Agricultural

Communication

Biosci. Biotech. Res. Comm. 9(3): 399-405 (2016)

Biodegradation of naphthalene, phenanthrene and

anthracene (PAHs) with bacteria in the oily soil of Tabriz

Kh. Sadighbayan,

* M. Mazaheri Assadi

, A. Farazmand

, A. R. Monadi

and

N. Aliasgharzad

*Student of Ph D, Department of Biotechnology, Iranian Research Organization for Science and Technology,

Tehran, Iran

Professor, Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran

Assistant Professor, Department of Biotechnology, Iranian Research Organization for Science and

Technology, Tehran, Iran

Associate Professor, Department of Microbiology, College of Medicine, Tehran Medicine University, Tehran, Iran

Professor, Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

ABSTRACT

Most of polycyclic aromatic hydrocarbons (PAHs) are highly toxic, mutagenic, carcinogenic and teratogenic in nature

and exposure to these PAHs represents public health risks and raises environmental concerns. Signi cant interest in the

potential for bioremediation of PAHs on the contaminated sites has resulted in considerable progress in understand-

ing biodegradation of PAHs and degradation pathways. Much of this research has focused on individual compounds,

whereas on contaminated sites PAHs usually occur as mixtures of compounds. Four triplicate batch tests were used

to establish the conditions for maximum degradation of naphthalene, phenanthrene and anthracene by changing the

following four factors one at a time: temperature, pH, reaction time and pollutant concentrations. The optimal reactor

conditions for the degradation of PAH, at the concentration of 1000 mg/L, were found to be 28ºC in pH= 7, under the

reaction time of 7 days. The degradation of PAHs by these bacteria was con rmed by GC-MS analysis and UV- spectros-

copy. These bacteria were capable of degrading a mixture of PAHs with low and high molecular weight and degradation

ef ciency was found tobe 57.1% for naphthalene, 82.1 % for phenanthrene and 55.2 % for anthracene at the end of 7

days. Fourteen types of bacteria were isolated and in 1000 mg/L of PAH destruction level, they achieved 3.3-82.1% of

these compounds. Therefore this method is capable of removing PAHs from contaminated oily soil.

KEY WORDS: BIODEGRADATION, PAHS, POLLUTANT, DEGRADATION, OILY SOIL

399

ARTICLE INFORMATION:

*Corresponding Author: sadighbayan@yahoo.com

Received 27

July, 2016

Accepted after revision 10

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

400 BIODEGRADATION OF NAPHTHALENE, PHENANTHRENE AND ANTHRACENE (PAHS) WITH BACTERIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Sadighbayan et al.

INTRODUCTION

Nowadays, Petroleum products are one of the most

widely used chemicals in society. With the massive

quantity of fuel required to power automobiles and heat

homes, and the number of times each gallon of petro-

leum is stored, transported or transferred, accidents and

leakages are unavoidable (Andrea et al., 2001). Petro-

leum contamination results from leaking aboveground

and underground storage tanks, spillage during trans-

port of petroleum products, abandoned manufactured

gasoline sites, other unplanned releases, and current

industrial processes (Gilchrist et al., 1998). As petroleum

contains hazardous chemicals such as benzene, toluene,

naphthalene, anthracene and phenanthrene (PAHs), this

contamination can be hazardous to the health of plants,

animals, and humans. PAHs are a group of chemicals

consisting of carbon and hydrogen, arranged in the

form of two or more aromatic rings. The metabolism

of PAHS in human body produces epoxide compounds

with mutagenic and carcinogenic properties and in some

cases, lung, intestinal, liver, pancreas and skin cancers

have been reported (Zhang et al., 2004).

