Detection of β- lactam Genes in Pseudomonas aeruginosa Isolates

Transcription

Jornal ofthBabylon Univ. Special Issue - Proceding of 5th International Conference of Environmental Scince University of Babylon / Environmental Research Center 3-5 December 2013
5 International Conference for Environmental Researcuys-Environmental Researc
Detection of β- lactam Genes in Pseudomonas aeruginosa Isolates in
Some Hospitals in Bagdad Governorate by using Multiplex PCR
Prof. Amina N. AL-Thwani
Genetic Engineering and Biotechnology Institute for Post graduate
Abstract
Multidrug resistant (MDR) P.aeruginosa had emerged in some burn‘s hospitals in Baghdad
and seen mainly in nosocomial infections due to the selective pressure by overused of
antibiotics. This study was to delineate prevalence of MDR P.aeruginosa in nosocomial
infection burn units , and to screen for ESBLs producing P.aeruginosa . Samples from
hospital environment were collected. Antibiogram of P. aeruginosa isolates were determined.
MDR and ESBLs P. aeruginosa were detected. Pseudomonas aeruginosa is a bacterium
responsible for severe nosocomial infections, Plasmid DNA analysis and encoded many types
of genes responsible for beta-lactameases , to determine some of this type of genes in P.
aeruginosa strains isolated from 100 swabs of burn‘s units environment, using a molecular
methods (Multiplex PCR) by primers specific to ESBLs (bla ) gene (OXA-10) and (VEB-1).
The results revealed that 15 strains were isolated from environment, and all of the 15 (100%)
were positive OXA-10 and 6 (40%) VEB-1 gene was found in isolates.
Introduction
Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen, is the
most common gram-negative bacterium found in nosocomial infections. A nosocomial, or
hospital-acquired, infection is a new infection that develops in a patient during
hospitalization. Nosocomial infections (NI) are a worldwide problem that occur both in
developed and in developing countries (Valles and Ferrer, 2009). Nosocomial infection is one
that develops during a hospital stay that is, patients was not infected when admitted, the word
nosocomial is derived from Greek word hospital. Contaminated inanimate surfaces in hospital,
especially those frequently touched by hand, can contribute to the spread of healthcareassociated pathogens. Transmission can occur either indirectly when a healthcare worker‘s
hands or gloves become contaminated by touching contaminated surfaces after which they
1
touch patients, or when a patient comes in direct contact with a contaminated surface (Tortora
et al., 1986; Kramer et al., 2006).
Hospital environment is contaminated by a variety of pathogenic and nonpathogenic
microorganisms that can persist on surfaces for prolonged periods; numerous studies showed
that hospital surfaces and frequently used medical equipment become contaminated by a
variety of these microorganisms. The acquisition of nosocomial pathogens by a patient and the
resultant development of infection depend on a multifaceted interplay between the
environment, a pathogen and a susceptible host (Branson et al., 2010). Besides transmission
through fomites and vectors, bacterial flora can be carried into a hospital by the patient and
can be an important source of infection for the same individual after injury. Regarding
multidrug resistance (Hsueh et al ., 1998). Pseudomonas aeruginosa is responsible for 1015% of nosocomial infections worldwide. The β-lactam group of antibiotics which include
Penicillins, Cephalosporins, Monobactams and Carbapenems are mainly used to treat
infections caused by Gram negative bacteria. The widespread use of antibiotics put
tremendous selective pressure on bacteria which develop new mechanisms to escape the lethal
action of the antibiotics. (Washington et al., 2006).
Materials and Methods
Swabs Collection
This study last for nine months starting from beginning of August 2012, till the end of
April 2013. One hundred environmental swaps were collected burn's units in three
hospitals in Baghdad, Al-Kindi General Teaching Hospital / Rusafa (40 swabs), AlYarmuk General Teaching Hospital / Karkh (35 swabs) and Al-Imam Ali Hospital / Rusafa
(25 swabs). The study included collected from burn‘s units environmental swabs (gloves,
beds, floors, benches, walls and washing baths) in mentioned three hospitals.
