Isolation of plastic waste decomposing bacteria from the soils of different areas of Anbar province

Abstract

The current study focused on the possibility of isolating and diagnosing plastic-decomposing bacteria from soil samples in different areas of Anbar province, the pureAkar PVC medium (polyvinyl chloride) and the pure PS polyester acar medium were used to isolate the plastic-decomposing bacteria, as 10 bacterial isolates capable of growing on the pure PVC (polyvinyl chloride) medium and the pure PS polyester acar medium were obtained to isolate well characterized 3 Of which with its ability to grow efficiently on both mediums, and the method of measuring the diameter of the colony or the transparent area around the colony was used to measure the growth efficiency on both mediums and choose the most efficient isolation of which is the isolation that was given the local symbol C1 isolated from the soil of plastic landfills in the Sufi area of Ramadi, after which the isolation was diagnosed by morphological and biochemical tests that appeared to belong to the bacteria Kytococcucsedentarius and the diagnosis was confirmed by the phyticdevice Vitek 2 compact system used three types of plastic waste (nylon bags, water bottles, water cups) The results showed that nylon bags were the best carbon source used by bacteria as the only source of carbon and energy at a temperature of 30 ° C pH 7.

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NeuroQuantology|August 2022|Volume20|Issue10|Pag e 3579-3586|doi:10.14704/nq.2022.20.10.NQ55347
Al-Rawi et al/ Isolation of plastic waste decomposing bacteria from the soils of different areas of Anbar province
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Isolation of plastic waste decomposing
bacteria from the soils of different areas
of Anbar province
Al-Rawi, Dhafer Fakhri Rawnaq Aadil Ibrahim
Biology Department, College of Education for Pure Sciences, Al-anbar University,
Ramadi, Al-anbar, Iraq
mrawnak84@gmail.com
Abstract :
The current study focused on the possibility of isolating and diagnosing plastic-
decomposing bacteria from soil samples in different areas of Anbar province, the pureAkar
PVC medium (polyvinyl chloride) and the pure PS polyester acar medium were used to
isolate the plastic-decomposing bacteria, as 10 bacterial isolates capable of growing on the
pure PVC (polyvinyl chloride) medium and the pure PS polyester acar medium were
obtained to isolate well characterized 3 Of which with its ability to grow efficiently on both
mediums, and the method of measuring the diameter of the colony or the transparent area
around the colony was used to measure the growth efficiency on both mediums and choose
the most efficient isolation of which is the isolation that was given the local symbol C1
isolated from the soil of plastic landfills in the Sufi area of Ramadi, after which the isolation
was diagnosed by morphological and biochemical tests that appeared to belong to the
bacteria Kytococcucsedentarius and the diagnosis was confirmed by the phyticdevice Vitek 2
compact system used three types of plastic waste (nylon bags, water bottles, water cups)
The results showed that nylon bags were the best carbon source used by bacteria as the
only source of carbon and energy at a temperature of 30 ° C pH 7.
DOINumber:10.14704/nq.2022.20.10.NQ55347
NeuroQuantology2022;20(10):3579-
3586
Introduction:
Plastic is defined as a polymer that
becomes mobile when heated, so it can
be poured into molds Plastic consists of
carbon, hydrogen, silicon, oxygen,
chloride and nitrogenn (Kale et.al., 2015),
plastic is a lightweight, strong, durable
and low-cost industrial or semi-synthetic
ring vehicle (Van Eygen et.al., 2017)
Plastic is used to produce a very
wide range of products and has quickly
moved to all aspects of human life
Plastics cover a wide range of synthetic
polymeric materials (such as
polypropylene, polyethylene, polyvinyl
chloride, polystyrene, nylon and
polycarbonate) produced a wide range of
different end products Most plastics are
derived from non-renewable raw
materials (such as natural gas, oil or coal)
and biorenewable materials such as
vegetable oils, carbohydrates and
vegetable proteins Starch, lignin,
celluloseand other workerscan be used in
plastic production (de Vargas Mores
et.al., 2018).
