Lichens arise from a symbiotic relationship between a fungus, the mycobiont, and one or more phototrophic algae and/or cyanobacteria, the photobiont(s). With more than 30,000 species, lichens represent 20% of the currently known global fungal biodiversity; they even occur in the most extreme ecosystems on earth. Lichens produce a magnitude of natural compounds. From the 700 lichen-based substances whose structures have been clarified, many are exclusively found in lichens; they include depsides, depsidones, dibenzofurans, quinones, chromones, carotenoids, poly- and monosaccharides, aliphatic acids and others [1]. These so-called lichen compounds may provide more than 40% of the lichen’s dry weight [2] and are partly excreted as crystals on the surface of the fungal hyphae of the lichen in a natural growth environment [1].

Substances from lichens have been used from ancient times on, e.g. as dyes or for medical purposes, as pH indicator (Litmus), or as fragrances. Examples are Evernia prunastri (oakmoss) which is an important basic fragrance in the perfume industry. The toxic Letharia vulpina was historically used as a dye, but also to empoison wolves [3, 4]; yellow dye of Xanthoria parietina has been used for dyeing textiles [5]. Many of the lichen substances have been recently shown to have anticancer and antimicrobial activities and thus lichens could play an important role for the production of pharmaceuticals [6,7,8]. However, due to their slow growth and sensitivity to changing environmental conditions, collecting larger quantities of lichen for human use (e.g. Iceland moss Cetraria islandica, Reindeer moss Cladonia subgen. Cladina, beard lichen Usnea) in nature becomes more and more problematic not only from an economic point of view, but also in view of nature conservation. Collecting most of the traditionally used lichen species is prohibited by law in the countries of Central Europe [e.g. 9]. Development of standards forthe cultivation of relevant species and for biotechnological production of economically interesting lichen compounds can therefore considerably gain importance in the future as a continuous, steady supply of various natural materials.

Up to now, it has been extremely difficult to mimic the lichen symbiosis in the laboratory in large quantities of biomass, and subsequently the production of lichen substances. One reason is that contaminants in the lichen thallus like bacteria and parasitic fungi, among others, grow faster in a nutrient-rich laboratory medium than the lichen symbionts themselves. To obtain a pure lichen culture, the separation of the individual lichen partners is a prerequisite. The respective isolates can be incubated on solid or liquid media with various carbon and nitrogen sources [10, 11]. The optimization of environmental parameters such as light, pH-value, nutrient supply, humidity and temperature regimes are particularly relevant for the growth of lichens.

Culture conditions vary greatly between species and have to be identified specifically [11]. Most studies with isolated mycobionts have been conducted in order to examine either lichen re-synthesis and thallus development under laboratory conditions or the production of secondary metabolites [12]. Some of the lichen substances can be obtained with axenic (i.e. one-species, pure) cultures of lichenized fungi (lichen-associated fungi) under artificial stresses such as simulated day-night cycles, temperature fluctuations, strong light intensity or specific moisture regimes [13]. Most of the published studies were carried out, however, under laboratory conditions with the purpose to demonstrate the principle feasibility. We are not aware of any publication, in which such methods have been further developed from a process-related view, either to ensure mass production of lichens or a specific substance from them.

While the isolation of the lichen photobiont, i.e. the algae or cyanobacterium, is relatively straightforward due to the nutrient-poor media, which can be applied, the isolation of the mycobionts is still a lengthy trial-and-error process, often with high probability of a contamination, long incubation times and low success rates. In our opinion, for a wider use of lichens as cell factories, it is important to critically evaluate and further improve the current isolation techniques and strategies, as well as the culture media used for isolation of mycobionts.

The studies about the isolation of lichen partners used either lichen thallus fragments or ascospores. The isolation of lichen thallus fragments was originally described by Yamamoto et al. [14] and subsequently used and modified by other authors [10, 15,16,17,18]. The isolation from spores was described by Ahmadijan [19] and Yoshimura et al. [20]. Recently Černajová and Škaloud [21] used soredia, and Zakeri et al. [22] used soredia and thallus without washing and homogenization to isolate lichen partners. There are some successful isolations described for lichen fungi from different groups, e. g. foliose, crustose and fruticose lichens [e.g. 2325]. The success of different methods for a specific species, however, was not compared yet. After obtaining axenic (i.e. pure) cultures of the lichen partners, isolates are normally cultivated on various solid and soft agar media with different nutrients, C- and N-source concentrations [10, 15, 16, 26]. Lichen symbionts grow much faster as cell aggregates in lab conditions than in nature, e.g. 6 g dry biomass of U. ghattensis on Petri dishes were achieved within 2 months [27]. Nevertheless, growth is still slower than that of many other microorganisms. To our knowledge, there are still no real systematic optimization studies that would be the basis for the development of industrial applications.

In order to facilitate access to the biotechnological exploitation of lichens, the aim of this current study was to describe the application and comparison of different methods for the isolation of the individual lichen symbionts. These methods comprise of the use of the special sexual and asexual structures in lichens (apothecia, pycnidia, thallus-fragment) and enable a cultivation of the mycobiont and photobiont of lichens in pure cultures. Our goal was to propose a simple, efficient and strategic approach for the selective isolation of lichen partners and their better growth in the biotech lab.

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