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Preservation of Penicillium species by lyophilization.


Genus Penicillium bears tremendous economic significance in human affairs. It is highly desired to establish the taxonomic position of Penicillium isolates due to extensive use of their products in the food and feed industry. It produces extra cellular enzymes, isozymes, acids, metabolites and other commercial products. Penicillium is one of the commonest moulds causing multifarious infections worldwide. The maintenance and production of reliable pure cultures with desirable quality is the key operation and the first significant stage in the success of any fungal identification.

Long-term preservation of Penicillium species is essential for further detailed research. However, both the viability and the stability of living cells should be ensured during the preservation period. Penicillium belongs to the phylum Ascomycota, while its taxonomic characterization is still a matter of discussion [1] and the difficulties in identifying most Penicillium species requires multidisciplinary approaches. Clarification of species concepts in the genus Penicillium is supported mainly by morphological characteristics.

Fungal isolates were usually preserved in water at room temperature [2], an easy and economical procedure introduced for fungi [3]. However, the stability of fungal cells is not ensured by this simple procedure. Other methods have been suggested, such as preservation in soil or on oil or in culture slants, cryopreservation either in liquid nitrogen or at low temperature (-20 and -70[degrees]C) and lyophilization (the freeze-drying procedure) [2,3,4,5,6,7,8,9,10,11,12,13].

Cryopreservation in liquid nitrogen and lyophilization are recommended and used by the American Type Culture Collection [12]. Lyophilization is used for dehydrating vast range of materials, including foodstuffs, pharmaceuticals, biotechnology products, vaccines, diagnostics and biological materials. Lyophilization (also known as Freeze-drying, or cryodesiccation) is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport.

Freeze-drying works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublime directly from the solid phase to the gas phase. Since World War II (WWII) lyophilization has been used to stabilize living cells and fungal isolates. Lyophilized mycelia increase the storage and the quality of DNA extracted for molecular studies [14]. Maintenance of vigor and genetic characteristics of a pure strain in the form of a culture is the main objective of Penicillium culture preservation. Therefore, the present study was conducted to assess the suitability of lyophilization (freeze drying) as simple, inexpensive and reliable method to preserve fungal cultures on large scale for longer period of time.



Fifty isolates of Penicillium species were isolated from different sources (Table 1) directly and by serial dilution method. The soil samples from specific sources were thoroughly mixed and 25 g of each sample was suspended in 225 ml of sterilized distilled water (SDW) in 1:10 ratio and 10 ml of this suspension were added to 990 ml of SDW (1:1000).

These suspensions were stirred for 20 min before making 7-fold falling dilutions then; 1 ml of the desired suspensions ([10.sup.-4]) was plated in triplicate on petri dishes containing 2% malt extract agar (MEA) composed of malt extract (20g) and agar (15g) in 1 L of distilled [H.sub.2]O (pH 7.2). The suspension was evenly spread on the surface of medium by a sterile glass rod while rotating the petri dish. Soil plates were prepared in triplicate by uniformly distributing 0.5 g of soil directly by sprinkle method on the surface of 2% MEA medium. The plates were incubated at 26[+ or -]2[degrees]C for six days before counting the growing fungal colonies. Individual fungal colonies were further isolated by sub-culturing onto new MEA plates.

Fungal isolates were purified and identified on the basis of morphological characteristics following protocols described by Pitt [15], and Domsch et al. [16]. Fungal isolates were collected in Punjab province from 2008 to 2010.

Preparation of the isolates for preservation

Spawn preparation: For the spawn preparation Pennisetum glaucum (Millet, Bajra) and Triticum aestivum (wheat) seeds were selected, washed and soaked in water overnight (18 hours). After removing excess water by spreading seeds on paper towel fifty grams of soaked seeds were sealed in each plastic bag and autoclaved. Sterilized seed bags were inoculated by fungal discs (8 mm) from 7 days old culture. Inoculated bags were kept at 25[+ or -]2[degrees]C for 15 days for spawn formation and were preserved at 4[degrees]C for further studies (Fig 1a, b).


