Kajian fisiologis dan molekular sifat resistensi bakteri terhadap tembaga

IRAWATI, Wahyu, Promotor Prof.Dr.Ir. Joedoro Soedarsono

2005 | Disertasi | S3 Bioteknologi

Pengolahan limbah dengan menggunakan mikrobia dalam bentuk lumpur aktif telah dilakukan di PT SIER Surabaya tetapi penelitian eksplorasi bakteri resisten tembaga yang terdapat di dalam lumpur aktif tersebut belum pernah dilakukan secara intensif. Penelitian ini bertujuan melakukan isolasi, seleksi, karakterisasi bakteri resisten tembaga yang terdapat di dalam lumpur aktif PT SIER, mengetahui mekanisme resistensi isolat bakteri dalam mengakumulasi tembaga, mengetahui ada tidaknya plasmid dalam sel isolat bakteri resisten tembaga, serta mengetahui letak gen pada genom yang menyandi sifat resistensi isolat bakteri terhadap tembaga. Bakteri resisten tembaga dalam penelitian ini diisolasi dari lumpur aktif pusat pengolahan limbah PT SIER dan limbah cair di pesisir pantai Kelurahan Sukolilo di Surabaya. Karakterisasi yang telah dilakukan meliputi pengujian biokimiawi, morfologi, dan fisiologi, serta analisis gen 16S rDNA. Mekanisme resistensi dapat di ketahui dengan mengukur jumlah tembaga yang diakumulasi sel, fraksi membran, dan sitoplasma selama pertumbuhan. Ada tidaknya plasmid dibuktikan dengan melakukan beberapa metode isolasi plasmid disertai dengan elektroforesis secara konvensional maupun dengan PFGE. Metode isolasi yang dilakukan meliputi metode lisis alkali, lisis SDS, lisis menurut Crosa dan Falcow, lisis menurut Kado dan Liu, lisis secara pemanasan, maupun lisis in situ. Deteksi gen yang menyandi sifat resistensi bakteri terhadap tembaga dilakukan dengan eliminasi plasmid, komplementasi genetik, dan amplifikasi gen resisten tembaga. Penelitian ini berhasil mengisolasi 9 isolat bakteri resisten tembaga. Tiga bakteri yang paling resisten dinamakan isolat C1, C2, dan C4 dengan nilai MIC (Minimum Inhibitory Concentration) masing-masing 6 mM, 6,5 mM dan 7 mM CuSO4. Berdasarkan nilai MIC tersebut, ketiga isolat mempunyai resistensi 2-3 kali lipat lebih tinggi dibanding bakteri lain yang telah ditemukan. Hasil analisis gen 16S rDNA menunjukkan bahwa isolat C1, C2, dan C4 masing-masing adalah Acinetobacter sp. C1, Acinetobacter sp. C2, dan Ralstonia sp. C4. Mekanisme resistensi isolat bakteri terjadi dengan mengakumulasi sebagian besar tembaga pada membran sel, dinding sel dan permukaan sel serta membatasi jumlah tembaga yang masuk ke sitoplasma. Acinetobacter sp. C1, Acinetobacter sp. C2, dan Ralstonia sp. C4 masing-masing dapat mengakumulasi tembaga sekitar 29 %, 50 %, dan 37 % dari berat kering sel bakteri. Kemampuan akumulasi tersebut lebih unggul 2-4 kali lipat dibanding bakteri lain yang pernah dilaporkan. Acinetobacter sp. C1, Acinetobacter sp. C2, dan Ralstonia sp.C4 memiliki plasmid yang berukuran besar, yaitu diatas 21,226 kb. Metode isolasi plasmid yang paling baik untuk ketiga isolat bakteri adalah metode lisis alkali.Telah berhasil dimodifikasi metode preparasi plasmid untuk PFGE ketiga isolat tanpa melalui lisis in situ. Perlakuan eliminasi plasmid menunjukkan bahwa sifat resistensi isolat bakteri terhadap tembaga disandi plasmid. Ada indikasi bahwa gen resisten tembaga sepanjang 0,9 kb pada Ralstonia sp. C4 dapat diamplifikasi dengan primer spesifik gen resisten tembaga L. lactis.

Sewage treatment by microorganism as activated sludge has been done at SIER Ltd.Co. However explorative research on copper-resistant bacteria in the activated sludge has not been done intensively. The objectives of these studies were to isolate, characterize the copper-resistant bacteria isolates in activated sludge of SIER Ltd.Co, to study the copper accumulation resistance mechanism, to examine the presence of plasmid in copper-resistant bacteria, and to establish the location of copper-resistant genes in bacterial isolates genom. Copper-resistant bacteria have been isolated from Surabaya Industrial Estate Rungkut and Sukolilo sea shore in Surabaya. Characterization of the isolates were conducted by employing biochemical, morphological and physiological tests as well as sequencing of 16S rDNA genes. Copper content of fractions of bacterial cell was determined for investigations of the copperresistance mechanism. Plasmid was isolated by several methods of cell lysis. Detection of copper resistant genes was carried out by plasmid curing, transformation, and amplification of copper-resistant genes. Nine copper resistant bacteria have been isolated. Three bacterial isolates were designated as C1, C2, and C4 isolates which had Minimum Inhibitory Concentration (MIC) of 6 mM, 6,5 mM, and 7 mM CuSO4, respectively. The level of copper-resistant in the three isolates were 2-3 times higher than copperresistant bacteria that have been known. Based on the 16S rDNA sequence analysis, the isolates were identified as Acinetobacter sp. C1, Acinetobacter sp. C2, and Ralstonia sp. C4. Copper resistance mechanism of these isolates was accomplished through the accumulation of copper in the inner membrane, the outer membrane and the surface of the cell as well as the protection the cellular cytoplasm from exposure to toxic levels. Acinetobacter sp. C1, Acinetobacter sp. C2, and Ralstonia sp. C4 accumulated copper 29 %, 50 %, and 37 % of dry weight of cells, respectively. These levels were 2-4 times higher than other bacterial copper accumulation level that have been published. These bacteria harbored plasmid with the size of more tha n 21 kb. The best method for plasmid isolation was lysis by alkali. A new method of DNA preparation for PFGE analysis has also been developed. Plasmid curing treatment indicated that the copper resistance genes were located on the plasmid. It was also shown that amplification of copper resistant genes was successful only on Ralstonia sp. C4 using specific primers complementary to copper resistant genes of L. lactis.

Kata Kunci : Mikrobia,Resistensi Bakteri,Tembaga, Acinetobacter sp. C1, Acinetobacter sp. C2, Ralstonia sp. C4, copper resistance bacteria, accumulation, plasmid