CJ
171N
II
172
None of the Streptomyces spp. (S. rochei A14, S. rochei A15, S.
chromofuscus A l l , S. diastaticus A12, and S. diastaticus A13) tested were able to decolourize Acid Yellow 9 (XXIV). However, five monosulfonated, two ring, mono azo dyes were decolourized by these bacteria. In all cases, these dyes had a hydroxyl group in the para- position, relative to the azo linkage, and at least one methoxy and/or one alkyl group in an ortho-position, relative to the hydroxyl group. Of the azo dye derivatives of naphthol studied, Streptomyces spp. decolourized Orange I (XI), but not Orange II (XII) or Orange 12 (XLVII).
Interestingly, P. chrysosporium decolourized all three. However, Orange II (XII) and Orange 12 (XLVII) were decolourized more effectively than Orange I (XI).
Although, most of the research, concerning the biodegradation of azo dyes by fungi, has focused on the use of P. chrysosporium, several Myrothecium spp. and several Ganoderma spp. were shown to be able to mediate substantial decolourization of Orange II and two relatively complex azo dyes, designated as RS(H/C) (XLVIII) and 10B(H/C) (XLIX) [50]. Of interest is the fact that many Myrothecium spp. and Ganoderma spp. are like P. chrysosporium, white rot fungi.
Very limited information is available, concerning the ability of other classes of fungi to remediate water contaminated with azo dyes. It is interesting to note [51] that the ascomycete, Neurospora crassa (strain 74A) was able to decolourize water containing the diazo dye, Vermelho Reanil, which was present in concentrations within the range (16-32 ~g ml -~) found in industrial effluents. It was unclear, however, if the fungus was able to metabolize the dye or if water decolourization was due solely to adsorption by fungal mycelium.
In an investigation concerning biodegradation of Reactive Red 22 (L) by bacteria [52], a stable consoritum of four bacterial species (Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Acinetobacter calcoaceticus, and Citrobacter freundii) was developed from a mixture of soil and sewage microorganisms, that was acclimated to Reactive Red 22 (L) under aerobic conditions. Reactive Red 22 (L) did not appear to serve as a sole carbon source for the bacterial consortium.
However, extensive decolourization occurred when glucose was present in culture. Seven metabolites of this dye were identified. Although no aromatic ring cleavage products were identified, the authors [52] suggested that complete biodegradation to carbon dioxide may occur in this systen~
In another study [53], three azo dyes, Diamira Brillian Orange RR (LI), Direct Brown M (LID, and Eriochrome Brown R (LIII), were decolourized aerobically by Pseudomonas S-42, isolated from activated sludge.
Decolourization also occurred in cell-free extracts by an enzyme purified from this bacterium. The enzyme, an azoreductase, appears to have a broader specificity than the one previously studied by Zimmermann et al. [22].
173
OCH3 OH
N~---'N
O=~ ~ SO3H
SOaNa
REACTIVE RED 22 L
OH
aO3S
$O3H OH2 I
CH2 I
SOaNa I
NHCOCOa
Na~
DIAMIRA BRILLIANT ORANGE RRLI
OH
... N .
DIRECT BROWN M LII
OH NH2
/ \
NO2 SOaNa
ERIOCHROME BROWN R LIII
174
C O N C L U S I O N S
Azo dyes, as a group, are resistant to biodegradation by microorganisms [2,18]. However, research during the past 10 years has clearly shown that a variety of microorganisms and approaches hold promise for the development of effective systems for the treatment of water (and possibly soils, sediments, and sludges), contaminated with these colourants. For bacterial systems, the most promising approach appears to utilize a sequential system in which the azo dye is initially reduced in anaerobic culture, and the aromatic amines, thus generated, are further metabolized under aerobic conditions [19]. Until recently, azo dyes were not thought to be degraded by microorganisms, unless an anaerobic step was included in the process. Although some specialized bacteria have been developed, which are able to degrade some simple azo dyes in aerobic culture [21], this was considered to be an exception. Furthermore, these unique bacteria are very specific and degrade only those azo dyes to which they have become adapted. The finding, that Pseudomonas S-42 [53] can decolourize three structurally diverse azo dyes, suggests that it may be possible to develop or discover other strains of bacteria, with an even broader spectrum of biodegradative abilitites. The discovery, that the white rot fungus P. chrysosporium degrades a wide variety of azo dyes, provides an entirely new approach to the study of azo dye biodegradation [33]. Possibly, the most interesting finding is that the initial degradation of many azo dyes, in this system, is not a reduction of the azo linkage, but rather an oxidation reaction, mediated by lignin peroxidases or Mn peroxidases that are secreted by the fungus during idiophasic metabolism.
