Journal of Microbiological Methods 47 2001. 307313 www.elsevier.

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Cryopreservation of basidiomycete strains using perlite

Ladislav Homolka) , Ludmila Lisa, Ivana Eichlerova, Frantisek Nerud
Institute of Microbiology AS CR, Vdenska 1083, 142 20 Prague 4, Czech Republic Received 6 July 2001; received in revised form 23 July 2001; accepted 20 August 2001

Abstract A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was successfully tested for cryopreservation of several basidiomycete species from different genera Armillaria, Pleurotus, Pluteus, Polyporus . which failed to survive or retain their properties in cryopreservation procedures routinely used in our laboratory. Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were either immediately after the freezing process or after a 6-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics. All cultures survived the cryopreservation procedure and no negative effects of cryopreservation by this method have been observed after 6 months of storage in liquid nitrogen. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Basidiomycetes; Cryopreservation; Liquid nitrogen; Perlite

1. Introduction Efficient microbiological and consequently also mycological. work requires a reliable source of cultures, i.e. well-defined and taxonomically determined starting material, which is ensured by its safe storage. Routine subculturing is not a very practical method of storing large numbers of fungal cultures. It is time-consuming, prone to contamination and does not prevent genetic and physiological changes

) Corresponding author. Tel.: q 420-2-475-2397; fax: q 420-2475-2384. E-mail address: homolka@biomed.cas.cz L. Homolka..

during long-term and frequent subculturing. Various storage methods have been developed in order to eliminate these disadvantages. Among them, the storage in liquid nitrogen has been considered the best and most widely applicable preservation technique available for filamentous fungi Smith, 1998.. It is a safe and perspective method of a long-term maintenance of most fungal species, especially those not amenable to freeze-drying. This storage technique seems to surpass all others in the ability to preserve genomic and phenotypic features and is, therefore, effective in most microbial biodiversity maintenance programmes Smith, 1982; Heckley, 1978; Prescott and Kernkamp, 1971.. This method, useful for both sporulating and nonsporulating fungal cultures, has several other important advantages: protection of cultures from contamination reduced

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number of transfers. and the saving of time, work and room. According to the literature and our personal experience, the maintenance of basidiomycetes is rather difficult. Many of these fungi do not form asexual spores, their dominant form is mycelium which is rather sensitive to environmental conditions, and also mycorrhizal relationships play often a certain role. Therefore, the most widely used method of culture maintenance in lyophilized state is rather limited in basidiomycetes, because most of them especially nonsporulating ones. do not survive the process Antheunisse, 1973; Hwang et al., 1976.. On the other hand, the method of cryogenic culture maintenance seems to be mostly successful, in particular with nonsporulating cultures Challen and Elliott, 1986; Homolka, 1976; Chvostova et al., 1995.. The cryogenic technique for long-term storage of large numbers of fungal species was first tested and then introduced to ATCC in the 1960s; the results have been highly satisfactory Hwang, 1960, 1966.. The technique was subsequently introduced to many other prominent collections, e.g. CAB International Mycological Institute Onions, 1971; Smith, 1983. etc. In some collections, e.g. IFO Institute for Fermentation Osaka., nonsporulating cultures of basidiomycetes are stored by cryopreservation at y80 8C in electric freezers Ito, 1996.. A comprehensive and detailed overview of the methods and results of cryopreservation of microorganisms, including basidiomycetes, was published by Hubalek 1996.. Mycelium andror spore suspensions with or without a cryoprotectant in sealed glass ampoules were originally used for cryopreservation of filamentous fungi. Later, glass ampoules were replaced with more safe polypropylene cryovials andror straws. Agar blocks immersed in an appropriate cryoprotectant were originally used as carriers of fungal mycelium for cryopreservation process Hwang, 1968.. A useful straw technique with agar miniblocks for the preservation of fungi in liquid nitrogen was developed by Elliott 1976. and improved by Stalpers et al. 1987.. Another technique using straws in cryotubes without a cryoprotectant solution was described by Hoffmann 1991.. Polystyrene beads as carriers were used for cryopreservation of sporulating Aspergillus fumigatus cultures at y80 8C by Belkacemi et al. 1997. and porous ceramic beads

