Journal of Research in Ecology
Journal of Research in Ecology
ISSN No: Print: 2319 –1546; Online: 2319– 1554
An International Scientific Research Journal
Short Communication
Effect of isolated bacteria from the contaminated areas in the decomposition of hydrocarbons and phenotypes of wheat plants
Triticum aestivum Authors: Iktifaa Naeem Jassim and Turki Muften Saad Institution: Soil and Water Resource Department, Agriculture College, Al-Muthanna University, Iraq. Corresponding author: Iktifaa Naeem Jassim
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 Web Address: http://ecologyresearch.info/ documents/EC0603.pdf Journal of Research in Ecology An International Scientific Research Journal
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
Jassim and Saad, 2018 INTRODUCTION
desirable starting point for the development of new
The 20th century was a period for high industrial
technologies under the concept of biodegradation and
dynamics and technological development, industrializa-
bioremediation. In this study, bacterial isolates were
tion, wars and expensive use of heavy metals and bio-
taken from the oil refineries located in Dhi Qar and
logical contaminants on a large scale, which led to many
Muthanna. The bacteria were isolated, purified and di-
environmental problems that must be seen with great
agnosed. The two most efficient isolates were used
seriousness and attention (Dzionek et al., 2016). The
through a series of laboratory experiments. Dating back
crude oil appeared as the most important source of pol-
to the genus (Klebsiella sp), the second isolation is tak-
lution and an indispensable material for the global eco-
en from the Samawah refinery and belongs to the genus
nomic and industrial growth at present, as a major
(Salmonella sp) and then cleared the soil of sterile
source of energy. Pesticides are also dangerous contami-
sieves and processed it for the cultivation of wheat
nants. These compounds were used in large quantities,
plant, and note the effect of these isolates on the growth
for long periods of time and for specific areas, which
of wheat plant. This study was conducted to achieve the
pose a risk to endemic organisms in the soil and to hu-
following objectives: Determining the amount of bio-
mans, because they are toxic to non-target organisms, as
degradation of crude oil by microorganisms for study
well as metabolites from pesticide decomposition which
sites and reclamation of soils which contaminated with
are also toxic and have significant pollution. Environ-
hydrocarbons of oil and effect of isolated bacteria on the
mental biodegradation has appeared as a technological
growth of wheat plant in its state of unilateral and syner-
and economic solution for the recovery of oil spills into
gy.
the environment. It is an efficient and environmentally friendly economic solution to treat oil spills on beaches,
MATERIALS AND METHODS
opposite the beaches and near the oil fields.
Crude oil samples
The effect of oil on soils depends on the size
Crude oil samples were obtained from the Sa-
and type of the flowing of oil the amount of soil cover
mawah Refinery and Plastic, sterile and sealed bottles
the annual chapter in which the flow occurred, as the
were used to collect five liter samples.
toxic effects of crude oil hydrocarbons to plants and
Soil samples
microbes in the soil. All microbiology stimulate meta-
Soil samples with mixed clay tissues were taken
bolic reactions and generally contribute to vital activity,
from the area devoted to agricultural research belonging
and each organism has a specific place to perform a
to the College of Agriculture, Al-Muthanna University,
certain function in nature (Marco-Urrea et al., 2015).
and some chemical, physical and alimentary analyses
The rate of biodegradation depends on the physiological
were carried out .Following standard protocol (Table 1).
state of micro-organisms that are sensitive to various
Isolation of bacteria from the soil samples
environmental factors and the chemical composition of
Samples taken from polluted and non-polluted
hydrocarbons. Modern studies were work hard to have
soils was innoculated on the brain heart infusion broth,
the microbial future designed as destroyers to deal with
to stimulate and isolate more species of bacteria. It is
many compounds, not only in terms of degradation or
incubated for 24 h at a temperature of 37°C. Activated
mineralization, and that the successful conversion of
samples were placed on nutrient agar and MacConkey
dangerous pollutants into usable molecules can be fur-
agar, and incubated for 24 h at 37°C and secondary im-
ther enhanced by a vital cell or catalyst cell that is a
plantation was carried out to develop colonies on the
1977
Journal of Research in Ecology (2018) 6(2): 1976-1986
Jassim and Saad, 2018 selective media (Cui et al., 2017).
