Optimization of Bacterial Biodegradation of Toluene and Phenol Under Different

Journal of Applied Sciences Research, 6(8): 1086-1095, 2010
© 2010, INSInet Publication
Optimization of Bacterial Biodegradation of Toluene and Phenol Under Different
Nutritional and Environmental Conditions
Reda, A. Bayoumi and Ashraf, T.Abul-Hamd
Botany &Microbiology Dept., Faculty of Science (Boys), Al-Azhar University, Cairo,
Egypt. P.O. 11884
Abstract: The widespread release of aromatic hydrocarbons through spillages and leakage from
underground tanks has caused extensive contamination of surface soils and sea and groundwater
environments. Of these hydrocarbons, this study focused on toluene and phenol because of their toxic and
carcinogenic potentials. Eight bacterial isolates (out of 109) utilized toluene and phenol as a sole source
of carbon and energy. Two isolates were selected as the most potent strains. Based on their morphological
and biochemical characterization, the eight bacterial isolates were identified as Micrococcus varians
EPRIS14, Bacillus subtilis-EPRS12, Pseudomonas alcaligens EPRIS11, Bacillus licheniformis
–EPRIS21,Bacillus laterosporus-EPRIS41, Pseudomonas putida-DAF1, Bacillus firmus-EPRIS22 and
Acientobacter sp. EPRIS32. RAPD-PCR used as a supporting tool in the identification of the eight
bacterial isolates. Influence of different nutritional and environmental parameters on the biodegradation
rate of toluene and phenol by Bacillus subtilis-EPRIS12 and Bacillus laterosporus-EPRIS41 were
investigated by measuring the growth rate by optical density and protein content of bacterial cell protein.
Ammonium chloride and diammonium hydrogen phosphate were found to be the best nitrogen and
phosphorous sources for the two bacterial strains, respectively. Concentration of NaCl at 2% was the
optimum salt concentration for both isolates. At pH 7 the two strains exhibited growth rate ranged from
(0.65-0.98) and protein content (0.44 and 0.45 µg/ml) while the optimum temperature was 30ºC at which
the growth ranged from (0.45-0.99) and the protein content of the cell ranged from (0.54-0.89µg/ml). Two
most potent Bacillus strains were suggestive being applied in toluene and phenol biodegradation under all
investigated nutritional and environmental condition in polluted soil and water.
Key words: Toluene, Phenol, RAPD-PCR, Hydrocarbon biodegradation, Bacillus, Pseudomonas,
Acinetobacter and Micrococcus.
INTRODUCTION
Monoaromatic pollutants are now considered as
wide spread contaminants of soil and groundwater.
Phenol is a highly toxic and carcinogenic compound
and its biodegradation is very important to meet the
environmental regulations. The use of microbial
metabolic potential for elimination of environmental
pollutants provides a safe and economic alternative to
their disposal in waste dump sites and to commonly
used physico-chemical strategies. Biodegradation has
been widely used over the last few years.
Bioremediation including biostimulation and
bioaugmentation is being used to enhance natural
biodegradation rates through optimization of limiting
environmental conditions and has been shown to be an
economic, versatile and ecologically acceptable cleanup
approach [12,13,22]. Among all remediation technologies
for treating toluene and phenol contaminated
groundwater and soil. Generally, the main issue of soil
contamination is ground and surface water pollution
which may a adversely affect the use of water for
drinking and technical purposes. Although petroleum is
comprised of a complex mixture of hydrocarbons,
monoaromatic hydrocarbons (toluene and phenol)
account for as much as 90 % of petroleum components
found in the water soluble fractions [31]. In view of
their high water solubility and their acute toxicity and
genotoxicity [10], toluene and phenol components are
classified as priority pollutants by the U.S.
Environmental Protection Agency [36]. Phenolic
compounds and toluene are toxic and undegradable
organic materials
discharged from chemical and
pharmaceutical factories. They are used as a
agrochemicals in farms and also as solvents in factories
and laboratories. Since factories, farmland and faculties
connect closely with water systems, rivers, lakes,
groundwater and seas, the phenol and toluene
Corresponding Author: Dr. Reda Ahmed Bayoumi, Botany &Microbiology Dept., Faculty of Science (Boys), Al-Azhar
University, Cairo, Egypt. P.O. 11884.
