Journal of Research in Ecology
An International Scientific Research Journal
Effect of isolated bacteria from the contaminated areas in the
decomposition of hydrocarbons and phenotypes of wheat plants
Authors:
Journal of Research in Ecology
Iktifaa Naeem Jassim and
Turki Muften Saad
Institution:
Soil and Water Resource
Department, Agriculture
College, Al-Muthanna
University, Iraq.
Corresponding author:
Iktifaa Naeem Jassim
Web Address:
http://ecologyresearch.info/
documents/EC0603.pdf
Journal of Research
in Ecology
An International
Scientific Research Journal
ISSN No: Print: 2319 –1546; Online: 2319– 1554
Short Communication
Triticum aestivum
ABSTRACT:
This study was conducted to investigate the effect of bacterial strains of
hydrocarbons isolated from the all regions (Dhi Qar and Muthanna) and their effect
on the growth characteristics of wheat with different levels of crude oil. This
experiment was carried out to demonstrate the effect of bacteria isolated from the
areas contaminated with crude oil in the degradation of hydrocarbons at three oil
levels (O0, O1 and O2), after the addition of first isolate (Klebsiella sp) and the second
isolate (Salmonella sp) and mixing of the two isolates (A1, A2 and A3). Three replicates
for each one were maintained and left a section of transaction as a comparison
without the addition of microbes (A0). The Completely Randomized Design (CRD) was
used for the agricultural experiment. All treatments at the oil level (O0) showed a
significant effect on all the studied wheat characteristics such as germination, plant
height, paper area, chlorophyll content and dry weight of the vegetative part. All the
transactions at the oil level (O1) of crude oil and all the studied characteristics of the
wheat plant mentioned above showed that the order is lower than the level of oil (O 0)
and above showed that the level of oil (O2). All transactions at the oil level (O2)
showed a decrease in the oil levels (O0 and O1) for all studied characteristics of wheat.
The isolation associated with the genus (Klebsiella sp) was taken from the Nasiriyah
refinery which is more efficient than the isolation of the genus (Salmonella sp) which
was taken from the Samawah refinery in stimulating the growth of the wheat plant
Ibaa (99). This study concluded with the possibility of reclamation of soil
contaminated with oil and cultivation using the bacteria isolated from the
contaminated soils to reduce the toxicity of hydrocarbons and to improve the
conditions for the growth of the plant which is grown on these soils.
Keywords:
Pollution, Bacteria, Bioremediation, Growth.
Article Citation:
Iktifaa Naeem Jassim and Turki Muften Saad
Effect of isolated bacteria from the contaminated areas in the decomposition of
hydrocarbons and phenotypes of wheat plants Triticum aestivum
Journal of Research in Ecology (2018) 6(2): 1976-1986
Dates:
Received: 21 July 2018 Accepted: 16 August 2018 Published: 18 Sep 2018
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
1976-1986| JRE | 2018 | Vol 6 | No 2
www.ecologyresearch.info

