24 (4) : 2944, 2002 29 1 2 Foxing Aspergillus Penicillium Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus ustus, Aspergillus spp.,penicillium citrinum, Penicillium chrysogenum, Penicillium spp.,chaetomium globosum, Trichoderma viride,fusarium solani, Cladosporium sp., Rhizopus sp., Phoma sp.,umbelopsis sp., Metarrhizium sp., Eurotium sp. 70 % 60 % Research paper Separation and Identification of Foxed Spots on Paperbased Cultural Relics Tsang-Chyi Shiah 1 Feng-Jyi Chang 2 AbstractFoxing of paper-based cultural properties could be caused by the biodegradations. Brown stains not only apparently decrease the life of rare books, paintings and art crafts, but also affect the preservation of cultural relics. The aim of this study is to explore the contributing factors of foxed spots showed on paper-based materials. The experimental results are summarized as follows Foxed spots usually showed yellowish color in the serface of paper artifacts. Hyphae and conidia of microorganisms were observed on the foxed area in SEM observation. Among 17 species of fungi isolated from paper-based materials of which the main species, were identified as the genera of Aspergillus and Penicillium. The foxing-causing fungi of Pineapple xuan paper were inhibited by less than 60% R.H. at room temperature, and over 70% R.H.,the fungal growth was very vigorous. 1. Assistant scientist, Division of Forest Chemistry, Taiwan Forestry Research Institute, Council of Agriculture. 2. Professor, Department of Forestry, NCHU.
30 Foxing or fox spots Florian,1994 Foxing Foxbrown Arai, 1992 Foxed Cain, 1984; Mori and Tachibana, 1992 50 Foxing-causing fungi Arai, 1984 100 Aspergillus sp.,alternaria sp.,penicillium sp.,stachybotrys sp.,stemphylium sp.,tricoderma sp.,chaetomium sp.,chrysosporium sp.,eurotium sp.1987 19971998 Szczepanowska, 1986 1999 1. 2 1887,1953 198 9 2
24 (4) : 2944, 2002 31 2 1941 NBKP 2. 1988; Tzean et al., 1990; 1994 (1)39 g PDAMerck 1000 ml (2)Mineral salt agar NH4NO33 g K2HPO4 1.4 gkcl 0.25 gmgso4 7H2O 0.25 g 10 g 1000 ml (3)CZ Czapek's agar NaNO3 3 g K2HPO41 gmgso47h2o 0.5 gkcl0.5 g FeSO47H2O0.01 gsucrose 30 g 10 g 1000 ml Aspergillus (4)CYA Czapek's yeast extract arar Czapek's extract 20 g Yest extract 5 gk2hpo4 1 gsucrose30 g 10 g 1000 mlpenicillium (5)MEA Malt extract agar Malt extract 20 g Peptone 1 gdextrose 20 g10 g 1000 ml Aspergillus Penicillium 3. PDA 25 (1)Aspergillus niger var. niger (2)Aspergillus flavus (3)Penicillium citrinum (4)Trichoderma sp. (5)Rhizopus sp. (6)Mucor sp. (7)Umbelopsis sp. (8)Metarrhizium sp. (9)Chaetomium globosum 4. 10 ml,, 10 l ml 10 4~5 1. 3 25 25 Arai,1984; 1987; Szczepanowska, 1986 1981991 19831999 2. ph (1) ph 67 8 ph ADVENTEC, TOYO, FILTER PAPER, No.1 PDA 100l L (2)
