International Biodeterioration 24 (1988) 289-298

Screening of Fungai Strains Employed in the Testing of Plastics Materials

J. Kel ley & P. A. Y a g h m a i e

CAB International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, UK

ABSTRACT

The problems of selecting test methods and suitable challenge organisms for plastics materials are discussed. Enzyme activities of fungal strains employed in the testing of plastics have been investigated and compared with isolates of these organisms held elsewhere. The ability of organisms to clear a polycaprolactone diol medium has been assessed semiquantitatively and the results discussed in relation to the selection of organisms.

I N T R O D U C T I O N

The susceptibility of plastics materials to microbial attack is not easy to establish unambiguously. Formulat ions contain many impurities such as residual oligomers, monomers , reagents and products of side reactions. There are also many compounds used as plasticizers and fillers. It is therefore difficult in a commercial plastics material to identify the components which may be supporting microbial growth. The heterogenous nature of most plastics materials must also be borne in mind when selecting suitable organisms to test their resistance to microbial attack.

Procedures

All microbial growth testing is based on 'the inducement of growth and measure of its degree' (Jones, 1968). To achieve this the material under

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investigation must be brought into contact with the organisms which may attack it. This is usually achieved via pure culture inoculation, mixed culture inoculation or soil burial. This paper concentrates on methods employing named identifiable organisms rather than techniques based on soil burial or tropical chamber incubation, and will consider the fungi currently recommended. Detailed reviews of methods of testing plastics have already been produced (Pantke, 1977; Osman & Klausmeier, 1977; Kelley, 1985).

Test organisms

Table 1 shows some of the organisms employed in standards testing and illustrates that many of them are common to a number of tests from various national standards organizations. It is unlikely that these organisms were chosen individually for each test to give the optimum 'performance'; the choice was probably made by selecting from known organisms quoted in other standards. Several of the isolates used in plastics testing were not isolated from synthetic polymeric materials but were adopted from other areas, for example textile testing. No systematic screening of organisms for activities has previously been carried out either on isolates from plastics materials or elsewhere.

A number ofthe organisms currently employed were first isolated 40 to 50 years ago (Table !) and were not deposited in a public culture collection until some years after isolation. The organisms may have changed their characteristics during fifty years of preservation - - much of which occurred before the advent of freeze-drying and liquid nitrogen storage procedures. Changes in the strain through spontaneous mutation, heterokaryosis, the parasexual cycle, or inadvertent selection during transfer may mean that the isolate loses its ability to degrade the test substrate. It is therefore essential that test strains should either be obtained fresh from freeze-dried or liquid nitrogen storage at regular intervals or they should be presented with the relevant substrates regularly, and should be checked and tested for enzyme activity.

As protocols are re-written and updated it is desirable to re-assess the organisms employed. However, the basic information available on many organisms is insufficient to allow informed decisions to be made and it may therefore be easier to retain traditional test strains. Many methods do not quote a particular strain for use, but simply recommend a species and possibly only give a numbered strain as an example. In order to increase the information available on fungi held in its culture collection, CMI has started an extensive data-enhancement programme. The methods used are simple, semiquantitative techniques that are designed

292 .I. Kelley. P. A. Yaghmaie

to give the maximum useful information on as many organisms as possible, as quickly as possible. The effort is concentrated on those having some applied significance. In addition, current investigations are designed to: first, build up information on organisms which may attack plastics materials, with a view to providing a much wider range of candidate species for use as test organisms; second, investigate the occurrence and variation of known activities within species; and third, study the variation in activity of designated test strains held in a number of public and private collections.

METHODS

Alpha amylase activity was assessed by a plate clearing technique using a starch agar followed by an iodine developing stain. Cellulase activity was measured using a modification of the method of Smith (1977) which involved assessing the release of dye from a cellulose azure medium, prepared using the product from Calbiochem. Protease activity was investigated by observing the increase in liquidity of a gelatin medium over an eight day period. Polycaprolactone degradation was used as an indication of depolymerase activity. The method employed was an amendment to that described by Shuttleworth & Seal (1985). Citric acid production was detected by a simple spot method visualized using Altman reagent (Gaffney et al., 1954).

All results were rated on a 0 to 5 scale, from zero activity/metabolite production to very high activity. Growth rates were assessed and designated A to E, indicating very good growth to no growth.

