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Aerial mycelium formation in rare thermophilic Actinobacteria on media solidified with agar and gellan gum

This study investigated aerial mycelium formation in 12 isolates of rare thermophilic Actinobacteria from Indonesia on four different media (International Streptomyces Project ISP 1, ISP 2, ISP 3, and Bennett’s solidified with agar and gellan gum). The results from media solidified with agar showed that aerial mycelium formation was observed on 9 isolates as follows: 3 isolates on ISP 1 agar (Amycolatopsis and Microbispora); 3 isolates on ISP 2 agar (Amycolatopsis and Microbispora); 9 isolates on ISP 3 agar (Actinoallomurus, Amycolatopsis, Microbispora, Thermobispora, and Streptoalloteichus); and 2 isolates of Amycolatopsis on Bennett’s agar. Aerial mycelium formation was not observed in 3 isolates (Microbispora and Nocardia) on all media solidified with agar. The results from media solidified with gellan gum showed that aerial mycelium formation was observed in all 12 isolates as follows: 8 isolates on ISP 1 gellan gum (Amycolatopsis, Microbispora, Nocardia and Thermobispora); 5 isolates on ISP 2 gellan gum (Amycolatopsis, Microbispora, and Nocardia); 11 isolates on ISP 3 gellan gum (Actinoallomurus, Amycolatopsis, Microbispora, Nocardia, Thermobispora, and Streptoalloteichus); and 5 isolates on Bennett’s agar (Amycolatopsis, Microbispora, Nocardia, and Streptoalloeichus). These results indicate that the media solidified with gellan gum induced aerial mycelium formation in larger number of rare thermophilic Actinobacteria isolates compared to media solidified with agar.


Introduction
The phylum Actinobacteria are Gram positive filamentous bacteria with high G+C content in their DNA [1]. Actinobacteria are frequently found in a wide range of habitats including soil, freshwater, marine, and endophyte on plant [2]. During the last few decades, researches have focused on isolating Actinobacteria from special habitats and extreme environments such as deep sea, salt-lake, and hot spring [3].
Actinobacteria are known as one of the most important sources of secondary metabolites such as antimicrobial, enzyme inhibitor, and biocontrol agent [1]. The genus Streptomyces produces almost 80 % of bioactive compounds known today [4]. Secondary metabolites of Actinobacteria are produced on late growth phase along with aerial mycelium production and sporulation [5]. Aerial mycelium production and sporulation is directly dependent on the quality and quantity of nutrient in the media [6].
Taxonomically, rare Actinobacteria are known as non-Streptomyces Actinobacteria and relatively difficult to isolate, cultivate, and maintain [7]. Rare Actinobacteria have gained attention related to their diversity and a promising source of secondary metabolites [8]. Various media and isolation techniques have been developed for the isolation, mycelium production (substrate mycelia and aerial mycelia), and sporulation of Actinobacteria [9]. According to Hamedi and Poorinmohammad [10], Actinobacteria form a substrate mycelium in both liquid and solid media, while aerial mycelia are formed specifically on solid media. Suzuki [11] reported that media with agar and gellan gum stimulated the formation of aerial mycelia as well as aerial spores of rare Actinobacteria.
In the previous study, Ningsih et al. [12] successfully isolated a novel thermophilic Actinobacteria genus and species, Gandjariella thermophila and another five genetically closely related isolates to this novel taxon from forest soil near geyser in Cisolok geothermal area. In another study, Ningsih et al. [13] obtained 25 isolates of thermophilic Actinobacteria from soil samples in the geyser of Cisolok and its vicinity. Their phylogenetic tree analyses based on 16S rRNA gene sequences showed that 15 out of 25 isolates are taxonomically regarded as rare Actinobacteria. In addition, we also obtained four isolates of rare Actinobacteria, e.g. three isolates from soil of Cisolok geyser and one isolate from litter sample of Galunggung geothermal area (unpublished data). However, the study of substrate and aerial mycelium formation of these rare thermophilic Actinobacteria isolates on various media solidified with agar and gellan gum has not yet been conducted.
This study investigated aerial mycelium formation in rare thermophilic Actinobacteria from geothermal area in Indonesia on four different media (International Streptomyces Project (ISP) 1, ISP 2, ISP 3, and Bennett's) solidified with agar and gellan gum. The results of this study will provide information about the effect of each solidifying agent on mycelium formation, in order to improve the methods of isolation, cultivation, and antimicrobial screening of rare thermophilic Actinobacteria.

Microorganisms
Twelve rare thermophilic Actinobacteria isolates from soil and litter samples in geothermal area of Cisolok and Galunggung, West Java were used in this study ( Table 1). All isolates were maintained on ISP 1 medium at room temperature, in 20 % (v/v) glycerol as agar blocks at -80 °C, and as lyophilized cells for long-term preservation [12].

Preparation of growth media
International Streptomyces Project (ISP) 1, ISP 2, ISP 3 medium was prepared according to Shirling and Gottlieb [14]. Bennett's medium was prepared according to Jones [15]. All media were solidified with agar and gellan gum with the addition of MgCl 2 .

Results and Discussions
The colony growth and formation of substrate mycelia as well as aerial mycelia of 12 isolates of rare thermophilic Actinobacteria were observed after seven days of incubation. The morphology of representative isolates on media solidified with agar and gellan gum incubated at 45 °C for five days is shown in Figure 1. The isolates showed formation of aerial mycelia as shown by the change of colony surface colour and formation of white sheath on the colony's surface, as similarly reported by Manteca et al. [16]. The ability of each isolate to produce substrate mycelia is shown in Table 2. All isolates were able to form substrate mycelia on media solidified with agar and gellan gum after seven days at 45 o C. The results indicated that all media solidified with either agar or gellan gum supported the growth of substrate mycelia. All isolates grown on all media produced substrate mycelia within two days of incubation.
The results showed that media solidified with gellan gum induced aerial mycelium formation in larger number of rare thermophilic Actinobacteria isolates compared to media solidified with agar. It was reported [17] that agar has been the dominant solidifying agent in microbial research. However, at high incubation temperature, agar-based media usually lose it strength and begin to melt or soften with the presence of significant amounts of surface water escaping from the gel.
Gellan gum has been known as an alternative solidifying agent. It has a potential as an ideal solidifying agent for isolation and growth medium of thermophilic microorganisms [18]. Gellan gum was better for cultivation and stimulating spore production of slow growing actinomycetes [19]. Media with gellan gum promoted better growth of various thermophilic bacteria such as Bacillus acidocaldarius, Thermus thermophilus, and Thermus aquaticus [17]. Media with gellan gum best stimulated the formation of aerial mycelia, as well as aerial spores, on Actinobispora yunnanensis IFO 15681 T , Sporichthya, Planobispora, and Planomonospora [11].
This study showed that rare thermophilic Actinobacteria isolated from geothermal area in Indonesia were able to grow on various media solidified with agar and gellan gum. Twelve rare thermophilic Actinobacteria isolates showed the ability to form substrate mycelia on both media solidified with agar and gellan gum. While, larger number of isolates formed aerial mycelia on gellan gum media compared to agar media. These results indicate that the media solidified with gellan gum induced aerial mycelia formation of rare thermophilic Actinobacteria isolates compared to media solidified with agar.