Review

Biodegradation of natural and synthetic rubbers: A review

Aamer Ali Shah
*
, Fariha Hasan, Ziaullah Shah, Nida Kanwal, Samia Zeb
Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
a r t i c l e i n f o
Article history:
Received 26 December 2012
Received in revised form
13 April 2013
Accepted 2 May 2013
Available online 6 June 2013
Keywords:
Biodegradation
Scanning electron microscopy
Attenuated total reectance-Fourier
transform infrared spectroscopy
Sturm test
Rubber degrading enzymes
Analysis of degradation
a b s t r a c t
Since polymeric materials do not decompose easily, disposal of waste polymers is a serious environ-
mental concern. Widespread studies on the biodegradation of rubbers have been carried out in order to
overcome the environmental problems associated with rubber waste. This report provides an overview
on the microbial degradation of natural and synthetic rubbers. Rubber degrading microbes, bacteria and
fungi, are ubiquitous in the environment especially soil. The qualitative data like plate assay, scanning
electron microscopy (SEM), attenuated total reectance-Fourier transform infrared spectroscopy (ATR-
FTIR) and Sturm test indicated that both natural and synthetic rubbers can be degraded by microor-
ganisms. It has conrmed that the enzymes latex clearing protein (Lcp) and rubber oxygenase A (RoxA)
are responsible for the degradation of natural and synthetic rubbers. Lcp was obtained from Gram-
positive bacterium Streptomyces sp. strain K30 and RoxA from Gram-negative bacterium Xanthomonas
sp. strain 35Y. Analysis of degradation products of natural and synthetic rubbers indicated the oxidative
cleavage of double bonds in polymer backbone. Aldehydes, ketones and other carbonyl groups were
detected as degradation products from cultures of various rubber degrading strains. This review em-
phasizes the importance of biodegradation in environmental biotechnology for waste rubber disposal.
2013 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146
2. Rubber as solid waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146
3. Biodegradation of polymeric materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146
4. Biodegradation of rubber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.1. Biodegradation of natural rubber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.1.1. Natural rubber degrading bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.1.2. Natural rubber degrading fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.2. Biodegradation of synthetic rubbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.2.1. Synthetic rubber degrading bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.2.2. Synthetic rubber degrading fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5. Analysis of natural rubber degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
5.1. Schiffs staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2. Scanning electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.3. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.4. CO
2
evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5.5. Effect of pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
6. Rubber degrading enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154
6.1. Latex clearing protein (Lcp) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
6.2. Rubber oxygenase A (RoxA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
7. Analysis of rubber degradation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .156
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
* Corresponding author. Tel.: 92 51 90643116.
E-mail addresses: alishah@qau.edu.pk, alishah_75@yahoo.com (A. Ali Shah).
Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation
j ournal homepage: www. el sevi er. com/ l ocat e/ i bi od
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http://dx.doi.org/10.1016/j.ibiod.2013.05.004
International Biodeterioration & Biodegradation 83 (2013) 145e157
1. Introduction
Civilization as we know it today is wholly dependent upon
rubber. It is a material of myriad uses, totally unlike anything the
world had previously known. It enters in a thousand ways into the
fabric of our daily lives. Rubbers have been drawing for a wide
range of application, the potential application areas include mining,
power generation, agriculture, transportation, paper industries.
The rubber insulation of cables and wires, however, is being
increasingly supplanted by plastic (PVC), but rubbers can also be
used for various equipment of livestock such as harness, collars,
horse-shoes, rubber saddles, milking machine hoses etc. Their use
in footwear industry and in rubber based textile goods are some of
the important applications. Various latex rubber articles are also
used in medicine and surgery, such as draw sheets, gloves, nger
stalls, teats, and hot water bottles. Rubber from used tyres and
tubes is used to make shoes, bushings, washers, gaskets, wheels,
containers, and a wide range of products for domestic, commercial
and industrial use (Ahmed et al., 1996).
Rubbers are elastomers, either of natural or non-natural origin.
Natural rubber (NR) is a biopolymer that is synthesized by more
than 2000 plant species, most of them belong to Euphorbiaceae or
Compositaceae, and by some fungi (Bode et al., 2001). Natural
rubber refers to a coagulated or precipitated product obtained
from the milky secretion (latex) of the rubber plants (Hevea bra-
siliensis), which forms non-linked but partially vulcanizable
polymer chains having molecular masses of about 10
6
Da with
elastic properties. Latex serves as a clogging material during
healing of wounds caused by mechanical injury of plants. Natural
rubber consists of C
5
H
8
units (isoprene), each containing one
double bond in the cis conguration (Tanaka and Sakdapipanich,
2001; Rose and Steinbuchel, 2005). The natural polymer synthe-
sized by the Hevea species is composed of 3 trans-isoprene units at
one end of the molecule followed by several hundred to a few
thousand cis-isoprene units (Bode et al., 2001). Although
approximately 2,000 plants synthesize poly(cis-1,4-isoprene), only
natural rubber of H. brasiliensis (99% of the world market) and
guayule rubber of Parthenium argentatum (1% of the world mar-
ket) are produced commercially (Tanaka and Sakdapipanich, 2001;
Rose and Steinbuchel, 2005). In 1998, the world production of
natural rubber was about 6.6 million tons; more than 70% of this
rubber was produced in only three countries (Thailand, Indonesia,
and Malaysia), and about 40% was purchased by only three
