Kegiatan Riset

Molecular Analysis of Polyaromatic Hydrocarbon- Dioxygenase Gene from Indonesia Marine Bacteria: Strategy for Biomonitoring Oil Spill : ITSF 2009

oilbacteria1Ahmad Thontowi, Nanik Rahmani and Yopi


Oil spills are also now becoming a frequent and major source of water and coastal contamination (US Environmental Protection Agency, 1990). Around five million tons of crude oil and refined oil enter the environments each year as a result of anthropogenic source such as oil spills (Hinchee and Kitte 1995). Petroleum is complex mixture of many compounds mainly consisting of carbon and hydrogen which potentially could be eliminated by microbial degradation.



Crude oil constituents are classified into four fractions: saturates (alkane), aromatics, resins and asphaltenes. Each of these fractions contains a large number of compounds. Polycyclic aromatic hydrocarbons (PAHs) are a class of fused-ring aromatic compounds that are ubiquitous environmental pollutants (Fernandez et al. 1999). Microorganisms play an important role in the degradation of PAHs in terrestrial and aquatic ecosystems, and microbial degradation is the main process in natural decontamination. Enhancement of this phenomenon is the basis of bioremediation technologies (Alexander 1999).

PAHs are a group of persistent, toxic, mutagenic and carcinogenic pollutants which pose a significant risk to human health and the environment (Menzie et al. 1992). They are converted to cis-dihydrodiols by bacterial dioxygenase, an enzymatic system consisting of ferredoxin, ferredoxin reductase, and a terminal ring-hydroxylating dioxygenase composed of a and b (Cerniglia 1992). The a subunit of the terminal dioxygenase is more conservative than other components and is thought to be critical for substrate recognition (Kauppi et al. 1998). Evaluating the particular gene expressing this subunit could improve our understanding of the genetics of PAH-degrading bacteria and assist in monitoring active microbial communities during bioremediation (Habe & Omori 2003; Dionisi et al. 2004).

oilbacteria2Several reports about the isolation of PAH-degrading bacteria and the biochemical and genetic analyses of PAH degradation have been cited (Cerniglia 1992). During the research time about diversity bacteria and functional genes in PAHs hydrolysis have conducted in subtropical sea. In the tropical sea environment, especially in Indonesia have been little reported (Widada et al. 2002; Harwati et al. 2007).

We have been reported diversity of oil-degrading bacteria from Muara Kamal Jakarta bay (Yopi et al. 2006). These bacteria have been known as alkane degrader (Thontowi et al. 2008), but have not been known their ability on PAHs degradation. From the bacteria collection expected can be obtained PAHs degrading bacteria. It is important of knowing the ability of PAHs degrading bacteria. In this case for the specific compound in PAHs. Besides that, the applications of dioxygenase enzyme for industry like polyphenylene (Romero et al. 2005), paint (indigo) (O'Connor et al. 1997), as well as an HIV disease inhibitor (Indivanir) (Gibson & Parales 2000; Boyd & Sheldrake 1998) very interestingly.

The aims of this research were selected and characterized PAHs-degrading bacteria, identified of potential PAHs degrading bacteria by partial analysis of 16S rDNA gene, and analyzed the diversity of PAHs-degrading gene from Indonesian sea water bacteria. From the expected targets to be obtained, bacteria and their character in PAHs-degrading activities, detected and data base form diversity of PAHs-degrading gene.

The eighteen bacteria from Indonesia Sea Water were analyzed base on partial sequence 16S rDNA gene, Polyaromatic hydrocarbons (PAHs) degradation ability and analyzed of PAHs dioxygenase gene. Homology analysis of partial 16SrDNA gene from 18 strain PAHs (phenanthrene, dibenzothiopene, fluorene, fluoranthene, phenothiazine and pyrene) degrader were acquired 14 genus bacteria and classified into two groups, a and g-proteobacteria (Fig 2). Phenantrene degradation result was remaining 10.5% after 4 days cultivation by strain 18. Dibenzothiophene degradation result was remaining 22% after 4 days cultivation by strain 5 (Fig 3). The sequences of PAHs dioxygenase gene of strain 5 and 8 showing 98 and 99% respectively identity with naphthalene dioxygenase gene from gene bank (Table 1).





Table 1. Sequence Homology of PAH Dioxygenase

Isolate number

Most similar 16S rDNA sequence

Sequence producing significant alignment



Pseudoalteromonas sp. KT 0812A

naphthalene dioxygenase



Marinobacter hydrocarbonoclasticus

naphthalene dioxygenase




From the results study bacteria and their character in PAHs-degrading activities, detected and data base form diversity of PAHs-degrading gene, we can manage of bioremediation and biomonitoring oil pollution. This research assumed that functional genes encode for specific enzymes in catabolic pathways such as key enzymes in xenobiotic degradation pathways. By assessing the abundance or the expression of key genes in environmental samples one can get a potential measure of the degradation activity. One way of assessing the abundance and expression of specific catabolic genes is by analyzing the metagenomic DNA and RNA from environmental samples.