INTRODUCTION
The 20th century was a period for high industrial
dynamics and technological development, industrializa-
tion, wars and expensive use of heavy metals and bio-
logical contaminants on a large scale, which led to many
environmental problems that must be seen with great
seriousness and attention (Dzionek et al., 2016). The
crude oil appeared as the most important source of pol-
lution and an indispensable material for the global eco-
nomic and industrial growth at present, as a major
source of energy. Pesticides are also dangerous contami-
nants. These compounds were used in large quantities,
for long periods of time and for specific areas, which
pose a risk to endemic organisms in the soil and to hu-
mans, because they are toxic to non-target organisms, as
well as metabolites from pesticide decomposition which
are also toxic and have significant pollution. Environ-
mental biodegradation has appeared as a technological
and economic solution for the recovery of oil spills into
the environment. It is an efficient and environmentally
friendly economic solution to treat oil spills on beaches,
opposite the beaches and near the oil fields.
The effect of oil on soils depends on the size
and type of the flowing of oil the amount of soil cover
the annual chapter in which the flow occurred, as the
toxic effects of crude oil hydrocarbons to plants and
microbes in the soil. All microbiology stimulate meta-
bolic reactions and generally contribute to vital activity,
and each organism has a specific place to perform a
certain function in nature (Marco-Urrea et al., 2015).
The rate of biodegradation depends on the physiological
state of micro-organisms that are sensitive to various
environmental factors and the chemical composition of
hydrocarbons. Modern studies were work hard to have
the microbial future designed as destroyers to deal with
many compounds, not only in terms of degradation or
mineralization, and that the successful conversion of
dangerous pollutants into usable molecules can be fur-
ther enhanced by a vital cell or catalyst cell that is a
1977
Jassim and Saad, 2018
desirable starting point for the development of new
technologies under the concept of biodegradation and
bioremediation. In this study, bacterial isolates were
taken from the oil refineries located in Dhi Qar and
Muthanna. The bacteria were isolated, purified and di-
agnosed. The two most efficient isolates were used
through a series of laboratory experiments. Dating back
to the genus (Klebsiella sp), the second isolation is tak-
en from the Samawah refinery and belongs to the genus
(Salmonella sp) and then cleared the soil of sterile
sieves and processed it for the cultivation of wheat
plant, and note the effect of these isolates on the growth
of wheat plant. This study was conducted to achieve the
following objectives: Determining the amount of bio-
degradation of crude oil by microorganisms for study
sites and reclamation of soils which contaminated with
hydrocarbons of oil and effect of isolated bacteria on the
growth of wheat plant in its state of unilateral and syner-
gy.
MATERIALS AND METHODS
Crude oil samples
Crude oil samples were obtained from the Sa-
mawah Refinery and Plastic, sterile and sealed bottles
were used to collect five liter samples.
Soil samples
Soil samples with mixed clay tissues were taken
from the area devoted to agricultural research belonging
to the College of Agriculture, Al-Muthanna University,
and some chemical, physical and alimentary analyses
were carried out .Following standard protocol (Table 1).
Isolation of bacteria from the soil samples
Samples taken from polluted and non-polluted
soils was innoculated on the brain heart infusion broth,
to stimulate and isolate more species of bacteria. It is
incubated for 24 h at a temperature of 37°C. Activated
samples were placed on nutrient agar and MacConkey
agar, and incubated for 24 h at 37°C and secondary im-
plantation was carried out to develop colonies on the
Journal of Research in Ecology (2018) 6(2): 1976-1986
Jassim and Saad, 2018
selective media (Cui et al., 2017).
Purification
For pure colonies of bacterial isolates of hydro-
carbons, individual colonies that were given specific
morphological characteristics were tested, the streaking
method was used for implantation on the surface of se-
lected cultivars of each bacterial species in dishes in
several forms using the loop. Under sterile conditions,
dishes were incubated for 48-24 h until colonies are
obtained. The isolated samples were then kept in the
nutrient broth medium at refrigerator temperature until
it is used (Kachienga and Momba, 2017).
Preparation of the bacterial vaccine and its use as
bio-fertilizers
Two isolates were selected from the hydroxylat-
ed samples viz: Klebsiella sp and Salmonella sp for use
in the field experiment. The bacteria were grown on the
liquid activated plant medium by placing 50 mL of this
medium in a 100 mL conical flask. After sterilization in
an autoclave with the mulch of a modern farm, samples
were inoculated and incubated at 30°C for 2-3 days. To
prepare a sufficient amount of vaccine for the field ex-
periment, conical flasks (250 mL) containing 100 mL of
the liquid activated plant medium was prepared and
after sterilization in the autoclave, each of them was
inoculated by adding one mL of the processed liquid
using sterile pipettes and incubated at a temperature of
30°C for a period 2-3 days. After preparing the liquid