Isolation and Identification of Bacteria
All swabs obtained were cultured directly on MacConkey agar and Blood agar, incubated
aerobically at 37°C for 24 hr and citrimide agar at 42˚C. Identification by traditional
biochemical methods and confirmed by API 20 E standardized identification system (Harly
and Prescott, 2002; Forbes et al., 2007 and Jawetz et al., 2007). The antimicrobial
susceptibility test fifteen isolation of P. aeruginosa against (12) antibiotic was conducted by
disc diffusions methods (DDM), (7 types β-lactam antibiotics and 5 types was non-β-lactam
antibiotics), (Table,1) as previously reported by Bauer et al. (1966).
Table -1: Types of Antibiotic susceptibility of Pseudomonas aeruginosa isolates
2
Molecular Detection of ESBLs
Genomic DNA and Plasmid DNA were extracted from isolates of burn‘s units samples by
using 2 Mini Kits extraction Genomic DNA and Plasmid DNA, Purification depending on
instruction of manufacturing company (Geneaid, Thailand). Multiplex PCR was used for
detection of ESBLs genes in P. aeruginosa isolated from burn‘s units (environmental
isolates), which were (bla OXA-10) and (bla VEB-1).
Multiplex PCR :
The primers sequence for ESBL genes showed in table (2).
Primer preparation: Lyophilized forward and reverse primers for OXA-10 and VEB-1 were
suspended with suitable volume of TE buffer as recommended by Bioneer Corporation
protocol, were dissolved in deionized water to give a final concentration of (100 pM/μl) (as
stock solution); to prepare 10μM concentration as work primer re-suspended 10 pM/μl in 90μl
of deionizer water to reach a final concentration 10μM These primers synthesized by Bioneer
company.
Table -2: The sequence forward and reverse primers of
List of β-lactam antibiotics
Antimicrobial class
Cephalosporin 3rd
generation
Cephalosporin 3rd
generation
Cephalosporin 3rd
generation
Carbapenem
Monobactam
Penicillin
(Urediopenicillin)
broad spectrum
Cephalosporin 4th
generation
List of non-β-lactam antibiotics
Agents included: generic
names
Antimicrobial class
Agents included:
generic names
Cefotaxime
Aminoglycoside
Amikacin
Ceftriaxone
Aminoglycoside
Gentamicin
Ceftazidime
Fluoroquinolone
Ciprofloxacin
Imipenem
Aztreonam
Chloramphenicol
Aminoglycoside
Chloramphenicol
Tobramycin
Piperacillin
Cefepime
blaOXA-10 and blaVEB-1 genes
Primer Name
OXA-10 (F)
OXA-10 (R)
VEB-1 (F)
VEB-1 (R)
5' –
Sequence
- 3'
TCA ACA AAT CGC CAG AGA AG
TCC CAC ACC AGA AAA ACC A
CGA CTT CCA TTT CCC GAT GC
GGA CTC TGC AAC AAA TAC GC
Detected gene
Product
size
blaOXA-10
277bp
blaVEB-1
643bp
In multiplex PCR two genes were detected, OXA-10 and VEB-1 in environmental isolates
of P. aeruginosa at the same time. The PCR reaction was conducted in a total volume of
20µl consisting of the following: 13 µl of deionizer water, lyophilized of PCR
3
master mix was dissolved by vortex, 2µl of each two primer (mixing together), and 3 µl
DNA templates (Table, 3), according to Nass et al. (2000).
Table -3: The mixture of Multiplex PCR working solution
Volume (µl)
Working solution
Water
13
Mix primers
4
DNA
3
Final volume
20 µl
PCR Protocol: The PCR program was adopted Strateva et al. (2007), PCR protocol included
initial denaturation at 95° C for 5 min, followed by 30 three-step cycles, including denaturation
at 94° C for 45 sec, annealing at 58°C for 1 min, extension at 72°C for 1 min and a final
extension at 72°C for 7 min as shown in table (4).
Table -4: PCR program for OXA-10 and VEB-1 gene fragment from
amplification by multiplex methods
No.
1.
2.
3.
4.
5.
Steps
Initial denaturation
Denaturation
Primmer annealing
Primmer extension
Final extension
Temperature (°C)
95
94
58
72
72
Time
cycles
5 min
45 sec
1 min
1 min
7 min
1
30
1
After the termination of these programs, the PCR amplification product of DNA were taken
and tested on agarose gel electrophoresis 1% with 70 volt/cm for 1.5 hr and visualized under
trans-illuminator machine .