countries (United States, China, and Japan). Most of the
natural rubber (75%) is used for production of automobile tyres
(Muller, 2000).
Natural rubber latex of H. brasiliensis origin is composed of 25e
35% (wt/wt) polyisoprene; 1e1.8% (wt/wt) protein; 1e2% (wt/wt)
carbohydrates; 0.4e1.1% (wt/wt) neutral lipids; 0.5e0.6% (wt/wt)
polar lipids; 0.4e0.6% (wt/wt) inorganic components; 0.4% (wt/wt)
amino acids, amides, etc.; and 50e70% (wt/wt) water
(Subramaniam, 1995). Only a few plant species synthesize poly-
isoprenes in the trans conguration. Chicle (Manikara zapota),
gutta-percha (Pallaquium gutta), and balata (Manikara bidentata)
are typical representatives of trans-polyisoprene-synthesizing
plants (Rose and Steinbuchel, 2005).
In 1839, Charles Goodyear discovered the vulcanization process
in which the addition of sulphur to natural rubber could dramati-
cally improve properties. The discovery of sulphur vulcanization is
used in current automotive tyres in order to give the desired
properties. Alternatively, vulcanization is also achieved by
employing organic peroxides (Coran, 1978) or radiation
(Subramaniam, 1995); such vulcanized materials have lower long-
term stability since the polymer chains are cross-linked solely by
carbon bonds (Rose and Steinbuchel, 2005).
Synthetic rubber is made of raw material derived from petro-
leum, coal, oil, natural gas, and acetylene. Although the rst syn-
thetic rubbers were produced at the beginning of the last century,
only after 1950, after the development of stereospecic catalysts,
polyisoprene be synthesized in the cis and trans congurations
(Tanaka and Sakdapipanich, 2001). Synthetic isoprene rubber (SR) is
a polymer of mainlycis-isopreneunits combinedby1,4-linkages and
10% trans-isoprene units with 3,4-linkages (Bode et al., 2001). Syn-
thetic polyisoprene can be produced with physical properties
similar to those of natural rubber with a purity of 98e99%. However,
the stress stability, process ability, andother parameters of synthetic
polyisoprene are still less satisfying than those of natural rubber
(Tanaka and Sakdapipanich, 2001). At the present time over 75% of
the rubber used in United States is synthetic, while on the world
basis about 65% of the rubber is synthetic. The various types of
synthetic rubber are styrene-butadiene rubber (SBR), acrylonitrile-
butadiene copolymers (NBR latex), ethylene-vinyl chloride co-
polymers (EVCL), polybutadiene, polychloroprene (neoprene)
(Dunn and Beswich, 2002).
2. Rubber as solid waste
Waste disposal management is one of the problems; mankind is
facing in this century. Since polymeric materials do not decompose
easily, disposal of waste polymers is a serious environmental
problem. Total world rubber production has increased to 24.3
million tonnes in 2010, a rise of 11.9% from 21.7 million tonnes in
2009 (IRSG, 2010). Large amount of rubbers are used as tyres for
aeroplanes, trucks, cars, two-wheelers etc. Almost the entire
amount of rubber fromthe worn out tyres is discarded, which again
need very long time for natural degradation due to cross linked
structure of rubbers and presence of stabilizers and other additives.
This poses two major problems: the wastage of valuable rubber and
the disposal of waste tyres leading to environmental pollution
(Adhikari et al., 2000). Many of the rubber additives (such as ac-
celerators, retarders, and antioxidants) used in different applica-
tions have been shown to be toxic to microorganisms, as a result
disposing and reprocessing the material safely is a challenge
(Christinasson et al., 2000; Bredberg et al., 2001a). Additional
problems arise from the presence of other natural biodegradable
compounds in natural rubber and latex or fromadditives which are
required for vulcanization or to inuence the material properties.
To avoid allocation of growth or CO
2
release to degradation of, e.g.,
proteins and lipids present in the material, growth and minerali-
zation experiments must be performed carefully. Additives can
promote (e.g., llers and stoppers) or inhibit (accelerators, antiox-
idants, and preservation material) biodegradation of rubber mate-
rial (MacLaghlan et al., 1966; Gomez and Moir, 1979). Billions of
discarded tyres are currently stockpiled around the world and
hundreds of millions are added every year. Those tyres stockpiles
present both health and environmental hazards (Liaskos, 1994;
Jang et al., 1998). Moreover, as landll sites for safe disposal of
rubber waste become limited, indiscriminate disposal are causing
water and land pollution problems. Burning of rubber waste gen-
erates large amount of heat and smoke which is one of the main
factors for global warming (Stevenson et al., 2008) (Table 1).
In this report, the current knowledge about the biodegradation
of natural and synthetic rubber hydrocarbon is summarized.
3. Biodegradation of polymeric materials
Microorganisms such as bacteria and fungi are involved in the
degradation of both natural and synthetic polymers, and very little
is known about the biodegradation of synthetic polymeric mate-
rials (Gu et al., 2000a). The reason is probably due to the recent
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 146
development and manufacture of this class of materials and the
relatively slow rate of degradation in natural environments. Poly-
meric materials are very unique in chemical composition, physical
forms, mechanical properties and applications. High versatility of
the carbon to carbon and carbon to non-carbon (CeC, CeR, CeH)
bonds and substituent groups, the possible congurations, stereo-
chemistry and orientation provide basis for variations of chemical
structures and stereochemistry. Very small variation in the chem-
ical structures may result in large differences in term of biode-
gradability (Odian, 1991). Biodegradation is governed by different
factors that include polymer characteristics, type of organism, and
nature of pretreatment. The polymer characteristics such as its
mobility, tacticity, crystallinity, molecular weight, the type of
functional groups and substituents present in its structure, and
additives added to the polymer all play an important role in its
degradation (Gu et al., 2000b; Artham and Doble, 2008). Dominant
groups of microorganisms and the degradative pathways associ-
ated with polymer degradation are often determined by the envi-
ronmental conditions. When O
2
is available, aerobic
microorganisms are mostly responsible for destruction of complex
materials, with microbial biomass, CO
2
, and H
2
O as the nal
products. In contrast, under anoxic conditions, anaerobic consortia
of microorganisms are responsible for polymer deterioration. The