E. mail: [email protected].
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J. Appl. Sci. Res., 6(8): 1086-1095, 2010
compounds in the wastewater pollutes the natural water
ecosystem. As the sea is the main water-receiving
ecosystem, the concentration of pollutants in the sea,
especially in coastal seawaters. Furthermore, crude oil
spills, petroleum stations and industrialized zones or
cities contribute to the increase of seawater pollution
by phenolic compounds. Feedstock chemicals are the
basis building blocks that serve as the raw materials
used to synthesize other chemicals, ranging from small
molecules to plastic and rubber, or that are used as
solvents in a variety of industrial processes. The
primary products of petroleum refining such as
ethylene, propylene, benzene, toluene and xylene are
the dominant feedstock chemicals for the chemical
industry. Phenol and toluene are starting materials for
synthesis of numerous important industrial chemicals.
These molecules are produced in huge amounts and are
used in fuels and as solvents and starting materials for
the production of plastics, synthetic fibers and
pesticides [7].
The aim of the present study is to isolate bacterial
isolates that able to degrade toluene and phenol from
contaminated environments by these two aromatic
compounds. This step followed by two features. One of
them is to characterize and identify the most potent
isolates. The second one is to investigate the influence
of different nutritional and environmental parameters
that affect on their biodegradation.
MATERIALS AND METHODS
I-Soil samples: Four crude and drill cutting polluted
soil samples were taken from gasoline, petroleum and
drill cutting storage region in EPRI, El-Hay Eltamin,
Madinet Nasr, Cairo, Egypt. Twenty grams were
collected per each soil sample in plastic bags and
transported immediately in cold storage containers to
laboratory for further work.
II-Hydrocarbons Utilizing Bacteriological Counts:
Drill cutting utilizing bacteria were determined by the
most probable number (MPN) method using tubes
containing 9 ml of a basal mineral salts prepared
according to [20] containing Na2HPO4:2.13 g l-1,
KH2PO4:1.3g l-1, NH4Cl:0.5 g l-1, MgSO4:0.2 g l-1
supplemented with drill cutting 3 ml l-1 and trace
elements solution (1 ml per liter) as described by
Bauchop and Flsiden [5]. All tubes were incubated at
30ºC for 21 days. One tenth ml of the turbid tubes
were inoculated in the previously mentioned mineral
salts medium supplemented with agar, 15.0 g l-1 and
drill cutting (0.2%). The number of viable colonies
were exhibited high growth were selected and reserved
in nutrient agar slants for completed other experiments.
III-Isolation and Identification of Phenol and
Toluene Biodegrading Bacteria: Enrichment and
isolation of drill cutting (DC) utilizing bacterial cultures
were done using mineral salts (MS) medium supplanted
with drill cutting as only source of carbon and energy
sources. Bacterial colonies producing clear zones were
selected and picked up from the plates after 21 days,
further purified by repetitive streaking on nutrient agar
plates. The isolated bacterial cultures were
characterized by morphological, physiological and
biochemical characteristics according to Krieg
[23]
,Sneath [35], Holt [16] and Brenner [6].
IV-DNA Isolation: Total DNA were extracted from 5ml samples of fresh overnight-MS broth cultures. The
final concentration of lysozyme used for cell lysis was
2 mg/ml. The quantity and purity of DNA were
assessed by determining the optical density at 260 and
280 nm, as described by Sambrook et al. [33].The final
DNA concentration of each sample was adjusted to 25
ng/µl. Each PCR reaction mixture contained 1 µl of
DNA, 5µl 10x PCR-buffer (100 mM Tris-HCl, pH 8.8,
500 mM KCl, 0.8% Nonidet P40, 25 mM MgCl2),
10µl dNTPs-mixture at a concentration of 200mM, 2µl
of the primer OPA-01 (5'-CAGGCCCTTC-3'), OPA-11
(5'-CAATCGCCGT-3', Operon Technologies, Alameda,
CA) at a concentration of 2 pmol/µl, 2µl Taqpolymerase (1U/µl, Life Technologies) and enough
sterile bidistilled water to bring the volume to 50µl.
