Abstract. Syafitri WA, Ningsih F, Setyaningsih PP, Rachmania MK, Sari DCAF, Yabe S, Yokota A, Oetari A, Sjamsuridzal W. 2019. Screening for amylolytic activity and characterization of thermophilic Actinobacteria isolated from a geothermal area in West Java, Indonesia. Biodiversitas 20: 1929-1938. In this study, we describe the screening for amylolytic activity of 17 thermophilic Actinobacteria isolates obtained from the soil of Cisolok geysers, a geothermal area in West Java, Indonesia. All isolates were screened for amylolytic activity by the starch-agar plate method at various temperatures. The results showed that all of isolates were able to grow at 45oC. The growth abilities of the isolates grown in ISP 1 medium varied at temperatures from 45 to 60oC. Fifteen of the 17 isolates showed amylolytic activity at 45oC, 13 showed such activity at 50oC, and four showed activity at 55oC. Only three isolates, designated SL1-2-R-2, SL1-2-R-3, and SL1-2-R-4, showed growth and amylolytic activity at 60oC. These three isolates were selected for molecular identification. The nearly full-length of 16S rRNA gene sequences data showed that these three isolates have a similarity of 99.93-100% with Actinomadura keratinilytica WCC-2265T and of 98.74-98.91% with A. miaoliensis BC 44T-5T. Phylogenetic tree shows that all three isolates are clustered together in a monophyletic group with the type strain of A. keratinilytica WCC-2265T as their most closely related species, with 100% bootstrap support. Based on sequencing of the 16S rRNA gene, phylogenetic comparison, and phenotypic characterization, the three isolates were identified as A. keratinilytica.

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.

Abstract. Ningsih F, Sari DCAF, Yabe S, Yokota A, Sjamsuridzal W. 2020. Potential secondary metabolite biosynthetic gene clusters and antibacterial activity of novel taxa Gandjariella. Biodiversitas 21: 5674-5684. Microbial resistance to available antibiotics has gained increasing attention in recent years and led to the urgent search for active secondary metabolites from novel microbial taxa. This study aimed to assess putative secondary metabolite biosynthetic gene clusters (BGCs) in the genome of a novel thermophilic Actinobacteria type strain Gandjariella thermophila SL3-2-4T and screen for its antibacterial activity. Four other related novel candidate Actinobacteria strains, isolated from forest soil in the Cisolok geothermal area (West Java, Indonesia), were also screened for antibacterial activity in various media solidified with gellan gum. The genome of the SL3-2-4T strain contained 21 antiSMASH-identified secondary metabolite regions harboring BGCs. These BGCs were for polyketide synthase, non-ribosomal peptide synthase, and ribosomally synthesized and post-translationally modified peptide family clusters. Three BGC regions displayed 50-100% similarity with known secondary metabolites. Thirteen and five regions displayed low (4-35%) and no similarity with known BGCs for secondary metabolites, respectively. Strains SL3-2-4T and SL3-2-7 on MM 2 medium solidified with gellan gum at 45 °C for 14 days demonstrated inhibitory activity against all Gram-positive, but not Gram-negative bacteria. Strain SL3-2-10 on ISP 3 gellan gum medium incubated for seven days only active against K. rhizophila NBRC 12078. Strains SL3-2-6 and SL3-2-9 did not exhibit any antibacterial activity against the tested bacterial strains on the three tested media. The results indicated that novel taxa have the potential for the discovery of active secondary metabolites.

Abstract. Rachmania MK, Ningsih F, Sari DCAF, Eshananda Y, Sakai Y, Yabe S, Yokota A, Sjamsuridzal W. 2022. Identification and screening of enzymatic abilities of Ktedonobacteria from forest soil of Cisolok Geothermal Area, Indonesia. Biodiversitas 23: 4686-4695. This study aimed to provide information on culturable Ktedonobacteria from forest soil in the Cisolok geothermal area and their potential as enzyme producers. Twelve ktedonobacterial isolates were obtained from this study and identified based on full-sequence of the 16S rRNA gene. Seventeen isolates (including five isolates from previous studies) were used for enzymatic screening and phylogenetic analyses. Screening of amylolytic (0.5% soluble starch) and cellulolytic (0.5% carboxymethylcellulose) (CMC) activities from ktedonobacterial isolates was performed on a ten-fold diluted R2A agar medium, and were incubated at 30 °C for 21 days. The EzBioCloud search revealed that all isolates showed low sequence similarities with Dictyobacter aurantiacus S-27T (97.82 to 98.18%) as their closest related species. The phylogenetic tree showed that all isolates belong to the genus Dictyobacter (family Dictyobacteraceae of the class Ktedonobacteria). All isolates formed a monophyletic group and were placed as the sister clade to D. aurantiacus, as supported by a very strong bootstrap value (99%). Screening of amylolytic and cellulolytic abilities showed that most isolates (88.23%) were able to degrade both 0.5% starch and 0.5% CMC as substrates. This study revealed the presence of Ktedonobacteria with amylolytic and cellulolytic abilities in the forest soil of Cisolok geothermal area.

Thermophilic Actinobacteria are known as potential producers of novel antimicrobial compounds. However, the optimum growth medium for antibacterial activity assessment of thermophilic Actinobacteria has rarely been reported. This study demonstrated the effects of nine different microbial growth media on antibacterial activity assessment of a thermophilic actinobacterium from the soil in Cisolok geysers, Sukabumi, West Java (Indonesia). The strain SL2-2-R-9 was identified as Streptomyces cellulosae based on 16S rRNA gene data (100% similarity). The antibacterial activity was examined by the agar plug diffusion method against five bacterial test strains. The result of antibacterial activity screening showed that SL2-2-R-9 grown on ISP 7 agar and Bennett’s gellan gum inhibited the growth of Bacillus subtilis, Staphylococcus aureus, and Kocuria rhizophila. Strain grown on ISP 3 gellan gum inhibited the growth of B. subtilis andS. aureus, while on 301 agar and TSA, inhibited only K. rhizophila. Strain grown on ISP 6 agar and modified Bennett’s gellan gum, inhibited onlyS. aureus. Strain grown on ISP 3 agar and SFM agar showed no inhibition zone against all tested bacteria. There was no inhibition observed against Gram-negative bacteria when the strain was grown on all media.