The uncontrolled use of plastics in
packaging, transportation, industry and
various uses in rural and urban areas over
the past few decades has led to an
increase in serious problems associated
with the disposal of plastic waste and
pollution, the effective decomposition of
plastic bags takes about 1000 years and
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plastic not only increases the problems of
waste disposal and landfill but also leads
to the release of carbon dioxide as well as
persistentorganic pollutants. When
combustion causing pollution and global
warming, common plastic disposal
methods have proven insufficient to
effectively manage plastic waste so there
is an increasing focus on the use of
effective microorganisms for
biodegradable synthetic polymers Kale
et.al., 2015 ) .
Population growth and industrial
activity in the world have led to an
increase in pollution caused by human
activities and plastic pollution has
become a concern , so plastic products
must be recycled or made biodegradable
Chemical and natural processing
methods are often energy consuming
and expensive in addition to that they are
not environmentally friendly and often
produce toxic by-products. Biological
treatment is a suitable option because it
is cost-effective and economical and
environmentally friendly The production
and consumption of plastics is increasing
every day and as a result more
microorganisms are exposed to these
non-biodegradable polymers so the study
of microorganisms effective in analyzing
plastic products and increasing the
understanding of their vitality can pave
the way for effective and possible plastic
processing processes (Delangizet.al., 2022
) . The increasing accumulation of
plastic waste has become a serious
environmental and social problem that is
necessary to develop innovative
approaches to the disposal of plastic
waste . In recent years reports have
emerged of the biodegradability of
synthetic plastics by microorganisms or
enzymes and these reports provide the
possibility of developing the
bioremediation technology of plastic
waste (Ru etal., 2020 ).
Although many studies have shed light on
the potential role of microorganisms in
the analysis of marine plastic debris, little
is known about the interaction of plastics
with fungi which are also important
degradation factors for complex organic
matter in the marine environment, in
fact recent research and studies have
shown that some fungal strains found in
the oceans such as Zalerionmaritimum
has the ability to decompose
polyethylene ( Zeghaletal., 2021 ).
Bacterial isolates decomposing
HDPE have been identified and most of
these bacterial isolates belong to the
genus Bacillusspp and Pseudomonas
sppRecent studies indicate that isolated
bacterial strains can be used as
environmentally friendly as they
represent a safe way to dispose of
polluting plastic waste (Devietal., 2019).
Materials and methodsof work:
Plastic Hydrolyzing Bacteria Isolation:
22 soil samples were collected from
different areas in Anbar province,
including soils planted with various field
crops and plastic landfills in order to
isolate bacteria that have the ability to
analyze plastics from the soil, (1 g) was
taken from the soil after cleaning and
purifying it from impurities and
conducting a series of decimal scarfs on it
in distilled water and used the method of
pouring dishes as it took (1 ml) of dilution
IV 10-4 and V 10-5 Each was placed in
sterile petri dishes after which both the
pure PVC (polyvinyl chloride) medium and
the pure PS polyester acar medium
which was prepared by dissolving the
substances 500 mg of K 2 HPO 4 , 400
mg of KH2PO4 and 100 mg of NaCl and
20 mg of CaCl 2, 200 mg of (NH 4)2 SO 4
,20 mg of MgSO 4 , 12 mg of FeSO4
and100 mg of MnSO 4 3.0g of Poly
Vinyl Powder Substance (PVC) Pure
Chloride and 15 g of Akar-Akarevi 1000
ml of distilled waterMaroof etal., (2021),
stirred the dishes towards and
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counterclockwise for the purpose of
ensuring the homogeneous distribution of
the sample in the dish, after which the
dishes planted at a temperature of 30° C
were incubated for 6 days, those isolates
were re-purified by replanting (sub-
culturing) ) after several transitions on
the pure PVC (polyvinyl chloride) medium
and pure PS polyester acar medium to
obtain pure single colonies, then the
selected insulation on both mediums was
sifted by planting them in the form of a
circle (1cm) in the center of the dish and
incubated at a temperature of 30° C for 5
The day after that it was revealed the
bacteria's ability to analyze the plastic by
measuring the diameter of the colony or
the transparent area around the
developing colony in the center of the
dish.