32 12 24 36 ph 6 78 28 3. (1) 66 cm 200 l L (2) 25 2030 (3) 80 Cupriethylene diamine 4. 50607078090% R.H. 1959 1 Aspergillus ochraceus, Aspergillus ustus, Penicillium chrysogenum, Chaetomium globosum, Trichoderma viride, Fusarium solani Cellulolytic microorganisms 200 Dhawan, 1986; Gallo, 1994 Penicillium, Aspergillus, Chaetomium, Mucor, Rhizopus, Fusarium, Stemphylium, Cladosporium,Stachibotrys, Alternaria, Trichoderma Czerwinska, 1979 1983 1981991; Hsieh et al., 1987; Tzean et al., 1990; 1994 1. Aspergillus flavus Link 1 Aspergillus flavus MEA Czapek's 25 14 5.5 cm PDA
24 (4) : 2944, 2002 33 1. Table 1. Identification of fungi from PDA cultivation of foxed spots found on paper-based cultural relics. Samples 1. 2. 3. 4. 5. 6.-1 7.-2 8. 9. 10. 11. 12. 13. 14. 15. 16.-1 17.-2 Species of fungi Phoma sp. Aspergillus niger var. niger Aspergillus niger var. niger Aspergillus niger var. niger Aspergillus niger var. niger Penicillium citrinum Penicillium citrinum Trichoderrma viride Aspergillus flavus Cladosporium sp. Aspergillus niger var. niger Aspergillus niger var. niger Aspergillus niger var. niger Umbelopsis sp. Metarrhizium sp. Aspergillus sp. Rhizopus sp. Aspergillus niger var. niger Eurotium sp. Aspergillus niger var. niger Remark 1887 1953 1985 NBKP 1984 1986 1986 1977 1979 1941 1. Aspergillus flavus Fig. 1. Colony morphology and SEM photographs of Aspergillus flavus. Topon MEA, at 2 for 14 daysleftaspergillumrightconidia
34 110780 5.423 m 11 60m Aspergilla 4.021.42.89.1 m 5.613.2.45.4 m 3.0 5.6 2.45.1 m 2. Aspergillus niger var. niger 2 Aspergillus niger MEA 25 Czapek's 14 5.2 cm Vesicle 170 m 22024604.424 m Aspergilla Biseriate Metulae 03 3.6 14.3 2.4 5.2mPhialidely4.817.2.4 6.7 m 3.24.8 m 3. Aspergillus niger var. awamori 25 Czapek's 14 4.8 cm 1 70 m 15015004.418.0 m Aspergilla 2. Aspergillus niger Fig. 2. Colony morphology and SEM photographs of Aspergillus niger. Topon MEA, at 2 for 14 daysleftaspergillumrightconidia
24 (4) : 2944, 2002 35 03 4.066.03.616 m 4.817. 2.46.7 m 2.8 5.2 m 4. Aspergillus ustus Thom et Church Czapek's 25 10 4.04.5 cm 4080 m 200450 m 1015 m 5.7.022.4 m 3.24.5 m 5. Aspergillus ochraceus Czapek's 25 10 3.03.5 cm 200300 m 10001500 m 1015 m 710.22.4 m 33.2 m 6. Pencillium chrysogenum Thom Czapek's 25 15 4.5.0 cm 12 mm 150 350 3 3.5 m 8 10 2.0 2.5 m 342.83.5 m 7. Pencillium citrinum Thom 3 Pencillium citrinum CYA 25 Czapek's 10 4.0 cm 4802.8 3.3 m 892.02.5 m 22.5 m 8. Trichoderma viride Pers. ex Gray 4 Trichoderma viride PDA MA 4.5 m 8142.43.0 m 3.64.5 m 9. Chaetomum globosum Kunze 1996 28 4.04.2 mm
