R +R − +R − − − grey dark brown RF 
                       
3818−5E S5S S. rochei − ± ± +R ± − +R − − grey dark brown SP 5−10 turns 
3829−17G M17R S. rochei − ± +R − − +R − ± − − grey dark brown SP 1−2 turns 
3840−31A G31C S. rochei ± ± +R ± − +R − − grey dark brown SP 5−10 turns 
3843−33B I33S S. rochei − ± − +R − − +R − − grey dark brown SP 5−10 turns 
3846−34B G34C S. rochei − ± +R − − +R ± − grey dark brown SP 5−10 turns 
3848−35A G35C S. rochei ± − ± − +R − − grey dark brown SP 5−10 turns 
                       
3844−33C I33S S. violaceoruber − ± − − +R ±R − +R ± grey brownish SP 1−3 
                       
3838−30B G30S Streptomyces sp. − − − ± ± − − +R − − grey dark brown SP 5−8 turns 
3828−17C M17R Streptomyces sp. − ± − − +R − − +R − − − grey dark yellow SP 5−10 turns 
3842−32D I32S Streptomyces sp. − ± ± +R ± − +R − − grey dark yellow SP 2−5 turns 
3850−38A G38S Streptomyces sp. − ± − ± ± − +R − ± − grey dark yellow SP 2−5 turns 
3834−25A M25WW Streptomyces sp. − ± ± +R − − +R − − grey dark yellow SP 2−5 turns 
3841−31B G31C Streptomyces sp. − ± +R − − +R − − grey dark yellow SP 5−10 turns 
3835−26A M26W Streptomyces sp. ± − ±R +R − +R − − − grey orange SP 1−3 open 
3883−32C I32S Streptomyces sp. − ± ± +R ± − +R − − grey yellow brown  
 
Bacterial isolates not yet identified: 
3845−33D I33S  − − +R ± − +R ± −    
4A S4S  ± − − − − − − ± − − − − − − −    
8A S8S  ± − − − − − +R − −    
18A M18WW  − − ± − − − ±R ± − − − − −    
20A M20S  − ± ± +R − − +R ± − − −    
21B M21S  − ± − − − − − +R − − − −    
33E I33S  − − − − − +R − − − − ± −    
39B G39S  − − ± − − − ±R − − − −    
 
Bacteria from culture collections: 
DSM40003  S. albogriseolus − − +R − − +R − −     
DSM40023  S. nitrosporeus − − − − ±R +R − +R − − ± − − − −     
DSM40066  S. griseobrunneus ± − − − +R − − +R − − − − −     
DSM40137  S. atroolivaceus − − − − +R − − − − −     
DSM40149  S. daghestanicus − − − − − − +R ± − ± − − −     
DSM40153  S. flavoviridis − − − − +R − − +R − −     
DSM40211  S. olivoviridis − − − − − +R − − − − −     
DSM40229  S. griseoviridis − − − − +R − − +R − − −     
DSM40456  S. griseoflavus − − +R − − +R ± − −     
DSM40478  S. albaduncus − − − − +R − − +R − −     
DSM40560  S. tauricus  − ± − − +R +R − +R −    
DSM40533  Streptomyces sp. − − − − ± − +R ± − − −     
DSM40441  Streptomyces sp. − − − − +R − − +R − −     
DSM40566  Streptomyces sp. − − − +R − − +R              
DSM43554  Actinomadura libanotica ± ± − − − − − +R − − − − −     
DSM43146  Actinoplanes italicus − − − +R − − +R − − −     
DSM43147  Actinoplanes utahensis − − − − ±R − − +R − − − −     
DSM43170  Micromonospora sp. − − − ± − − +R ± − − − −     
DSM43713  Micromonospora sp. − − − − − +R ± ± − − −     
DSM43423  Micromonospora sp. ± − − − ± − − +? − − − − −     
DSM43191  Nocardia sp. − − − +R − − +R − − − − −     
DSM43158  Dactylosporangiumthailandense − − − − − − +R − − ±     

aNotation: The first letter indicates the country of the sample (S Singapore, M Malaysia, G Germany, I Italy). The last letters indicate the type of sample (HB Hevea bark, S soil, R piece of aggregated natural rubber, WW waste water, LW lake water sediment, C compost), Micromonosp. Micromonospora, + good growth/halo formation; ± poor growth/halo formation; − same growth as on mineral medium without a carbon source/no halo; R repression of natural rubber degrading activity, citronellol, succinate, lactate and glucose were tested; RF spore chains rectus flexibilis; SP spiral spore chains; empty space in a column indicates that the value has not been determined.