vaccine, it was mixed with the betamus. The grains of
wheat (IBA 99 Strain) was contaminated at the surface
with betamus, and gum arabic was added at a concentra-
tion of 10% to increase the adhesion of bacteria with
wheat seeds. It was left for one and half hour to ensure
contamination of all seeds with the bacteria. Some seeds
were placed without the microbial treatments and served
as a control (Cui et al., 2017).
Pots experiment
Aerobic soil was dried, grinding and sifting was
sieved using a 4 mm diameter sieve. The soil was steri-
Journal of Research in Ecology (2018) 6(2): 1976-1986
lized in the autoclave at temperature 121°C and pressed
with 15 pounds/in2 and left to be cold and contaminated
with crude oil according to the treatments (0, 5 and 10
mL) and placed in the plastic pots, its capacity is 5 Kg
after sterilization with ethyl alcohol. The pots were
planted with sterile wheat seeds and vaccinated with
isolates. The bacteria were selected according to the
above-mentioned treatments, with 15 seeds/pot and then
eased to eight plants after one week of germination and
leaving some of the seeds without vaccine as a compari-
son and without the addition of crude oil. Fertilizers
were added according to the fertilizer recommendation
in the form of urea (46% N) in two phases viz., the first
at agriculture and the second at the elongation phase.
The recommended amount is 150 Kg N. ha-1. Phosphate
fertilizer is also added one batch before three days to
planting and in the form of the fertilizer superphosphate
(21% P). The recommended fertilizer level is 100 Kg.
ha-1. Potassium fertilizer has been added in batch one in
the form of potassium sulphate (41.5% K ) and the rec-
ommended level of fertilizer is 100 Kg.ha -1 (Ali et al.,
2014). The agricultural experiment was designed to
study the effect of inoculation on bacterial isolates of
hydrocarbons, with different levels of crude oil as well
as to study the effect of these isolate after the vaccina-
tion of wheat seeds and left a portion of these seeds
without adding the vaccine to them. Agriculture was
carried out on 6/11/2017 using two factors as given be-
low :
Agricultural experiment factors
The first factor
Crude oil in three different levels and the fol-
lowing treatments were used:
O0 : Without the addition of crude oil.
O1 : 5 mL of crude oil was added.
O2 : 10 mL of crude oil was added.
The second factor
Four different microorganisms were used at the
following treatments levels :
1978
 Jassim and Saad, 2018
A0 : Without adding microbes. Statistical analysis
A1 : Klebsiella sp was isolated and added. The data were statistically analyzed through the
A2 : Salmonella sp was isolated and added agricultural experimentation using The Complete Ran-
A3 : The two isolates viz., Klebsiella sp + Salmonella sp dom Design (CRD) Genstat version 12 (Payne, 2009).
were mixed and added together. The averages were compared using, the Least Signifi-
12 experimental units and three replicates were cant Difference (LSD) at the level Morality 0.05 (Al-
maintained, so that total number of experimental units Rawi and Klaf-Allah, 2000).
were 36 experimental units.
Appearance characteristics of wheat plant (plant RESULTS AND DISCUSSION
measurements) Isolation and diagnosis of Klebsiella sp
Germination The results of the laboratory tests, which includ-
The percentage of germination was calculated ed the biological and morphological tests of the bacteri-
according to the equation: (Aiyadurai, 1956) al colonies, that the isolation from the Nasiriyah refin-
ery was related to the genus Klebsiella sp as indicated
Number of plants growing
Germination rate = × 100 Makki (2013). It was observed that the isolates were
Total number negative chromatism, bony form and rounded ends, sur-
rounded by capsule, non-moving, aerobic and anaerobic
Plant height optional. It gives a negative reaction to the tests of in-
It was calculated from the flowers to the base of dole, methyl red, oxidase and non-producing H2S,
the plant touching the soil surface and taken into consid- which produces the catalase enzyme and is used for
eration for all the three plants in each pot. jackets (Jawetz et al., 2007).
Chlorophyll content Isolation and diagnosis of Salmonella sp
It was done at the flowering stage for three The second isolation selected for the agricultural
plants from each pot with a SPAD-502 m Chinese- experiment is taken from the Samawah Anaerobic Re-
made. The chlorophyll content is estimated in plant pa-
Table 1. Physical, chemical and biological
per quickly and without damage to leaves (Liu et al., characteristics of the soil
2014). S. No Trait Values
2
Paper area (cm ) 1 pH 8.00
The height and width of three plants from each 2 E.C.E (dsm-1) 4.20
3 O.M (g.Kg-1) 11.00
pot, were measured
4 Ca+ (mEq.L-1) 4.31
Paper area= Average length× Average width × Correc- 5 Mg+ (mEq.L-1) 11.40
tion Factor (0.95) (Thomas, 1975). 6 Cl- (mEq.L-1) 41.60
7 SO4= (mEq.L-1) 36.50
The dry weight of the vegetative part (g)
8 CO3= (mEq.L-1) Nil
It was calculated from the average weight of 9 HCO3- (mEq.L-1) 1.37
three plants after drying in the aerobic condition and 10 Soil texture Clay loam
then put in the oven at 62°C until weight stability was 11 Clay (g.Kg-1) 290
12 Silt (g.Kg-1) 470
seen and then weighed completely (stem, leaves and
13 Sand (g.Kg-1) 240
spikes). 14 Total bacterial count (CFU g-1 soil) 0.28×105
Source: (Aiyadurai, 1956)