Release of PAHs into the environment can lead to the

contamination of the natural resources. Their complex

molecular structure and low solubility in water, limit the

application of conventional remedial techniques (Abd El

Latif et al., 2009). Iran has the 9% of the world’s petrol

sources. Persian Golf and southern parts of Iran have

been polluted by160000 tons of petrol and petroleum

substances for years (Lakzian., 2007). In 1991, more than

5 tons of crude oil were spilled into Persian Golf waters

and caused the death of plants and sea side ecosystem of

the Persian Golf (Koochekzadeh., 2009). In Jan. 2010 an

oil well in Maroon of Khuzestan started to leak and more

than 20000 barrels of crude oil were spilled into the area

100 hectares of the surrounding area were contaminated.

But unfortunately there’s no accurate information about

these polluted areas (Koochekzadeh, 2009).

Bioremediation is a strategy for the removal of PAHs

because some microorganisms can metabolize PAHs to

inert substances, CO

and water. Microbial PAH degra-

dation occurs through successive oxidations catalyzed

by mono and dioxygenase enzymes that require iron as

cofactor. Bio- surfactant production by PAHs- degrading

bacteria may enhance the bioavailability of PAHs in the

environment (Samanta et al., 2002).

Although the aromatic hydrocarbons are generally

more resistant to biodegradation, some aromatic hydro-

carbons with low-molecular- weight such as naphtha-

lene, may actually be oxidized before they are saturated.

Mono aromatic hydrocarbons are toxic to some microor-

ganisms due to their solvent action on cell membranes,

but in low concentrations they are easily biodegrad-

able under aerobic conditions (Eder et al., 2006). PAHs

with 2–4 rings are less toxic and biodegradable at the

rates that decrease with the level of complexity. PAHs

with  ve or more rings can only be degraded through

co- metabolism, in which microorganisms fortuitously

transform non-growth substrates while metabolizing

simpler hydrocarbons or other primary substrates in the

oil (Jussara et al., 2015).

Alkylated aromatic are compounds degraded less

faster than their parent compounds; the more highly

alkylated groups are degraded less faster than less

alkylated ones. The metabolic pathways for the biodeg-

radation of aromatic compounds have been the subject of

extensive study (Li et al., 2006). The bacterial gradation

of aromatic compounds normally involves the formation

of a diol, followed by ring cleavage and formation of a

dicarboxylic acid. Fungi and other eukaryotes normally

oxidize aromatic compounds using mono-oxygenases

and forming trans-diols (Chanieau et al., 2005).

MATERIAL AND METHODS

MIXED BACTERIAL CULTURE PREPARATION

Different regions of contaminated soil in Tabriz re n-

ery were sampled in order to isolate effective bacteria.

After digging cavities of a depth of 30 cm in each spe-

ci c area, approximately 400 g soil was sampled. Each

sample was poured in an unclosed plastic bag and then

transferred to the laboratory. Information about sample

including sampling location, altitude and latitude was

attached on each packet (Curl et al., 2007).

After preparing 10

-1

to 10

-4

concentrations from sam-

ples in physiologic serum, 100 μl of concentrations were

cultured in a plate containing starch casein agar and

incubated at 28°C for one week. Strains of g

, g

and g

(from Tabriz Re nery soil) were already isolated

by enrichment culture technique on separate nutrient

agar plates (Glucose, Malt extract and yeast extract agar)

and incubated at 28 ºC for 24 h. Each colony of these

microorganisms was transferred by TSB broth in order

to prepare 0.5 Mac farland suspension (Burges., 2006;

Bardi et al., 2000). They were put separately into Erlen-

meyer  asks (250 ml) containing 100 ml of the Muller

Hinton broth, 10 mg of PAHs and 2ml of bacteria sus-

pension and incubated for 7 days at 28 ºC on an orbital

shaker at 100 rpm, then culture medium was centrifuged

for 15 min. PAH residues were separated with 5ml tolu-

ene and decantation (Bardi et al., 2000).

SPECTROSCOPIC ANALYSIS

Cultured  uid without algae incubated under the same

conditions since the test specimens were used as the

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIODEGRADATION OF NAPHTHALENE, PHENANTHRENE AND ANTHRACENE (PAHS) WITH BACTERIA 401

Sadighbayan et al.