lized in the autoclave at temperature 121°C and pressed
Purification
with 15 pounds/in2 and left to be cold and contaminated
For pure colonies of bacterial isolates of hydro-
with crude oil according to the treatments (0, 5 and 10
carbons, individual colonies that were given specific
mL) and placed in the plastic pots, its capacity is 5 Kg
morphological characteristics were tested, the streaking
after sterilization with ethyl alcohol. The pots were
method was used for implantation on the surface of se-
planted with sterile wheat seeds and vaccinated with
lected cultivars of each bacterial species in dishes in
isolates. The bacteria were selected according to the
several forms using the loop. Under sterile conditions,
above-mentioned treatments, with 15 seeds/pot and then
dishes were incubated for 48-24 h until colonies are
eased to eight plants after one week of germination and
obtained. The isolated samples were then kept in the
leaving some of the seeds without vaccine as a compari-
nutrient broth medium at refrigerator temperature until
son and without the addition of crude oil. Fertilizers
it is used (Kachienga and Momba, 2017).
were added according to the fertilizer recommendation
Preparation of the bacterial vaccine and its use as
in the form of urea (46% N) in two phases viz., the first
bio-fertilizers
at agriculture and the second at the elongation phase.
Two isolates were selected from the hydroxylat-
The recommended amount is 150 Kg N. ha-1. Phosphate
ed samples viz: Klebsiella sp and Salmonella sp for use
fertilizer is also added one batch before three days to
in the field experiment. The bacteria were grown on the
planting and in the form of the fertilizer superphosphate
liquid activated plant medium by placing 50 mL of this
(21% P). The recommended fertilizer level is 100 Kg.
medium in a 100 mL conical flask. After sterilization in
ha-1. Potassium fertilizer has been added in batch one in
an autoclave with the mulch of a modern farm, samples
the form of potassium sulphate (41.5% K ) and the rec-
were inoculated and incubated at 30°C for 2-3 days. To
ommended level of fertilizer is 100 Kg.ha -1 (Ali et al.,
prepare a sufficient amount of vaccine for the field ex-
2014). The agricultural experiment was designed to
periment, conical flasks (250 mL) containing 100 mL of
study the effect of inoculation on bacterial isolates of
the liquid activated plant medium was prepared and
hydrocarbons, with different levels of crude oil as well
after sterilization in the autoclave, each of them was
as to study the effect of these isolate after the vaccina-
inoculated by adding one mL of the processed liquid
tion of wheat seeds and left a portion of these seeds
using sterile pipettes and incubated at a temperature of
without adding the vaccine to them. Agriculture was
30°C for a period 2-3 days. After preparing the liquid
carried out on 6/11/2017 using two factors as given be-
vaccine, it was mixed with the betamus. The grains of
low :
wheat (IBA 99 Strain) was contaminated at the surface
Agricultural experiment factors
with betamus, and gum arabic was added at a concentra-
The first factor
tion of 10% to increase the adhesion of bacteria with
Crude oil in three different levels and the fol-
wheat seeds. It was left for one and half hour to ensure
lowing treatments were used:
contamination of all seeds with the bacteria. Some seeds
O0 : Without the addition of crude oil.
were placed without the microbial treatments and served
O1 : 5 mL of crude oil was added.
as a control (Cui et al., 2017).
O2 : 10 mL of crude oil was added.