1979 Journal of Research in Ecology (2018) 6(2): 1976-1986

Jassim and Saad, 2018

Aver-
finery, moved by peripheral luminescent, bronchial, and

78.4

84.2
82.5
87.6
age

-
-
have the ability to alter phenotype and genotype.

46.60±0.23 55.50±0.44 42.20±0.17 53.30±0.48 57.70±0.51 71.10±0.65 86.60±0.44 66.60±0.57 71.10±0.53 88.60±0.44 75.60±0.56 71.10±0.53
65.60±0.37 66.60±0.51 35.50±0.11 82.20±0.66 66.60±0.43 68.80±0.52 82.20±0.51 68.80±0.44 75.00±0.51 93.30±0.51 84.40±0.71 75.00±0.55
42.20±0.19 46.60±0.32 42.10±0.20 84.40±0.54 68.80±0.62 59.90±0.44 91.10±0.32 83.10±0.71 71.00±0.47 91.10±0.32 83.10±0.75 73.30±0.62
73.30±0.21 35.50±0.15 44.40±0.16 77.60±0.46 72.20±0.54 59.90±0.36 86.60±0.15 79.90±0.62 73.30±0.39 100.0±0.15 86.60±0.66 76.40±0.49
Response to surrounding changes, not made up of op-

73.90
2

-
tional aerobic or anaerobic spores, it gave a positive

Crude oil levels %

W4(fourth week)
reaction to the red blood test and had the ability to pro-
duce H2S, react negatively to the indole and Voges–

82.40
1

-
Proskauer tests and do not have the ability to produce
and form gelatin. Dark brown colonies appear to have a

93.20
black status due to H2S production (Makki, 2013).