Lyophilization: Efficacy of lyophilization was evaluated by direct lyophilization of the fungal mat and lyophilization of the fungal spawn. For direct method 7-10 days old fungal colony mat (5-6 cm diameter) was used. Fungal mat was prepared in 2% Malt Extract (ME) broth. Each fungal mat was washed three times with distilled sterile water and dried in paper towel for two minutes. Fungal mat was transferred into sterile falcon tube for lyophilization. Neither stabilizer nor skimmed milk was used in fungal material to protect it from sticking with the walls of the container. Nylon mesh (200[micro]m) was used in order to close the mouth of falcon tube. Falcon tubes were placed in the vacuum bottles. Fungal spawn was directly lyophilized by placing spawn bags in vacuum bottles. Material was lyophilized in freeze dryer (TFD5505, Ilshin, Korea) under vacuum at [less than or equal to] 50[degrees]C (Fig. 2).


The lyophilizer was allowed to run for 6-8 hours. Samples were removed from vacuum bottles when pressure reached at 25 bar. Lyophilized samples were kept at 4 [degrees]C and room temperature (28[degrees]C). After 1, 2, and 3 months, viability of lyophilized and spawn cultures were checked on MEA medium (Fig. 3). Each fungal isolate was considered viable if the rate of growth present was the same as that of the original culture and if the morphology of the colony matched the fungal identification documented for each species.



Thirty two Penicillium species were isolated from 25 variable soil and other samples of different types of plants parts, growing in ten districts of Punjab, Pakistan. Three species belong to subgenus Aspergilloides, twelve of Furcatum, fourteen species of Penicillium and three species belong to subgenus Biverticillium. Overall prevalence showed that Penicillium oxalicum was the most frequent species with six isolates followed by P. simplicissimum (4 isolates), P. implicatum and P. melinii (3 isolates each). Whereas P. canescens, P. italicum, P. atrovenetum, P. granulatum P. expansum and P. chrysogenum were there with two isolates, respectively. On the other hand the rest of the species were isolated once from different samples amongst the genus Penicillium.

The viability and the purity of the fungal species by fungal spawn and lyophilized fungal mat were monitored immediately after storage, at 1, 2 and 3 months at 4[degrees]C. The fungal isolates were compared in terms of rate of growth, colony morphology and sporulation after rejuvenation. Each of the isolates was also matched with the parent cultures for the above said features. The cultures isolates were subcultured for verification of their viability. It was recorded that all lyophilized Penicillium species were viable after 1-3 months of storage. The rate of recovery and percentage viability was equally good for fungal isolates recovered from both type of lyophilized material, fungal spawn and fungal mat showing complete compatibility with the initial colony characteristics and growth rates.


The genus Penicillium is considered as a group of "opportunistic fungi" being found everywhere; it is unavoidable in life. The purpose of present study was to isolate species from climatically diverse zones of Punjab and long term preservation of the species of Penicillium so that they may be used for teaching, research and industry.

Storage procedures and recovery methods exhibit considerable differences in response to stresses by different taxonomic groups and even strains within a given species [17]. Important parameters such as rate of cooling, size of propagules, and thickness of cell wall [18, 19, 20] are not considered in this study and they may explain intra and interspecific variability of fungal cultures after different storage periods.

Several methods have been used for long term preservation of fungus cultures [5, 21, 22]. The appropriate selection and success of preservation method varies with the fungal species being preserved. Studies confirm that lyophilization is much simpler, effective and convenient for preservation of fungal cultures in the laboratory at large scale as compared to conventional freeze-drying methods [23, 24, 25]. Viability of lyophilized cultures has also been reported 100% with minimal cultural variations [26]. Modified method of Penicillium spawn preservation by lyoplilization has been found cost effective in contrast to liquid nitrogen preservation.


The present study has shown that fungal spawn lyophilization is much easier, convenient, economical, and effective tool for the long term preservation of fungal isolates. In case of other microscopic and macroscopic fungi, longer monitoring of preserved isolates and storage will be further validated for the reliability of freeze dry preservation. Furthermore, stability of fungal cells will also be assessed through molecular parameters.


Authors are highly obliged to the University of the Punjab for providing funds to First Fungal Culture Bank of Pakistan, Institute of Agricultural Sciences to facilitate this research.


