Biochemical Properties of Mercuric Reductase from Local Isolate of Bacillus sp for Bioremediation Agent

Mercuric reductase is the important enzyme which catalyzes a reduction of a toxic Hg2+ to non-toxic Hg0. The enzyme which has been potentially used as mercury bioremediation agent is produced by mercury resistant bacteria. These research aims are to determinate the resistance level of a local Bacillus sp to HgCl2 in media, to determine the mercuric reductase activity from the bacteria, and to determine the biochemical properties of the mercuric reductase. The Bacillus sp was grown in the Nutrient Broth media with various of 0; 20; 40; 60; 120; and 160 µM HgCl2 to know the response of the bacteria against mercury, The cell growth of Bacillus sp was measured by optical density (OD) method of at λ 600 nm. The mercuric reductase activity was assayed in the solution of MRA (Mercury Reductase Assay), then the oxidized NADPH was observed by the spectrophotometry method at λ340 nm. The result showed that the Bacillus sp has been resistant to media containing mercury at 120 µM, but the microbial growth was decreased by 50% in media containing mercury 80 µM. The Bacillus sp could produce highly the mercuric reductase enzyme at 16 hours of growth time with enzyme activity as 0.574 Unit/µg. The mercuric reductase from the bacteria has an optimum activity at pH 6 and temperature 37 °C

Identifikasi Bakteri Resisten Merkuri Menggunakan Metode 16SrRNA terhadap Plak Gigi pada Pasien Pengguna Tumpatan Amalgam

Amalgam is a popular dental filling due to its cheaper price than other dental fillings. Basically, amalgam is an alloy, consists of two or more metals; one of them is mercury. The unfavorable thing about this alloy is that its vapor in the oral cavity can trigger the development of mercury-resistant bacteria. This type of bacteria has an enzyme called mercury reductase that can reduce Hg2+ to Hg0. 16SsRNA is a gene that contains important information to describe the prokaryotic type. This study was aimed to identify the type of mercury-resistant bacteria from dental plaque of patients with amalgam fillings. Samples were taken from the dental plaques. Isolation of DNA, sequensing of 16SsRNA gene by using PCR, and online BLAST through GenBank NCBI, and finally looking for the closest relative using a phylogenetic tree were performed in the Pharmacy Laboratory, Faculty of Mathematics and Science. The result of BLAST showed 4 types of bacteria, and the closest relative is B. thuringiensis. Conclusion: The type of mercury-resistant bacteria found in dental plaques was Bacillus thuringiensis.Keyword: amalgam, mercury resistant bacteria, dental plaques, 16SsRNA, PCR Abstrak: Amalgam adalah suatu logam campuran yang terdiri dari dua atau beberapa logam yang salah satunya adalah merkuri atau air raksa. Amalgam sebagai bahan tumpatan sampai saat ini masih banyak digunakan oleh dokter gigi karena harganya yang relatif murah. Namun penggunaan amalgam ini dapat melepaskan uap merkuri selama berada di dalam rongga mulut. Penggunaan amalgam ini memicu munculnya bakteri yang resisten terhadap merkuri dimana bakteri dapat mereduksi Hg2+ menjadi Hg0 melalui enzim yang menginduksi merkuri reduktase. 16SsRNA adalah gen yang menampung informasi-informasi penting agar mendeskripsikan jenis-jenis prokariotik. Penelitian ini bertujuan untuk mengidentifikasi bakteri resisten merkuri pada plak gigi pasien pengguna tumpatan amalgam menggunakan metode PCR. Jenis penelitian ialah deskriptif observatif. Sampel diambil dari plak gigi pasien pengguna tumpatan amalgam di Laboratorium Farmasi Fakultas MIPA. Dilakukan langkah-langkah untuk isolasi DNA, sekuensing gen 16SsRNA menggunakan PCR, kemudian dilakukan BLAST secara online melalui GenBank NCBI lalu dicari kekerabatannya menggunakan pohon filogenetik. Hasil BLAST mendapatkan 4 jenis bakteri, dan kekerabatan terdekatnya ialah Bacillus thuringiensis. Simpulan: Jenis bakteri resisten terhadap merkuri pada plak gigi ialah Bacillus thuringiensis.Kata kunci: amalgam, bakteri resisten merkuri, plak gigi, 16SsRNA, PCR

