Identifikasi Molekuler Bakteri Lipolitik Yang Diisolasi Dari Sedimen Mangrove Teluk Kendari

Molecular identification of lipolytic bacteria isolated from Kendari Bay mangrove sediments

Abstract

The study aims to isolate and molecularly identify lipolytic bacteria from mangrove sediment in Kendari Bay, Southeast Sulawesi. Lipolytic bacteria were inoculated into Nutrient Agar Rhodamin B containing an olive oil emulsion and 2% NaCl by spread plate, then incubated at room temperature for 24-48 hours. Genomics DNA was extracted using the saline tris-edta (STE) method, while 16S rRNA gene was amplified using PCR technique involving universal primers 63F and 1387R. PCR conditions consisting of pre-denaturation (94ºC for 5 min), denaturation (95ºC for 30 sec), primer annealing (55ºC for 30 sec), extension (72ºC for 1 min), and post extension (72ºC for 7 min) were carried out 35 cycles. Bioinformatics analysis of 16S rRNA gene include similarity analysis using NCBI Blast, genetic distance matrix as well as phylogenetic tree reconstruction. The results revealed that two bacterial isolates (LMA1 and LMB3) exhibited lipolytic activity and had 16S rRNA gene sequence lengths of 1292 and 1283 bp, respectively. According to the results of BLAST analysis, the LMA1 isolate had a similarity value of 99.69% and a genetic distance of 0.0023 with Vibrio fluvialis, whereas LMB3 had a similarity value of 99.69% and a genetic distance of 0.0008 with Acinetobacter junii. This is also compatible with the phylogenetic tree classification of the two isolates. As a result, based on the partial 16S rRNA gene sequence, isolates LMA1 and LMB3 have the most genetic resemblance and/or are the same species as Vibrio fluvialis and Acinetobacter junii, respectively.

Key words: Lipolytic bacteria, 16S rRNA, mangrove sediment