The PCR program comprised 35 cycles of denaturation
for 1 min at 97°C, annealing for 1 min at 35°C, and
extension for 2 min at 72°C. The cycles were preceded
by denaturation at 96°C for 4 min and followed by
extension at 72°C for 5 min (30). PCR products were
separated by electrophoresis at 120 V on a 1.5%
(wt/vol) agarose gel (Gibco BRL, Life Technologies,
Milan, Italy), and the DNA was detected by UV
transillumination after staining with ethidium bromide
(0.5 mg/ml). The molecular sizes of the amplified DNA
fragments were estimated by comparison with a 100bp
ladder DNA (Gibco BRL, Life Technologies).
V- Inoculums Preparation: The bacterial culture(Fresh
culture,12 h old) were inoculated in MS medium
supplemented with 1% DC, 0.1 % phenol and 1%
toluene as each only source of carbon and energy. The
inoculated flasks were incubated under shaking
conditions at 100 rpm at 30ºC for 7 days. The growth
was measured through cell densities by using
spectrophotometer at 600nm.
Vi-Parameters Controlling the Biodegradation of
Toluene and Phenol: The toluene and phenol utilizing
bacterial isolates were selected for investigating the
effect of pH, temperature, different sodium chloride
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J. Appl. Sci. Res., 6(8): 1086-1095, 2010
concentrations and substrates concentrations on the
growth of the most potent bacterial isolates on toluene
and phenol.
Effect of different initial pH values on bacterial
growth in toluene and phenol media: The effect of
initial pH in the growth of the two most potent toluene
and phenol utilizing bacterial isolates of 1% toluene
and phenol was investigated. MS medium supplemented
with 1% toluene or phenol was prepared at pH 6,7,8
and 9 using 1N HCl and 1N NaOH. The inoculated
flasks were incubated under shaking conditions at 100
rpm at 30ºC. The bacterial growth was determined
spectrophotometricaly at 600nm and protein content by
Lowry et al., [26].
Effect of different temperatures on the growth of
toluene and phenol utilizing bacterial isolates: The
effect of different temperatures (20,30,35 and 40 ºC) on
the growth of the two selected toluene and phenol
utilizing bacterial cultures was investigated using MS
medium supplemented with 1% toluene or phenol at
pH 7. The bacterial growth was determined as
previously mentioned.
Effect of different toluene and phenol
concentrations on the growth of two bacterial isolates:
The influence of different concentrations of toluene or
phenol (0.025, 0.050, 0.1, 0.15, 0.2 and 0.25) were
carried out at 30 ºC and pH 7. The bacterial growth
was determined as previously mentioned.
Effect of different nitrogen sources: Sodium nitrate,
ammonium chloride, ammonium monohydrogen
phosphate, ammonium sulphate and potassium nitrate
on the growth of the two bacterial isolates on the
toluene and phenol MS media. The nitrogen sources
were added at equimolecular amount by nitrogen
content located in ammonium chloride (Control). All
flasks were incubated at 30ºC for 7 days at shaking
conditions at 100 rpm. The bacterial growth was
determined as previously mentioned.
Effect of different phosphorous sources: Potassium
dihydrogen phosphate, dipotassium monohydrogen
phosphate, disodium monohydrogen phosphate and
diammonium hydrogen phosphate. The
different
phosphorus sources were added at an equimolecular
phosphorous content located in control. All flasks were
incubated at shaking conditions at 100 rpm at 30ºC for
7 days. The growth was determined at 600nm as
previously mentioned.
Effect of different sodium chloride concentrations:
The different sodium chloride concentrations viz.
2,4,6,8,10 and 12% were added with MS medium
supplemented with toluene or phenol as only carbon
sources. The inoculated flasks were incubated under all
obtained optimal conditions under shaking conditions
for 7 days at 30 ºC. The growth was determined at
600nm as previously mentioned.
RESULTS AND DISCUSSIONS
The isolation procedure resulted in one hundred
and nine pure bacteria cultures able to grow in MS
medium supplemented with drill cutting, toluene and
phenol as only source of carbon and energy sources.