primary products will be microbial biomass, CO
2
, CH
4
and H
2
O
under methanogenic (anaerobic) conditions or H
2
S, CO
2
and H
2
O
under suldogenic conditions (Barlaz et al., 1989; Gu et al., 2000a;
Gu, 2001; Gu and Mitchell, 2001).
4. Biodegradation of rubber
4.1. Biodegradation of natural rubber
The microbial susceptibility of natural rubber (NR) either in the
raw or in the vulcanized state was sufciently examined and
reviewed (Zyska, 1981; Seal, 1988). Soil is a rich source of micro-
organisms, both bacteria and fungi, which can degrade natural as
well as synthetic rubbers and utilize it as carbon and energy source
(Imai et al., 2011). Several microorganisms were isolated from such
experiments, and pure cultures were tested for their rubber-
degrading potential. Results showed that actinomycetes were
almost the only organisms able to considerably decompose NR and
to use the rubber hydrocarbon as a carbon source (Tsuchii et al.,
1985; Heisey and Papadatos, 1995; Jendrossek et al., 1997; Linos
and Steinbuchel, 1998).
Several serious difculties hamper investigation of microbial
degradation of rubber. Rubber biodegradation is a slow process,
and the growth of bacteria utilizing rubber as a sole carbon source
is also slow. Therefore, incubation periods extending over weeks or
even months are required to obtain enough cell mass or degrada-
tion products of the polymers for further analysis. This is particu-
larly true for members of the clear-zone forming group. Periods of
10e12 weeks have to be considered for Streptomyces coelicolor 1A
(Bode et al., 2001), Thermomonospora curvata E5 (Ibrahim et al.,
2006), or Streptomyces sp. strain K30 (Rose et al., 2004); the only
exception is Xanthomonas sp. strain 35Y (Tsuchii and Takeda, 1990).
Although members of the non-clear-zone-forming group exhibit
slightly faster growth, cultivation periods of at least 4e6 weeks are
also required for Gordonia westfalica (Broker et al., 2004) and Ba-
cillus sp. S-10 (Shah et al., 2009) to determine whether a putative
mutant is able to grow on the polymer.
As a natural product, NR is subjected to biological mineralization
cycles, and many reports on the biodegradation of rubbers have
been published since the study of Sohngen and Fol (1914), even
cross-linked rubbers (e.g., vulcanized rubber, latex gloves) have
been shown to be biodegradable (Tsuchii et al., 1985; Heisey and
Papadatos, 1995). Spence and van Niel (1936) were the rst to
use latex overlay plates in mineral agar resulting in an opaque
medium, for the isolation of rubber-degrading bacteria. Growth of
many rubber degrading microorganisms resulted in visible clearing
zone formation around the colonies. Jendrossek et al. (1997) used
the same clear zone method and isolated 50 newrubber-degrading
bacteria. Enrichment techniques with solid (natural or cross-
linked) rubber led to the isolation of several rubber-degrading
bacteria that had high rubber degrading activity but do not
necessarily form clearing zones on latex agar. Two Actinomycetes,
which formed colonies directly on the puried NR material, were,
identied as Actinomyces elastica and Actinomyces fuscus (Linos
et al., 1999, 2000a).
Table 1
The potential environmental impacts of materials used in rubber products.
Material Source Application Potential impacts
Natural rubber From the sap of the
Hevea brasiliensis tree
Currently makes up about 30e40%
of the total rubber used.
Loss of habitat in tropical forests,
impacts from transportation to markets
and from processing including odour.
Synthetic rubber Petrochemicals Makes up approximately 60e70%
of the total rubber used.
Resource depletion of petroleum.
Energy consumption and waste
during manufacture.
Other reinforcing fabrics Predominantly
sourced from
petrochemicals.
Used for structural strength and rigidity.
Makes up about
5% of a radial tyre.
Impacts during production and transport.
Carbon black Generally sourced from
petroleum stock.
Imparts durability and resistance to degradation.
Makes up about 28% of a passenger tyre.
Impacts during production and transport.
Zinc oxide To provide resistance to UV degradation,
control vulcanization.
Zinc oxide makes up about 1.2% of a passenger tyre.
Impacts during manufacture and
disposal of waste tyres.
Sulphur (including compounds) Sulphur is used to
vulcanize the rubber.
Makes up about 1% of a passenger tyre. Impacts during production and
combustion for energy recovery.
Other additives and solvents:
age resistors, processing aids,
accelerators, vulcanizing agents,
softeners and llers
The additives make up about 8% by weight
of a passenger tyre.
Impact in manufacture and transportation.
Emissions from tyres in use.
Recycled rubber Recovered from
used tyres or
other rubber products.
Used in manufacture of new rubber products. Impacts from energy use in production.
(http://www.environment.gov.au/settlements/publications/waste/tyres/national-approach/tyres5.html).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 147
4.1.1. Natural rubber degrading bacteria
Many bacterial strains that are able to utilize rubber as the sole
source of carbon and energy, have been described during the last
hundred years (Rose and Steinbuchel, 2005; Rose et al., 2005).
These bacteria can be divided into two groups, which follow
different strategies to degrade rubber (Linos et al., 2000b). Mem-
bers of the rst group form translucent halos if they are cultivated
on solid media containing dispersed latex particles, indicating the
excretion of rubber-cleaving enzymes. The most potent represen-
tatives in this group belong to actinomycetes from Actinoplanes,
Streptomyces, and Micromonospora (Imai et al., 2011). Members of
the second group do not form halos and do not grow on latex
plates; they require direct contact with the polymer. They grow
adhesively at the surface of rubber particles in liquid culture, and
they represent the most potent rubber-degrading bacterial strains.
This group comprises mycolic acid-containing Actinobacteria
belonging to the genera Gordonia, Mycobacterium, and Nocardia.
Some new rubber-degrading strains belonging to the Corynebac-
terium, Nocardia, Mycobacterium group, such as Gordonia poly-
isoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and
Mycobacterium fortuitum strain NF4, were isolated (Arenskotter
et al., 2001, 2004; Linos et al., 2002). All rubber-degrading species
described so far are mesophilic species, with only one exception,
identied as a Streptomyces sp. closely related to Streptomyces
albogriseolus and Streptomyces viridodiastaticus, which was able to
growat temperatures up to 50

