Biodegradation of Polycyclic Aromatic Hydrocarbon Compound by Bacterial Cultures

: In the present study biodegradation of Polycyclic Aromatic Hydrocarbon (PAH) compound Naphthalene by four bacterial cultures Bacillus subtilis PD6, Bacillussp. PD9, Enterobactersp. PD11 and Bacillussp. PD14 has been targeted. Biodegradation of Naphthalene by these four selected bacterial cultures was analysed by HPLC (High Performance Liquid Chromatography) technique. HPLC analysis revealed biodegradation of naphthalene by all the four bacterial cultures within a span of six days. Highest biodegradation 78.1% has been shown by Bacillus subtilis PD6 while other bacterial cultures Bacillus sp. PD9 has shown 77.90%, Enterobacter sp. PD11 showed 74.4% and Bacillus sp. PD14 exhibited 73.5% biodegradation of naphthalene.

PAHs in combination with global transport phenomena result in their worldwide distribution. Hence, the need to develop practical bioremediation strategies for heavily impacted sites is evident ( Fantroussi et al., 2005). PAH concentrations in the environment vary widely, depending on the proximity of the contaminated site to the production source, the level of industrial development, and the mode(s) of PAH transport. Soil and sediment PAH concentrations at contaminated and uncontaminated sites ranging from 1 µg/kg to over 300 g/kg have been reported (Potter,1999;Wilson, 1993).
Naphthalene is released into the environment in complex mixtures of coal tar and coal tar products such as creosote. Many diverse groups of bacteria that degrade naphthaleneare widely distributed in nature (Cerniglia, 1980;Eaton, et al., 1992, Van Hamme et al., 2003. Various types of PAH compounds are released into the environment from different sources such as petroleum products. Crude oil (Chikere et al., 2009), automobile vehicular washing are those sources which contain large number of different PAH compounds present. Toxic effects of these PAH compounds are well known and bring about drastic changes in the gene structure by intercalating between the nucleotide base pairs, leading to cancer.
Over a period of time microorganisms have developed a mechanism to utilize PAH compounds as carbon and energy source. Many different bacteria are known to be capable of degrading, and in many cases, completely mineralizing, various individual xenobiotic compound such as (PAHs) making them good candidate species for site remediation applications. The ability to degrade low molecular-weight PAH compounds, such as naphthalene and phenanthrene, is widespread, and numerous researchers have identified bacteria capable of utilizing these compounds for growth. Growth on PAH containing four fused aromatic rings (e.g. chrysene, fluoranthene, pyrene, benz[a]anthracene) is somewhat more rare, although organisms are known that can utilize each of these as growth substrates (Churchill et al., 1999). Currently, only a few bacterial isolates have been reported to degrade five-ring PAHs (e.g. benzo[a]pyrene); furthermore, this generally occurs through co-metabolism, during growth on simpler substrates (Bogan et al., 2003).

Toxicity of Naphthalene
Naphthalene, also known as naphthalin, is a crystalline, aromatic, white, solid hydrocarbon with formula C10H8 and structure of two fused benzene rings ( Figure 1). It is best known as the traditional, primary ingredient of moth balls. It is volatile, forming an inflammable vapour, and readily sublimes at room temperature, producing its characteristic odour. It's insoluble in water, somewhat soluble in methanol/ethanol, soluble in organic solvents and very soluble in ether, chloroform, or carbon disulfide (Franco, 2009).

Figure 1: Structure of Naphthalene
Naphthalene is a toxic substance and its toxic effects vary from individual to individual (in adults, ingestion of 6 grams has led to significant toxicity or no symptoms at all; in children, absorption occurs rapidly, a reported dose of 2 grams has been fatal) and they act at on both local and a systemic level. The systemic effects of naphthalene occur chiefly in the erythrocytes causing haemolysis, with subsequent blocking of renal tubules by precipitated haemoglobin. Haemolysis is more likely to occur in individuals with a hereditary deficiency of glucose-6-phosphate dehydrogenase, sickle cell anaemia and sickle cell trait. Besides that, Naphthalene has noxious effects on other targets such as the gastroenteric system, the liver (hepatic necrosis may occur), the urinary system, the brain and the eye (cataract). Local effects are not that serious. It's all about the skin (Contact Dermatitis) and the cornea (corneal lesion). Exposure to large amounts of naphthalene may damage or destroy red blood cells. Humans, particularly children, have developed this condition, known as hemolyticanemia, after ingesting mothballs or deodorant blocks containing naphthalene. Symptoms include fatigue, lack of appetite, restlessness, and pale skin. Exposure to large amounts of naphthalene Naphthalene has been classified as possibly carcinogenic to humans and animals (Group 2B) by the International Agency for Research on Cancer (IARC). According to the IARC acute exposure to naphthalene may result in cataracts in humans, rats, rabbits, and mice. It may also result inhemolytic anemia in children and infants after oral or inhalation exposure or after maternal exposure during pregnancy. Under California's Proposition 65, naphthalene is listed as "known to the State to cause cancer"(ATSDR