The observation, that other white rot fungi (Myrotheeium spp. and Ganoderma spp.) degrade azo dyes, suggests that this ability may be widespread among such fungi [50].
AC K N O W L E D G E M E N T S
Research in the author's laboratory is supported by NIEHS grant ESO 4492.
R E F E R E N C E S
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Biotransformations: Microbial Degradation of Health Risk Compounds Ved Pal Singh, editor
9 1995 Elsevier Science B.V. All rights reserved. 177
Microbial d e g r a d a t i o n of natural rubber Akio Tsuchii
National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba city, Ibaragi 305, Japan
INTRODUCTION
Natural rubber (NR) was once the only source of the elastomeric materials required by a wide range of products. Nowadays, a much greater amount of synthetic rubbers than NR is used in an even larger number of products.
Many synthetic polymers, like plastics and rubbers, are highly resistant to microbial degradation. As a result, such polymers are accumulating in the environment in huge quantities. This has led to a growing interest in the development of degradable plastics with enhanced bio-degradability and photo-degradability in landfills and composts [I].
NR is today obtained mainly from Hevea trees on plantations in tropical Asia, from where it is exported throughout the world as an industrial raw material. As noted by Thomas Edison, however, many plants in temperate climates, like the goldenrod and the dandelion, also contain rubber in small amounts [2,3]. Consequently, rubber- degrading microorganisms can be expected to be present widely in the natural environment.
NR is also degraded by solar ultraviolet rays [4]. Thus, NR has been degraded since prehistoric times by the action of both microorganisms and sunlight. The fact that NR is not only a renewable resource but also an enviromentally degradable material would be appreciated more.
RUBBER-DEGRADING MICROORGANISMS
Actinomycetes play a major role in degradation of NR [5], while some strains of fungi and bacteria are also known to attack rubber.
Microorganisms, capable of degrading NR, cannot degrade synthetic rubbers other than synthetic isoprene rubber [6,7]. Although there have been a number of reports concerning microbial degradation of various synthetic rubbers, degradation of the hydrocarbon polymer has not been demonstrated yet.
178
M I C R O B I A L D E G R A D A T I O N OF UNVULCANIZED R U B B E R Microbial attack of raw rubber was first reported early in 1914. Thin films of NR, floating on an aqueous medium, were shown to be disintegrated by some actinomycete strains to a certain extent, that could not be ascribed to the disappearance of impurities from the rubber [8]. Spence and van Niel [9] reported that NR, in the latex state, was degraded by some actinomycete strains, and that a rubber weight loss of up to 70%
was observed a t ~ r a 28-day cultivation period [9]. Thin films of NR on agar plates were also found to be degraded by strains of Streptomyces and Nocardia, and the weight loss reached 52% after the cultivation period of a month and a half [10]. It was reported that thin strips of NR and synthetic isoprene rubber, with a diameter of 0.5 ram, were decomposed completely by a strain of Nocardia in 56 days [11].
Blake and Kitchin [12] found that colonies of Gram-positive micrococci developed on thin films of NR, and that the surface was extremely pitted by action of bacteria as well as actinomycetes [12]. A 20 per cent weight loss of rubber films by the action of a bacterial strain has also been reported [10].
Unlike rubber-degrading actinomycete colonies, fungal colonies on latex-agar plates are not surrounded by transparent zones [5]. Superficial growth of fungi on NR films has been observed with a negligible consumption of the rubber [10]. Growth of the molds on rubber may have proceeded at the expense of the non-rubber constituents, like proteins in these cases.