RESISTANCE LEVEL OF Pseudomonas stutzeri AGAINST MERCURY AND ITS ABILITY IN PRODUCTION OF MERCURY REDUCTASE ENZYME

Mercury reductase is an enzyme that is able to reduce Hg2+ to Hg0 non toxic. This enzyme is usually produced by mercury resistant bacteria. The research wanted to determine the resistance of indigenous Pseudomonas stutzeri isolate toward mercury and to explore the mercury reductase activity which is produced by the bacteria. The results of resistance assay of the Pseudomonas stutzeri toward mercury ion showed that the isolate could survive in media containing HgCl2 up to a concentration of 80 µM. The bacteria could produce mercury reductase optimally at the 24th of fermentation time. The enzyme showed optimum activity at pH 7 and temperature of 45 oC

MERCURY REDUCTASE ACTIVITY OF AN INDIGENOUS MERCURY RESISTANT BACTERIAL ISOLATE (Bacillus sp. S1) FROM KALIMAS-SURABAYA AS A POTENTIAL REDUCING AGENT FOR MERCURIAL ION (HG2+)

<p>Mercury reductase activity and Hg2+ lowering capacity of a Mercury Resistant Bacteria (MRB) (Bacillus sp. S1) which was isolated from Kalimas River of Surabaya, Indonesia were studied. The activity was determined by Mercury Reductase Assay System (MRAS) in a solution mixture contained 50 mM PBS (pH ± 7.0), 0.5 mM EDTA, 200 µM MgSO4, 0.1% (v/v) ß-merchaptoethanol, 200 µM NADH2 and 25 mg/L HgCl2 and one volume of crude extract incubated at room temperature for various interval period of time. Mercury reductase activity was measured spectrophotometrically at 340 nm. One unit of reductase activity was defined as one molar of oxidized NADH2 produced per total cell per minute. Results of study showed that, the isolate could resist the concentration of HgCl2 up to 11 mg/L. At 30 minute incubation period at room temperature, the highest mercury reductase activity and the Hg2+ lowering capacity was found to be 0.006 unit/109cell and <br />1.48 mg/L/109cell/minute, respectively with the reduction efficiency of Hg2+ to Hg0 of 0.18% per minute. Therefore, it can be concluded that the Bacillus sp. S1 isolate could be assumed to be exellent mercury bioremediation agent since it was found to be highly mercury resistant and very efficient to reduce cationic mercury (Hg2+) to elemental mercury (Hg0). </p><p><strong>Keywords</strong>: Kalimas-Surabaya, Bacillus sp. S1, Mercury resistant, Mercury reductase</p>