Pots experiment
The second factor
Aerobic soil was dried, grinding and sifting was sieved using a 4 mm diameter sieve. The soil was steriJournal of Research in Ecology (2018) 6(2): 1976-1986
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 maintained, so that total number of experimental units
cant Difference (LSD) at the level Morality 0.05 (AlRawi and Klaf-Allah, 2000).
were 36 experimental units. Appearance characteristics of wheat plant (plant
RESULTS AND DISCUSSION
measurements)
Isolation and diagnosis of Klebsiella sp The results of the laboratory tests, which includ-
Germination The percentage of germination was calculated according to the equation: (Aiyadurai, 1956)
al colonies, that the isolation from the Nasiriyah refinery was related to the genus Klebsiella sp as indicated
Number of plants growing Germination rate =
ed the biological and morphological tests of the bacteri-
× 100 Total number
Makki (2013). It was observed that the isolates were negative chromatism, bony form and rounded ends, surrounded by capsule, non-moving, aerobic and anaerobic optional. It gives a negative reaction to the tests of in-
Plant height 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 characteristics of the soil
per quickly and without damage to leaves (Liu et al., 2014). 2
Paper area (cm ) The height and width of three plants from each pot, were measured Paper area= Average length× Average width × Correction Factor (0.95) (Thomas, 1975). The dry weight of the vegetative part (g) It was calculated from the average weight of three plants after drying in the aerobic condition and then put in the oven at 62°C until weight stability was seen and then weighed completely (stem, leaves and spikes).
S. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Trait pH E.C.E (dsm-1) O.M (g.Kg-1) Ca+ (mEq.L-1) Mg+ (mEq.L-1) Cl- (mEq.L-1) SO4= (mEq.L-1) CO3= (mEq.L-1) HCO3- (mEq.L-1) Soil texture Clay (g.Kg-1) Silt (g.Kg-1) Sand (g.Kg-1) Total bacterial count (CFU g-1 soil)
Values 8.00 4.20 11.00 4.31 11.40 41.60 36.50 Nil 1.37 Clay loam 290 470 240 0.28×105
Source: (Aiyadurai, 1956) 1979
Journal of Research in Ecology (2018) 6(2): 1976-1986
A3 (synergistic treatment) had the highest germination rate (87.6) and percentage increase of 11.7% than the A0 treatment (without vaccine). A1 gave a germination rate of 84.2%, with percentage increase of 7.3% than the A0. A2 gave the rate of 82.5%, with percentage increase of 5.2% (Al-Rikabi, 2017). This is due to the fact that the germination of seeds and variation of germination ratio according to the isolates used and their efficiency, so that the treatment gave without a bacterial vaccine the Journal of Research in Ecology (2018) 6(2): 1976-1986
-
87.6
82.5
84.2
78.4
73.90 82.40 93.20 72.60 74.60 86.60 64.90
2 -S. Isolates No
presence of bacteria has effectively contributed to the
-
a level of probability at 0.05% in seed germination(%),
A×O 18.82
bacterial inoculation on selected isolates showed that at
O 9.41
The results of Table 2 indicate that the effect of
A 10.87
Seeds germination (%)
6 LSD 0.05
tween bacterial isolates.
66.30
case of mixing, and showing the effect of synergy be-
74.30
that can be relied upon in the biological experiment in
41.00
between the two isolates and is an important indicator
51.00
trol (Al-Rikabi, 2017). There found no inconsistency
56.90
es, the properties of these bacteria in the biological con-
Average
as the number of siderophores and all of these substanc-
5
cin, phenazine, carboxylic acid and pyoluteorin, as well
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
cond (Salmonella sp) produce antibiotics such as oomy-
0
The first efficient isolation (Klebsiella sp) and the se-
0
and containing the center of the solid nutrient lacquer.
W3(third week) Crude oil levels % 1
48 hours of incubation of isolates on the same petri dish
W2 (second week) Crude oil levels % 1 2
ed in the study (Klebsiella sp and Salmonella sp) after
W1(first week) Crude oil levels % 0 1 2
potential adverse effects between the two isolates select-
Table 2. The percentage (%) germination of wheat seeds in the pots
The results of the antimicrobial test showed no
A3
Antimicrobial resistance test
4
black status due to H2S production (Makki, 2013).