The latex of NR was found to be attacked by some AspergiUus and Penicillium strains, with a 32% weight reduction after a month cultivation [13]. Williams [14] reported that a 2 mm thick smoked sheet of NR was degraded by a strain of Penicillium and lost approximately 13% of its dry weight over 56 days' cultivation [14]. A fungal growth of 4.1 mg protein/ mm 2 was obtained at the same time. It was observed that ground particles of NR (between 0.8 and 2 mm in diameter) were attacked by a stroJn of Cladosporium, and that 6 successive treatments of 20 days each caused a decrease in molecular weight, estimated by gel permeation chromatography (GPC), from 2 x 10 e to 1 x 105 [15].
Production of 7.6 mg of protein from 5 g of nitrile rubber by a mixed culture of bacteria was reported [16]. A strip of styrene-butadiene rubber (1.5 x 2.5 x 0.2 cm) was buried in soil, and a significant growth of fungi (2.8 ~g protein/ram 2) was detected after a 9-month incubation [14]. From these observations, however, it was very difficult to estimate the degradation of polymer itself.
Antoine et al. [17] reported t h a t a terpolymer of acrylonitrile, methylacrylate, and butadiene was bioconverted by a strain of either Nocardia or Penicillium [17]. They found that, after 6 months of incubation, the terpolymer was transformed into both a lower molecular weight
179 form and a second form insoluble in dimethylsulphoxide. It was observed that a strain of Moraxella grew on a 1,4-type polybutadiene with an average molecular weight of 2,500 and degraded 44% of the oligomer in 5 days [18]. However, a polymer of butadiene with average molecular weight of 17,000 was not attacked by the bacterial strain.
MECHANISMS OF NR DEGRADATION
I n f r a r e d spectroscopy analysis of microbiologically deteriorated vulcanized NR by Cundell and Mulcock [19] indicated the following structural and chemical changes: the presence of the hydroxyl and carbonyl structures at 3,500 cm -1 and 1,720 cm -1, respectively [19];
decrease in unsaturation at 890 cm -~, and appearance of a broad peak in the 1,000 cm -~ region, which might represent ether, epoxide, or peroxide group. Biochemical oxidation of NR tire tread, ground to about 100 mesh sieve size, was also reported [20]. After bioexposure of the ground particles in a perfusion reactor for 30 and 60 days, the infrared curve showed a great increase of structures related to CffiO in acid, aldehyde and ketone.
Spence and van Niel [9] noted that, when a sterile latex of NR was degradad by soil microorganisms, the relative viscosity of the dilute rubber solution became lower [9] : it dropped from 20 sec (control) to 3 sec, showing that the residual rubber was thoroughly deteriorated, soft and "dead."
It was observed that isoprene oligomers, with molecular weight from 108 to 104, accumulated during microbial growth on a latex glove. From the chemical structure of the oligomers, it was supposed that the strain of Nocardia cleaved NR at the double bond shown by a wavy line in the formula in Figure 1 [11]. The net-work of the vulcanizate was considered to have been attacked directly by the biological action during microbial degradation of the glove, with the oligomers produced by the scission of polymeric chains being used by the organism as growth substrate.
CH 3 CH 3 CH 3
I I 2 I
-CH2-C = CH-CH2--CH2- C :~ CH-CH2- CH2-C = CH-CH2-
CH 3 CH 3 CH 3
I I I
-CH 2- C=CH 2- CH2-C= O --I- O = CH-CH2-CH 2-C =CH--CH 2-
Figure 1. Schematic representation of natural rubber-cleaving reaction.
180
The formation of clear zones surrounding actinomycete colonies on a latex-agar plate can be regarded as an indication of extracellular enzymatic decomposition [9].
Quite recently, Tsuchii and Takeda [21] have reported that rubber- degrading enzyme was secreted in the extracellular culture medium by a strain of Xanthomonas [21]. The latices of natural and synthetic isoprene rubber are degraded by the crude enzyme, but no reaction is observed on the latices of other kinds of synthetic rubber. Isoprene oligomers, with average molecular weight of 104 (acetonyl polyprenyl acetoaldehyde, ALPnAt), are produced by random scissions of NR in endowise form, and ALPHA t iS further degraded to form mainly 12-oxo- 4,8-dimethyltrideca-4,8-diene-l-al (acetonyl dipreny! acetoaldehyde, ALP2At).
MICROBIAL DEGRADATION OF VULCANIZED RUBBER
Rubber products, made of NR, are known to be rather susceptible to biological attack, and synthetic rubbers are preferred in certain types of rubber goods to be used in moist air, the domestic water supply system, and soils [22].