Forty nine, forty three and seventeen bacterial isolates
were obtaining from drill cutting, toluene and phenol
when used as only source of carbon sources
respectively. Eight bacterial isolates were selected on
the basis of morphological characteristics on agar
media, their capabilities to grow on DC, toluene and
phenol as their sole carbon sources and exhibited good
growth as well as forming clear zone on DC. The eight
DC, toluene and phenol utilizing bacterial isolates were
identified on the basis of morphological, physiological
and biochemical characteristics as Micrococcus varians
EPRIS14, Bacillus subtilis-EPRS12, Pseudomonas
alcaligens EPRIS11, Bacillus licheniformis
–EPRIS21,Bacillus laterosporus-EPRIS41, Pseudomonas
putida-DAF1, Bacillus firmus-EPRIS22
and
Acientobacter sp. EPRIS32.
Pattern in figure 1-A (lanes 3 &6) developed eight
DNA identical bands from the RAPD PCR reaction of
their genomic DNA with a similarity matrix (calculated
by Dice coefficient) of 100. RAPD-PCR along with the
biochemical characterization resulted in the
identification of isolates (EPRIS11 and DAF1) as
Pseudomonas alcaligenes and Pseudomonas putida.
However, identical DNA pattern were developed in
case of isolates 26, 72 and 73 except for a single DNA
band which developed in case of isolate 73 at ~530 bp
(fig 1B, lane7) and in case of isolate 72 at ~1400bp
(lane8 fig 1B). This result supported the suggestion of
their conventional identification as Bacillus subtilis,
Bacillus firmus and Bacillus licheniformis, respectively.
Parameters Controlling the Growth and
Biodegradation of Both Toluene and Phenol :- It is
necessary to carry out the influence of different
parameters on the biodegradation rate of toluene and
phenol by most potent bacterial strains viz. Bacillus
sutilis-EPRIS12 and Bacillus laterosporus –EPRIS41.
Six parameters were studied effect of different initial
pH values, temperatures, substrates concentrations,
nitrogen sources, phosphorous sources and sodium
chloride concentrations. All the previously mentioned
parameters were determined by bacterial growth rate
(OD at 600nm) and protein content (Lowry method at
750nm) [26].
1.
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Effect of initial pH values: This experiment was
performed to examine the optimum pH for the two
most potent bacterial isolates at which the highest
degree of toluene and phenol biodegradation can
J. Appl. Sci. Res., 6(8): 1086-1095, 2010
2.
3.
4.
be obtained. It was obvious from the data
presented graphically in figs(2A&2B) that the
optimum pH value for both Bacillus subtilisEPRIS12 and Bacillus laterosporus –EPRIS41 was
7 at which these bacterial strains gave high growth
rate (0.98 and 0.65) and protein content (0.45 and
0.44 µg/ml) on toluene biodegradation respectively.
The optimum initial pH value for both B.subtilisEPRIS12 and B.laterosporous-EPRIS41 was also 7
at which these two bacterial strains gave high
growth rate (0.98 and 0.99) and protein content
(0.85 and 0.32µg/ml) on phenol biodegradation
respectively(Figs 2A&2B). Below and above this
optimal initial pH values, the growth rate and
protein content in both strains decreased gradually.
Effect of different temperatures: This experiment
was performed to examine the optimum
temperature for both bacterial strains at which the
growth and protein content in toluene and phenol
biodegradation. Data presented graphically in
figs(2C&2D) showed that the optimum temperature
for toluene biodegradation by both B.subtilisEPRIS12 and B.laterosporous-EPRIS41 was found
to be 30ºC exerting the highest growth rate
(0.89&0.84) and protein content (0.46& 0.89
µg/ml) in toluene biodegradation respectively. The
optimum temperature for the previously mentioned
both bacterial strains was 30ºC at which the
growth (0.45&0.99) and protein content (0.54 and
0.89µg/ml) in phenol biodegradation respectively
(Figs 2C&2D). Below and above the optimal
temperatures for the growth rate and protein
content for both bacterial strains in toluene and
phenol biodegradation decreased gradually.
Different substrate concentrations: It could be
concluded from the results presented graphically in
figures (2E&2F) the increasing in toluene and
phenol concentrations were decreased gradually the
biodegradation by B.subtilis-EPRIS12 and
B.laterosporous-EPRIS41.