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
and form gelatin. Dark brown colonies appear to have a
A2
Proskauer tests and do not have the ability to produce
3
duce H2S, react negatively to the indole and Voges–
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
reaction to the red blood test and had the ability to pro-
A1
tional aerobic or anaerobic spores, it gave a positive
2
W4(fourth week) Crude oil levels % 0 1 2
Response to surrounding changes, not made up of op-
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
have the ability to alter phenotype and genotype.
A0
finery, moved by peripheral luminescent, bronchial, and
1
Average
Jassim and Saad, 2018
1980
Jassim and Saad, 2018 Table 3. The height of wheat plant (cm) at 50% flowering Isolates A0 A1 A2 A3 Average LSD0.05
Crude oil levels % 0 1 2 39.40±0.12 22.60±0.12 15.30±0.06 42.70±0.06 27.60±0.06 17.80±0.04 43.50±0.14 25.60±0.14 16.50±0.08 41.50±0.16 28.70±0.16 21.10±0.05 41.50 26.10 17.60 A 1.70 O 1.40 A×O 2.90
Table 4. Chlorophyll content in wheat plant (SAPD) at 50% flowering Isolates A0 A1 A2 A3 Average LSD0.05
Crude oil levels % 0 1 2 9.50±0.06 2.40±0.01 1.40±0.01 10.40±0.01 3.20±0.02 1.90±0.01 9.70±0.02 2.60±0.02 1.70±0.01 11.40±0.03 3.80±0.01 2.40±0.01 10.20 3.00 1.80 A 0.20 O 0.20 A×O 0.40
Average 4.40 5.10 4.60 5.80 -
lowest reading. The effect of the oil level on the experi-
availability of phosphorus through the production of
mental parameters had a significant effect on seeds ger-
organic acids and soluble phosphates, which increase
mination. The oil level (O0) showed the highest percent-
the readiness of phosphorus in soil Hwangbo et al.
age of germination (93.25%), with percentage increase
(2003) as well as their effectiveness in the production of
of 13.1% than the oil level O1 and percentage increase
growth regulators, indole acetic acid and giberillins
of 26.0% than O2. The absence of hydrocarbons that
affecting the elongation of the cells and their division .
may be difficult to decompose and their toxic effect on
These mechanisms increase the protein content which is
plant growth has helped to increase seed germination,
positively reflected in the rate of plant height (Hasan,
and the presence of bacteria that contribute to the disas-
2002; Jat and Shaktwat, 2003).
sembly of crude oil components has reduce these nega-
The results of Table 3 indicate that the increase
tive effects of crude oil to some extent (Kachienga and
in crude oil levels led to a decrease in the rate of plant
Momba, 2017).
height. The oil level O0 gave the highest rate 41.5 cm
The effect of the interference among the oil lev-
with percentage increase of 59.0% from the oil level O 1
els and the effect of bacterial inoculation in a level of
and 135.7% from the oil level O2, which recorded the
probability (0.05%) in seed germination (%). The treat-
lowest height of the plant that reached 17.6 cm. The
ment A3 (synergistic treatment) gave the highest germi-
decrease in plant height by increasing oil levels is due to
nation rate to 100% at the oil level O0, while the treat-
the toxic effects of multiple oil hydrocarbons which
ment A0 gave the lowest germination rate of 71.1% at
effects negatively the rate of plant growth and this is in
the oil level O2. This is consistent with what was re-
line with what was pointed out by Hatem (2007) In the
ferred by Cui et al. (2017).
effect of irrigation with contaminated water hydrocar-
Plant height (cm)
bons provide the availability of nutrient elements. The
As shown in Table 3 the effect of bacterial inoc-
effect of the interference between bacterial inoculation
ulation led to high morality and increased in a level of
and oil levels on plant height was also shown in the ta-
probability (0.05%) in Plant height rate of the local iso-
ble 3. Given the treatment A0 (without adding bacterial
lates (Klebsiella sp and Salmonella sp) alone or double
vaccine), rates are lower than the treatments added to
on the A0 (without vaccine), (Das and Kumar, 2016).