were employed for cryopreservation of several sporulating fungal cultures and for a Saccharomyces cereisiae culture at y70 8C by Palagyi et al. 1997.. Perlite is a unique aluminosilicate volcanic mineral holding and retaining substantial amounts of water, which can be released as needed. Expanded perlite is being used in many applications, particularly in the construction, horticulture and other various industrial fields. It is recommended as an efficient purifying agent and as a carrier for pesticides, feed concentrates, herbicides and other similar applications. It is also used as a solid support in solid-state fermentations Kerem and Hadar, 1993.. We have not found any report on using solid carriers for cryopreservation of nonsporulating cultures of filamentous fungi and any report concerning the use of such carriers for preservation of any fungal cultures in liquid nitrogen. A principal requirement for successful maintenance of production strains is the full preservation of their important properties e.g. production of antibiotics, enzymes and other metabolites.. Routine tests carried out in large collections of microorganisms to estimate the success of surviving the process of cryopreservation usually involve growth and sporulation tests together with morphology assessment of fungal colonies. Data on the production of metabolites or other features are relatively rare Hubalek, 1996.. The effect of cryopreservation on the production of the antibiotic mucidin by the basidiomycete Oudemansiella mucida was studied by Homolka 1976., who found that the production ability was practically unaffected. In our previous successful experiments with some white-rot basidiomycetes, we found no negative effect of cryopreservation or the cryoprotective used on the production of ligninolytic enzymes Stoychev et al., 1998.. A need for an alternative method of cryopreservation arose after several partially or completely unsuccessful attempts to preserve some of our basidiomycete strains or their specific properties by our current routinely used procedure using straws in cryovials and by a standard cryotechnique using agar discs in polypropylene vials with a cryoprotectant solution. This papers deals with four such strains and the culture of Pleurotus ostreatus CCBAS 472 serving as a reference strain which can be successfully preserved by all tested techniques. We also tested if

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the cryopreservation of different basidiomycete species on perlite had any negative effect on their essential characteristics. 2. Materials and methods 2.1. Organisms and cultiation Fungi obtained from the CCBAS collection Institute of Microbiology, Prague, Czech Republic. P. ostreatus Jacq.:Fr.. Kumm. CCBAS 472, P. ostreatus var. columbinus Quel. in Bres.. Quel. CCBAS 462, Armillaria socialis DC.:Fr.. Herink CCBAS 331, Pluteus petasatus Fr.. Gill. CCBAS 483 and Polyporus squamosus Huds..:Fr. CCBAS 850were maintained by serial transfers and kept on wort agar slants at 4 8C. These cultures have not been previously stored in LN. Static submerged cultivation for growth and enzyme estimation was carried out in 100-ml Erlenmeyer flasks containing 10 ml of N-limited Kirk medium Tien and Kirk, 1988.. The flasks were inoculated with two agar plugs 6 mm diameter. cut from the actively growing part of a colony on a Petri dish containing wort medium wort 48 Balling, 1.5 % agar Difco. from cultures before or after cryopreservation. 2.2. Preparation of organisms and freezingr thawing protocols Fungal cultures for cryopreservation experiments with perlite were grown directly in sterile plastic Nunc CryoTube Vials 363401 1.8 ml. with 200 mg of perlite Agroperlit, agricultural grade, 0.5 mm, purchased from Keramik, Prague. moistened with 1 ml of wort 48 Balling. enriched with glycerol Sigma, final concentration 5%. as a cryoprotectant, inoculated with an agar plug 6 mm diameter. prepared as above and then incubated for 10 days at 24 8C. The cryovials with perlite overgrown by the mycelium were frozen in a programmable freezer IceCube 1800 SyLab to y70 8C at a freezing rate of 1 8C per minute. They were then placed in liquid nitrogen in a HARSCO TW-5K container. This procedure we named Aperlite protocolB PP. Our current routine freezing protocol control protocolCP., which is a slightly modified Hoffmanns