C and belong to the so-called clear-
zone-forming group of rubber-degrading bacteria (Gallert, 2000).
Genes encoding rubber-cleaving enzymes from mesophilic clear-
zone-forming bacteria have been reported (Braaz et al., 2004;
Rose et al., 2005). The author has reported on the existence of holes
in the rubber material, beneath the adhering Actinomycetes col-
onies, both in liquid mineral culture and on agar plates coated by
puried NR lms, and concluded that under these conditions in-
crease in biomass could only have taken place at the expense of the
rubber hydrocarbon. A new rubber degrading strain Bacillus sp. AF-
666, was isolated through enrichment and NR latex overlay (latex
lm plates) in the authors laboratory (Shah et al., 2012). One
disadvantage of latex overlay agar plates is that all rubber-
degrading bacteria cannot be cultivated in this way, because most
of them do not form halos on agar plates. Besides this, a very small
amount of polyisoprene is locally available to allow formation of
visible colonies by these organisms (Linos et al., 2002).
4.1.2. Natural rubber degrading fungi
De Vries (1928) was the pioneer in examining the possible
decomposition of rubber hydrocarbon by fungi. A decade later,
Kalinenko reported fungal strains from Aspergillus and Penicillium
as rubber degraders (Kalinenko, 1938). Kwiatkowska et al. (1980)
performed soil burial tests of NR vulcanized sheets of denite
composition and detected substantial weight losses reaching up to
40% of the initial weight after 91 days. These authors isolated a
fungal strain Fusarium solani from the surface of rubber and
claimed its degradation by this strain. In a study by Williams in
1982, the author isolated a fungal strain, Penicillium variabile from
deteriorated NR sample after soil burial. Spore suspensions inocu-
lated on to NR smoked sheet in a humidity cabinet led to a suc-
cessive increase of biomass on the NR surface, as shown by cell
protein determination every 14 days, and was accompanied by a
weight loss of rubber strips up to 13% after 56 days. However,
further increase in biomass and in weight loss beyond this time
period could not be determined. Using solution viscosity mea-
surement as analytical tool, the author estimated a 15% reduction in
the molecular weight of polyisoprene after 70 days (Williams,
1982). Several rubber-deteriorating fungi were isolated from min-
eral agar plates containing powdered NR as sole substrate and also
from the surface of deteriorated tyre material. The fungal strains
F. solani, Cladosporium cladosporioides and Paecilomyces lilacinus
were grown on the surface of rubber after 20 days of incubation.
Using GPC, a relative reduction of the molecular weight up to 20%
was detected and even a decrease in intrinsic viscosity up to 35%
(Borel et al., 1982).
4.2. Biodegradation of synthetic rubbers
4.2.1. Synthetic rubber degrading bacteria
Nocardia sp. strain 835A, was one of the rst strains that was
investigated in detail with regard to synthetic rubber biodegrada-
tion, and it was postulated that there was oxidative cleavage of
poly(cis-1,4-isoprene) at the double-bond position, resulted in
weight losses of the rubber material used of 75 and 100% (wt/wt)
after 2 and 8 weeks of incubation, respectively (Tsuchii et al., 1985).
NR-latex gloves, synthetic rubbers as well as other acyclic iso-
prenoids, phytol, squalane, squalene and prostane degrading
Nocardia sp. strain MBR was isolated from old tyre samples in
Alexandria, Egypt (Berekaa, 2006). A Moraxella strain capable of
utilizing 1,4-type polybutadiene as a sole source of carbon was
isolated from soil. About 40% of polybutadiene having an average
molecular weight (Mn) of 2350 was degraded and assimilated by
the strain in 5 days. A polybutadiene sample with an Mn of 16,100,
however, was hardly degraded. It was found that the size of the
polymer emulsion might have a great inuence on the growth rate
and vinyl branchings in the polybutadiene prevented its microbial
degradation (Tsuchii et al., 1984). In case of non-vulcanized syn-
thetic rubber, it was observed by GPC and by weight loss mea-
surement that Pseudomonas citronellolis was able to cleave the
carbon backbone of synthetic poly (cis-1,4-isoprene) and utilize the
low molecular weight degradation products for growth.
P. citronellolis was isolated on the basis of its ability to grow on
linear terpenes (Bode et al., 2000).
Biodegradation of vulcanized rubber material is also possible,
although it is even more difcult due to the inter-linkages of the
poly(cis-1,4-isoprene) chains, which result in reduced water ab-
sorption and gas permeability of the material (Seal and Morton,
1986). During vulcanization, polyisoprene chains in rubber are
covalently linked with sulde bridges ranging from monosuldic
and disuldic bridges to polysuldic bridges (Aprem et al., 2003).
The polymers in tyre rubber can be devulcanized by the activity of
aerobic and anaerobic microbes and both the metabolic pathways
and enzymes involved are becoming better known. Removal of
sulphur facilitates the recycling of rubber (Rose and Steinbuchel,
2005; Ibrahim et al., 2006).
Two Streptomyces strains were isolated fromvulcanized gaskets
of cement water tubes, which were the cause of 1.5-mm-diameter
holes in the material after 12 months of incubation (Rook, 1955).
Continuation of these studies led to development of the so-called
Leeang test bath, in which rubber material is examined in a
steady aquatic stream with regard to its stability against microbial
degradation (Leeang, 1963). In order to test whether rubber-
degrading bacteria might also be able to degrade vulcanized rub-
ber, in which the polymer chains have been cross linked by sulphur
bridges, weight loss experiments, were performed with 100-mg
pieces of vulcanized latex gloves. In contrast to a non-inoculated
control culture or a culture containing Streptomyces lividans strain
1326, weight losses of 10e18% were obtained in the presence of
degrading bacteria within 6 weeks of inoculation (Bode et al.,
2000).
Several species of Thiobacillus, such as Rhodococcus rhodocrous
and Sulfolobus acidocaldarius have been tested for desulphurization
of rubber material (Torma and Raghavan, 1990; Romine et al., 1995;
Christiansson et al., 1998). These microorganisms oxidize sulphur
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 148
to sulphate and may break the sulphur bridges in the rubber ma-
terial. Fiala and Stetter (1986) investigated the use of anaerobic
sulphur reducing archaeon, Pyrococcus furiosus, a thermophilic
heterotroph. The specicity of the microorganisms for sulphur
gives the benet of leaving the hydrocarbon chains intact during
treatment. This quality is enhanced in anaerobic process, where
oxidation of the hydrocarbon chains is avoided (Bredberg et al.,
2001b). Kanwal (2009) reported that Bacillus sp. S-10 can degrade
not only the natural but also synthetic rubber. She treated the
rubber tyre pieces with Bacillus sp. S-10 in mineral salt medium
with no extra carbon source, for 28 days at different temperatures.
The SEM micrographs in Fig. 1 indicate the appearance of pits and
erosions after treatment with strain S-10. The results were further
conrmed by Sturm test where high amount of CO
2
was evolved in
case of test as compared to control (Kanwal, 2009).
4.2.2. Synthetic rubber degrading fungi
Vulcanized rubber degrading fungal strain, Paecilomyces variotii
strain SFA-25 was isolated fromhot spring of Balakot, Pakistan (Zeb,
2009). SEM micrographs indicated the strain SFA-25 hyphal
network on the surface of tyre piece, and surface erosion can easily
be seen belowthe network, as showin Fig. 2. Fungi attack materials
with a damaged or rough surface more successfully than materials
with smooth surfaces because propagules are retained more easily
at the surface (Verran et al., 2000). Rough surfaces or those with
cracks may concentrate nutrients and moisture more easily,
providing favourable conditions for fungal attachment and growth.
Once established on the surface, and deriving nutrients from
components of the material or from debris accumulated at the
surface, fungi usually start to penetrate into deeper layers via
micro-cracks or damaged areas. The use of environmental carbon
sources by microorganisms may enable them to attack a material
more vigorously (Jenings and Lysek, 1996). The degradation was
conrmed by comparing the attenuated total reectance-fourier
transform infrared spectroscopy (ATR-FTIR) results of both test and
control. Presence of aldehydic group in ATR-FTIR spectra along with
increase in unsaturation intensities indicated the degradation of
rubber tyre pieces. Formation of aldehydes was conrmed by the
appearance of new peaks at 1680 and 3401 cm
1
. Increase in in-
tensity of the peak at 1680 cm
1
indicated an increase in unsatu-
ration due to biodegradation. The peak at 1590 cm
1
formed by the
stretching of >C]O indicated the formation of aldehyde group as a
result of biodegradation of rubber. Increase in the intensity of the
peak at 1634 cm
1
, corresponding to unsaturation, was also
observed (Fig. 3) (Zeb, 2009).
Numerous techniques have been proposed for recycling of waste
rubbers, including mechanical, thermo-mechanical, cryo-mechan-
ical, microwave, ultrasound and microbial desulphurization tech-
niques (Debapriya et al., 2005).
5. Analysis of natural rubber degradation
In the authors laboratory, the degradation of pieces of NR latex
gloves, by Bacillus sp. AF-666, was investigated. The degradation
was monitored by staining of rubber pieces with Schiffs reagent at
the end of experiment. Scanning electron micrographs (SEM) and
attenuated total reectance-Fourier transform infrared spectros-
copy (ATR-FTIR) was also performed to conrm the degradation
(Shah et al., 2012).
5.1. Schiffs staining
Staining with Schiffs reagent revealed the presence of aldehyde
groups and gave purple colour indicating the presence of degra-
dation products containing aldehyde groups (Shah et al., 2012). The
isoprene oligomers, aldehyde and ketone groups formation after
Fig. 1. Scanning electron micrographs of tyre pieces after treatment with Bacillus sp. S-10 in mineral salt medium for 28 days at different temperatures. Rubber pieces were washed
with sterilized distilled water before SEM. Abiotic control (a); surface erosion and pits formation after treatment with strain S-10 at 25