Table 1: Percentage of degradation of different PAHs by

different bacteria

Degradation %

PAHs Day g1 g2 g3 g4 g5

Naphthalene 7 41.4 35.3 30.1 24.1 57.1

Phenanthrene 7 23.4 29.1 33.3 30.3 82.1

Anthracene 7 28.6 29.2 6.2 12.6 55.2

control. The percentage of PAH destruction by bacteria

was calculated by the following relationship:

Destruction percentage =

100

: hydrocarbon absorption before destruction

: hydrocarbon absorption after destruction by

microorganism

GC MASS ANALYSIS FOR DETERMINATION OF

METABOLITES

The GC mass analysis was preformed on Shimadzu GC

2010 equipped with spilt (rate: 100) as an injection mode

in order to determine the metabolites resulted from bac-

terial degradation. The CPED1-M25-025 column was

used and the length of column was 24.9 m. Temperature

within column which was 8°C for 9 minutes, increased

by 150°C. The maximum temperature was 325°C (tem-

perature program begins from 80°C) and system condi-

tions were as follows:

Hold time = 1 min, Detection system FID= 310°C,

Injection system STL= 300°C (Kishore et al., 2007). The

wavelengths of PAHs were as follows, Naphthalen (288

nm), Phenanthrene (296 nm), and Anthracene (360 nm).

Three complete metabolites from most biologi-

cal destructions were identi ed by GC-Mass (Farina-

zleen., 2004). The  ndings of this research reveal that

by improving and optimizing the conditions, having

effective bacteria, analyzing and identifying produced

metabolites from destruction processes and employing

a pilot study, the recti cation of contaminated soil by

PAHs and production of useful metabolites such as dif-

ferent acids, alcohols and other harmless substances can

be carried out.

RESULTS AND DISCUSSION

The present results showed that different viable species

belonging to bacteria possess varied activities in bio-

logical biodegradation of a variety of PAHs depending

on the type of the aromatics, its concentration and the

bacteria species. Table 1 and Fig. 1-3 indicate biodegra-

dation of Naphthalene, Phenanthrene and Anthracene

by g

, g

and g

and exhibit ability to degradate of

10 ppm PHAs after 7 days.

Gas chromatography analysis of the PAHs (Fig. 4-6)

suggest that the high biodegradation activity in 7 days,

which could be a consequence of synergistic interac-

tions among different strains composing the bacteria,

allowed degradation of aromatic compounds with high

molecular weight.

In order to study, bacteria were isolated from farm-

land sludge of Tabriz Re nery which had potential deg-

radation mechanisms. The bacteria with codes of g

, g

and g

increased biodegradation rate of the PAHs

from oily soil. Inoculation of the oil-degrading bacteria

complimented oil degradation and thus enhanced bio-

degradation of PAHs. The synergetic interaction among

the oil degrading bacteria degraded higher amount of

the polycyclic aromatic hydrocarbons. The bacteria iso-

lated from different locations can be used to formulate

bacterial consortia. The purpose of the present study was

to assess the ability of an indigenous amount of bacteria

from a petroleum re nery waste site to degrade PAHs

and to emulsify other hydrocarbon substrates.

Bioremediation is a natural process by which pollut-

ants are recycled rather being buried. Furthermore, from

the public point of view, bioremediation is more desir-

able and most of the world organizations disseminate

this method to correct and improve damaged regions

by removing environmental contaminants (Kishore et

al., 2007). One of the best bioremediations are biologic

methods and the use of microorganisms. Bacteria are

of the greatest importance compared with other micro-

organisms because of their different reductive enzymes

(Chanieau et al., 2005). Considering the results of the

present study and conducted studies, soil bacteria, more

or less, have the potential of reduction and destruction

of polycyclic aromatic hydrocarbons.