-
Antimicrobial resistance test
The results of the antimicrobial test showed no

72.60
Table 2. The percentage (%) germination of wheat seeds in the pots
potential adverse effects between the two isolates select-

-
ed in the study (Klebsiella sp and Salmonella sp) after

Crude oil levels %

W3(third week)
48 hours of incubation of isolates on the same petri dish

74.60
and containing the center of the solid nutrient lacquer.

-
The first efficient isolation (Klebsiella sp) and the se-
cond (Salmonella sp) produce antibiotics such as oomy-

86.60
cin, phenazine, carboxylic acid and pyoluteorin, as well

-
as the number of siderophores and all of these substanc-
es, the properties of these bacteria in the biological con-

64.90
2

trol (Al-Rikabi, 2017). There found no inconsistency

-
Crude oil levels %
W2 (second week)

between the two isolates and is an important indicator

that can be relied upon in the biological experiment in

66.30
1

case of mixing, and showing the effect of synergy be-

-
tween bacterial isolates.
Seeds germination (%)
74.30
0

The results of Table 2 indicate that the effect of

bacterial inoculation on selected isolates showed that at
A×O 18.82
41.00

a level of probability at 0.05% in seed germination(%),

2

A3 (synergistic treatment) had the highest germination

Crude oil levels %
W1(first week)

rate (87.6) and percentage increase of 11.7% than the A0

O 9.41
51.00

treatment (without vaccine). A1 gave a germination rate

1

of 84.2%, with percentage increase of 7.3% than the A0.

A2 gave the rate of 82.5%, with percentage increase of
A 10.87
56.90

5.2% (Al-Rikabi, 2017). This is due to the fact that the

0

presence of bacteria has effectively contributed to the

6 LSD 0.05
Average
Isolates

germination of seeds and variation of germination ratio

A0
A1
A2
A3

according to the isolates used and their efficiency, so

that the treatment gave without a bacterial vaccine the
No
-S.

1
2
3
4
5

Journal of Research in Ecology (2018) 6(2): 1976-1986 1980

 Jassim and Saad, 2018

Table 3. The height of wheat plant (cm) at 50% Table 4. Chlorophyll content in wheat plant (SAPD) at
flowering 50% flowering
Crude oil levels % Crude oil levels %
Isolates Isolates
0 1 2 0 1 2 Average
A0 39.40±0.12 22.60±0.12 15.30±0.06 A0 9.50±0.06 2.40±0.01 1.40±0.01 4.40
A1 42.70±0.06 27.60±0.06 17.80±0.04 A1 10.40±0.01 3.20±0.02 1.90±0.01 5.10
A2 43.50±0.14 25.60±0.14 16.50±0.08 A2 9.70±0.02 2.60±0.02 1.70±0.01 4.60
A3 41.50±0.16 28.70±0.16 21.10±0.05 A3 11.40±0.03 3.80±0.01 2.40±0.01 5.80
Average 41.50 26.10 17.60 Average 10.20 3.00 1.80 -
LSD0.05 A 1.70 O 1.40 A×O 2.90 LSD0.05 A 0.20 O 0.20 A×O 0.40 -

lowest reading. The effect of the oil level on the experi-
mental parameters had a significant effect on seeds ger-
mination. The oil level (O0) showed the highest percent-
age of germination (93.25%), with percentage increase
of 13.1% than the oil level O1 and percentage increase
of 26.0% than O2. The absence of hydrocarbons that
may be difficult to decompose and their toxic effect on
plant growth has helped to increase seed germination,
and the presence of bacteria that contribute to the disas-
sembly of crude oil components has reduce these nega-
tive effects of crude oil to some extent (Kachienga and
Momba, 2017).
The effect of the interference among the oil lev-
els and the effect of bacterial inoculation in a level of
probability (0.05%) in seed germination (%). The treat-
ment A3 (synergistic treatment) gave the highest germi-
nation rate to 100% at the oil level O0, while the treat-
ment A0 gave the lowest germination rate of 71.1% at
the oil level O2. This is consistent with what was re-
ferred by Cui et al. (2017).
Plant height (cm)
As shown in Table 3 the effect of bacterial inoc-
ulation led to high morality and increased in a level of
probability (0.05%) in Plant height rate of the local iso-
lates (Klebsiella sp and Salmonella sp) alone or double
on the A0 (without vaccine), (Das and Kumar, 2016).
The isolation A3 (synergistic treatment) gave the highest
rate of plant height of 31.0 cm and percentage increase
of 20.6% compared to the treatment A0 (without vac-
cine) which reached 25.7 cm. This is due to the viability
of these organisms in the case of synergy to increase the
availability of phosphorus through the production of
organic acids and soluble phosphates, which increase
the readiness of phosphorus in soil Hwangbo et al.
(2003) as well as their effectiveness in the production of
growth regulators, indole acetic acid and giberillins
affecting the elongation of the cells and their division .
These mechanisms increase the protein content which is
positively reflected in the rate of plant height (Hasan,
2002; Jat and Shaktwat, 2003).
The results of Table 3 indicate that the increase
in crude oil levels led to a decrease in the rate of plant
height. The oil level O0 gave the highest rate 41.5 cm
with percentage increase of 59.0% from the oil level O 1
and 135.7% from the oil level O2, which recorded the
lowest height of the plant that reached 17.6 cm. The
decrease in plant height by increasing oil levels is due to
the toxic effects of multiple oil hydrocarbons which
effects negatively the rate of plant growth and this is in
line with what was pointed out by Hatem (2007) In the
effect of irrigation with contaminated water hydrocar-
bons provide the availability of nutrient elements. The
effect of the interference between bacterial inoculation
and oil levels on plant height was also shown in the ta-
ble 3. Given the treatment A0 (without adding bacterial
vaccine), rates are lower than the treatments added to
the bacterial vaccine. Treatment A3 recorded the highest
rate of plant height of 43.4 cm at the oil level O0, while
A0 was the lowest (15.3 cm O2). This indicated to the
positive effect of the bacterial vaccine in reducing the
toxicity of hydrocarbon by decomposing and processing
the plant with nutrients elements and according to the