Results and Discussion
Out of the 100 environmental swabs collected from three hospitals, revealed that 38 (38%) of
swabs gave positive result for bacterial growth and the rest 62 (62%) were negative. Fifteen
isolate successfully were diagnosed as P. aeruginosa, representing 39.50% of total isolates
and it is a highest percentage from all isolates was obtained from burn‘s units (gloves, beds,
floors,
benches,
walls
and
washing
baths),
followed
by
S. aureus8(21.0%) while K. pneumonia came thirdly 6(15.7%), then E. coli 3(7.8%), P.
putida, E. aerogenes recovered in similar rate 2(5.26%) ,the least isolated microorganism were
A.baumannii and P. mirabilis as 1(2.63%) for each, (Table -5).
4
Table -5: Types of environmental isolates from burn's units
Number & frequency
Isolate
Pseudomonas aeruginosa
Staphylococcus aureus
Klebsiella pneumonia
Escherichia coli
Pseudomonas putida
Enterobacteraerogenes
Acinetobacterbaumannii
Proteus mirabilis
Total No.
Single
Isolates
Mixed
Isolates
11
6
4
3
2
2
1
29
4
2
2
1
9
Total
No.
Percentage
(%)
15
8
6
3
2
2
1
1
38
39.50
21.0
15.7
7.8
5.26
5.26
2.63
2.63
100
P. aeruginosa is the third most-common pathogen associated with hospital-acquired
infections (Moreau-Marquis et al., 2008). Gram negative organisms were found to be more
incidence as nosocomial infections, P. aeruginosa was found to be the most common isolate
followed by S. aureus, E.coli, and Klebsiella spp. (Church et al., 2006). These organisms do
not have fastidious growth requirements and can grow at various temperatures and pH
conditions prevalent in the hospital environment, and in addition, are able to exploit varieties
of carbon and energy sources. These properties explain the ability of these pathogens to
persist for a reasonable time in either dry or moist conditions in the hospital environment,
thereby causing disease. These hard line posture combined with their intrinsic resistance to
many antimicrobial agents, contribute to the organisms fitness and enable them to spread in
the hospital environment (Hart and Kariuki, 1998; El-Mahmoodet al., 2010).
One of the most worrisome characteristics of P. aeruginosa is its low antibiotic
susceptibility, which is attributable to a concerted action of multidrug. Pseudomonas
aeruginosa is naturally resistant to a large range of antibiotics and may demonstrate additional
resistance after unsuccessful treatment; also it have ESBLs enzymes that mediate resistance to
extended-spectrum (third generation) cephalosporins (ceftazidime, cefotaxime, and
ceftriaxone) and Monobactam (Aztreonam) but do not affect Carbapenem (meropenem or
imipenem), (Lee et al., 2005).
Antimicrobial susceptibility was performed on 15 P.aeruginosa isolates against 12
antibiotics 7 of them were ESBLs represented by Cefotaxime, Ceftriaxone, Ceftazidime,
Imipenem, Aztreonam, Piperacillin and Cefepime, and to 5 antibiotics were non ESBLs
represented by Aminoglycoside (Amikacin, Gentamicin and Tobramycin), Chloramphenicol
and Fluoroquinolone (Ciprofloxacin), by the disc diffusion method (DDM), as described by
(Bauer et al., 1966). The antibiogram for studied isolates was revealed that all isolates
(100%) resist to Ceftrixone, Cefepime, Chloramphenicol and Tobramycin, and this resistance
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became 93.3% against Gentamicin, while each for 86.6% against Cefotaxime, Ceftazidime
respectively, followed by 80% for Piperacillin, and lower resistance 60% for Aztreonam,
Amikacin respectively and 46.5% for Ciprofloxacin. Pseudomonas aeruginosa are becoming
resistant to commonly used antibiotics and gaining more and more resistance to newer
antibiotics (Rajat et al., 2012). This study found that Imipenem is the drug of chose in
treatment of P. aeruginosa , because 66.6% of isolates were susceptible to it and only five
isolate were exhibit resistance (Table-6).
Table -6: Antibiotic susceptibility of Pseudomonas aeruginosa isolates
Antibiotics
Disk content
Cefotaxime
Ceftrixone
Ceftazidime
Imipenem
Aztreonam
Amikacin
Gentamicin
Ciprofloxacin
Piperacillin
Cefepime
Chloramphenicol
Tobramycin
30µg
30µg
30µg
10µg
30µg
30µg
10µg
5µg
100µg
30µg
30µg
10µg
Resistant
No.