Genes involved in NaphthaleneCatabolism
It is amazing to observe that a single bacterial species can utilize number of different xenobiotic compounds. Some are in soluble state and some are sparingly soluble or completely insoluble. For the utilization of compounds occurring in different physical state in the soil or water as a contaminant, bacteria have developed various mechanisms to break it down and derive energy from it. The basis of these mechanisms lies in the presence of various catabolic genes on chromosome or catabolic plasmids (Sayler Gary S. et. al.,1990).
The catabolic gene clusters responsible for the degradation of various xenobiotic compounds are located on the bacterial chromosome and catabolic plasmids carried by the bacterial cell (Widadaet. al., 2002).
Large catabolic gene clusters are also found on mobile elements integrated into bacterial chromosomes as genomic islands or conjugative transposons.
Bacterial catabolic plasmids carry various genes that empower their host cells to utilize number of natural and xenobiotic compounds as sole sources of carbon, nitrogen, and energy. Most such plasmids are large (>50 kb) and carry genes for their conjugal transfer to other bacterial strains. Such transfer events across taxa, and subsequent mutations and rearrangements of the catabolic genes, have contributed to the rapid adaptation of bacteria to novel chemical compounds. Recent studies have shown that genes for the degradation of xenobiotic compounds, such as atrazine, haloacetate, and 2,4-dichlorophenoxyacetate, are predominantly carried on the incompatibility group P-1 (IncP-1) plasmids, whereas genes that encode the degradation of natural aromatic hydrocarbons, such as phenol, naphthalene, and toluene/xylenes, are usually found on IncP-2, IncP-7, and IncP-9 plasmids.

Experimentation and Result Degradation of Naphthalene by selected bacterial cultures
Degradation potential of selected four bacterial cultures towards polycyclic aromatic hydrocarbons was assessed as follows.
 To assess the degradation capability of Bacillus subtilis PD6, Bacillus sp. PD9, Enterobacter sp. PD11 and Bacillus sp.
PD14 total 15conical flasks of 150 ml, each containing 100 ml of 0.1X M9 medium were prepared. After autoclaving 25 ppm of naphthalene was added to each flask. Experiment was carried out in triplicate and appropriate controls were included in the experimental setup.
 Each of the four bacterial cultures was inoculated in the 150 ml flask containing 100 ml of 2% nutrient broth. All flasks were incubated at 28 o C at 100 rpm till the development of desired growth. Sufficient amount of growth was observed within 24 h after inoculation. ODs of these cultures were measured spectrophotometrically at 600 nm. Sufficient amount of culture was withdrawn aseptically,washed with 0.1X M9 minimal medium in order to remove the traces of nutrient medium. This bacterial culture was inoculated into the experimental flask at a concentration of 0.02 OD/ml of 0.1X M9 minimal medium containing naphthalene as a sole source of carbon.
 All experimental and control flasks were kept for incubation at 28 o C for 6 days at 120 rpm.  Samples were withdrawn at regular intervals of 24 h from each flask for the HPLC analysis (Perkin Elmer, USA).

High Performance Liquid Chromatography (HPLC)
Biodegradation of naphthalene by Bacillus subtilis PD6,Bacillus sp. PD9, Enterobacter sp. PD11 andBacillus sp. PD14 was analysed by HPLC technique.          In the present study, Enterobacter sp. PD11 belonging to family Enterobacteriaceae was isolated and tested for the PAH degrading capability. Enteric bacteria are mainly inhabitants of gut and PAH degrading capability by these bacterial cultures seem to be unusual. However, though few, but reports are available which show that the enteric bacteria are involved in the utilization of aromatic compounds. Such bacteria include genera Klebsiella, Enterobacter, Escherichia, and Hafnia (Sarmaet al., 2005).

CONCLUSION
Four bacterial cultures Bacillus subtilis PD6, Bacillus sp. PD9, Enterobacter sp. PD11 and Bacillus sp. PD14 with the ability to biodegrade polycyclic aromatic hydrocarbon compounds were successfully assessed for naphthalene degradation. All the four bacterial cultures were found to be efficiently eliminating significant amount of naphthalene within a period of six days. Two bacterial cultures Bacillus sp. PD9 and Bacillus sp. PD14 were found to solubilise naphthalene completely within 24 hrs. Thus four cultures with catabolic potential towards polycyclic aromatic hydrocarbons were isolated and tested successfully.