1981 Journal of Research in Ecology (2018) 6(2): 1976-1986

Jassim and Saad, 2018
nature of the hydrocarbon ingredients. The plant roots
usually absorb nutrients with small molecular weights,
whereas it cannot absorb the large molecular weight
substances easily. Also environmental conditions and
plant characteristics such as plant size, root type, etc.
can be a significant factor to facilitate absorption by
roots. And that the enzymes secreted by the roots are
active on an area of 1 mμ from the root region. In this
case the enzymes have the most effective ability to de-
compose and break down oil carbohydrates and thus
benefit the roots from their degradation products in met-
abolic processes within the plant cells (Al-Jader, 2006 ).
The roots are working to provide the root secretions for
microbiology which contains important food compo-
nents for microorganisms such as sugars, enzymes and
nucleic acids. The microorganisms then get the material
which they need through the root and provide the nutri-
ents and organic needs by microorganisms that lead to
support and stimulate large number of microbiology in
the soil and thus increase their effect on the decomposi-
tion of hydrocarbons.
Chlorophyll SPAD content in wheat plant at flower-
ing stage
The results of Table 4 showed significant differ-
ences in the chlorophyll content when inoculation with
isolates Klebsiella sp and Salmonella sp were done.
These synergy between them compared with the non-
addition of the bacterial vaccine (A0). The treatment
was superior with two types of bacterial vaccine A3.
Highest rate is 5.8 SPAD, with the percentage increase
of 31.8% compared to the measurement treatment of A0,
Table 5. The area of leaf of wheat plant (cm2) at 50%
flowering
Crude oil levels %
Isolate
0 1 2 Average
A0 8.14±0.02 3.08±0.02 1.18±0.01 4.31
A1 10.50±0.01 3.62±0.02 2.26±0.02 5.46
A2 9.87±0.02 3.21±0.01 1.50±0.01 4.86
A3 11.80±0.01 4.21±0.01 2.42±0.01 6.14
Average 10.07 3.53 2.42 - Average
LSD0.05 A 0.86 O 0.74 A×O 1.48 - LSD0.05
Journal of Research in Ecology (2018) 6(2): 1976-1986
which gave the lowest rate in chlorophyll content,
which reached 4.4 SPAD, followed by treatment A1 ,
with a rate of 5.1 SPAD, while A2 recorded the lowest
rate of 4.6 SPAD. This may be due to the role of micro-
organisms in the secretion of giberillins, which affect
the production of auxiliary enzymes in the formation of
pigments (Weiss et al., 1990). The reason for the in-
creased chlorophyll content was that microorganisms
had processed enough nitrogen, especially NH4 , as well
as an increase in the root total absorption, which helped
to absorb nutrients elements (Buddhika et al., 2012).
The results of Table 4 also showed that the ef-
fect is uneven when crude oil was added, which resulted
in significant differences at the level of 0.05. The oil
level (O0) with a higher chlorophyll rate from O1 and
O2, had 10.2 SPAD with a percentage increase of 2.4
and 4.46% from O1 and O2 respectively. This confirms
the negative effect of crude oil on plant growth. The
second oil level at O1 with a higher chlorophyll rate
with the average 3.0 spade and a percentage increase of
66.6 than the third levels of oil (O2).
As for the effect of the interaction between the
bacterial vaccine and the oil levels, the results of the
table showed significant differences in the content of
chlorophyll. The treatment of vaccination was given in
isolation A3 at the oil level O0. Highest rate of chloro-
phyll content observed was 11.4, while A0 (without vac-
cine) recorded the lowest rate of average chlorophyll
content at O2 with 1.4 SPAD. The reason for the in-
creased chlorophyll content in the plant may be due to
the presence of microorganisms in the form of a vaccine
Table 6. The dry weight (g.plant-1) at 50%
flowering
Crude oil levels %
Isolate
0 1 2 Average
A0 2.851±0.02 0.539±0.004 0.239±0.001 1.209
A1 3.169±0.02 0564±0.003 0.274±0.002 1.335
A2 3.051±0.01 0.543±0.005 0.256±0.002 1.283
A3 3.615±0.02 0.571±0.002 0.487±0.002 1.557
3.171 0.554 0.314 -
A 0.324 O 0.281 A×O 0.562 -
1982