%
13
86.6
15
100.0
13
86.6
5
33.3
9
60
9
60
14
93.3
6
40
12
80
15
100.0
15
100.0
15
100.0
Intermediate
No.
%
1
6.6
1
6.6
1
6.6
-
Sensitive
No.
%
2
13.3
1
6.6
10
66.6
6
40
5
33.3
1
6.6
9
60
2
13.3
-
This is called multiple drugs resistance (MDR). MDR P. aeruginosa has been previously
reported (Loureiro et al., 2002).This resistance results from the complex interaction of several
mechanisms, which tend to inactivate the antibiotics or prevent their intracellular
accumulation to inhibitory levels (Hancock and Speert, 2000).
The outcome of PCR amplification of ESBLs (bla) genes in environmental P. aeruginosa
DNA clarified that almost all isolates were ESBLs producer.
The bla OXA-10 genes was detected in all isolates 15(100%), while blaVEB-1 gene in
6(40%) isolates were positive for gene by used multiplex PCR technique (Figure -1). OXA-10
β-lactamase also possesses the ability to hydrolyze cephalosporins, hydrolyzing cefotaxime,
ceftriaxone, and aztreonam at low levels but sparing ceftazidime, cephamycins, and
carbapenems . The blaOXA-10 gene is encountered in a large variety of gram-negative
species, being the bla OXA gene identified most frequently in P. aeruginosa (Mirsalehian et
al., 2010). VEB-1 (for Vietnamese extended spectrum beta – lacctamase) was first reported in
1996 in relation to an E. coli isolate from a Vietnamese patient, is plasmid- and integronlocated , VEB-1 has highest amino-acid identity with PER-1 and PER- 2 (38%), and confers
high-level resistance to ceftazidime, cefotaxime and aztreonam. The blaVEB-1 gene was
detected in two P. aeruginosa isolates from Thailand, where it was chromosomal- and
integronlocated (Girlich et al., 2002). The structure of the P. aeruginosa genome is a mosaic
to be the result of multiple acquisitions from different donors during its evolution, to
horizontal gene transfer includes the presence of genes or remnants of genes associated with
6
mobile elements (i.e., insertion sequences, bacteriophages or plasmids) and the presence of
numerous genomic islands (Kulasekara and Lory, 2004).
1
2
3
4
5
M
6
643 bp
600 bp
300 bp
277 bp
100 bp
Figure -1: Gel electrophoresis for Multiplex PCR product for detection of ESBL
blaVEB-1, and OXA-10 gene. Using 1%agarose for 90 minutes at 70 v\cm
(1). M: Marker DNA lader Size (100bp).
(2). Lanes (1-6) positive multiplex PCR for blaOXA-10 (277bp) and blaVEB-1
(643 bp).
7
Many researchers are using molecular methods such as pulsed-field gel electrophoresis
(PFGE) to examine epidemiology with the strains involved in outbreaks of infections caused
by ESBLs . Other methods for studying the epidemiology of these strains include plasmid
profiles, random amplified polymorphic DNA (RAPD), and arbitrarily primed PCR , these
outbreaks often start in an ICU and then spread to other parts of the hospital by the usual
transmission routes .Very often, the exact source of outbreaks caused by ESBL-producing
organisms is never identified (Bermudes et al., 1997).
Multiplex PCR because is a widespread molecular biology technique for amplification
of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target
sequence can be amplified by using multiple primer pairs in a reaction mixture. As an
extension to the practical use of PCR, this technique has the potential to produce considerable
savings in time and effort within the laboratory without compromising on the utility of the
experiment (De Vos et al., 1997; Curran et al., 2004).
Multiplex PCR technique was used to distinguish to genes of P. aeruginosa isolate,
which were, firstly the OXA-10 gene, which represent by band appeared of 277bp and VEB1 gene which appeared of 643bp, the results confirmed our previous results (monoplex
PCR), and showed a high degree of specificity because the DNA of isolate did not represent
any other amplification when examined with UV eliminator .
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10
Effect of Different Parameters for the Enhancement Removal of Methylene Blue
Dye from aqueous Solutions by using Attapulgite Clay Surface.