The morphology of the colonies and fatty acid pattern analysis of the NR-degrading isolates showed that the isolates fell into two main clusters: members of cluster I (8 strains) exhibited an orange (young cells) to black-colored (old cells) substrate mycelium phenotype, and no aerial mycelium was formed. In combination with the fatty acid pattern this is consistent with the genus Micromonospora. Since the similarity indices were low a classification of the species according to the fatty acid pattern was not possible. Members of cluster II (33 strains) produced a yellow to grey aerial mycelium with rectus flexibilis or spiralic spore chains which is diagnostic for the genus Streptomyces. Morphological characterization of aerial mycelium and spore chains in combination with fatty acid pattern justified the classification of 5 strains as the species S. coelicolor, 9 strains as S. griseus, 4 strains as S. halstedii, 6 strains as S. rochei, and 1 strain as S. violaceoruber. Additional subclusters could not be identified as a species due to low similarity indices (Table 2).

Growth of selected strains in liquid mineral medium with solution-cast films of NR (0.2%) as a carbon source resulted in significant weight loss (10–30%) and reduction of the weight average molecular mass of the residual polymer from 640.000 to 25.000 as determined by gel permeation chromatography (data not shown).

Regulation of NR degradation

When clearing zone formation was studied on NR plates, which contained one additional soluble carbon source, evidence for inhibition of NR-degrading enzyme expression was obtained. Carbon sources that allowed good growth, e.g. glucose or succinate, repressed the NR-degrading enzymes in most strains (Table 2). The extent of inhibition varied with strains and with and substrates. In S. griseus 3814-1D clearing zone formation was hardly affected by the presence of soluble substrates (constitutive expression). In some other Streptomycetes halo formation was repressed by glucose but not by succinate. Linear oligoterpenes such as racemic citronellol, farnesol or squalene did not significantly support growth of the bacteria and also had no effect on halo formation on latex agar. For some strains these compounds (applied as vapor) were slightly toxic.

Discussion

A great variety of NR-degrading bacteria was identified by halo formation on NR-containing media in 30 of 33 samples taken from various ecosystems in South East Asia and Europe. The greatest numbers (up to ∼105 NR-degrading bacteria/g) were obtained from soil samples of Hevea brasiliensis plantations and from waste water ponds of a rubber-producing company in Malaysia. Apparently, NR-degrading bacteria are widely distributed in nature.

Interestingly all NR-degrading strains belong to the actinomycetes and despite much effort no Gram-negative bacterium was enriched or isolated with NR as carbon source. In the literature on microbial rubber degradation Gram-positive bacteria dominate also ([7] and references cited therein). The only example of a Gram-negative, a Xanthomonas species, has been published by Tsuchii et al. [5]. The screening for NR degradation of a large number of Gram-negative bacteria from culture collections including 45 Xanthomonas strains, 115 Pseudomonas strains, 16 Alcaligenes strains and many hydrocarbon-utilizing bacteria revealed no positive one (Table 1). We conclude that degradation of purified NR as sole carbon source is a privilege of mycelium-forming microorganisms. Gram-negative NR-degrading bacteria appear to be the exception. However, we cannot exclude NR degradation capabilities encoded by Gram-negative bacteria in general. Potential Gram-negative NR degraders might just require additional growth factors or degrade NR by co-metabolism.

In contrast to the catabolism of oligoisoprenoids or other methyl-branched compounds [10, 11], the biochemical mechanism of NR degradation has not been investigated. Since NR is a high molecular mass compound that is too large to be taken up by bacteria, the polymer has to be cleaved extracellularly as a first step. The extracellular nature of such an enzyme system was shown by the appearance of translucent halos on latex-containing solid media. Tsuchii et al. [5] demonstrated 18O incorporation from 18O2 into oligomeric isoprenoid intermediates produced by the above mentioned Xanthomonas strain. The reduction of the average molecular mass of partially degraded NR by a factor of more than 10 indicated an endo-cleavage mechanism of NR degradation but the additional presence of an exo-type activity cannot be excluded. Most of the NR-degrading bacteria were unable to utilize alkanes such as hexadecane (4 exceptions), octane, or hexane, unlike Pseudomonas oleovorans and Acinetobacter calcoaceticus (controls). The latter synthesize specific mono- or dioxygenases, respectively, which initiate alkane degradation [13, 14]. We conclude that NR-degrading actinomycetes apparently have other, highly substrate-specific polyisoprenoid oxygenases that are responsible for the first oxidation step of NR. None of the tested NR-degrading isolates showed significant growth on oligomeric isoprenoids such as citronellol, farnesol, or squalene, which contrasts with Pseudomonas citronellolis and Pseudomonas mendocina which were used as controls ([12] and references cited therein). Therefore these compounds are not likely to be intermediates of NR degradation.

Acknowledgements

We are grateful to H.Y. Yeang and A. Ikram from the Rubber Research Institute of Malaysia for providing latex, hospitality and helpful advice and G. Muth and W. Wohlleben (Universität Tübingen) for testing 200 actinomycetes of their culture collection. We also thank R.-J. Müller (GBF Braunschweig) for performing GPC measurements. This work was supported by the Deutsche Forschungsgemeinschaft.

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