protocol Hoffmann, 1991., is the following: strains are grown on Petri dishes on MEYA medium malt extract 2%, yeast extract 0.2%, Difco agar 1.5%. supplemented with glycerol at a final concentration of 5% vrv. at 24 8C. Sterile thin diameter 1 mm. plastic straws, open at both ends, are used to punch the mycelium with agar from the colony. This is repeated until the straws are filled with agar plugs containing the mycelium. These filled straws are then transferred to the sterile cryovials, which are tightly sealed with screw stoppers provided with silicone gaskets and frozen as above. Another tested freezing protocol disc protocol DP. using agar discs with mycelium submerged in 10% glycerol solution in polypropylene cryovials was performed according to Butterfield et al. 1978.. Thawingreactivation of cultureswas common for all tested protocols and was carried out by transferring the cryovials to warm water 38 8C.. After thawing, the perlite particles overgrown with mycelium were at least partially separated by shaking, the content of the cryovials was divided into three approximately equal aliquots and these were plated onto wort solid medium in Petri dishes and incubated at 24 8C. In case of CP or DP, three individual straws or agar discs were used instead of perlite particles. Prior to opening the surface of the cryovials was disinfected with alcohol. All experiments were carried out twice in 10 replicates. 2.3. Growth estimation Submerged growth in liquid medium was evaluated by determining the dry mass of mycelia. The mycelia were harvested from the cultivation flasks which were incubated as above, washed with distilled water, dried at 105 8C for 24 h and weighed. All measurements were done in quadruplicate. Enzyme activities and overall extracellular H 2 O 2 production were measured on the 10th and 14th days of cultivation in the filtrates from three parallel flasks after separation of the mycelia. 2.4. Enzyme and hydrogen peroxide assays Enzyme activities were measured in the filtrates from four replicate flasks after removing the

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mycelium by filtration. Activities of extracellular laccase and manganese peroxidase MnP. were determined spectrophotometrically by monitoring the absorbance increase at 425 nm laccase. or 590 nm MnP. in medium filtrates. Laccase activity was determined according to Bourbonnais and Paice 1990. by monitoring the oxidation of 2,2X-azinobis3-ethylbenzothiazoline-6-sulfonic acid. ABTS.. Determination of MnP activity was based on the method of Ngo and Lenhoff 1980. modified according to Daniel et al. 1994.. 3-methyl-2-benzothiazolinone hydrazone MBTH. and 3-dimethylaminobenzoic acid DMAB. were oxidatively coupled to give a purple indamine dye product by the action of the enzyme in the presence of added H 2 O 2 and Mn2q ions. One unit of enzyme activity U. was defined as catalyzing the production of 1 mmol of green or purple dye per milliliter per minute. The overall production of extracellular hydrogen peroxide was measured using the phenol red method according to Pick and Keisari 1980..

3. Results and discussion Five basidiomycete strainsP. ostreatus CCBAS 472, P. ostreatus var. columbinus CCBAS 462, A. socialis CCBAS 331, Plu. petasatus CCBAS 483 and Pol. squamosus CCBAS 850were tested for the ability to grow on perlite with nutrients, to survive the cryopreservation in liquid nitrogen with glycerol as a cryoprotective and to maintain their important characteristicsgrowth rate, morphology, production of hydrogen peroxide and enzymes in-

volved in lignin modification laccase and manganese peroxidase.. The cultures were tested before and immediately after the freezing process and then after a 6-month storage in liquid nitrogen. The above-listed strains were chosen because the attempts to preserve them by our routinely used procedure CP. using open straws in cryovials and by a cryotechnique according to Butterfield et al. 1978. using agar discs in polypropylene vials with a cryoprotectant solution DP. were partially or completely unsuccessful. Moreover, in case of P. ostreatus var. columbinus CCBAS 462, the culture lost its ability to produce laccase after freezing. The failure of A. socialis CCBAS 331 to survive the process of freezing by our routine protocol CP could be partially ascribed to the fact that the fungus forms thick rhizomorphs which make difficult to cut out the miniblocks using a thin straw as a plunger. The culture of P. ostreatus CCBAS 472 served as a reference strain which can be successfully preserved by all tested techniques. The recovery of the tested strains subjected to the three different cryopreservation protocols is summarized in Table 1, their growth and biochemical characteristics are shown in Table 2. The cultures were checked for their viability and other characteristics immediately after the cryopreservation process and then after 6 months of storing under liquid nitrogen. The principal criteria for a successful recovery of fungal strains were the ability to survive the cryopreservation process expressed as an ability to retain the original growth and macro- and micromorphological characteristics. and the ability to retain the selected biochemical characteristics production of lac-