that helped to provision the nutrients elements, includ-
ing nitrogen, which increased the duration of the repre-
sentation and made it more effective, leading to the
increased chlorophyll content in the leaves
(Vaguseviciene et al., 2012). The low chlorophyll con-
tent with the high levels of crude oil and at non-addition
of bacterial vaccine may be due to the reduction of soil
nitrogen content or that the crude oil inhibited the effec-
tiveness of other bacterial species found in the soil, be-
cause crude oil has toxic components on organisms,
found in the soil, by reducing their numbers and thus
their effectiveness and efficiency in the conversion of
nitrogen to the plant (Golafarin et al., 2017).
2
Area of the leaf of wheat plant when flowering (cm )
The results of Table 5 showed that bacterial
inoculation had a significant effect on the increase of
the paper area of the wheat plant compared to the non-
addition of the vaccine A0 (comparison treatment). Bac-
terial infection was recorded with the inoculation of
Klebsiella sp And Salmonella sp and the synergies be-
tween the two isolates are higher than the paper area.
Treatment A3 gave the highest rate of paper area (6.14
2
cm ). The isolation of A3 excelled the rest of isolates
with the ratio of increase (12.4%) compared to A1 isola-
tion which recorded an average of 5.46 cm2, while isola-
2
tion of A2 recorded a rate of 4.86 cm and that is due to
the role of bacterial vaccine in the process of decompo-
sition. The soil and its role in the dismantling of hydro-
carbons added , results in an increase in the food pro-
cessed plant and then increase the paper area and listed
according to the added vaccine and its role (Das and
Kumar (2016) and referred to Abo Noktta and El-Shater
(2011) to demonstrate the role of bacteria in increasing
the nutrient elements availability, including nitrogen,
which plays an important role in the formation of cell
protoplasm and cellular membranes and in the for-
mation of enzymatic enzymes and enzymes such as
NADH, NADPH and vitamins, which are the complex
B group. In some growth regulators, the chlorophyll is
1983
Jassim and Saad, 2018
an important part of the process of photosynthesis and
in some nucleic acids such DNA and RNA.
Table 5 showed that the increase in the oil levels
led to the reduction of the paper area of the plant. The
first oil level (O0) gave the highest rate in the paper area
(10.07 cm2) with the ratio of increase (185.2%) at the oil
level O1 and 447.2% at the oil level O2. It may be due to
the decrease of paper area by increasing the levels of oil
to the type of components of crude oil of Samawah re-
finery and it contains hydrocarbons which vary in the
speed of disintegration according to their chemical com-
position and to the role of bacteria added vaccine in the
processes of degradation of hydrocarbons. It has been
observed that the paper area has increased in case of the
use bacterial vaccine (Al-Rikabi, 2017).
The interference between the bacterial vaccine
and the oil levels added significantly affected the paper
area. The highest rate in paper area when insemination
with isolation A3 and oil level O0 (11.8 cm2) and the
lowest plant height (without vaccine) A0 and oil level
O2. The rate (1.18 cm2) is consistent with the findings of
Varjani and Upasani (2017), which referred to the role
of bioremediation in reducing the negative effects of
spilled hydrocarbons on the soil and the possibility of
reducing their effect or the use of bacteria in reclama-
tion after studying the environmental factors and the
type of oil components and the type of bacteria added
their numbers and their role in the degradation of hydro-
carbons.
Dry weight (g.plant-1) for vegetative part of wheat
plant at (50%) flowering
The results of Table 6 indicate that the vaccina-
tion with local isolates resulted in the significant in-
crease of the average mean dry weight compared with
the measurement of the treatments. Treatment A3 ( Mix
the two isolates) (Klebsiella sp and Salmonella sp) gave
highest dry weight (1.557 g.plant-1) with the increase
(28.7 %) compared to A0 (without vaccine), that record-
ed the lowest rate of dry weight (1.209 g.plant -1). Fol-
Journal of Research in Ecology (2018) 6(2): 1976-1986
Jassim and Saad, 2018
lowed by, treatment A1 , which gave the average 1.335
-1
g.plant and then treatment A2, which recorded an aver-
-1
age 1.283 g.plant . The reason for the dry weight gain
of the vegetative group may be due to increased num-
bers of bacteria added in the form of a vaccine and then
increase of the dismantling of hydrocarbons and the
addition of more nutrients elements, which led to the
increased plant growth and accumulation of dry matter
as well as increasing the number of branches of the
plant that led to the increase the dry weight of the plant.
These results are consistent with (Al-Jader, 2006). The
dry weight of the plant increased with the addition of
the bacterial vaccine, reduce the spread of oil carbohy-
drates by reducing or eliminating the biological effec-
tiveness of contaminants to another area. Carbohydrates
do not need to be broken for getting minimized. Plants
have several ways to reduce the spread of contaminants.
Plant roots work to attract oil carbohydrates to its root
area as a result of its absorption of water, so it acts as an
organic pump to attract organic carbohydrates and iso-
late them from the rest of the soil is not contaminated,
and the root is actually indirect in reducing the spread of
carbohydrates when the root provides for carbohydrate
enzymes. Latter is associated with carbohydrates in the
soil and is known as humus, and the plant benefits from
the breakdown of carbohydrates and the production of
emulsified substances as a source of carbon and nitro-
gen in the form of amino acids polymerized by the en-
zyme of peroxidase which is produced by roots, and the
root can polymerize the oil (Al-Jader, 2006).
Also the results of Table 6 showed that the dry
weight of the vegetative group was reduced by the high
levels of oil. Oil level O0 gave the highest rate of 3.171
g.plant-1 with the ratio of increase 909.8% from the oil
-1
level O2, which gave the lowest rate 0.314 g.plant . The
level of oil O1 gave higher than the level of oil O2 which
-1
recorded an average of 0.554 g.plant on oil level ( O2).
The reason behind the low proportion of dry matter in
the plant at increasing the levels of oil may be because
Journal of Research in Ecology (2018) 6(2): 1976-1986
of the components of crude oil also it contains difficult
decomposition compounds that has caused inhibition of
the activity of bacteria because of its toxic effect on the
plant and then reduced plant growth and reduced dry
weight, or non-disintegration of the carbon that has
completely reduced the food additive, affecting plant
growth and thus the dry weight or that the non-
disintegration of hydrocarbons completely reduced the
added nutrient elements, which affected the growth of
the plant and therefore on dry weight. Crude oil has a
negative impact on the vital activities of the plant,
which affect the plant hormones and enzymatic activi-
ties, which hinder the growth of the plant and reduces
the dry weight of the plant if not completely decom-
posed and this is due to the types of bacteria used and
their molecular structure and the type and components
of crude oil used.
The effect of interference between inoculation
with local isolates and oil levels in the dry weight of the
plant, showed that the results superiority of isolation A3
at the oil level O0 on the rest of the isolates in the dry
weight of the vegetative group, with the rate of 3.615.
While the lowest average dry weight of the vegetative
group was recorded in the measurement treatment of A 0
at the oil level O2, which recorded 0.239 g.plant -1, that
may be the superiority of this isolation to the other iso-
lates ,because this isolation was the synergy between
two isolates that proved their ability to break down hy-
drocarbons. The microbial activity in the decomposition
has doubled due of their presence together. Which
means that this isolation is adaptive to the presence of
crude oil (Mingming et al., 2017).
CONCLUSION
The inability of bacteria isolated from non-
polluted areas to decompose and deteriorate crude oil.
Differences in the efficiency and species of bacteria
isolated from oil-contaminated areas in the decomposi-
tion of the oil hydrocarbons degradation. The isolates
1984