Aseel M. Aljeboree
1
2
1
Abbas Noor Alshirafawi Ayad F. Alkaim ()
1
Babylon university/ college of science for women/ Chemistry Department/Hilla, Iraq
2
Babylon university/ college of science / Chemistry Department/Hilla, Iraq
Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher)
ABSTRACT
The present study shows that the HCl-modified attapulgite clay powder can be used as a potential adsorbent
for the removal of methylene blue dye from aqueous solutions. Experiments were carried out as a function of
concentration, pH, particle size and dosage, the equilibrium was attained in 60 min. The amount of dye
uptake (mg/g) was found to decrease with increase in pH, mass dosage, and particle size. The applicability of
the isotherm's model for the present data follows the order: Freundlich > Langmuir > Temkin .
KEYWORDS
Adsorption; attapulgite clay; particle size; methylene blue.
1. Introduction
Textile dyes are the essential cause of color in the discharged wastewaters[1]. Dye molecules
present a considerable structural diversity, allowing them to be classified by several manners [2]:
either according to their chemical structures or their applications with suitable type of fiber. They
can also be classified with respect to their solubility in water[3]. Acid, basic, reactive and direct
dyes are soluble dyes, while dispersed pigments and oxidized dyes are insoluble in water.
Address correspondence Aseel M. Aljeboree, [email protected] ; Ayad F. Alkaim, [email protected]
11
The wastewater treatment is required by various obligations which are environmental or
concerning with public health. Dye wastewaters represent the most amounts of the discharged
industrial water [4].
Several physical or chemical processes are used to treat dye laden wastewaters. These
processes include flocculation, precipitation, ion exchange, membrane filtration, electrochemical
destruction, irradiation and ozonation. However, these processes are costly and lead to generation
of sludge or formation of by-products[5]. Among the physical methods available, adsorption
process is one of the most efficient methods to remove dyes from wastewater, especially if the
adsorbent is inexpensive and readily available [6]. Activated carbon is the most widely used
adsorbent for dye removal, but it is too expensive [7], consequently, numerous low-cost
alternative adsorbents have been proposed including: peat [8], sepiolite [9], montmorillonite,
chitosan and nanocomposite [10], and pine sawdust [11]. Clays are mostly used as available
adsorbent and catalyst[12].
Methylene blue (MB), an organic dye, has wide applications including cottons or wools dyeing,
paper coloring, temporarily coloring hair, and coating for paper stock[13]. Due to its known strong
adsorption onto solids, MB often serves as a model compound for removing dyes and organic
contaminants from aqueous solutions.[14] Although not strongly poisonous, MB can have some
harmful effects on human beings [15].
Attapulgite (AT, or palygorskite as it often called), as a natural nanostructure material can
be used as a matrix [16, 17]. AT is a crystalline hydrated magnesium aluminum silicate with
reactive –OH groups on its surface[18]. AT has advantages of specific features in dispersion, high
temperature endurance, salt and alkali resistance, high adsorption and penetrability due to its
regular structure and large specific surface area. Because of its unique structure, AT has been used
as adsorbent [19], and drilling muds[20].
Clay based adsorbents have also emerged as promising adsorbents for wastewater
treatment [21]. Clays possess certain properties which make them an ultimate choice for
adsorption process such as low cost, high availability, and environmentally friendly material.[22,
23]
The mechanism of interaction between clay mineral and dye molecule in liquid phase is
more complex than that of traditional materials considering its unique structure, and is the
research hotspot of nano-mineralogy, geology and environmental science [24].
12
Therefore, the objective of this investigation was to explore the potential of attapulgite clay
as a low cost adsorbent for the removal of MB dye from aqueous solutions. The present study
describing the effects of initial solution pH, adsorbent dose, particle size, and initial dye
concentration.
2. Adsorption experiments
2.1 MATERIALS
Attapulgite clay used in this study was obtained from the general company for geological
survey and mining, Baghdad, Iraq, have the general structure consisting mainly
2[(OH2)4(Mg,Al,Fe)5(OH)2Si8O20]4H2O, the chemical analysis of attapulgite is listed in Table 1.
Attapulgite used in this study was washed with (5% HCl) to remove soluble materials after
washing with distilled water for several times. The product was dried at 105 °C and sieved until
similar-sized particles were collected, Then clcinated in an oven at (3000C) for 1 hour to get a
constant weight. Dry Attapulgite was crashed into powder and sieved to (75μ) particle size, then
preserved in the desiccators for use.
