Bioakumulasi merkuri oleh bakteri sedimen dari Sungai Sangon- D.I. Yogyakarta

RETNOWATI, Yuliana, Prof.Dr. Endang Sutariningsih, M.Sc

2005 | Tesis | S2 Biologi

Sedimen sungai tercemar merkuri pada umumnya merupakan habitat bakteri heterotrofik tertentu, khususnya bakteri pengguna merkuri. Bakteri tersebut mempunyai arti penting dalam pembersihan lingkungan (bioremediasi) tercemar logam berat. Penelitian ini bertujuan untuk mendapatkan bakteri pengguna merkuri dan mempelajari karakteristik bakteri tersebut berdasarkan profil protein. Bakteri pengguna merkuri diisolasi melalui teknik kultur diperkaya dengan menggunakan medium Nutrient Cair yang dimodifikasi ditambah 0,1 mg/L metil merkuri klorida (CH3HgCl) sebagai sumber karbon dan tenaga. Setelah beberapa kali sub-kultur, bakteri pengguna merkuri diisolasi melalui pengenceran seri dan ditanam pada medium agar yang dimodifikasi ditambah 0,1 mg/L CH3HgCl menggunakan tehnik taburan. Isolat yang diperoleh diseleksi berdasarkan kemampuan tumbuh pada berbagai konsentrasi CH3HgCl, yang dinyatakan dalam waktu generasi (g) dan kecepatan tumbuh spesifik (μ). Pengaruh CH3HgCl pada pertumbuhan bakteri dinyatakan sebagai Konstanta hambatan (Ki). Tiga tipe bakteri dengan waktu generasi tertinggi, sedang, terendah dan Ki tertentu dipilih untuk percobaan kultivasi. Tiap interval waktu tertentu pertumbuhan bakteri ditentukan secara spektrometri (A600nm) dan merkuri yang terakumulasi ditentukan menggunakan spektrofotometri absorpsi (cold vapor atomic absorption spectrometry; A353 nm). Aktivitas bakteri dideteksi melalui visualisasi protein dengan elektroforesis gel poliakrilamida-SDS (12% PAGE-SDS), dengan kultur tanpa merkuri sebagai kontrol. Isolat bakteri terpilih diidentifikasi berdasarkan metoda standar. Penelitian ini berhasil memperoleh empatbelas isolat bakteri pengguna merkuri dari sedimen sungai dengan populasi sebanyak 133,5x106 CFU/ml. Hanya empat isolat yang mampu tumbuh pada medium mengandung 2-4 mg/L CH3HgCl dengan waktu generasi bervariasi dari 1,3–5,5 jam dan kecepatan tumbuh spesifik (μ) 0,1–0,5 /jam. CH3HgCl menghambat pertumbuhan bakteri yang dinyatakan dalam Ki, bervariasi dari 1,03–1,67 mg/L. CH3HgCl memacu pembentukan kapsula yang lebih tebal, sehingga memungkinkan merkuri terakumulasi. Bakteri tersebut mampu mengakumulasi merkuri 71,7 – 252,6 μg/g biomass dengan enzim spesifik yang ditunjukkan berupa pita protein pada gel elektroforesis dengan berat molekul 22-24 kDa dan 59-66 kDa. Protein tersebut mempunyai berat molekul yang sama dengan enzim organomerkuri liase dan merkuri reduktase. Hasil identifikasi keempat isolat adalah Bacillus sp. (strain YLN002), Alcaligenes sp. (strain YLN004 dan strain YLN005) dan Pseudomonas sp. (strain YLN006).

Mercurial contaminated river sediments are the most suitable habitat for spesific heterotrophic bacteria, especially mercury utilizing bacteria. The aims of the research were to obtain mercury utilizing bacteria from river floor sediment and to study of the characteristics of the bacteria based on their protein profile. Mercury utilizing bacteria were isolated through batchwise enrichment culture techniques using modified nutrient broth containing 0.1 mgL-1 methyl mercury chloride (CH3HgCl) as carbon dan energy sources. After several subcultures, those bacteria were isolated through serial dilution and plated on the growth medium using pour plate methods. Selection of isolates was carried out through the growth experiment based on their abilities to grow on different methyl mercury concentrations, in terms of generation time (g) and specific growth rates (μ). The effects of CH3HgCl on the bacterial growth were observed and determined as inhibition constants (Ki). Three types of bacteria showing the highest, moderate and the lowest generation time and Ki specific were selected and purified for futher cultivation experiments. During cultivation experiment at every interval of time, the bacterial growth was monitored spectrophotometrically (A600nm) and the accumulation of mercury was measured using cold vapor atomic absorption spectrometer (A353nm). The activity of mercury utilizing bacteria was detected using gel electroforesis (PAGE-SDS) based on the protein profile and bacterial culture without methyl mercury used as control. The selected isolates were identified using standard methods. The results revealed that fourteen mercury utilizing bacteria were succesfully isolated from river sediment (133.5x106 CFU/ml). Only four isolates were selected because they were able to grow on growth liquid medium containing 2–4 mg L-1 at generation times of 1.3–5.5 h and specific growth rates of 0.1–0.5 generation h-1. CH3HgCl demonstrated its inhibition on those bacterial growth with inhibition constants (Ki) of 1.03–1.67 mg L-1. Despite, CH3HgCl stimulated bacterial cells to produce more mucilage mass as external capsules. The capsules enabled mercury to be accumulated. The mercury accumulation occured up to 71.7 – 252.6 μg g-1 biomass. The activity of mercury utilizing bacteria was demonstrated on PAGE-SDS as bands located at 22 – 24 kDa and 59 – 66 kDa. Those bands were assumed as organomercurial lyase and mercury reductase enzymes. The four selected isolates were identified as Bacillus sp. (strain YLN002), Alcaligenes sp. (strain YLN004 and strain YLN005) and Pseudomonas sp. (strain YLN006).

Kata Kunci : Bioremediasi,Bakteri Pengguna Merkuri, heterotrophic, growth, accumulation, specific growth rate, generation rate, inhibition constant