the bacterial vaccine. Treatment A3 recorded the highest
The isolation A3 (synergistic treatment) gave the highest
rate of plant height of 43.4 cm at the oil level O0, while
rate of plant height of 31.0 cm and percentage increase
A0 was the lowest (15.3 cm O2). This indicated to the
of 20.6% compared to the treatment A0 (without vac-
positive effect of the bacterial vaccine in reducing the
cine) which reached 25.7 cm. This is due to the viability
toxicity of hydrocarbon by decomposing and processing
of these organisms in the case of synergy to increase the
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
which gave the lowest rate in chlorophyll content,
usually absorb nutrients with small molecular weights,
which reached 4.4 SPAD, followed by treatment A1 ,
whereas it cannot absorb the large molecular weight
with a rate of 5.1 SPAD, while A2 recorded the lowest
substances easily. Also environmental conditions and
rate of 4.6 SPAD. This may be due to the role of micro-
plant characteristics such as plant size, root type, etc.
organisms in the secretion of giberillins, which affect
can be a significant factor to facilitate absorption by
the production of auxiliary enzymes in the formation of
roots. And that the enzymes secreted by the roots are
pigments (Weiss et al., 1990). The reason for the in-
active on an area of 1 mμ from the root region. In this
creased chlorophyll content was that microorganisms
case the enzymes have the most effective ability to de-
had processed enough nitrogen, especially NH4 , as well
compose and break down oil carbohydrates and thus
as an increase in the root total absorption, which helped
benefit the roots from their degradation products in met-
to absorb nutrients elements (Buddhika et al., 2012).
abolic processes within the plant cells (Al-Jader, 2006 ).
The results of Table 4 also showed that the ef-
The roots are working to provide the root secretions for
fect is uneven when crude oil was added, which resulted
microbiology which contains important food compo-
in significant differences at the level of 0.05. The oil
nents for microorganisms such as sugars, enzymes and
level (O0) with a higher chlorophyll rate from O1 and
nucleic acids. The microorganisms then get the material
O2, had 10.2 SPAD with a percentage increase of 2.4
which they need through the root and provide the nutri-
and 4.46% from O1 and O2 respectively. This confirms
ents and organic needs by microorganisms that lead to
the negative effect of crude oil on plant growth. The
support and stimulate large number of microbiology in
second oil level at O1 with a higher chlorophyll rate
the soil and thus increase their effect on the decomposi-
with the average 3.0 spade and a percentage increase of
tion of hydrocarbons.
66.6 than the third levels of oil (O2). As for the effect of the interaction between the
Chlorophyll SPAD content in wheat plant at flower-
bacterial vaccine and the oil levels, the results of the
ing stage The results of Table 4 showed significant differ-
table showed significant differences in the content of
ences in the chlorophyll content when inoculation with
chlorophyll. The treatment of vaccination was given in
isolates Klebsiella sp and Salmonella sp were done.
isolation A3 at the oil level O0. Highest rate of chloro-
These synergy between them compared with the non-
phyll content observed was 11.4, while A0 (without vac-
addition of the bacterial vaccine (A0). The treatment
cine) recorded the lowest rate of average chlorophyll
was superior with two types of bacterial vaccine A3.
content at O2 with 1.4 SPAD. The reason for the in-
Highest rate is 5.8 SPAD, with the percentage increase
creased chlorophyll content in the plant may be due to
of 31.8% compared to the measurement treatment of A0,
the presence of microorganisms in the form of a vaccine
Table 5. The area of leaf of wheat plant (cm2) at 50% flowering Crude oil levels % 0 1 2 A0 8.14±0.02 3.08±0.02 1.18±0.01 A1 10.50±0.01 3.62±0.02 2.26±0.02 A2 9.87±0.02 3.21±0.01 1.50±0.01 A3 11.80±0.01 4.21±0.01 2.42±0.01 Average 10.07 3.53 2.42 LSD0.05 A 0.86 O 0.74 A×O 1.48 Isolate
Average 4.31 5.46 4.86 6.14 -
Journal of Research in Ecology (2018) 6(2): 1976-1986
Table 6. The dry weight (g.plant-1) at 50% flowering Crude oil levels % 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 Average 3.171 0.554 0.314 LSD0.05 A 0.324 O 0.281 A×O 0.562 Isolate
1982
Jassim and Saad, 2018 that helped to provision the nutrients elements, includ-
an important part of the process of photosynthesis and
ing nitrogen, which increased the duration of the repre-
in some nucleic acids such DNA and RNA.