Table 1: Chemical composition of attapulgite sample[25]
Component
SiO2
MgO
Al2O3
Fe2O3
CaO
TiO2
MnO
K2O
Na2O
P2O5
SO3
Weight (%)
66.43
13.52
10.25
5.51
1.82
0.53
0.32
1.12
0.11
0.26
0.02
2.2 Chemicals
Methylene blue (99.9% from Aldrich) was used as model adsorbate. A stock solution of
1000 mg L−1 was prepared by dissolving an appropriate quantity of MB (1 gm) in a liter of distilled
13
water. The working solutions were prepared by diluting the stock solution with distilled water to
give the appropriate concentration of the working solutions.
2.3 Analysis
The methylene blue concentration in the supernatant solution was analyzed using a UV
spectrophotometer by monitoring the absorbance changes at a wavelength of maximum
absorbance of 663 nm .[26]
2.4 Adsorption studies
Batch adsorption experiments were performed in a set of Erlenmeyer flasks (250 mL) each one
containing 100 mL of different initial concentrations of MB (2–10 mg L−1) together with 0.5 g of
adsorbents at a particle size of 75 μm. A mechanical magnetic stirrer was used at a desired
temperature and pH. To reach equilibrium a time contact equal to 60 min was fixed for all
experiments. After 60 min the filtrate of the solution is recovered to be analyzed by a UV/vis
spectrophotometer with λ = 663 nm, which is appropriate to the maximum absorption of the light
for different time, while the pH was gradually adjusted by adding small amounts of diluted HCl or
NaOH solutions (0.1 mol L−1).
The adsorbed amount of MB at equilibrium; qe (mg g−1) was calculated by the following equation,
Eq. (1): [27]
The amount of dye adsorbed per gram of adsorbent (qe) is given in Eq. (1), and the percentage
removal (R) was calculated by using Eq. (2).
(
)
( )
( )
( )
where Co and Ce are the initial and equilibrium CV concentrations, respectively (mg L−1), V is the CV
solution volume (L), m is the mass of the adsorbent (g).
2.5 Effect of pH
The effect of pH on the amount of color removal was analyzed over the pH range from 3 to 10.
The pH was adjusted using 0.1N NaOH and 0.1N HCl solutions. In this study 100mL of dye solution of
14
2-10 mg/L was agitated with 0.5g of attapulgite for 1h, which is more than sufficient to reach
equilibrium. The samples were then centrifuged and the left out concentration in the supernatant
solution were analyzed using UV Spectrophotometer by monitoring the absorbance changes at a
wavelength of maximum absorbance (663 nm).
2.6 Effect of adsorbent dose
100 mL of dye solution was prepared in different conical flasks with dye concentrations of (210 mg.L-1) and adsorbent doses of (0.5, 1.0 and 2.0 g/100ml). The solutions were kept inside the
shaker at 100 rpm and 25
0
C. The final concentration of dye was measured using a
spectrophotometer.
2.7 Effect of particle size
The study was carried out with different particle size (100,150, and 200) mesh, (0.5,1.0,2.0
gm)dose of adsorbent attapulgite mixing with (100 ml) of (2-10) mg.L-1 aqueous solution of dye
concentration, and the sample was shaking a period for (1 hour) at a fix temperature (25 0C) at
pH5.
3 Results and Discussion
3.1 Effect of particle size
The particle size of adsorbent inversely influences the adsorption process. Smaller the particles,
higher the adsorption efficiency and capacity due to the large total surface area of the adsorbent
[28]. The particle size of attapulgite clay was varied from 300 mesh to 100 mesh as shown in Figs.
(1, and 2). The smallest particle size (300 mesh) resulted in the maximum adsorption capacity
(1.8011 mg/g) and the highest dye removal (90.05 %), respectively. This is due to the increase in
the available surface area with the decrease in particle size. [26]
15
3.2 Effect of adsorbent dosage:
Adsorbent dosage is an important parameter because this factor determines the capacity of an
adsorbent for a given initial concentration of the adsorbate[29, 30]. The effect of mass dossage on
the uptake of MB onto attapulgite clay was studied at T = 298 K and C0 10 mg/l and the results are
shown in Figs. (1, and 2). The removal of MB was found to be increasing with an increase in the
mass from 0.5 to 2 gm. Initially, rapid increase in adsorption with the increase in adsorbent dose
can be attributed to greater surface area and the availability of more adsorption sites [31-33]. Also
it can be observed that the MB adsorption capacity decreases with increasing adsorbent. This is
because the quantity of dye adsorbed per unit weight of the adsorbent is reduced causing a
decrease in the utility of active sites [34, 35].