(Klebsiella sp and Salmonella sp) showed more efficacy
than other isolates. The use of these bacteria has a sig-
nificant effect (0.05) on all wheat characteristics at
flowering, when added without the presence of oil .The
use of synergy between two efficient bacterial isolates
has a significant effect on the growth of wheat plant.
REFERENCES
Abo Noktta F and El-Shater MS. 2011. Soil Fertiliza-
tion and Fertilization. Theoretical Part Publications of
Damascus University, Faculty of Agriculture.
Aiyadurai SG. 1956. Observations on germination of
dry seed coconuts. Madras Agricultural Journal, 43(9):
464-466.
Ali NS, Salman H and Abdul Razzaq AW. 2014. Soil
fertility, Dar al-Kuttab al-Alami for printing and pub-
lishing.
Al-Jader BMS. 2006. Effect of fuel oil on the activity
of risobium bacteria, growth and yield of beans and
resistance to antibiotics, Master Thesis, Faculty of Agri-
culture - University of Baghdad. Soil Section.
Al-Rawi KhM and Kalaf Allah AAM. 2000. Design
and analysis of agricultural experiments. Faculty of Ag-
riculture, University of Al Mosul. 2nd ed. Ministry of
Higher Education and Scientific Research. Iraq.
Al-Rikabi SJ. 2017. Effect of bacterial fungal mem-
brane as fertilizer in growth and yield of wheat
(Triticum aestivum L.), PhD thesis, Faculty of Agricul-
ture, University of Baghdad.
Buddhika UVA, Seneviratne G and Abayasekara
CL. 2012. Biofilmed biofertilizers for maize (Zea mays
L.): effect on plant growth under reduced doses of
chemical fertilizers. Proceedings of the abstracts of Jaf-
fina University International Research Conference
(JUICE-2012).
1985
Jassim and Saad, 2018
Cui Z, Zhang X, Yang H and Sun L. 2017. Bioreme-
diation of heavy metal pollution utilizing composite
microbial agent of Mucor circinelloides, Actinomucor
sp and Mortierella sp. Journal of Environmental
Chemical Engineering, 5(4): 3616-2621.
Das AJ and Kumar R. 2016. Bioremediation of petro-
leum contaminated soil to combat toxicity on Withania
somnifera through seed priming with biosurfactant pro-
ducing plant growth promoting rhizobacteria. Journal of
Environmental Management, 174: 79-86.
Dzionek A, Wojcieszynska W and Guzik UD. 2016.
Natural carriers in bioremediation: a review. Electronic
Journal of Biotechnology, 23: 28-36.
Golafarin C, Abbas A, Mohammad M and
Mohmmad D. 2017. Bioremediation capability and
characterization of bacteria isolated from petroleum
contaminated soils in Iran. Sustainable Environment
Research, 27(4): 195-202.
Hasan HA. 2002. Gibberellin and auxin-indole produc-
tion by plant root-fungi and their biosynthesis under
salinity-calacium interaction. Acta Microbiologica et
Immunologica Hungarica, 49(1): 105-118.
Hatem ZAH. 2007. Effect of Irrigation on Water Con-
taminated with Hydrocarbons in Preparedness of N, P
and K in Soils and Growth of Maize Plant, MA Thesis,
Faculty of Agriculture, Basrah University.
Jat BL and Shaktwat MS. 2003. Effect of residual
phosphorus, sulphur and biofertilizers on productivity,
economics and nutrient content of pearl millet
(Pennisetum glacum L.) in fenugreek (Trigonella
Foenum-Gracceum L.) pear millet cropping sequence.
Indian Journal of Agricultural Science, 73(3):134-137.
Jawetz E, Melnick JL and Adelberge EA. 2007. Med-
ical Microbiology 24th ed. Churchill Living
stone .London.
Journal of Research in Ecology (2018) 6(2): 1976-1986
Jassim and Saad, 2018
Kachienga L and Momba MNB. 2017. Kinetics of
petroleum oil biodegradiation by a consortium of three
protozoau isolates (Aspidisca sp, Trachelophyllum sp
and Peranema sp). Biotechnology Reports, 15: 125-131.
Liu B, Yue YM, Li R, Shen WJ and Wang KL. 2014.
Plant leaf chlorophyll content retrieval based on a field
imaging spectroscopy system. Sensors, 14: 19910-
19925.
Makki MA. 2013. Bacteriological study of Lake Sawa
water and coastal culture. Master degree, Faculty of
Agriculture, Plant Production, Al-Muthanna University.
tion using rhamnolipid as a model biosurfactant.
Bioresource Technology, 232: 389-397.
Weiss D, Van Tunen AJ, Halevy AH, Mol JN and
Gerats AG. 1990. Stamens and gibberellic acid in the
regulation of flavonoid gene expression in the corolla of
petunia hybrid. Plant Physiology, 94(2): 511-515.
Hwangbo H, Park RD, Kim YW, Rim YS, Park
KH, Kim TH, Suh JS and Kim KY. 2003.
2-Ketogluconic acid production and phosphate solubili-
zation by enterobacter intermedium. Current
Microbiology, 47(2): 87-92.

Marco-Urrea E, Garcia-Romera I and Aranda E.

2015. Potential of non-ligninolytic fungi in bioremedia-
tion of chlorinated and polycyclic aromatic hydrocar-
bons. New Biotechnology, 32(6): 620-628.

Mingming S, Kuan L, Yuanchao Z, Da T, Nao Y,

Manqiang L, Jiaguo J and Xin J. 2017. Effect of bac-
terial-feeding nematodes grazing and tea saponin addi-
tion on the enhanced bioremediation of pyrene-
contaminated soil using polycyclic aromatic hydrocar-
bon-degrading bacterial strain. Pedosphere, 27(6): 1062
-1072.

Payne RW. 2009. "Genstat". Wiley Interdisciplinary

Reviews: Computational Statistics, 1(2): 255–258.

Thomas H. 1975. The growth response of weather of

simulated vegetative swards of single genotype of Loli-
um perenne. The Journal of Agricultural Sciences, 84 Submit your articles online at ecologyresearch.info
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