Different nitrogen sources: This experiment was
performed to examine the optimum nitrogen
sources for the toluene and phenol biodegradation
by B.subtilis-EPRIS12 and B.laterosporousEPRIS41. Data presented graphically in
figs(3G&3H) showed that, ammonium chloride,
sodium nitrate, potassium nitrate, ammonium
hydrogen phosphate and ammonium sulphate were
achieved high growth rates(1.89, 1.58, 0.99,092
and 0.76) and protein content (0.65,0.68,0.45,0.79
and0.89 µg/ml) respectively in toluene
biodegradation by B.subtilis-EPRIS12. Data
presented graphically in figs.(2G&2H) showed that,
ammonium chloride, ammonium hydrogen
phosphate, sodium nitrate, ammonium sulphate and
5.
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potassium nitrate were achieved high growth rates
(1.99,1.97,1.88,0.89 and 0.66) and protein content
(0.54,0.58,0.45,0.94 and 0.5 µg/ml) respectively in
toluene biodegradation by B. laterosporousEPRIS41. Data presented graphically in
f i g . ( 3 G & 3 H ) s h o w e d t h a t a mmo n i u m
monohydrogen phosphate, sodium nitrate,
ammonium chloride, sodium nitrate, ammonium
sulphate and potassium nitrate were achieved high
growth rate (0.99,0.95, 0.88,0.76 and 0.66) and
protein content (0.44,0.56,0.65,0.89 and 0.99
µg/ml) respectively in phenol biodegradation by
B.subtilis-EPRIS12. Finally data presented
graphically in figs.(3G&3H) showed that
ammonium chloride, sodium nitrate, ammonium
monohydrogen phosphate, potassium nitrate and
ammonium sulphate were achieved high growth
rate(2.0,1.89,1.65,0.89 and 0.68) and protein
content (0.45,0.54,0.8,0.82 and 0.42 µg/ml)
respectively in phenol biodegradation by B.
laterosporus –EPRIS41.
Different phosphorous sources: This test was
performed to examine the best phosphorous source
at which the maximum biodegradation of both
toluene and phenol could be detected by B.subtilisEPRIS12 and B.laterosporous-EPRIS41. Data
presented graphically in fig.(3I&3J) showed that
diammonium hydrogen phosphate, disodium
hydrogen phosphate, potassium dihydrogen
phosphate and dipotassium monohydrogen
phosphate were achieved high growth rate (2.0,
1.98,1.89 and 1.59) and protein content
(2.1,0.65,0.86 and 1.59 µg/ml) respectively in
toluene biodegradation by B. subtilis–EPRIS12.
Data presented graphically in figs.(3I&3J) showed
that diammonium monohydrogen phosphate,
potassium dihydrogen phosphate, dipotassium
hydrogen phosphate and disodium monohydrogen
phosphate were achieved high growth rate (2.05,
1.99,1.77 and 1.55) and protein content
(0.87,0.7,0.57 and 1.55 µg/ml) respectively in
toluene biodegradation by B. laterosporus
–EPRIS41. Data presented graphically in
figures(3I&3J) showed that diammonium hydrogen
phosphate, disodium hydrogen phosphate,
dipotassium hydrogen phosphate and potassium
dihydrogen phosphate were achieved high growth
rate (2.8,2.3,1.99 and 1.88) and protein content
(0.87,0.7,0.95 and 0.88 µg/ml) respectively in
phenol biodegradation by B. subtilis-EPRIS12.
Data presented graphically in figs. (3I&3J) showed
that diammonium hydrogen phosphate , disodium
hydrogen phosphate, potassium dihydrogen
phosphate and disodium phosphate were achieved
high growth rate(2.01,0.95,0.88 and 0.65) and
J. Appl. Sci. Res., 6(8): 1086-1095, 2010
6.
protein content (0.99,0.87,0.42 and 0.7 µg/ml)
respectively in phenol biodegradation by B.
laterosporus –EPRIS41.
Different sodium chloride concentrations: This
experiment was performed to examine the optimum
sodium chloride concentrations for the two most
potent selected bacterial strains at which the
highest degree of toluene and phenol
biodegradation can be obtained. Results presented
graphically in figure (3K) showed that the growth
rate and protein content were decreased with
sodium chloride concentration increased in all
cases for biodegradation of toluene and phenol by
both bacterial strains viz. B.subtilis-EPRIS12 and
B.laterosporous-EPRIS41.