BAKTERI RESISTEN MERKURI (Hg) PADA PLAK GIGI PASIEN DENGAN TUMPATAN AMALGAM DI PUSKESMAS BAHU

Amalgam is an alloy of mercury with various metals which used for dental amalgam fillings since 150 years ago. It is still popular because of it’s strength and durability. However, it also has issue about it’s safety due to releasing of mercury inside oral cavity. Long time of exposure to mercury will result in resistant mercurial. Bacteria have ability to reduce Hg2+ to Hg0 by mercury reductase enzyme, changing toxicity to non-toxic form. We obtained the dental plaque from 5 patients who has been using amalgam for 5 years or more at Puskesmas Bahu. The dental plaque were stored inside the sterile glass tube with 0.9% NaCl solution and then tested for mecury resistant bacteria and identified bacteria. We founded 15 isolates were resistant to mercury. Then we did physiology, morphology, and biochemistry tests. There are 8 genus of bacterias which has ability to reduce mercury from dental plaque of patient with amalgam fillings. Keywords: Amalgam, Mercury Resistant Bacteria, Dental Plaque Abstrak: Amalgam merupakan campuran logam, yang diantaranya adalah merkuri sudah digunakan sejak 150 tahun yang lalu oleh kedokteran gigi sebagai penambal gigi berlubang dan sampai sekarang amalgam masih digunakan oleh dokter gigi karena merupakan bahan tambalan yang kuat dan tahan lama. Namun resiko utama amalgam adalah pelepasan uap merkuri yang mungkin terjadi selama penggunaannya di rongga mulut. Penggunaan amalgam ini memicu munculnya bakteri resisten terhadap merkuri dimana bakteri mampu untuk mereduksi ion Hg2+ menjadi Hg0 oleh enzim merkuri reduktase, yang sebelumnya bersifat toksik menjadi kurang toksik. Tujuan dari penelitian untuk mengetahui bakteri resisten merkuri pada plak gigi pasien dengn tumpatan amalgam. Desain penelitian adalah metode deskriptif eksploratif. Sampel yang diambil dalam penelitian ini adalah 5 koloni bakteri resisten merkuri pada plak gigi dari pasien yang telah menggunakan tumpatan amalgam minimal 5 tahun yang berkunjung ke Puskesmas Bahu. Spesimen yang didapatkan dimasukkan ke dalam tabung reaksi steril yang berisi larutan NaCl 0.9% segera dibawa ke laboratorium untuk dilakukan uji resistensi merkuri dan identifikasi bakteri. Isolasi isolat bakteri resisten merkuri pada 5 sampel, diperoleh 15 isolat. Selanjutnya dilakukan identifikasi bakteri melalui uji morfologi, uji fisiologi, dan uji biokimia. Hasil uji yang dilakukan ditemukan 8 genus bakteri yang dapat diidentifikasi. Kata Kunci: Amalgam, Bakteri Resisten Merkuri, Plak Gigi

BAKTERI RESISTEN MERKURI (Hg) PADA PLAK GIGI PASIEN DENGAN TUMPATAN AMALGAM DI PUSKESMAS BAHU

Amalgam is an alloy of mercury with various metals which used for dental amalgam fillings since 150 years ago. It is still popular because of it’s strength and durability. However, it also has issue about it’s safety due to releasing of mercury inside oral cavity. Long time of exposure to mercury will result in resistant mercurial. Bacteria have ability to reduce Hg2+ to Hg0 by mercury reductase enzyme, changing toxicity to non-toxic form. We obtained the dental plaque from 5 patients who has been using amalgam for 5 years or more at Puskesmas Bahu. The dental plaque were stored inside the sterile glass tube with 0.9% NaCl solution and then tested for mecury resistant bacteria and identified bacteria. We founded 15 isolates were resistant to mercury. Then we did physiology, morphology, and biochemistry tests. There are 8 genus of bacterias which has ability to reduce mercury from dental plaque of patient with amalgam fillings. Keywords: Amalgam, Mercury Resistant Bacteria, Dental Plaque Abstrak: Amalgam merupakan campuran logam, yang diantaranya adalah merkuri sudah digunakan sejak 150 tahun yang lalu oleh kedokteran gigi sebagai penambal gigi berlubang dan sampai sekarang amalgam masih digunakan oleh dokter gigi karena merupakan bahan tambalan yang kuat dan tahan lama. Namun resiko utama amalgam adalah pelepasan uap merkuri yang mungkin terjadi selama penggunaannya di rongga mulut. Penggunaan amalgam ini memicu munculnya bakteri resisten terhadap merkuri dimana bakteri mampu untuk mereduksi ion Hg2+ menjadi Hg0 oleh enzim merkuri reduktase, yang sebelumnya bersifat toksik menjadi kurang toksik. Tujuan dari penelitian untuk mengetahui bakteri resisten merkuri pada plak gigi pasien dengn tumpatan amalgam. Desain penelitian adalah metode deskriptif eksploratif. Sampel yang diambil dalam penelitian ini adalah 5 koloni bakteri resisten merkuri pada plak gigi dari pasien yang telah menggunakan tumpatan amalgam minimal 5 tahun yang berkunjung ke Puskesmas Bahu. Spesimen yang didapatkan dimasukkan ke dalam tabung reaksi steril yang berisi larutan NaCl 0.9% segera dibawa ke laboratorium untuk dilakukan uji resistensi merkuri dan identifikasi bakteri. Isolasi isolat bakteri resisten merkuri pada 5 sampel, diperoleh 15 isolat. Selanjutnya dilakukan identifikasi bakteri melalui uji morfologi, uji fisiologi, dan uji biokimia. Hasil uji yang dilakukan ditemukan 8 genus bakteri yang dapat diidentifikasi. Kata Kunci: Amalgam, Bakteri Resisten Merkuri, Plak Gigi

