Amplifikasi Gen Resistensi Tembaga (Cur) pada Bakteri yang Diisolasi dari Limbah Industri di Surabaya
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Abstract
ABSTRACT
Copper pollution is one of the serious environmental problems in Indonesia. Higher concentration of copper is toxic so that threat living organism. Bio-remediation using copper resistant bacteria is effective for solving heavy metals pollution because the bacteria adapt easily when applied in environment. Using bacteria containing gene encoded copper resistance could help effort of copper bio-remediation. The purpose of this research is to isolate and characterize copper resistant bacteria from industrial sewage in Surabaya also to amplification of gene encoded resistance to copper (Cur). Bacteria was grown on Salts Base Solution medium with addition of appropriate concentration of copper. Resistance to copper was determined based on Minimum Inhibitory Concentration of CuSO4. Molecular characterization was done based on 16S rDNA gene analysis using universal primer. Amplification of Cur gene was done using specific primer of gene orf3, orf4, orf5 from Lactoccocus lactis. Three highly copper resistant bacteria have been isolated with the MIC of 2.5-7.5 mM CuSO4 encoded IrC1, IrC2, and IrC4. Isolate IrC4 is the highest copper resistant bacteria with the MIC of 7.5 mM. Resistance mechanism may be through accumulation copper inside the cells with the total of 371 mg/g dry weight of cells. The three bacteria have plasmid with the size of 21 kb. Isolate IrC4 have 96.99% similarity with Cupriavidus pauculus. Amplification of copper-resistance (Cur) gene demonstrated that a single band of 0.9 kb was obtained from isolate C4. The finding of indigenous resistant bacteria encoded Cur gene may give better solution for pollution problem in Indonesia.
Keywords: bacteria, copper, Isolate IrC4, Lactococcus lactis, resistant
ABSTRAK
Pencemaran tembaga di Indonesia merupakan salah satu masalah lingkungan yang serius sehingga perlu diatasi. Tembaga pada konsentrasi yang tinggi bersifat toksik sehingga mengancam kehidupan organisme. Bioremediasi menggunakan bakteri resisten tembaga yang diisolasi dari lingkungan tercemar terbukti efektif mengatasi pencemaran logam berat karena lebih mudah beradaptasi ketika diterapkan di lingkungan. Pemanfaatan bakteri yang mengandung gen penyandi resistensi tembaga dapat menunjang keberhasilan usaha bioremediasi tembaga. Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi bakteri resisten tembaga dari limbah industri di Surabaya serta melakukan amplifikasi gen yang menyandi resistensi bakteri tersebut terhadap tembaga (Cur).  Isolasi bakteri dilakukan menggunakan medium Salt Base Solution dengan penambahan CuSO4. Uji resistensi ditentukan berdasarkan nilai Minimum Inhibitory Concentration (MIC) terhadap CuSO4. Karakterisasi molekular dilakukan berdasarkan analisis gen 16S rDNA menggunakan primer universal. Amplifikasi gen Cur dilakukan dengan menggunakan primer yang spesifik terhadap gen orf3, orf4, orf5 dari gen Cur pada Lactobacillus lactis. Hasil penelitian menunjukkan bahwa terdapat tiga isolat bakteri yang paling resisten dengan nilai MIC= 6.5-7.5 mM, yaitu isolat IrC1, IrC2, dan IrC4. isolat IrC4 merupakan isolat bakteri yang paling resisten dengan nilai MIC sebesar 7.5 mM. Mekanisme reistensi isolat IrC4 adalah dengan cara mengakumulasi tembaga di dalam sel selitar 371 mg/g berat kering sel. Ketiga isolat memiliki plasmid berukuran sekitar 21 kb. Analisis gen 16S rDNA menunjukkan bahwa isolat IrC4 memiliki kemiripan 96,99% dengan Cupriavidus pauculus. Gen Cur pada isolat IrC4 sepanjang 0.9 kb berhasil diamplifikasi dengan menggunakan primer spesifik gen orf3 yang menyandikan pengikatan tembaga. Penemuan bakteri indigen indonesia yang menyandikan gen resisten tembaga diharapkan dapat menunjang keberhasilan penanganan masalah pencemaran tembaga di Indonesia.
Kata kunci: Â bakteri, isolat IrC4, Lactobacillus lactis, resisten, tembaga.
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