36 3. Pencillium citrinum Fig. 3. Colony morphology and SEM photographs of Penicillium citrinum. Topon CYA, at 25 for 14 daysleftpenicillusright conidia 4. Trichoderma viride Fig. 4. Colony morphology and SEM photographs of Trichoderma viride. Topon PDA, at 25 for 14 daysleftphialides showing formation of conidiarightconidia
24 (4) : 2944, 2002 37 146.0-201.4215.0-248.2219.44-252.44273.30-365.0m 8 7.54-7.587.62-9.049.04-9.39.54-12.06m 10. Cladosporium sp PDA 24 10 11. Fusarium solani 24 PDA 4 3.7 cm 5.16.5 1.15.5 m 1-5 3 4 19. 51 4.1 6.2m 9.211.8 8 10.3 m ph 1. 2 24 36 24 Trichoderma viride, Penicillium citrinum Cladosporium sp. 12 35 3037 2229 Szczepanowska, 1986 2436 5 CZCYA MEA Aspergillus niger Aspergillus flavus Penicillium citrinum 32 12 2. ph Szczepanowska ph Szczepanowska, 1992 3 ph ph 5 ph 6 7 100 % ph 7 ph 8 30 Trichoderma virvide 30 62.7 % Chaetomium globosum 75.2 % Metarrhiziumn sp. 83.6 % 10 % 6 30 50 % Trichoderma viride415.1metarrhizium sp. 448.4Aspergillus niger 457.9 Chaetomiun globosum510.7 Thakre
38 2. Table 2. Effect of temperature on the growth of fungi and stain formation on inoculated filter paper. Fungi 12 24 36 Penicillium citrinum Umbelopsis sp Metarrhizium sp Aspergillus flavus Aspergillus niger Trichoderma sp Cladosporium sp Chaetomium globosum Growth 2 2 3 + 4 5 + Stain 1+ yellowish 1 1 yellowish 1 2 Growth 3 4 Stain 1 greenish 3 reddish 3 blackish 3 1 yellowish 3 yellowish 3 yellowish Growth 4 4 4 Stain 3 reddish 3 blackish 1 yellowish 3 3 brownish Fungi were grown for 7 days on filter paper added with mineral salt agar at 12,24,36 in dark incubator respectively. Growthon paper surface no fungi colony growth 1paper was covered with 20 % of fungi colonies 2paper was covered with 40 % of fungi colonies 3paper was covered with 60 % of fungi colonies 4paper was covered with 80 % of fungi colonies paper was covered with 100 % of fungi colonies Stainon paper surface colorless 1stained noticeably 2stained appreciably 3stained apparently Cellulolytic fungi Thakre and Bhajbhuje, 1991Thakre 7 60 % R.H. 2. 3.8 % 2.5 365 nm 60 % R.H. UV SEM 70 %
24 (4) : 2944, 2002 39 5. Aspergillus niger,aspergillus flavus Penicillium citrinum Fig. 5. Effect of temperature on the mycelia growth of Aspergillus niger,aspergillus flavus on CZ and MEA mediaand Penicillium citrinum on CYA and MEA media. 3. ph Table 3. Effect of ph on the growth of fungi and stain formation on inoculated filter paper. Fungi ph 5 ph 6 ph 7 ph 8 Penicillium citrinum Umbelopsis sp Metarrhizium sp Aspergillus flavus Aspergillus niger Mucor sp Cladosporium sp Chaetomium globosum Trichoderma sp Growth Stain 2 brownish 1 brownish 1 1 1 1 1 Growth 4 4 Stain 2 brownish 1 brownish 1 1 1 1 1 yellowish Growth Stain 1 brownish 2 1 2 brownish Growth 4 Stain 2 1 2 brownish Symbols see Table 2 shown.