Table 1 Recovery in percent. of different fungal strains frozen using three alternative cryoprotocols after 6-month storage in cryovials 20 replicates of each strain were tested in two experiments. Freezing protocol DP CP PP Fungal strain P. ostreatus CCBAS 472 100 100 100 P. ostreatus v. columbinus CCBAS 462 0 60a 100 A. socialis CCBAS 331 55 0b 100 Plu. petasatus CCBAS 483 30 65 100 Pol. squamosus CCBAS 850 50 70 100

CP s control protocol, DP s disc protocol, PP s perlite protocol see Section 2.. a The recovered cultures lost their ability to produce laccase. b Mycelia with rhizomorphs were recalcitrant to punching and entering the straws.

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Table 2 Growth and biochemical characteristics of fungal cultures before freezing, immediately after freezing and after a 6-month storage in liquid nitrogen LN. Experimental conditions Strains P. ostreatus v. columbinus Nonfrozen culture Dry mass mgrml. Laccase mUrml. MnP mUrml. H 2 O 2 nM. Dry mass %. Laccase %. MnP %. H 2 O 2 %. Dry mass %. Laccase %. MnP %. H 2 O 2 %. 3.65 " 0.56 31.69 " 6.02 1.29 " 0.15 5.96 " 0.26 109.95 112.95 98.52 136.14 100.50 110.45 88.51 102.45 P. ostreatus 4.65 " 1.01 55.39 " 5.10 12.84 " 1.12 7.37 " 1.89 102.26 101.45 104.21 82.08 108.44 111.28 96.29 118.11 A. socialis 4.05 " 0.48 15.64 " 3.94 0.48 " 0.19 2.32 " 0.09 95.58 98.06 93.25 110.44 105.12 95.65 108.15 92.00 Plu. petasatus 3.95 " 0.60 8.81 " 0.86 1.19 " 0.06 0.88 " 0.05 104.16 110.18 115.26 98.50 97.16 99.87 117.00 95.84 Pol. squamosus 1.70 " 0.02 19.28 " 4.16 5.44 " 1.45 10.91 " 2.26 96.11 95.46 89.22 96.59 98.00 105.42 111.82 101.25

Immediately after freezing

After 6-month storage in LN

case, Mn peroxidase and hydrogen peroxide. unchanged. Twenty replicates of each fungal strain were tested with each protocol; the recovery was counted as percentage of surviving replicates, the growth and biochemical characteristics as percentage of original values. Unlike the other two protocols tested, the perlite protocol was fully successful in cryopreservation of all tested strains. In our experience, the process of recovery using the disc protocol can be complicated in some fungal strains due to the separation of the mycelial mat from the discs in the cryopreservation solution. The growth rates of all strains tested were virtually unaffected by the process of freezing and thawing. The activities of all enzymes before cryopreservation and after the revival did not differ substantially and the differences between the obtained values did not exceed the usual fluctuation. The same held for the macro- and micromorphological characteristics. Even after another two transfers on fresh solid media the results were almost the same data not shown.. No contamination was detected during the whole study. The improved cryopreservation procedure has several additional advantages. One cultivation step is saved by using the cryovials directly for the cultivation of cultures; the transparency of the cryovials makes it possible to check the growth of the culture

inside and to prevent possible problems with insufficient inoculation and contamination. The mycelium on perlite grows continuously and its damage caused by punching it from the agar plate and by subsequent handling is prevented and specific mycelial structures rhizomorphs etc.. can be preserved more easily. Special features of perlite retention and release of water and air. affect the ice formation and thaw-

Fig. 1. Freezing curves of P. ostreatus CCBAS 472 cultures frozen using perlite protocol PP, bold line. or control protocol CP, thin line..

