Diversity of antibacterial substances producing actinomycetes isolated from Calcareous Cave

ISMAEL, Dr.Ir. Ngadiman, M.Si

2008 | Tesis | S2 Bioteknologi

Aktinomisetes merupakan mikroorganisme penghasil senyawa bioaktif paling banyak khususnya antibiotik. Mikroorganisme ini ditemukan hampir di seluruh lingkungan seperti tanah, laut, gua, gunung, kompos. Penelitian ini bertujuan untuk mempelajari keragamaan aktinomisetes penghasil senyawa antibakteri yang berasal dari gua kapur. Sedimen, stalagtit, dinding keras dan dinding lunak gua kapur digunakan sebagai sumber isolat aktinomisetes. Isolasi dilakukan pada medium starch nitrate agar menggunakan metode taburan (pour plate). Isolat-isolat aktinomisetes diseleksi berdasarkan kemampuannya dengan menghambat pertumbuhan bakteri Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis dan Escherichia coli. Isolat yang mempunyai kemampuan antibakteri selanjutnya diidentifikasi berdasarkan morfologi koloni dan sel, keragamaan genetik menggunakan metode Rep-PCR dengan primer BOXA1R. Disamping itu, isolat-isolat terpilih diuji keragaman metabolit sekunder hasil ekstraksi dengan etil asetat menggunakan KLT (Kromatografi lapis tipis) dan pembacaan TLC dengan scanning densitometry. Sebanyak 78 isolat aktinomisetes telah berhasil diperoleh dari sampel gua kapur, akan tetapi hanya 8 isolat yang mempunyai aktivitas antibakteri tinggi terhadap 4 spesies bakteri yang diujikan. Diantara 8 isolat-isolat tersebut, hanya isolat BL3 dan BL5 yang mempunyai daya penghambatan terhadap semua bakteri yang diujikan. Sementara, isolate DAHP4 diketahui mempunyai aktivitas antibakteri yang spesifik terhadap P. aeruginosa. Berdasarkan uji morfologi, koloni sel, pola genetik dan pola metabolit sekunder pada KLT menunjukkan bahwa 8 isolat tersebut berbeda satu sama dengan lain. Analisis KLT menunjukkan bahwa isolate BL3 menghasilkan macam metabolit sekunder yang paling banyak yaitu 9 senyawa. Uji bioautografi isolat BL3 sedikitnya ada 3 senyawa (Rf: 0.87; 0.94; 0.97) yang memiliki aktivitas antibakteri terhadap S. aureus.

Actinomycetes are main microorganism producing bioactive compounds such as antibiotic. This microorganism can almost be found in every environment such as soil, deep sea water, cave, forest, mountain, compost. This research was aimed to study the diversity of antibacterial substances producing actinomycetes isolated from calcareous cave in Imogiri, Bantul , Yogyakarta province. Sediment, stalagtite, hard wall and soft wall of calcareous cave were used as source of actinomycetes isolates. Isolation was done by growing microbes at Starch nitrate agar using pour plate method. The isolates were selected based on the ability to inhibit the growth of pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli. The selected isolates were then identified according to colony and cell morphology, genetic diversity using Rep-PCR method with BOXA1R. In addition, the secondary metabolites diversity extracted by ethyl acetate of the selected isolates was analyzed by TLC (Thin layer chromatography) and scanning densitometry. The research resulted that 78 isolates were obtained from calcareous cave samples, but only 8 isolates showed higher antagonistic activity toward the four tested bacteria. Among the 8 selected isolates, only BL3 and BL5 isolates were able to inhibit all tested bacteria. Whereas, DAHP4 isolate demonstrated a specific antibacterial activity to P. aeruginosa. Based on morphology, repetitive gene profile and secondary metabolite analysis indicated that the 8 selected isolates were different characteristics from each other. TLC analysis showed that BL3 isolate produced highest number of secondary metabolites (9 substances) on scanning densitometry and at least 3 compounds which run at Rf: 0.87; 0.94; 0.97 showed high antibacterial activity to S. aureus.

Kata Kunci : Aktinomisetes,Antibakteri,Gua Kapur, Calcareous cave, actinomycetes, antibacterial, diversity.