sentation and made it more effective, leading to the increased
chlorophyll
content
in
the
Table 5 showed that the increase in the oil levels
leaves
led to the reduction of the paper area of the plant. The
(Vaguseviciene et al., 2012). The low chlorophyll con-
first oil level (O0) gave the highest rate in the paper area
tent with the high levels of crude oil and at non-addition
(10.07 cm2) with the ratio of increase (185.2%) at the oil
of bacterial vaccine may be due to the reduction of soil
level O1 and 447.2% at the oil level O2. It may be due to
nitrogen content or that the crude oil inhibited the effec-
the decrease of paper area by increasing the levels of oil
tiveness of other bacterial species found in the soil, be-
to the type of components of crude oil of Samawah re-
cause crude oil has toxic components on organisms,
finery and it contains hydrocarbons which vary in the
found in the soil, by reducing their numbers and thus
speed of disintegration according to their chemical com-
their effectiveness and efficiency in the conversion of
position and to the role of bacteria added vaccine in the
nitrogen to the plant (Golafarin et al., 2017).
processes of degradation of hydrocarbons. It has been 2
Area of the leaf of wheat plant when flowering (cm ) The results of Table 5 showed that bacterial
observed that the paper area has increased in case of the use bacterial vaccine (Al-Rikabi, 2017).
inoculation had a significant effect on the increase of
The interference between the bacterial vaccine
the paper area of the wheat plant compared to the non-
and the oil levels added significantly affected the paper
addition of the vaccine A0 (comparison treatment). Bac-
area. The highest rate in paper area when insemination
terial infection was recorded with the inoculation of
with isolation A3 and oil level O0 (11.8 cm2) and the
Klebsiella sp And Salmonella sp and the synergies be-
lowest plant height (without vaccine) A0 and oil level
tween the two isolates are higher than the paper area.
O2. The rate (1.18 cm2) is consistent with the findings of
Treatment A3 gave the highest rate of paper area (6.14
Varjani and Upasani (2017), which referred to the role
2
cm ). The isolation of A3 excelled the rest of isolates
of bioremediation in reducing the negative effects of
with the ratio of increase (12.4%) compared to A1 isola-
spilled hydrocarbons on the soil and the possibility of
tion which recorded an average of 5.46 cm2, while isola-
reducing their effect or the use of bacteria in reclama-
2
tion of A2 recorded a rate of 4.86 cm and that is due to
tion after studying the environmental factors and the
the role of bacterial vaccine in the process of decompo-
type of oil components and the type of bacteria added
sition. The soil and its role in the dismantling of hydro-
their numbers and their role in the degradation of hydro-
carbons added , results in an increase in the food pro-
carbons.
