Effect of isolated bacteria from the contaminated areas in the decomposition of hydrocarbons and phe

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-

than other isolates. The use of these bacteria has a sig-

diation of heavy metal pollution utilizing composite

nificant effect (0.05) on all wheat characteristics at

microbial agent of Mucor circinelloides, Actinomucor

flowering, when added without the presence of oil .The

sp and Mortierella sp. Journal of Environmental

use of synergy between two efficient bacterial isolates

Chemical Engineering, 5(4): 3616-2621.

has a significant effect on the growth of wheat plant.

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