Competency Test of Elected Isolations:
The most efficient isolation in
plastic analysis was elected using the
method of measuring the diameter of the
colony or the transparent area around it,
as the ten selected and dissolved plastic
isolates were replanted on the pure PVC
(polyvinyl chloride) agar medium and the
pure PS polyester acar medium in the
form of a circle (1cm) in the center of the
dish and incubated for 5 days at a
temperature of 30 After that, the
diameter of the colony or the transparent
area around the colony was measured in
measuring the growth efficiency of the
isolates used, and three isolates were
elected, which were the largest diameter
among the other isolators, these three
isolates were replanted on three
environmental waste mediums, namely
the center of solid nylon bags, the center
of solid water bottles and the center of
solid water cups in the same way as the
previous by making a circle of 1 cm in
diameter and showed high growth
efficiency on all mediums and it turned
out that the highest rate The diameter of
growth on the center of the solid nylon
bags reached 5.5 cm and based on the
results of these analysis, only one
isolation was selected that was the most
efficient of the three, namely the
bacterial isolation with the local symbol
C1, which reached the diameter of the
colony and the transparent area of
decomposition 4.5.
Identification of Bacterial Isolates
The elected bacterial isolation with
the highest decomposition rate was
diagnosed depending on its phenotypic
characteristics involved (colony's shape,
height, color, edge and texture) and then
studied the characteristics of bacterial
cells microscopically after dyeing them
with Gram's stain dye according to Holt
etal., (1994) After which the diagnosis
was confirmed using the Vitek 2 compact
system equipped by the French company
BioMerieux.
Election of the best local carbon source
used by elected isolation:
To determine the best local carbon
source used by bacteria as the only
source of carbon and energy, three local
carbon sources were used, namely nylon
bags, water bottles and water cups,
where the center of the solid nylon bags,
the center of the hard water bottles and
the medium of the hard water cups were
prepared by adding3.0 mg of each of the
nylon bags, water bottles and water bags
to 500 mg of K 2 HPO 4 and 400 mg of
KH2PO 4 100 mg of NaCl, 20 mg of CaCl
2, 200 mg of (NH 4)2 SO 4, 20 mg of MgSO
4 , 12 mg of FeSO 4 and100 mg of
MnSO4 15 g of Agar-Agar 1000 ml of
distilled water, then sterilized the
mediums with the repellent and poured
the media after cooling them into petri
dishes and left to harden, then planted
the elected bacterial isolation by making a
circle 1 cm in diameter in the center of
the dish by three repeaters for each
source and incubated at a temperature of
30° C and for 5 The day after that, the
best environmental residue was elected
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that bacteria used as the sole source of
carbon and energy according to the
measurement of the diameters of
bacterial colonies.
Results and discussion:
Isolation of plastic-decomposing
bacteria:
The results of the primary isolation
in Table (1) showed obtaining 6 bacterial
isolates capable of growing efficiently on
the pure PVC (polyvinyl chloride) medium
and pure PS polyester acar medium out
of 30 bacterial isolates isolated from 22
soil samples brought to the laboratory,
these isolates varied on their ability to
grow and use plastics used in the study as
the sole source of carbon and energy
based on their growth density on the
insulation medium. After the transplant
and incubation of these bacterial isolates
on the pure PVC (polyvinyl chloride)
medium and the pure Acaralbolester PS
medium, the colonies appeared with clear
growth diameters on both mediums, as
the bacteria showed a good ability to
grow in the two mediums and exploit
plastics in it as the only source of carbon
and energy, and a discrepancy appeared
in the diameter of the colonies on both
mediums and that the variation in the
growth of bacterial isolates is due to the
difference in their ability to consume
plastics as well as to the environment
from which these bacteria were isolated.