cessed plant and then increase the paper area and listed
Dry weight (g.plant-1) for vegetative part of wheat
according to the added vaccine and its role (Das and
plant at (50%) flowering
Kumar (2016) and referred to Abo Noktta and El-Shater
The results of Table 6 indicate that the vaccina-
(2011) to demonstrate the role of bacteria in increasing
tion with local isolates resulted in the significant in-
the nutrient elements availability, including nitrogen,
crease of the average mean dry weight compared with
which plays an important role in the formation of cell
the measurement of the treatments. Treatment A3 ( Mix
protoplasm and cellular membranes and in the for-
the two isolates) (Klebsiella sp and Salmonella sp) gave
mation of enzymatic enzymes and enzymes such as
highest dry weight (1.557 g.plant-1) with the increase
NADH, NADPH and vitamins, which are the complex
(28.7 %) compared to A0 (without vaccine), that record-
B group. In some growth regulators, the chlorophyll is
ed the lowest rate of dry weight (1.209 g.plant -1). Fol-
1983
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
of the components of crude oil also it contains difficult decomposition compounds that has caused inhibition of
age 1.283 g.plant . The reason for the dry weight gain
the activity of bacteria because of its toxic effect on the
of the vegetative group may be due to increased num-
plant and then reduced plant growth and reduced dry
bers of bacteria added in the form of a vaccine and then
weight, or non-disintegration of the carbon that has
increase of the dismantling of hydrocarbons and the
completely reduced the food additive, affecting plant
addition of more nutrients elements, which led to the
growth and thus the dry weight or that the non-
increased plant growth and accumulation of dry matter
disintegration of hydrocarbons completely reduced the
as well as increasing the number of branches of the
added nutrient elements, which affected the growth of
plant that led to the increase the dry weight of the plant.
the plant and therefore on dry weight. Crude oil has a
These results are consistent with (Al-Jader, 2006). The
negative impact on the vital activities of the plant,
dry weight of the plant increased with the addition of
which affect the plant hormones and enzymatic activi-
the bacterial vaccine, reduce the spread of oil carbohy-
ties, which hinder the growth of the plant and reduces
drates by reducing or eliminating the biological effec-
the dry weight of the plant if not completely decom-
tiveness of contaminants to another area. Carbohydrates
posed and this is due to the types of bacteria used and
do not need to be broken for getting minimized. Plants
their molecular structure and the type and components
have several ways to reduce the spread of contaminants.
of crude oil used.
Plant roots work to attract oil carbohydrates to its root
The effect of interference between inoculation
area as a result of its absorption of water, so it acts as an
with local isolates and oil levels in the dry weight of the
organic pump to attract organic carbohydrates and iso-
plant, showed that the results superiority of isolation A3
late them from the rest of the soil is not contaminated,
at the oil level O0 on the rest of the isolates in the dry
and the root is actually indirect in reducing the spread of
weight of the vegetative group, with the rate of 3.615.
carbohydrates when the root provides for carbohydrate
While the lowest average dry weight of the vegetative
enzymes. Latter is associated with carbohydrates in the
group was recorded in the measurement treatment of A 0
soil and is known as humus, and the plant benefits from
at the oil level O2, which recorded 0.239 g.plant -1, that
the breakdown of carbohydrates and the production of
may be the superiority of this isolation to the other iso-
emulsified substances as a source of carbon and nitro-
lates ,because this isolation was the synergy between
gen in the form of amino acids polymerized by the en-
two isolates that proved their ability to break down hy-
zyme of peroxidase which is produced by roots, and the
drocarbons. The microbial activity in the decomposition
root can polymerize the oil (Al-Jader, 2006).
has doubled due of their presence together. Which
Also the results of Table 6 showed that the dry weight of the vegetative group was reduced by the high
means that this isolation is adaptive to the presence of crude oil (Mingming et al., 2017).
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
CONCLUSION
level O2, which gave the lowest rate 0.314 g.plant . The
The inability of bacteria isolated from non-
level of oil O1 gave higher than the level of oil O2 which
polluted areas to decompose and deteriorate crude oil.
-1
recorded an average of 0.554 g.plant on oil level ( O2).
Differences in the efficiency and species of bacteria
The reason behind the low proportion of dry matter in
isolated from oil-contaminated areas in the decomposi-
the plant at increasing the levels of oil may be because
tion of the oil hydrocarbons degradation. The isolates
Journal of Research in Ecology (2018) 6(2): 1976-1986
1984
Jassim and Saad, 2018 (Klebsiella sp and Salmonella sp) showed more efficacy
Cui Z, Zhang X, Yang H and Sun L. 2017. Bioreme-
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