C (b); 35

C (c) & 40

C (d). Strain S-10
caused maximum degradation of rubber pieces within the temperature range 35e40

C. Bars, 5 mm (Kanwal, 2009).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 149
incubation of latex gloves with G. polyisoprenivorans, were detected
by staining with Schiffs reagent, whereas the decrease in the
number of double bonds in the polyisoprene chain was measured
by ATR-FTIR spectroscopy (Ibrahim et al., 2006). Rook (1955)
observed the colonization behaviour of rubber degrading Gordinia
sp. strain Kb2 on the surface of pretreated NR latex glove through
Schiffs reagent. Actively growing colonies on the rubber surface
were clearly visualized by staining with this reagent and gave a
clear purple colour thus providing evidence for the occurrence of
degradation products containing aldehyde groups during the
degradation.
5.2. Scanning electron microscopy
There are different ways to monitor the changes in rubber pieces
as a result of microbial treatment such as visual observations
(roughening of the surface, formation of holes or cracks, defrag-
mentation, changes in colour or formation of biolms on the sur-
face), scanning electron microscopy (SEM) or atomic force
microscopy (AFM) or changes in the mechanical properties and
molar mass (Erlandsson et al., 1997). Shah et al. (2012) reported the
detailed analysis of the growth behaviour of Bacillus sp. AF-666 on
NR latex gloves by means of SEM. During colonization, the strain
was tightly adhered and embedded into the rubber matrix, forming
characteristic penetration craters on the surface (Fig. 4). Linos et al.
(2000a,b) reported about the isolation of a Gram-negative bacte-
rium from foul tyre water, which was taxonomically characterized
as Pseudomonas aeruginosa AL98. SEM analysis also revealed the
effective colonization of the rubber surface by the strain AL98, and
formation of a biolm during cultivation. However, visible disin-
tegration of the rubber started only after 2 3 weeks (Linos et al.,
2000a,b).
5.3. Attenuated total reectance-Fourier transform infrared
spectroscopy (ATR-FTIR)
Chemical changes that arose directly on the rubber surface as
result of the biodegradation process were determined using ATR-
FTIR spectroscopy. This comprised the ATR technique a method
which allows non-destructive, in-situ analysis of surfaces coated by
microbial biolms (Linos et al., 2000a). The author has presented
the ATR-FTIR analysis of the NR latex gloves pieces after treatment
with Bacillus sp. AF-666. There was decrease in peaks at the region
1200e1400 cm
1
in rubber pieces treated with bacterial culture as
compared to control, indicating the breakdown of important
functional groups like C]C, carbonyl, methyl and ester bonds. A
peak at 3034 cm
1
, which was present in control, disappeared after
microbial treatment in case of test, also corresponds to CH
2
stretching. A peak at 835 cm
1
which corresponds to cis-1,4 double
bond decreased to 834 cm
1
after treatment with Bacillus sp. AF-
666. Changes in the range of 1160 cm
1
indicate formation of al-
dehydes in case of test pieces as compared to control (Fig. 5) (Shah
et al., 2012). Linos et al. (2000a) veried Tsuchiis proposed cleavage
mechanism for the rubber degrading Gordonia strains by implying
ATR-FTIR spectroscopy directly on the surface of biodegraded NR
latex glove material in the absence of cells. After proving me-
chanical removal of the biolm of Gordonia cells from the sample
surface, the spectra detected revealed signals corresponding to
those known from the literature for cis-1,4-polyisoprene in Hevea-
NR. These spectra exhibited the following changes when compared
with non-inoculated controls: (1) a decrease in the number of cis-
1,4 double bonds; (2) the appearance of ketone and aldehyde
groups; and (3) the formation of two different bonding environ-
ments (Linos et al., 2000a).
Cundell and Mulcock (1972) suggested that the overall degra-
dation process was oxidative, and resembled chemical ageing. The
authors assumed initiation of the biodegradation by a mono-
oxygenase, as was indicated by the additional occurrence of CeO
and C]O groups in the spectra of biodeteriorated rubber samples.
All the studies with various microorganisms indicated that during
rubber degradation oxidative cleavage of the double bond in the
poly (cis-1,4-isoprene) backbone must occur as the rst step.
Fig. 2. Scanning electron micrographs of the rubber pieces after treatment with Pae-
cilomyces variotii strain SFA-25 in mineral salt medium for 28 days. Rubber pieces were
washed with sterilized distilled water before SEM (a) un-treated (abiotic control);
(b&c) Hyphal network of strain SFA-25 can be seen on the surface of rubber pieces.
Hyphae are deeply rooted in the rubber pieces resulted in surface erosion and
breakdown. Bars, 5 mm (Zeb, 2009).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 150
Furthermore, most of the degradation products detected (Tsuchii
et al., 1985; Rose et al., 2005) contained aldehyde and keto
groups. This can be explained by oxygenases like RoxA. Even
enzyme mediator systems yielded in vitro degradation products
containing aldehyde or keto groups (Enoki et al., 2003; Sato et al.,
2003).
5.4. CO
2
evolution
CO
2
evolution/O
2
consumption (Puchner et al., 1995) or the
formation of carbon dioxide (Sturm test) is the most often used
method to measure biodegradation in laboratory tests as it gives
direct information on the bioconversion of the carbon backbone of
Fig. 3. ATR-FTIR spectra of rubber pieces after incubation with P. variotii strain SFA-25 in mineral salt medium for 28 days. (a) Abiotic control; (b) Presence of aldehydic group in
ATR-FTIR spectra were conrmed by appearance of peaks at 1680 and 3401 cm
1
. Increase in the intensity of the peaks at 1634 and 1680 cm
1
, corresponding to unsaturation due to
degradation. Appearance of a peak at 1590 cm
1
corresponds to stretching of >C]O indicated the formation of aldehyde group as a result of biodegradation of rubber (Zeb, 2009).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 151
the polymer to metabolic end product (Pagga et al., 2001). Deter-
mination of biogas, compared with the formation of CO
2
in the
presence of oxygen, anaerobic microorganisms produce predomi-
nantly a mixture of CO
2
and methane (called biogas) as one
extracellular product of their metabolic reactions (Gartiser et al.,
1998; Reischwitz et al., 1998; Abou-Zeid et al., 2001). The amount
of CO
2
liberated was determined during the cultivation of cells in
the mineral salts latex medium in which latex was the sole source
of carbon provided (Berekaa et al., 2000). The released CO
2
was
trapped in a solution of 1 N NaOHand was quantied by titrating of
the remaining NaOH with 0.1 N HCl (Alvarez, 2003). CO
2
evolution
as a result of rubber biodegradation by Bacillus sp. AF-666, was
determined by Sturmtest, in authors laboratory (Fig. 6) (Shah et al.,
2012). SturmTest was used to determine CO
2
evoluted as a result of
degradation of rubber by strain AF-666. Approximately 500 mg of
rubber pieces were added to the culture bottle (Test bottle) con-
taining 300 ml of MSM without any other carbon source. Both test
and control bottles were inoculated with overnight grown culture
up to 0.08 OD
600
, but the control bottle was without any rubber
substrate. Sterilized air was pretreated to remove dissolved CO
2
by
passing it through pretreatment chamber consisting of two bottles
having KOHsolution (3 M). The test and control bottles were stirred
continuously by placing them on the magnetic stirrer. The test was
performed at roomtemperature (30

