Identification Of Isolate Gram-Negative Bacteria From The Hot Mud Flow Of Mount Semeru Using Phylogenetic Approach

Rompas, Daniel Joshua (2022) Identification Of Isolate Gram-Negative Bacteria From The Hot Mud Flow Of Mount Semeru Using Phylogenetic Approach. Other thesis, Institut Teknologi Sepuluh Nopember.

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Abstract

Penelitian terhadap bakteri ekstremofilik semakin hari semakin sering untuk dilakukan karena dari kelompok bakteri tersebut bisa enzim yang bersifat termostabil yang dapat diaplikasikan di berbagai bidang industri. Penelitian ini bertujuan untuk mengidentifikasi isolat bakteri yang diambil dari wilayah terdampak erupsi Gunung Semeru di Lumajang, Jawa Timur. Proses identifikasi dilakukan dengan analisis sequencing gen 16S-rRNA dengan PCR (polymerase chain reaction). Isolat didapatkan dari lokasi dengan titik koordinat 9096’126.959”LU, 7208’12.318”BT kemudian ditumbuhkan dan dimurnikan pada media ISP2 dengan teknik streak plate dan kemudian diinkubasi selama 48 jam pada suhu 37℃. Media yang sudah ditumbuhi dengan koloni bakteri murni kemudian dilakukan pewarnaan gram untuk mengetahui jenis gram pada bakteri pada isolat tersebut. Selain itu setelah didapatkan koloni murni dari isolat kemudian sampel juga dianalisis sekuens dengan gen 16S-rRNA fragmen bakteri dengan sepasang primer universal, primer forward 27F (5’-AGATTTGATCATGGCTCAG-3’) dan primer reverse 1492R (5’-CACGGATCCTACGGGTACCTTGTTACGACTT-3’) dengan menggunakan PCR. Hasil dari PCR kemudian dilakukan elektroforesis dan didapatkan sampel JN1 dan JN4 menunjukan pita tebal pada daerah ±1500 bp dan sampel JN2 dan JN3 tidak menunjukan adanya pita pada daerah tersebut sehingga hanya sampel JN1 dan JN4 yang dilanjutkan ke proses sequencing DNA. Setelah melakukan sequencing DNA hasil sekuens kemudian dimasukan kedalam menu BLAST pada website NCBI (National Center of Biotechnology Information) untuk mencari kekerabatan yang mirip. Hasil BLAST kemudian dibuat menjadi pohon filogenetik dengan bantuan software MEGA-X. Hasil pohon filogenetik dari sampel JN1 dan JN4 menunjukan bahwa sampel JN1 yang merupakan kode untuk isolat LS-ISP2-JN1 memiliki kemiripan 96,89% dengan Uncultured pseudomonas sementara sampel JN4 yang merupakan kode.
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Research about extremophilic bacteria nowadays has become more and more massive because of the advantage of using extremophilic bacteria's enzymes that have a special ability to resistant to hot conditions or commonly called thermostable. This study aimed to identify an isolate taken from an area affected by Mount Semeru's hot mud flow at Lumajang, East Java. The isolate identification method uses 16S-rRNA gene sequencing with PCR (polymerase chain reaction). The isolate was taken at 90°96'126.959" LU, 72°08'12.318" BT, then the isolate was regenerated and purified in ISP 2 media with streak plate technique and incubated at 37℃ for 48 hours. The gram staining results suggested that the isolate was a gram-negative bacteria. And then, the purified colony was brought to another lab for a PCR test. The colony was extracted and amplified using 16S-rRNA gene sequence. This study uses forward primer 27F (5'-AGAT TTGATCATGGCTCAG-3') and reverse primer 1492R (5'-CACGGAT CCTACGGGTACCTTGTTACGACTT-3'). The results of the PCR were then carried out by electrophoresis and the samples JN1 and JN4 showed thick bands in the ±1500 bp area, and samples JN2 and JN3 did not show any bands in that area, so only samples JN1 and JN4 were continued to the DNA sequencing process. After performing DNA sequencing, the results of the sequences are then entered into the BLAST menu on the NCBI (National Center of Biotechnology Information) website to look for similar kinship. The BLAST results were then made into a phylogenetic tree with the help of MEGA-X software. Based on phylogenetic tree analysis using software MEGA-X, 16S-rRNA gene sequencing of isolate JN1 and JN4 is, JN1 that a code for isolate LS-ISP2-JN1 have the similarity about 96,89% with Uncultured pseudomonas and JN4 that a code for isolate LS-ISP2-JN4 have the similarity about 99,54% with Bacterium strain BS2049.

Item Type:	Thesis (Other)
Additional Information:	RSKi 579.3 Rom i-1 2022
Uncontrolled Keywords:	16S-rRNA, Ekstremofilik, Elektroforesis, Gunung Semeru, Identifikasi Bakteri, PCR, Pohon filogenetik. 16S-rRNA, bactery identification, electrophoresis, extremophilic, Mount Semeru, PCR, phylogenetic tree.
Subjects:	Q Science > QR Microbiology > QR74.8 Bacteria
Divisions:	Faculty of Science and Data Analytics (SCIENTICS) > Chemistry > 47201-(S1) Undergraduate Thesis
Depositing User:	Mr. Marsudiyana -
Date Deposited:	04 May 2026 08:06
Last Modified:	05 May 2026 05:35
URI:	http://repository.its.ac.id/id/eprint/132968

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