Table (1) showing the diameters of plastic decomposition in secondary screening
Pronounced like
Symbol of isolation
Diameter of transparent area (cm)
1
C1
4.5
2
CH
3.8
3
F2
3.1
4
G1
3.3
5
N2
3.4
6
R1
2.4
7
T
2.0
8
X1
2.1
9
Y
2.3
10
S
2.0
Test the efficiency of the selected
isolates in plastic analysis:
After obtaining pure single colonies
that have the ability to analyze plastic
waste, replant the 30 selected bacterial
isolates on the pure PVC acar medium
and the pure PS polyester medium by
making a circle 1 cm in diameter at the
center of the dish and incubated for 5
days to choose the most efficient
insulation in plastic analysis by measuring
the decomposition diameters of these
isolates whose efficiency in plastic
analysis varied as the diameter of the
colony and the transparent area ranged
from 4.5–2) ) cm, 10 isolates were elected
that were the most efficient in plastic
analysis Table(1), then those isolates
were replanted and the same conditions
were applied to them after which three
bacterial isolates that carry local g-
rheumatozoaC1 - N2 -CH were elected in
their ability to analyze plastics and the
three isolates were replanted under the
same conditions and the average
diameter of the transparent area around
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the colony was 3.8 -3.4 -4.5) ) cm
respectively and also replanted these
three isolates on three environments of
environmental waste, namely the center
of nylon bags, the center of water bottles
and the center of water cups in the same
way as the previous by making a circle
with a diameter of 1 cm and showed high
growth efficiency on all media and it was
found that the highest growth diameter
rate on the center of nylon bags
amounted to 4.5 cm and based on the
results of these decomposition only one
isolation with high efficiency in plastic
analysis was chosen , namely C1.
Figure (1) shows the secondary screening of the selected isolates on the purePVC medium
Diagnosis:
The results of implant, microscopic
and biochemical examinations of the
elected bacterial isolation and based on
Holt et.al., (1994) showed that the
bacterial isolation given the local code C1
bears the characteristics of the bacteria
Kytococcucsedentarius Table (2), (3) and
this was confirmed by the diagnosis of
isolation using the VITEK2 Compact
device .
Table (2) Implant and microscopic qualities of elected bacterial isolation with local
code C1
Pronounced
like t
Diagnostic trait
C1
Pronounced
like t
Diagnostic trait
C1
1
Shape
Pie
6
Height
Elevated
2
Color
White
7
Cell shape
spherical
3
Textures
solid
8
Cell
aggregation
Pairs or
quatrains
4
The Edge
Round
9
Tincture of
Cram
Positive
5
Transparency
Dark
10
Production of
dyes
Does not
produce dyes
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Table (3) Biochemical Tests for Kytococcussedentarius
Kytococcussedentarius
Type of test
N
-
Oxidase
1
+
Catalase
2
-
Indole
3
-
Methyl red
4
-
Voges-Proskauer
5
-
Motility
6
+
Urease
7
-
Simmon Citrate
8
-
Hymolysis
9
-
Gelatin hydrolysis
10
+
Arginine
11
+
Gram stain
12
( + ) Presence of growth or positive for the test ( - ) Lack of growth or negative for the test
Selection of the best environmental
residue as a carbon source of
Kytococcucsedentarius :
The results shown in Table (6-4)
showed the variation of the isolation of
Kytococcussedentarius in its ability to
analyze plastic waste and in its ability to
exploit carbon sources represented by
nylon bags, water bottles and water cups,
as it was found that nylon bags are the
best used carbon source, which gave the
highest rate of decomposition diameter,
and amounted to 4.5 cm for the selected
bacterial isolation.
This result is attributed to the type
and nature of the materials from which
transparent nylon bags are made and the
sheet of these bags and the increase in
their surface area led to an increase in
their exposure to decomposing bacteria
which made them easier to consume by
these bacteria, that the abundance of
microplastics formed in the
decomposition medium increases with
the thinness and decreasing size of the
plastic piece (Wilby, 2019 ).
This finding is consistent with
several studies that have used nylon bags
as a carbon source, if Kale et.al., (2015)
referred to the use of nylon bags as the
best carbon source, and Patil (2018) also
used polyethylene bags as the sole
source of carbon and energy in his study.
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Figure (2) represents the diameter of the colony and the transparent area on the center of
the solid nylon bags at a temperature of 30° C and for a period of incubation (5) days
Figure 3: Rate of diameters of the bacterial colony on carbon sources
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