Discussion: Toluene and phenol are classified as main
pollutants by the US Environmental Protection Agency
because they are common organic contaminants. The
appearance of toluene and phenol compounds in natural
environments is usually associated with the spill or
discharge of petroleum products and synthetic
chemicals in the form of herbicides, pesticides and
industrial effluents [41,27]. Microorganisms such as
bacteria, fungi and microalgae play a key role in
monoaromatic removal through in situ bioremediation
processes [28,34]. Monoaromatic pollutants act as carbon
source for microorganisms. Also, they require macro
nutrients (nitrogen and phosphorous), micronutrients,
electron acceptor and optimum environmental
conditions for growth (temperature, pH, salinity,
presence of inhibitors and of a nitrogen source
[17,25,32,34,37]
. However, little information on bacteria with
a high toluene and phenol tolerance and high
metabolically activity is available. Therefore, there is
still the need to isolate new toluene and phenoldegrading bacteria that can aerobically grow at elevated
concentration.
One hundred and nine pure bacterial culture
capable of growing on and degrading of drill cutting,
toluene and phenol as a sole source of carbon and
energy were isolated from crude oil polluted soil
samples. The most potent bacterial strains were isolated
after serial enrichment with toluene and phenol as a
sole source of carbon and energy. Initial subculture
took longer to degrade toluene and phenol, whereas
subsequent subcultures degraded toluene and phenol
more quickly and had high numbers of the total
previously mentioned compounds degrading colonies
when plated on MSM containing toluene and phenol.
Given that growth of the two Bacillus strains viz.
Bacillus subtilis and Bacillus laterosporus were verified
by demonstrating an increase in bacterial cell biomass
concomitant with a decrease in the tested substrate
concentrations, biodegradation of toluene and phenol
were directly correlated with bacterial growth. In this
study, different controlling the both toluene and phenol
biodegradation by Bacillus subtilis and Bacillus
laterosporus. Results showed that, the optimum pH for
bacterial degradation of toluene and phenol was 7.
Generally, the optimum pH for microbial degradation
in the range 6 to 8. The solubility of phosphorous, an
important nutrient in biological systems, is maximized
at pH 6.5. Results showed that, the optimum
temperature was 30ºC. These results are in agreement
with Das and Mukherjee [8,9] who stated that
temperature affects the rates of microbial metabolism
as well as physical state of hydrocarbons. It also
affects the solubility of the substrate where some of
which undergo solid-liquid phase transitions within the
range of temperature changes in natural ecosystems. Li
et al., [24] found that Planococcus sp. strain ZD22 full
degraded 2mM of benzene at temperature between 8
and 30ºC for 5 days, with an optimal biodegradation
temperature of 20ºC. Biodegradation was faster at 15
and 25ºC that at 30ºC and was observed at a
comparable rate at 15 and 25ºC. Given that mesophilic
and thermophilic microorganisms are severely limited
at or below 10ºC [21]. Biodegradation of benzene,
toluene and xylene by Pseudomonas spp. with
amendments of inorganic nutrients was studied at
temperatures of 20ºC to 28ºC and pH of 7-7.5 in sandy
medium [18,19]. Biodegradation of hydrocarbons declines
with lowering temperatures because of some factors,
such as the change in viscosity and the retention of
potentially toxic light hydrocarbons, which well have
a greater effect than a temperature-mediated drop in
enzyme activity. Finally most hydrocarbon metabolic
pathways were shown to attain its maximum typically
within the range of 30 to 40ºC, above which the
member toxicity by hydrocarbon increases. AbuHamed
et al., [1,2,3,4] observed that the maximum concentrations
of benzene, toluene and phenol consumed by
Pseudomonas putida F1 (ATCC 700007) in singlephase (only aqueous phase) were only 880,880 and 200
mg l-1 respectively. Yang and Lee [39] isolated phenoldegrading strains from enriched mixed cultures,
monitoring the variations of species during the
enrichment period. They were identified as
Pseudomonas resinovorans strain P-1 and Brevibacillus
sp. The optimum growth temperatures for Pseudomonas
resinovorans and Brevibacillus sp. were 31 and 39ºC
respectively. Ying et al., [40] isolated a new phenoldegrading bacterium with high biodegradation and high
tolerance of phenol Acinetobacter sp. strain PD12 from
activated sludge. This strain was capable of removing
500 mg phenol/l in liquid medium by 99.8 % within 9
h and metabolizing phenol at concentration up to 1100
mg/l.