Existence of Ferrous Iron-Dependent Mercury Reducing Enzyme System in Sulfur-Grown A. Ferrooxidans MON-1 Cells

Iron-grown Acidithiobacillus ferrooxidans MON-1 cells are highly resistant to organomercurial compounds as well as mercuric chloride (HgCl2). Existence of a novel Hg2+-reducing enzyme system, in which mercury resistant aa3-type cytochrome c oxidase catalyzes the reduction of Hg2+ with reduced mammalian cytochrome c or Fe2+ as an electron donor to give Hg0, has been shown in iron-grown MON-1 cells. There has been no reports on the mechanism of Hg2+ reduction by sulfur-grown A. ferrooxidans cells. The level of mercury resistance in sulfur-grown A. ferrooxidans MON-1 cells was compared with that of iron-grown MON-1 cells. Strain MON-1 was able to grow in 1% elemental sulfur medium (pH 2.5) containing 10 μM of Hg2+ or 0.2 μM phenylmercury acetate (PMA), suggesting that the levels of mercury resistance to inorganic and organic mercurial compounds are nearly the same in iron- and sulfur-grown MON-1 cells. Activity levels of Hg0 volatilization from HgCl2, PMA, and methylmercury chloride (MMC) were also nearly the same in iron- and sulfur-grown cells and these activities were markedly activated by 100 mM of Fe2+, but strongly inhibited by 1 mM of sodium cyanide, indicating that sulfur-grown MON-1 cells has the activity of ferrous iron-dependent mercury reducing enzyme system containing aa3-type cytochrome oxidase. aa3-type cytochrome c oxidase purified partially from sulfur-grown MON-1 cells showed both the iron oxidase and mercury reductase activities in the presence, but not in the absence, of rusticyanin and c-type cytochromes (Cyc1 and Cyc2) partially purified from iron-grown MON-1 cells.

Normalization of Quantitative Real-Time PCR Data of Identified Genes from an Industrial Bioleaching Operation

A strategy for the monitoring and control of genetic expression in an industrial bioleaching process of copper sulphide minerals is developed in order to understand more fully this process and investigate optimization possibilities. The aim of this research is to find a group of housekeeping genes to normalize the genetic expression data associated to the metabolic functions from industrial bioleaching samples obtained through specific real-time PCR. The data includes the quantification of the previously identified genes in the industrial process in samples from different strips (heap sections) and times. Two studies were carried out, one with the gene expression data associated to each species and the other with the expression data from all the genes detected in the industrial process, during a period of 18 months, using the VBA applet geNorm [1]. The first analysis showed that for the archaea Ferroplasma acidiphilum, the gene with the most stable expression codifies to the ribosomal protein S4 (rpS4) and secondly the mercury reductase gene (merA). In the case of Acidithiobacillus ferrooxidans DM and D2 strains, genes merA and pyruvate dehydrogenase (pdhA) presented similar factors of stability. In the community analysis it was concluded that the gene with the most stable expression was pdhA of A.ferrooxidans. A new analysis was done including other genes using geNorm to define the most stable genes for the whole community. In the community analysis it was concluded that the gene with the most stable expression was pdhA of A.ferrooxidans and merA of F.acidiphilum.