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Ibatsam K * (1), Rukhsana B (1) and G Nasim (1)

* Corresponding author email:

(1) Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan
Table 1: Isolation of Penicillium species from different substrates

Sr.No.   FCBP NO.        Penicillium species

01       FCBP-IK-1003    Penicillium sp.
02       FCBP-IK-1010    Penicillium oxalicum
03       FCBP-IK-1028    Penicillium canescens
04       FCBP-IK-1029    Penicillium simplicissimum
05       FCBP-IK-1030    Penicillium italicum
06       FCBP-IK-1037    Penicillium canescens
07       FCBP-IK-1038    Penicillium oxalicum
08       FCBP-IK-1039    Penicillium sp.
09       FCBP-IK-1040    Penicillium atrovenetum
10       FCBP-IK-1041    Penicillium verrucosum
11       FCBP-IK-1050    Penicillium steckii
12       FCBP-IK-1052    Penicillium verrucosum v cyclopium
13       FCBP-IK-1057    Penicillium lanosum
14       FCBP-IK-1062    Penicillium sp.
15       FCBP-IK-1066    Penicillium implicatum
16       FCBP-IK-1067    Penicillium granulatum
17       FCBP-IK-1069    Penicillium sp.
18       FCBP-IK-1075    Penicillium oxalicum
19       FCBP-IK-1080    Penicillium granulatum
20       FCBP-IK-1082    Penicillium digitatum
21       FCBP-IK-1083    Penicillium rubrum
22       FCBP-IK-1100    Penicillium citrinum
23       FCBP-IK-1101    Penicillium simplicissimum
24       FCBP-IK-1102    Penicillium expansum
25       FCBP-IK-1106    Penicillium bilaii
26       FCBP-IK-1107    Penicillium rolfsii
27       FCBP-IK-1108    Penicillium italicum
28       FCBP-IK-1109    Penicillium implicatum
29       FCBP-IK-1110    Penicillium oxalicum
30       FCBP-IK-1111    Penicillium expansum
31       FCBP-IK-1112    Penicillium simplicissimum
32       FCBP-IK-1113    Penicillium atrovenetum
33       FCBP-IK-1114    Penicillium simplicissimum
34       FCBP-IK-1115    Penicillium rugulosum
35       FCBP-IK-1116    Penicillium oxalicum
36       FCBP-IK-015     Penicillium oxalicum
37       FCBP-IK-016     Penicillium claviforme
38       FCBP-IK-017     Penicillium miczynskii
39       FCBP-IK-018     Penicillium implicatum
40       FCBP-IK-019     Penicillium melinii
41       FCBP-IK-022     Penicillium chrysogenum
42       FCBP-IK-024     Penicillium viridicatum
43       FCBP-IK-025     Penicillium melinii
44       FCBP-IK-026     Penicillium chrysogenum
45       FCBP-IK-027     Penicillium griseofulvum
46       FCBP-IK-028     Penicillium spinulosum
47       FCBP-IK-029     Penicillium janthinellum
48       FCBP-IK-030     Penicillium melinii
49       FCBP-IK-031     Penicillium waskmanii
50       FCBP-IK-032     Penicillium velutinum

Sr.No.   Substrate

01       Malus domestica
02       Rhizosphere of Hibiscus esculenta
03       Rhizosphere of Eucalyptus camaldulensis
04       Rhizosphere of Psidium guajava
05       Rhizosphere of Pennisetum americanum
06       Rhizosphere of Dalbergia sissoo
07       Effluent of Amar Fabrics
08       Rhizosphere of Vitis vinifera
09       Rhizosphere of Eucalyptus camaldulensis
10       Effluent of Amar Fabrics
11       Effluent of Amar Fabrics
12       Effluent of Textile industry
13       Effluent of Textile industry
14       Effluent of Textile industry
15       Effluent of Textile industry
16       Effluent of Pak polyester
17       Bread
18       Rhizosphere of Dalbergia sissoo
19       Malus domestica
20       Effluent of Amar Fabrics
21       Punica granatum
22       Bread
23       Allium cepa
24       Allium sativum
25       Punica granatum
26       Effluent of Amar Fabrics
27       Lemon
28       Punica granatum
29       Rhizosphere of Vitis vinifera
30       Pods of Albizia procera
31       Vitis vinifera
32       Canal water
33       Zingiber officinale
34       Effluent of Glass industry
35       Air
36       Soil
37       Citrus sinensis
38       Citrus sinensis
39       Allium cepa
40       Allium cepa
41       Allium cepa
42       Bread
43       Bread
44       Orange
45       Bread
46       Punica granatum
47       Rhizosphere of Citrus sinensis
48       Rhizosphere of Citrus sinensis
49       Rhizosphere of Citrus sinensis
50       Rhizosphere of Citrus sinensis
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Author:Ibatsam, K.; Rukhsana, B.; Nasim, G.
Publication:African Journal of Food, Agriculture, Nutrition and Development
Article Type:Report
Geographic Code:9PAKI
Date:May 1, 2012
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