RESULTS AND DISCUSSION

Organisms showing clearing of plates containing polycaprolactone diol

Table 1 gives the source oforganisms used previously in testing and gives their rating after testing on a polycaprolactone medium. Aureobasidium pullulans IMI 45533 and Giiocladium virens IMI 45553 (formerly Trichoderma viride) showed extensive clearing, while Chaetomiurn globosum IMI 16203 produced no clearing whatsoever and very little growth. The two organisms (IMI 108007 and IMI 114933) that were actually isolated from synthetic polymeric materials showed clearing but were not of the highest levels. Aureobasidium puilulans IMI 45533 (showing high activity) is not currently employed in standards testing of

Screening of fungal strains used in plastics testing 293

TABLE 2 Number of Strains Within Species Showing Clearing of Polycaprolactone Medium

Organism Number of strains tested Number showing clearance

Aspergillus niger 54 54 Paecilomyces variotii 16 16 Fusarium solani 37 36 Chaetomium globosum 14 7 Trichoderma harc.ianum 43 43 Gliocladium roseum i 2 I 1 Penicillium funiculosum 15 15

plastics materials while C. globosum IM145550 (showing little activity) is. Benedict et al. (1983) have shown that this strain of C. globosurn will, however, break down relatively high molecular weight polycaprolactones. Fields et al. (1974) found that a strain (not identified) of A. pullulans would, on the other hand, degrade low molecular weight polycapro- lactones but nothing over a molecular weight of 15 000. Benedict et al. (1983) concluded that degradation depends upon the molecular weight and crystallinity ofa polycaprolactone. Shuttleworth & Seal (1985) found that all organisms clearing a polycaprolactone medium did not necessarily break down all polyurethanes. However, a positive result indicates a potential degrader, which can then be investigated further using more sophisticated techniques. The results indicate that a strain isolated from one plastics material will not necessarily be useful as a test organism for all plastics materials.

Variation in activities within species

Table 2 shows the results oftesting a number of strains of a single species for their ability to clear a polycaprolactone medium. Some showed remarkable consistency, for example Aspergillus niger, Trichoderma

TABLE 3 Number of Strains of Trichoderma viride Showing Cellulase

and Amylase Activity

Enzyme activity No. of strains tested No. showing activity

Cellulase 33 28 Amylase 33 I !

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harzianum and Penicillium funicu/osum, while others like Chaetomium g/obosum gave wide variations.

The ability within species to produce amylase and cellulase activity also showed a wide variation, for example in Trichoderma viride (Table 3). Figure i illustrates that not only the ability to produce enzyme activity varies but also the amount of activity varies greatly under these conditions.

Variation in activities between strains held by different collections

Table 4 gives the activity ratings of two collections of strains with the same isolation details (held at Centraalbureau voor Schimmelcultures,

Screening of fungal strains used in plastics testing 297

Netherlands and at CMI in the UK). Organisms from other sources are currently under investigation. The activities are consistent and illustrate that careful main tenance and preservation will retain the desired qualities and activities of fungal strains.

These results suggest that the activities of organisms currently in use should be looked at carefully before employing them in updated or new protocols. The materials likely to be tested should be fully considered in order to help select the best 'package' of organisms to employ. The variation in activities found suggests that it is important to designate numbered strains for use in standards. It is inadequate to quote a number as an example and allow any isolate named as that organism to be employed, unless clear guidance is included explaining that non-designated strains should be tested fully against suitable substrates before being used as a test organism.

There is clearly scope for a more critical review and laboratory experimentation in the selection of test strains than is currently the practice in many standards organizations.

R E FE R E NC ES

Benedict, C. V., Cook, W. J., Jarrett, P., Cameron, J. A., Huang, S. J. & Bell, J. P. (1983). Fungai degradation of polycarpolactones. Journal of Applied Polymer Science, 28, 327-34.

Fields, R. D., Rodriguez, F. & Finn, R. K. (1984). Microbial degradation of polyesters: polycaprolactone degraded by P. pullulans. Journal of Applied Polymer Science, 18, 3571-9.

Gaffney, G. W., Schreier, K., Diferrante, N. & Altman, K. (1954). The quantitative determination of hippuric acid. Journal of Biological Chemistry, 206, 695-8.

Jones, E. S. L. (1968). Some problems posed by quality screening for biodeterioration. In Biodeterioration of Materials, ed. A. H. Waiters & J. J. Elphick. Elsevier Publishing Co., London, pp. 188-95.

Kelley, J. (1985). Testing of plastics for resistance to microorganisms. In Biodeterioration and biodegradation of plastics and polymers, ed. K. J. Seal. Biodeterioration Society, Kew, Surrey, UK, pp. 111-24.

Osman, J. L. & Klausmeier, R. E. (1977). Techniques for assessing biodetefioration of plastics and plasticizers. In Biodeterioration Investigation Techniques, ed. A. H. Waiters. Applied Science Publishers, London, pp. 77-94.

Pantke, M. (1977). Test methods for evaluation of susceptibility of plasticized PVC and its components to microbial attack. In Biodeterioration Investigation Techniques, ed. A. H. Waiters. Applied Science Publishers, London, pp. 51-75.

Shuttleworth, W. & Seal, K. J. (1985). The fungal degradation of polycaprolactone

298 J. Kelley, P. AL Yaghmaie

polyurethane elastomers. In Biodeterioration and biodegradation of plastics and polymers, ed. IC J. Seal. Biodeterioration Society, Kew, Surrey, UK, pp. 70-6.

Smith, 1~ E. (1977). Rapid tube test for detecting fungal cellulase production. Applied and Environmental Microbiology, 33 (4), 980-1.