An Evaluation about the Role of Microorganisms Isolated from the Soil in the Biological Removal of Toxic Petroleum Pollutants

Polycyclic aromatic hydrocarbons (PAHs) such as anthracene, naphthalene, and phenanthren are the most toxic and carcinogenic pollutants which cause severe damages on soil and water resources. In order to remove these materials, biological methods and using potential of local microorganisms of oil contaminated soils are preferred because of cheapness and availability. Existing soil microorganisms use these hydrocarbons as carbon and energy and finally produce water, CO₂, biomass, and harmless material sources. In the present study, the sampling was conducted from different points in soil of Tabriz and oil-polluted soils from oil materials of Tabriz refinery. The samples were cultured microbial in YGM and starch case in agar. About 100 microbial colonies and isolations were obtained. 1000 mg/l of anthracene hydrocarbons were obtained in Muller Hilton broth, and then fixed amounts of these bacteria were added separately. They were treated and incubated by shaker in 130 RPM, at 28˚C for one week. The rate of mentioned hydrocarbons’ destruction was evaluated by the spectrophotometer that determined reliability of primary aromatic compounds by TLC method. Up to 90 anthracene reducing bacteria were isolated that their destroying rates were 3.4-82.6%. Some samples from secondary metabolites of each hydrocarbon which had the most destroying percentage were under GC-Mass analysis in order to be identified. Then, some toxic-free mediatory substances were obtained because of anthracene biological reduction. By improving the growth and proliferation of effective bacteria and identifying obtained metabolites, it can be possible to clear polluted spoils from PAHSs and to produce useful metabolites and other harmless materials in semi-industrial and practical pilots.
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are composed of two or more rings of six members. These rings bind to each other by sharing a pair of carbon atoms among rings. Aromatic hydrocarbons are among oil-pollutants which enter water and soil ecosystems by different sources like petrochemical, industrial and household sewages, oil exploitation, pharmaceutics, colour,  plastic, insecticide. They can transmit to human being directly so cause some problems such as cancer. Anthracene (C₁₄H₁₀) is  toxic and carcinogenic material. Its polluting rate in the air is 0.02 mg/l. The main ways of absorption of Aromatic hydrocarbons in human body are inhalation and skin. The rate of its toxicity depends on its physicochemical compounds, natural toxicity, metabolites, and clinical medicines. The target organs for aromatic compounds are nervous system, liver, kidneys, skin, lungs, mucous membrane of the respiratory tract, and eyes. Crude oil, with 340 products, is one the most important energy source and worldwide economical power. Some bacteria and microorganisms can cause oil material reduction in soil. Bioremediation is one of the main ways for environmental clearance. In this method living creature specially bacteria, fungi and plants are used in order to reduce environmental contaminants as well as to change them into nontoxic compounds.
 Various microorganisms have a role in this process, the most important of which are bacillus- pseudomonas- proteus- cloestridium- staphylococcus- acinetobacter- mycobacterium- rhodococcus- micrococcus. No microorganism, essentially, is able to reduce completely the oil- hydrocarbons to carbon dioxide and water as final products. The aim of the present study is at evaluating the ability of soil-isolated organisms from different regions of Tabriz to reduce phenanthrene, anthracene as a polycyclic aromatic hydrocarbon.
 
Materials and Methods
Soil in different region of Tabriz and outside as well as contaminated soil of Tabriz refinery were sampled in order to isolate effective bacteria. Followings prepare concentrations of 10^-1 to 10^-4 from samples in physiologic serum, 100 µl of concentrations was cultured in a plate containing starch casein agar and incubated at 28°C for a week. Then, grown colonies were fixed according to the incubation condition in yeast glucose malt agar for fortification and purification. 25 mg of pure anthracene  was weighted and added to 25 ml Muller Hilton broth in capped Falcon tubes in sterile condition. Then, a suspension equal to 05-Mac Farland standard was obtained from purred bacteria in Tryptic Soy Broth; then, 0.5 ml of the suspension was added to Falcon tubes. Falcon tubes were fixed in a shaker incubator at 28°C and 130rpm in one week to reduce anthracene by bacteria .
After the mentioned period, contains of Falcon tubes was transferred to a 100 ml decantation funnel in a sterile condition. Organic solvent of toluene was used in order to isolate the remaining anthracene. Two phases were formed by adding 10cc toluene to the funnel with mixing it. Lower phase is consisted of culture medium and bacteria, and upper phase consisted of remained toluene and metabolite . The upper phase was collected in a capped bottle and was kept in a refrigerator at 4°C until the rate of OD is read. In order to evaluate OD at first anthracene λ_max must be determined. For this purpose, various concentration standards in toluene were obtained and their OD was determined using a double beam spectrophotometer (made by Shimadzu, Japan) compared with blank solution and λ_max was determined. Then, the samples of OD were evaluated at this wavelength. Considering the drawn curve, the rate of anthracene reduction in different samples was observed . The percentage of anthracene destruction of bacteria was calculated by the following relationship:          Destruction percentage =
A₁: anthracene absorption before destruction
A₂: anthracene absorption after destruction by microorganism
Results
100 stubs of purred bacteria were obtained in order to culture different soils. 14 microbial stubs associated to the Tabriz refinery with the codes of g₁ to g_14, and 86 isolated stubs are from different regions of Tabriz. 90 bacterial stubs had the ability to reduce and destruct anthracene in in-vitro condition following treatment of the isolated bacteria. λ_max was determined 360nm by measuring anthracene standard OD against blank solution in 250-500nm wavelength. Absorbed spectra by samples in wavelength of 360nm were recorded following the treatment of specific amounts of anthracene with different bacteria and exploitation of remaining anthracene (curve 1). The percentage of anthracene destruction in different microorganisms was obtained by  . Findings suggest that different destruction percentages of 3.4 to 82.6 have primary anthracene.
Discussion
Bioremediation is a natural process by which pollutants are recycled rather than burying. Furthermore, from the public point of view, bioremediation is more desirable and in most of the world organizations are disseminated by this method for remediation of damaged regions using environmental contaminants. One of the best bioremediations is biologic method and using microorganisms. Bacteria have the most importance compared with other microorganisms because of their different reductive enzymes. Considering the results of the present study and other conducted studies, soil bacteria, more or less, have the potential of reduction and destruction of polycyclic aromatic hydrocarbons. In the present study, stable hydrocarbons with high toxicity were used and it was observed that from 100 isolated stubs only 90 bacteria had the destruction power between 3.4 – 82.6% and among them, 12 stubs had over 50% destruction power. In the present study, also, polluted soil bacteria of Tabriz refinery were used that 14 stubs of bacteria were isolated. By evaluating their results about the destruction of hydrocarbons this destructive potential of polluted soil bacteria can easily be understood.
Using micro organisms as an inoculation is the main consideration in purifying the polluted soils with oil substances; because 1. They are able to degrade hydrocarbons, 2. They are durable and adaptable to environment; 3. They are able to compete with endogen microorganisms; 4. Most of them are not pathogen. Therefore, several studies are conducted based on isolation and evaluation of capabilities of environmental microorganisms.
 
Curve 1. absorbed spectra associated with anthracene treated samples.