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L. Homolka et al.r Journal of Microbiological Methods 47 (2001) 307313 Chvostova, V., Nerud, F., Homolka, L., 1995. Viability of wood inhabiting basidiomycetes following cryogenic preservation. Folia Microbiol. 40, 193197. Daniel, G., Volc, J., Kubatova, E., 1994. Pyranose oxidase, a major source of H 2 O 2 during wood degradation by Phanerochaete chrysosporium, Trametes ersicolor, and Oudemansiella mucida. Appl. Environ. Microbiol. 60, 25242532. Elliott, T.J., 1976. Alternative ampoule for storing fungal cultures in liquid nitrogen. Trans. Br. Mycol. Soc. 67, 545546. Heckley, R.J., 1978. Preservation of microorganisms. Adv. Appl. Microbiol. 24, 153. Hoffmann, P., 1991. Cryopreservation of fungi. World J. Microbiol. Biotechnol. 7, 9294. Homolka, L., 1976. On the problem of maintenance and cultivation of higher fungi. Folia Microbiol. 21, 189. Hubalek, Z., 1996. Cryopreservation of Microorganisms. Academia Publishing House, Prague. Hwang, S.-W., 1960. Effects of ultra-low temperatures on the viability of selected fungus strains. Mycologia 52, 527529. Hwang, S.-W., 1966. Long-term preservation of fungus cultures with liquid nitrogen refrigeration. Appl. Microbiol. 14, 784 788. Hwang, S.-W., 1968. Investigation of ultra-low temperature for fungal cultures: I. An evaluation of liquid nitrogen storage for preservation of selected fungal cultures. Mycologia 60, 613 621. Hwang, S.-W., Kwolek, W.F., Haynes, W.C., 1976. Investigation of ultra-low temperature for fungal cultures: III. Viability and growth rate of mycelial cultures following cryogenic storage. Mycologia 68, 377387. Ito, T., 1996. Preservation of fungal cultures at the Institute of Fermentation, Osaka IFO.. In: Samson, R.A., Stalpers, J.A., van der Mei, D., Stouthamer, A.H. Eds.., Culture Collections to Improve the Quality of Life. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands, pp. 210211. Kerem, Z., Hadar, Y., 1993. Effect of manganese on lignin degradation by Pleurotus ostreatus during solid-state fermentation. Appl. Environ. Microbiol. 59, 41154120. Ngo, T.T., Lenhoff, H.M., 1980. A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions. Anal. Biochem. 105, 389397. Onions, A.H.S., 1971. Preservation of fungi. In: Booth, C. Ed.., Methods in Microbiology, vol. 4. Academic Press, New York, pp. 113115. Palagyi, Z., Nagy, A., Vastag, M., Ferency, L., Vagvolgyi, C., 1997. Maintenance of fungal strains on cryopreservative-immersed porous ceramic beads. Biotechnol. Tech. 11, 249250. Pick, E., Keisari, Y., 1980. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. Immunol. Methods 38, 161170. Prescott, J.M., Kernkamp, M.F., 1971. Genetic stability of Puccinia graminis tritici in cryogenic storage. Plant Dis. Rep. 55, 695696. Smith, D., 1982. Liquid nitrogen storage of fungi. Trans. Br. Mycol. Soc. 79, 415421. Smith, D., 1983. Cryoprotectants and the cryopreservation of fungi. Trans. Br. Mycol. Soc. 80, 360363.

ing and this can be useful for culture protection and preservation. Another potential advantage of the procedure is the partial prevention of a large increase in the temperature of the frozen sample around the crystallization point. This can be seen in Fig. 1, where the temperature course curves of the samples frozen on perlite and in a straw with agar miniblocks are compared. The presence of perlite seems to partially counteract the generation of heat in the sample and the corresponding curve is flattened. The described method is evidently suitable for different fungal strains requiring special treatment and the authors believe that it is generally applicable to most fungal cultures. This expectation is supported by the promising results of preliminary testing of the method on another 60 fungal strains from 19 different genera and 36 species 14 strains of whiterot basidiomycetes from eight species of two genera Inonotus and Pholiotawere also successfully cryopreserved on perlite without visible changes and further 46 strains from 28 species of 17 genera were at least able to grow on perlite and survive the process of freezing and thawing; they are at present under further study..

Acknowledgements This work has been supported by Institutional Research Concept no. AV0Z5020903.

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L. Homolka et al.r Journal of Microbiological Methods 47 (2001) 307313 Smith, D., 1998. The use of cryopreservation in the ex-situ conservation of fungi. Cryo-Lett. 19, 7990. Stalpers, J.A., de Hoog, A., Vlug, I.J., 1987. Improvement of the straw technique for the preservation of fungi in liquid nitrogen. Mycologia 79, 8289. Stoychev, I., Homolka, L., Nerud, F., Lisa, L., 1998. Activities of

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