In the study conducted by our colleagues on reduc-

tive microorganisms of PAHs, they succeeded to identify

a yeast (AH70) isolated from oil-polluted soils which

was determined as a 100% alkaline sequence homo-

logue in determining genomic sequence of 26S rRNA.

This organism is able to destruct Naphthalene at the rate

of 89.76%, Phenanthren at the rate of 77.21%, Pyrene

at the rate of 60.77%, and Benzopyrene at the rate of

55.53% during 10 days (Abd El Latif et al., 2009).

Andrea R.Clement et al. reported that two yeast spe-

cies are identi ed among studied soil microorganisms

that the species 984 was able to destruct Anthracene

at the rate of 64%±10 and the species 870 was able to

destruct Naphthalene at the rate of 69%±10 (Andrea et

al., 2001).

C.H.Chaineau et al. in their research titled “The effects

of nutritions on crude oil biological destruction by soil

402 BIODEGRADATION OF NAPHTHALENE, PHENANTHRENE AND ANTHRACENE (PAHS) WITH BACTERIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Sadighbayan et al.

FIGURE 1. Absorption spectra of biodegradation of Naphthalene

FIGURE 2. Absorption spectra of biodegradation of Phenanthrene

FIGURE 3. Absorption spectra of biodegradation of Anthracene

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIODEGRADATION OF NAPHTHALENE, PHENANTHRENE AND ANTHRACENE (PAHS) WITH BACTERIA 403

Sadighbayan et al.

FIGURE 4. Gas chromatography analysis for biodegradation of Naphthalene

FIGURE 5. Gas chromatography analysis for biodegradation of Phenanthrene

Sadighbayan et al.

404 BIODEGRADATION OF NAPHTHALENE, PHENANTHRENE AND ANTHRACENE (PAHS) WITH BACTERIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

microbial population in a farm soil”, increased the rate

of these materials’ reduction from 47% to 62% (Cha-

nieau et al., 2005).

Zhang.H.Kallimanis et al. demonstrated that a species

of Pseudomonas was able to solve 35 mg/l Phenanthrene

at the presence of surfactant produced by that bacte-

rium and  nally caused the destruction of Phenanthrene

(Zhang et al., 2004).

In the present study, isolation of some of soil bacte-

ria from polluted soil of Tabriz re nery was conducted;

then, treatment was done and  nally the destruction of

hydrocarbons PAHs by these microorganisms was per-

formed, and different percentages of destruction were

observed and reported. Considering the results and

 ndings of the present study, isolated bacteria from

soil have the potential to reduce the oil hydrocarbons

invitro. Rich farmlands which have signi cant resources

of phosphorous, nitrogen and sulfur, also con rm this

 nding.

Bhattacharya et al. isolated 150 stubs of oil-hydro-

carbons destructive bacteria from India oil-polluted soils

and demonstrated that Ps.citronellolis are dominant

considering the destructive ability of aromatic and ali-

phatic compounds (Bhattacharya et al., 2003).

Barati and Vasudevan in their research titled “using

oil-hydrocarbons by isolating Ps.  uorescence from oil-

polluted soils” demonstrated that the microbial stub had

the signi cant power to destruct short and long-chain

alkalenes (Barathi et al., 2001).

Eder et al. isolated a stub of Pseudomonas from oil-

polluted soil of re nery which had the 72% of destruc-

tion power by producing surfactants (Eder et al., 2006).

Considering the mentioned studies, the present study

has had similar and con rmative results and innovation

aspects due to the use of native and wild microorgan-

isms of Tabriz re nery soils.

ACKNOWLEDGEMENTS

This study was adopted from a research plan which

was supported  nancially by Tabriz Petroleum Re n-

ery. Authors of this essay acknowledge the Research

Vice President of Re nery and Laboratory Technicians

of Chemical Science Faculty of University of Tabriz for

their sincere cooperation.

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