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J. Appl. Sci. Res., 6(8): 1086-1095, 2010
Fig. 1: (A) RAPD-PCR patterns of the eight bacterial strains by OPA-1 primer. Lane M, 100pb ladder (Gibco
BRL). Molecular sizes indicated (right) are in base pairs. lanes 1 to 8, isolates, Bacillus pumilus,
Pseudomonas alcaligenes or nautical, Bacillus subtilis, Micrococcus lylae, Pseudomonas putida Biovar
A or Burcholderia cepacia, Bacillus licheniformis, respectively. (B). RAPD-PCR pattern of the eight
strains by OPA-10 primer.
(A)
(B)
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J. Appl. Sci. Res., 6(8): 1086-1095, 2010
(C)
(D)
(E)
(F)
Fig. 2: Role of different pH values(A&B), temperatures(C&D) and substrate concentrations (E&F) on the toluene
and phenol bacterial degradation by B.subtilis-EPRIS12 and B.laterosporous –EPRIS41.
(G)
(H)
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J. Appl. Sci. Res., 6(8): 1086-1095, 2010
(I)
(J)
(K)
Fig. 3: Role of different nitrogen(G&H), phosphorous sources(I&J) and sodium chloride concentrations (K) on
the toluene and phenol bacterial degradation by B.subtilis-EPRIS12 and B.laterosporous –EPRIS41.
The experiments on toluene and phenol
biodegradation by Bacillus subtilis and Bacillus
laterosporus were performed at different nutrient levels.
The results indicate that an increase in the
concentration of nutrients would promote both toluene
and phenol biodegradation. Significant biomass
increases were observed with an increase in the
concentrations of phosphate and sulphate. Comparison
of experimental results show that toluene appears to be
the most biodegradation followed by phenol. Jean et
al., [19] found that the biodegradation of benzene,
toluene and xylene (BTX) by Pseudomonas spp. in a
laboratory porous media sand aquifer model. They also
found that the increase of nutrient levels resulted in
enhanced bacterial growth and BTX degradation. The
addition of nutrients (N/P) might be effective in
increasing the biodegradation of organic compounds
because these amendments effectively
stimulate
bacterial growth [8,9,15,29,38]. The degradation of toluene
could be enhanced when nitrate is amended as an
electron acceptor [11,14]. There is considerable interest to
assess the effects of different inorganic nutrients on
toluene and phenol biodegradation by microorganisms.
To determine the capacity of two bacterial strains to
degrade of toluene and phenol in media with different
salinities, a series of tests using salt concentrations
ranging from 0 to 10 % were performed. No
degradation occurred at NaCl concentrations above 10
%.
Conclusion: This study investigated the biodegradation
of toluene and phenol represent a monoaromatic
compounds by two Bacillus subtilis and Bacillus
laterosporus and the following conclusions were drawn:
[1]
Both toluene and phenol degrading bacterial strains
were high biodegradation activity and high tolerance of
toluene and phenol, Bacillus subtilis and Bacillus
laterosporus were isolated from crude oil polluted
soil samples. These strains were capable of utilizing
both toluene and phenol at concentrations up to
0.1/100ml(vol/vol) or 0.1/100ml (wt/vol) respectively.
The optimal culture conditions for biodegradation
by two bacterial strains were found that: pH, 7;
temperature, 30°C ; Ammonium chloride
and
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J. Appl. Sci. Res., 6(8): 1086-1095, 2010
diammonium hydrogen phosphate were found to be the
best nitrogen and phosphorous sources for the two
bacterial strains and sodium chloride, 2%.
REFEERNCES
1.
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2. AbuHamed, T., E. Bayraktar, T. Mehmetoglu and
Mehmetoglu, U. (2004a): Kinetics model for
growth of Pseudomonas putida F1 during benzene,
toluene and phenol biodegradation. Process
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