C) for 30 days. After 30 days of

culturing, the change in viable cell count and the amount of CO
2
produced was calculated in the test and control bottles. CO
2
evolved
as a result of rubber utilization was trapped in the absorption
bottles containing KOH (1 M). Barium chloride solution (0.1 M) was
added to the CO
2
containing KOHbottles and as a result precipitates
of barium carbonate (BaCO
3
) were formed. CO
2
was calculated out
gravimetrically from the CO
3
precipitates evolved by addition of
BaCl
2
. Difference in the amount of precipitates in the test and
control was determined (Muller et al., 1992). The microorganism
breakdown the polymers and the carbon is used for their metabolic
activities and result in release of carbon dioxide as end product.
More amount of CO
2
was produced in case of test due to the utili-
zation of rubber as a carbon source by microorganisms during their
growth. The increased CFU/ml in case of test also proved the ability
of microorganisms to utilize rubber and increase in number. Sturm
test is cheap and easy to perform but laborious because regular
monitoring is required to check the continuous and controlled
supply of CO
2
free air to the test and control vessels, and to mini-
mize any chances of air leakage. Sturm test (Sturm, 1973) was
commonly employed for evaluation of the biodegradability of
polymer materials (Calmon et al., 2000). Various modications of
this test have been used for the measurement of carbon dioxide
evolution during degradation of biodegradable polymers (Muller
et al., 1992), and the aliphatic and aromatic compounds (Kim
et al., 2001). Similar kind of test was performed by Warneke
et al. (2007) for determining the percentage of mineralization of
poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene). They found
that the evidence of biodegradation of hydrocarbon chain to CO
2
was obtained by determination of respiratory CO
2
released during
cultivation of cells in the presence of polyisoprene as sole carbon
source. Keeping in view the ndings of these researchers we have
employed the similar analytical method to determine the degra-
dation of rubbers and were able to conclude that Sturmtest can also
be used to check degradation of rubbers.
Additional problems arise from the presence of other biode-
gradable compounds in natural rubber and latex or from additives
which are required for vulcanization or to inuence the material
properties. To avoid allocation of growth or CO
2
release to de-
gradation of, e.g., proteins and lipids present in the material,
growth and mineralization experiments must be performed care-
fully. Additives can promote (e.g., llers and stoppers) or inhibit
Fig. 4. Scanning electronmicrographs of the polyisoprene rubber pieces after treatment
with Bacillus sp. AF-666 in mineral salt mediumat 37

C for 21 days. Rubber pieces were

washedwithsterilizeddistilled water before SEMinorder tomake the surface clear from
the growth of strain AF-666. (a) Abiotic control (500); (b&c) Strain AF-666 attached to
the surface of rubber piece, degraded the rubber and resulted in surface erosion and pits
formation (500 & 1000 respectively). Bars, 10 mm (Shah et al., 2012).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 152
(accelerators, antioxidants, and preservation material) biodegrada-
tion of rubber material (MacLaghlan et al., 1966; Gomez and Moir,
1979). The inhibitory effect of antioxidants, extracted from syn-
thetic polyisoprene, on the growth of G. westfalica was demon-
strated by Berekaa et al. (2000). It was also shown that extraction of
latex gloves with organic solvents before incubation enhanced the
growth of some rubber-degrading strains (Berekaa et al., 2000).
5.5. Effect of pretreatments
Brune et al. (2000) studied the effect of pretreatment of
different rubber materials on its biodegradability by several bac-
teria. The effect of pretreatments on biodegradation of several cis-
1,4-polyisoprene containing rubbers was also examined by Berekaa
and colleagues and isolated different bacterial genera like Gordonia,
Fig. 5. ATR-FTIR spectrum of polyisoprene rubber piece after treatment with Bacillus sp. AF-666 in mineral salt medium at 37