Microbial degradation of synthetic rubbers will be a subject of further study. A rubber product is made from a number of complex ingredients, and smaller molecules in a synthetic polymer (e.g., stearate, process oils, and waxes in vulcanized synthetic rubber) may be decomposed by microorganisms. A clear distinction must be made between the superficial growth of microorganisms on non-rubber constituents in a synthetic polymers and the biodegradation of the rubber hydrocarbon [23].
Many components in vulcanizates, such as accelerators, fillers, oils, and antioxidants, are known to affect microbial activities, and protection of rubber goods against microorganisms, by the addition of chemicals with microbiocidal activities, has been an important research area.
However, the use of microbiocides is beyond the scope of this review, and extensive references have been given by Zyska [24,25]. The present review mainly covers the degradation of the polymer itself.
D E T E R I O R A T I O N OF R U B B E R P R O D U C T S
As far as it is known, the microbial attack of vulcanized rubber was first investigated in 1942, when oxygen consumption and carbon dioxide production were observed during microbial growth [26,27]. When buried in soil, the rubber insulation of electric cables loses its insulating properties [12,28]. With NR compounds, loss of electrical resistance of the insulation is accompanied by visible pitting caused by actinomycetes, and fungal hyphae penetrating the insulation.
181 Microbial corrosion of vulcanized NR sealing rings was observed in the underground pipelines for water supply in areas of Holland, Australia, New Zealand, and America. Since then, a number of investigations have been made to protect the rubber gaskets from the attack of microorganisms.
Pure cultures of certain strains of Streptomyces apparently attack thin strips of vulcanized NR, leading to a marked decrease in tensile strength after 12 months [5]. The presence of large population of Streptomyces
spp. was found in the deteriorated rubber rings [6].
In 1968, Leeflang tested different rubber compounds for their resistance to biological attack [29]. The strips were immersed in a basin, through which a slow and constant flow of potable water was maintained, and a piece of deteriorated ring was placed on the bottom of the basin as a source of Streptomyces. It appeared that all NR compounds tested were susceptible to corrosion in the long run, but with the exception of synthetic polyisoprene, synthetic rubber compounds were resistant. On the other hand, the addition of 5% of casein to nitrile rubber did not make it susceptible to attack by Streptomyces [29]. The method, first used by Leeflang, was an excellent way to estimate the resistance of many compounds in vivo under laboratory conditions, and was used by many investigators as a standard [30]. In areas of Holland, where corrosion was most pronounced, high phosphate level in the dune water and the absence of deliberate chlorination were thought to contribute to the ability of actinomycetes to proliferate [31].
In 1975, Hutchinson et al. reported that the population of actinomycetes, isolated from deteriorated rubber rings, was 5x10S-4x106/g, while that of the organism, isolated from undeteriorated rings was 3x108-4x104/g [32]. Thiobacflli were also isolated from the rubber rings in the pipelines of municipal sewage, and found to have a population of 22x104/g [32].
Up to 40% loss in weight of a strip of vulcanized NR (0.07 mm thick) after 91 days of soil burial test was reported by Kwiatkowska et al. [33].
In 1986, Williams buried vulcanized NR sheet (15x15x0.2 cm) in soil for 6 months and observed a 4.5% loss in weight, accompanied by a 66% loss in tensile strength [34]. According to a report by Simpson, the weight of a 2 mm thick strip of NR vulcanizate decreased by 10.7% after an immersion period of 2 years, under accelerated test conditions of Leeflang's test basin [35]. In 1988, Kwiatkowska and Zyska found the weight loss of NR vulcanizate sheet to be 8.5% after 28 days of exposure to an
Aspergillus strain [36], while in 1991, Kajikawa et al. found that thin film of a commercial glove (0.2 mm thick) was completely degraded by a strain of Nocardia after a 20-day cultivation period [37].
Of the 31 references published between 1942 and 1972, 94% gave evidence of susceptibility of NR to microbial attack, while 24 to 50% of them reported the resistance of various synthetic rubbers [38].
Blake et al. reported that, when synthetic rubber insulation made of styrene-butadiene rubber was buried in soil, invisible micropores and