40 6. Fig. 6. Reduction of filter paper DP after 30 days foxing-induced fungi inoculation. A B C D E 7. 60 % R.H. 2.5 Fig. 7. Observation of paper samples after 2.5 years storage at 60 % R.H. Anaked eyes, Bunder UV light, C ESEM photographs
24 (4) : 2944, 2002 41 5.59 % 75 % 5.79 % 90 % 8.96 % 80 % 7.33 % 75 % 5.79 % 70 % 5.59 % UV SEM 8 90 % R.H. 2.5 8.96 % Arai 7 94 % 235 Arai, 1987Dhawan 75 % R.H. 235 Dhawan, 1986 Gallo 65 % 10 % 20 Gallo, 1994 18971997 23 80% Craig 1318 560 % Craig, 1986 1. Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus ustus, Aspergillus spp., Penicillium citrinum, Penicillium chrysogenum, Penicillium spp., Chaetomium globosum, Trichoderma viride, Fusarium solani, Cladosporium sp., Rhizopus sp., Phoma sp., Umbelopsis sp., Metarrhizium sp., Eurotium sp., Aspergillus Penicillium 2. 24 36 12 ph ph 7, 8 3. Trichoderma viride Chaetomium globosum, Metarrhizium sp. Aspergillus niger 4. 2.5 60 % UV SEM 70 % 90 %80 %75 % 70 % R.H. 5. 60 %
42 A B C D E F 8. 90 % R.H. 2.5 Fig. 8. Observation of paper samples after 2.5 years storage at 90 % R.H. Anaked eyes, Bunder UV light, C FSEM photographs
24 (4) : 2944, 2002 43 219 1983 101-1 198 Trans. mycol. Soc. R.O.C., 1(2)77-9 1988 335-37 1991 355-62 1996 1997 109-123 1998 47(3)1-18 1999 34(4)481-488 1999 1969 100-107 197 1433-43 1987 99-117 Arai, H. (1984) Microbiological studies on the conservation of paper and related cultural properties, (part1) Isolation of fungi from the foxing on paper. Science for Conservation 2333-39. (in Japanese) Arai, H. (1987a) Microbiological studies on the conservation of paper and related cultural properties. (part 5) Physiological and morphological characteristics of fungi isolated from foxing, foxing formation mechanisms and countermeasures.science for Conservation 2643-52.(in Japanese) Arai,H. (1987b) Control of biodeterioration on the foxing-causing fungi. ICOM Committee for Conservation, working group 21165-1167. Arai,H. (1992) Relationship between fungi and brown spots found in various materials. 2nd Int.Conference on Biodeteriation of Cultural Property, Yokohama 5-8 October pp.78-81. Barnett, H.L. and B.B. Hunter (1987) Illustrated Genera of Imperfect Fungi. Fourth Edition, Macmillan publishing company, New York. Czerwinska, E. and R. Kowalik (1979) Microbiodeterioration of audiovisual collections an art Protection of audiovisual records against destructive microflora. Part. Microbial problems in photographic print collections. RESTAURATOR 363-80. Cain,C.E., and B.A.Miller (1984) Proposed classification of foxing. AIC 10th annual meeting pp.29-30. Craig, R. (1986) Alternative approaches to the treatment of mold biodeterioration -an International problem. The paper conservation: Journal of the institute of paper conservation 1027-30. Coleman, M.D., C.S. Bledsoe and W. Lopushinsky (1989) Pure culture response of ectomycorhizal fungi to imposed water stress
44. J. Can. Bot. 6729-39. Dhawan, H. 1986 Microbial deterioration of paper material (A literature review). Publication ManagementKhan, M. M. India. pp.1-18. Florian, M-L. E. (1994) Conidia fungi mould, mildewbiologya basis for logical preservation, eradication and treatment for museum and archival collections. Leather Conservation News 101-28. Hsieh, H.M., H.J. Su and S.S. Tzean (1987) The genus Penicillium in Taiwn. Two new taxa of synnematous Penicillium. Trans. Mycol. Soc. R.O.C.. 2 (2) 157-168. Mori,W.and Tachibana,N., (1992) Foxing found in books and other paper materials. 2 nd Int. Conference on Biodeterioration of Cultural Properties p.77. Szczepanowska, H. (1986) Biodeterioration of art objects on paper. The Paper Conservator 1031-39. Tzean, S.S., J.L. Chen, G.Y. Liou, C.C. Chen and W.H. Hsu (1990) Aspergillus and related teleomorphs from Taiwan. Food industry Research Development Institute, Taiwan. Tzean, S.S., S.C. Chiu, J.L. Chen, S.H. Hseu, G.H. Lin, G.Y. Liou, C.C. Chen and W.H. Hsu (1994) Penicillium and related teleomorphs from Taiwan. Food industry Research Development Institute, Taiwan.