C for 21 days (a) Abiotic control (b) After bacterial
degradation, a number of peaks have been decreased in the region 1200e1400 cm
1
. A peak at 3034 cm
1
has been disappeared after microbial treatment corresponds to CH
2
stretching. The peak at 835 cm
1
which corresponds to cis-1,4 double bond decreased to 834 cm
1
after treatment with Bacillus sp. AFe666. Changes in the range of 1160 cm
1
indicates formation of aldehydes (Shah et al., 2012).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 153
Micromonospora, Mycobacteria and Pseudomonas (Berekaa et al.
2000). All species were able to use natural rubber (NR) as well as
NR latex gloves as sole carbon source. Further analysis by scanning
electron microscopy (SEM) clearly demonstrated the enhanced
colonization efciency of these bacteria towards pretreated NR la-
tex gloves. Colonization was additionally visualized by staining of
overgrown NR latex gloves with Schiffs reagent, and the purple
colour produced in the area of degradation was an evidence for the
accumulation of aldehydes containing oligomers (Berekaa et al.,
2000). Shah et al. (2009) reported the effect of acetone, UV and
sunlight pretreatments on biodegradation of rubber by measuring
the growth of Bacillus sp. strain S-10 by taking absorbance at
600 nmafter every four days interval. Absorbance data showed that
Bacillus sp. strain S-10 was better able to utilize acetone pretreated
natural rubber latex gloves as a sole source of carbon as compared
to UV, sunlight and un-pretreated rubber pieces. In IR containing
the antioxidant 6PPD (N-(1,3-dimethyl-butyl)-NP-phenyl-p-
phenylene diamine), the 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 species on IR was only possible
after acetone-extraction of the material. This nding suggested also
to use treated IR as enrichment substrate to isolate additional
rubber degrading bacteria (Berekaa et al., 2000).
Lee et al. (1991) studied the effects of UV pretreatment on the
change in molecular weight and biodegradation of polymeric ma-
terial that is polyethylene. UV light is a known initiator of poly-
ethylene oxidation, and photo-oxidant activity is enhanced by the
addition of transition metals such as cobalt, manganese, nickel, and
zinc, which are also used as pro-oxidant catalysts. Almost no change
in molecular weight was observed for the zero control after 2 weeks
of UV irradiation, whereas, an average 2.5-fold increase in molec-
ular weight was demonstrated for all incubated lms (inoculated
and uninoculated) (range, 1.7e3.3-fold increase) when compared
with zero time samples. However, the 4-week UV treatment did
accelerate bacterial biodegradation when compared with its cor-
responding control. Streptomyces badius and Streptomyces setonii
caused the reduction in molecular weight of polyethylene lmup to
31 and 36%, respectively, after pre-treatment with UV light for 4-
week (Lee et al., 1991).
6. Rubber degrading enzymes
It is very important to have information about the enzymes
involved in catalysing cleavage of the rubber backbone and disin-
tegrate it into intermediate products. The latex clearing protein
(Lcp) from the Gram-positive Streptomyces sp. K30 (Rose and
Steinbuchel, 2005) and a rubber cleaving deoxygenase RoxA from
the Gram-negative Xanthomonas sp. 35Y (Jendrossek and
Reinhardt, 2003) have been identied and characterized. Both of
these bacteria belong to the clear zone forming group of rubber-
degrading bacteria, and both of the Lcp and RoxA are the extra-
cellular enzymes responsible for the formation of translucent halos
on the natural rubber latex. Both the Lcp and RoxA are apparently
completely different polypeptides with no signicant amino acid
similarities (Yikmis and Steinbchel, 2012a).
6.1. Latex clearing protein (Lcp)
Bacteria differ in their mechanisms of polymer degradation in
three possible ways: (i) the secretion of extracellular enzymes or
factors resulting in halo formation on latex agar (e.g., Xanthomonas
sp. and many actinomycetes); (ii) also involves in the secretion of
enzymes or factors but without halo formation on latex agar; (iii)
the third requires surface colonization of solid rubber materials
with or without solubilization of solid rubbers to water-soluble
products and subsequent degradation as it has been described for
Gordonia strains (Linos et al., 2000a). According to the recent re-
ports, Lcp is an essential constituent of a protein complex respon-
sible for clear zone formation on latex agar overly plates by cleavage
of poly(cis-1,4-isoprene). This relationship was conrmed after
expression of Lcp from Streptomyces sp. strain K30 in strains of
S. lividans and Saccharopolyspora erythraea (Yikmis and
Steinbchel, 2012a). The Lcp secretion in the extracellular me-
diumwas conrmed by detection of Lcp-eGfp fusion proteins in the
supernatant employing anti-eGfp and anti-Lcp antibodies. The Lcp
Fig. 6. Schematic diagram of Sturm test (Shah et al., 2012).
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 154
from Streptomyces sp. strain K30 has been cloned and expressed in
different Streptomyces species by protoplast transformation. All
protein solutions were obtained from cell-free supernatant of cells
of S. erythraea, S. lividans TK23, and TK24 grown in LB medium at
30

C. The expression of Lcp in these species was analysed by SDS-
PAGE and identied by Western blotting employing anti-Lcp-IgG
antibodies. Approximately 43-kDa Lcp protein was nally recog-
nized (Yikmis and Steinbchel, 2012b). The Lcp gene encoding latex
clearing protein from Streptomyces sp. strain K30 has been identi-
ed and characterized by Braaz et al. (2005). Analysis of the amino
acid sequence encoded by lcp revealed a twin arginine motif,
indicating that Lcp is a substrate of the twin-arginine translocation
(Tat) pathway (Yikmis et al., 2008).
The function of enzyme Lcp from Streptomyces sp. strain K30
was investigated by disrupting lcp gene and the knock out lcp
mutant was successfully obtained. The resulting mutant Strepto-
myces sp. K30_lcpUKm exhibited reduced growth in liquid mineral
salts media containing poly(cis-1,4-isoprene) as the sole carbon and
energy source. Additionally, there was no detectable Lcp activity on
latex overlay agar plates. The heterologous expression of Lcp from
Streptomyces sp. strain K30 in strains TK23 and TK24 of S. lividans
and a strain of S. erythraea with plasmid pIJ6021::lcp was success-
ful. The recombinant strains acquired the ability to cleave synthetic
poly(cis-1,4-isoprene), conrming the involvement of Lcp in initial
polymer cleavage (Yikmis and Steinbchel, 2012b).
6.2. Rubber oxygenase A (RoxA)
Rubber oxygenase A (RoxA) is an extracellular rubber oxidizing
enzyme isolated from cell free culture supernatant of latex grown
Xanthomonas sp. strain 35Y. This enzyme degraded both natural
rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by
oxidative cleavage of the double bonds of poly(cis-1,4-isoprene)
(Braaz et al., 2004; Hambsch et al., 2010). RoxA cleaves poly(cis-1,4-
isoprene) to give 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al
(ODTD) as a major degradation product, with a homologous series
of minor metabolites that differ from the major degradation prod-
uct only in the number of repetitive isoprene units between ter-
minal functions, CHOCH
2
e and eCH
2
eCOCH
3
(Braaz et al., 2004).
The latex degrading activity of RoxA was analysed in vitro by
growing Xanthomonas sp. on mineral salt mediumwith puried latex
for about 7e9 days. When aliquots of fresh latex was added to cell-
free concentrated supernatant and incubated at 30

C, latex was
coagulated or degraded within 2 days, whereas the control mixture
remained milky. It was concluded that apparently an enzyme was
responsible for coagulation or degradation of latex. The latex
degrading enzyme RoxA was puried to homogeneity by dia-
ltration and subsequent chromatography on Q-Sepharose and
MonoP (chromatofocusing). Sodium dodecyl sulphate-
polyacrylamide gel electrophoresis indicated a RoxA protein of
approximately 65 kDa showed strong absorption at 406 nm (Braaz
et al., 2004). The properties of the puried protein were studied
and it was concluded that RoxA puried protein caused an oxidative
cleavage of rubber. It did not require the addition of any soluble co-
factors or metal ions for its activity. The optimal pHand temperature
for degradation was 7.0 at 40

C, respectively. Carbon monoxide and
cyanide inhibited the reaction. Peroxidase activity was negative, and
addition of catalase had no effect. RoxA was insensitive to high
concentrations of chelating agents such as EDTA. Analysis of the
amino acid sequence deduced from the cloned gene (roxA [rubber
oxygenase]) revealed the presence of two haem-binding motifs
(CXXCH) for covalent attachment of haem to the protein. Spectro-
scopic analysis conrmed the presence of haem, and approximately
2 mol of haem per mol of RoxA was found (Braaz et al., 2004). It has
cleared from the details that RoxA is not a peroxidase and that it
apparently uses a different reaction mechanism for polyisoprene
cleavage. RoxA causes the oxidative cleavage of a carbonecarbon
double bond of polyisoprene and not the reduction of hydrogen
peroxide or another compound (Schmitt et al., 2010).
Since the double bonds of poly(cis-1,4-isoprene) are cleaved by
incorporation of oxygen, a monooxygenase- or dioxygenase-based
reaction mechanism is possible. Tsuchii and Takeda (1990) sug-
gested a dioxygenase cleavage mechanismof poly(cis-1,4-isoprene)
based on preliminary experiments using cell-free culture uid of
Xanthomonas sp. Linos and Steinbuchel (2001) assumed a common
oxidative cleavage mechanism for all types of rubber-degrading
organisms and proposed a metal-dependent dioxygenase mecha-
nism. A monooxygenase-based mechanism of poly(cis-1,4-
isoprene) cleavage appears not very likely since RoxA is an extra-
cellular enzyme and functions in vitro without any cofactors such
as NADH or related compounds. However, experimental data that
conrmed the presence of a dioxygenase mechanism or excluded a
monooxygenase mechanism did not exist (Braaz et al., 2004). Braaz
et al. (2005) investigated the oxidative cleavage of poly(cis-1,4-
isoprene) by rubber oxygenase RoxA puried from Xanthomonas
sp. in the presence of different combinations of
16
O
2
,
18
O
2
, H
2
16
O,
and H
2
18
O. 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD; m/z
236) was the main cleavage product in the absence of
18
O-
compounds. Incubation of ODTDwith H
2
18
Oin the presence of
16
O
2
indicated that the carbonyl oxygen atoms of ODTD signicantly
exchanged with oxygen atoms derived from water. The isotope
exchange was avoided by simultaneous enzymatic reduction of
both carbonyl functions of ODTD to the corresponding dialcohol
(12-hydroxy-4,8-dimethyltrideca-4,8-diene-1-ol (HDTD; m/z 240)
during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In
the presence of
18
O
2
, H
2
16
O, and alcohol dehydrogenase/NADH,
incorporation of two atoms of
18
O into the reduced metabolite
HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a
dioxygenase mechanism (Braaz et al., 2005).
7. Analysis of rubber degradation products
Analysis of the degradation products of disintegrated latex
gloves revealed several compounds, which could be separated by
high-performance thin-layer chromatography. Three of the com-
pounds isolated were identied by one- and two-dimensional 1H
NMR spectroscopy as 2,6-dimethyl-10-oxo-undec-6-enoic acid,
5,6-methyl-undec-5-ene-2,9-dione, and 5,9-6,10-dimethyl-penta-
dec-5,9-diene-2,13-dione. Several Gram-positive as well as Gram-
negative bacteria such as Acinetobacter calcoaceticus, Xanthomo-
nas sp., and P. citronellolis (Bode et al., 2000) were reported as
rubber degraders in terms of molecular weight distribution during
degradation. An endo-type reaction was responsible for degrada-
tion of polymer. However, the biochemical reactions, which cleave
the polymer and the subsequent reactions leading to the central
metabolism, are still unknown (Bode et al., 2001). An oxidative
cleavage of the polymer for initiation of microbial rubber degra-
dation has been suggested (Tsuchii et al., 1985; Tsuchii and Takeda,
1990). This assumption was based on the identication of acetonyl
diprenyl acetoaldehyde and related oligomers in the culture uid of
a rubber-grown culture of Xanthomonas sp. 35Y or Nocardia sp.
835A. Bode and co-workers isolated the three low molecular
weight rubber degradation products which were identied by
comparative HPTLC (Bode et al., 2000, 2001). Additional low mo-
lecular weight degradation products were also found (Bode et al.,
2000). The majority of the accumulated molecules could be
degraded by b-oxidation, and propionyl-coenzyme A (CoA) would
be generated. After two cycles of b-oxidation one isoprene unit
would be metabolized to acetyl-CoA and propionyl-CoA. The last
isoprene unit of the primary oxidation product containing the
A. Ali Shah et al. / International Biodeterioration & Biodegradation 83 (2013) 145e157 155
carbonyl function of the primary degradation product would be
cleaved into acetyl-CoA and pyruvate. The principle degradability
of R-methyl-carboxylates to acetyl-CoA and propionyl-CoA is
known from the isoleucine degradation pathway. If propionyl-CoA
is a metabolite of rubber degradation, one would expect that
rubber-degrading bacteria generally are able to grow with propi-
onate as a sole source of carbon and energy. The b-oxidation
pathway resulting in a sub-sequential cleavage of one isoprene unit
in to one molecule of acetyl-CoA and propionyl-CoA each. Addi-
tional evidence for the involvement of a functional b-oxidation
pathway was obtained for S. coelicolor 1A by the selective inhibition
of rubber degradation by the b-oxidation-specic inhibitor acrylic
acid. It is unknown whether the identied metabolites 2,6-
dimethyl-10-oxo-undec-6-enoic acid, 6-methyl-undec-5-ene-2,9-
dione and 6,10-dimethyl-pentadec-5,9-diene-2,13-dione, are dead
end products or whether they can be degraded. Enzymatic studies
will be necessary to elucidate which of the proposed biochemical
reactions is present in rubber degrading bacteria (Bode et al., 2001).
8. Conclusions
Accumulation of knowledge on the biodegradation of natural
and synthetic rubbers during the past century was mainly accom-
panied by progress of the analytical equipment needed for these
investigations. The simple visual and microscopic observations
used during the early stages were in turn replaced by electron
microscopy and spectroscopy approaches, enabling detailed illus-
tration to be made of microbial rubber colonization and the
chemical structure of rubber degradation products to be elucidated.
It is important to work further in understanding the biochemical
pathways of rubber biodegradation and putting even more efforts
on the characterization of enzymes involved in rubber biodegra-
dation so that engineered systems could be generated to overcome
these rubber waste management problems more effectively.
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