% of removal
20.0
100 mesh
17.5
15.0
12.5
200 mesh
% of removal
80
75
70
% of removal
96
300 mesh
93
90
87
0.5
1.0
1.5
2.0
mass of Adsorbent
Fig.1 : Effect of particle size on the removal percentage of MB dye in the presence of different
mass dosage.
16
0.260
100 mesh
qe/ mg.g
-1
0.208
0.156
0.104
200 mesh
qe/ mg.g
-1
1.28
0.96
0.64
0.32
qe/ mg.g
-1
1.88
300 mesh
1.41
0.94
0.47
0.5
1.0
1.5
2.0
mass of adsorbent/ g
Fig.2: Effect of particle size on the adsorption capacity of MB dye in the presence of different
mass dosage.
17
3.3 Effect of initial pH
One of the main variables affecting the adsorption process is pH [32, 36] influencing not only the
surface charge of the sorbent, the degree of ionization of the material present in the solution and
the dissociation of functional groups on the active sites of the sorbent, but also the solution dye
chemistry[37]. In this work, we investigated the effect of four initial solution pH (i.e., 3, 5, 8 and 10)
on the removal rate of MB dye by attapulgite clay. The adsorption capacity for dye was decreased
when the initial pH increased from 3 to 10 results are shown in Fig.3. The pH effect on dye
adsorption observed in this study may be explained by protonation of clay surfaces and
electrostatic interaction between clay and dye molecules. Another hypothesis would be the
preliminary protonation of the coloring agent in acid environment and then the binding on the clay
by cationic exchange[38]. Other studies they are finding that the adsorption process observed no
significant improvement beyond pH > 5. Nandi et al. [31] and Shirsath et al. [39] had also optimized
MB adsorption on clay at pH 5.
adsorption capacity
% of Removal
1.9
100
1.8
qe/ mg.g
-1
80
1.7
70
1.6
60
1.5
50
2
4
6
8
% of Removal
90
10
pH
Fig.3: effect of solution pH on the adsorption capacity, and removal percentage of MB dye.
3.4 Equilibrium isotherms studies
The analysis of the equilibrium adsorption isotherm model is a prerequisite for predicting the
adsorption uptake of the adsorbent, which is one of the main parameters required for designing an
optimized adsorption system.
18
Several isotherm models have been used to predict validity of the experimental data. In the present
study, three of the most commonly used models, namely the Langmuir, Freundlich and Tempkin
isotherms were used to describe the adsorption equilibrium.
The non-linear form of the Langmuir isotherm model[40] is given as:
( )
where KL (L.mg-1)
is
the
Langmuir
adsorption
constant
related
to
the
energy
of
adsorption, qmax and qe (mg/g) are the maximum and equilibrium adsorption capacity, respectively.
Langmuir constants generated from adsorption data plot of qe against Ce shown in (Fig. 4) are
summarized in Table 2.
The Freundlich isotherm is based on the premise that adsorption occurs on rare heterogeneous
surfaces sites with different energy of adsorption and are also non-identical. The non-linear form of
the Freundlich isotherm was used to investigate the adsorption process adherence to the model
[41]:
( )
kf can be defined as the adsorption or distribution coefficient and represents the quantity of
dye adsorbed onto adsorbent for unit equilibrium concentration. 1/n is the heterogeneity factor and
n is a measure of the deviation from linearity of adsorption. Its value indicates the degree of nonlinearity between solution concentration and adsorption as follows: if the value of n is equal to unity,
the adsorption is linear; if the value is below to unity, this implies that adsorption process is
chemical; if the value is above unity adsorption is a favorable physical process.[42] The values of the
model parameters obtained from the plot of qe against Ce shown in (Fig. 4) are presented in Table 2.
The non-linearized form of Temkin isotherm[43] is represented by Eq (5):
(
)
( )
19
where b Tempkin constant related to the heat of adsorption(kJ/mol), R : Gas constant (8.314 J.mol 1
.K-1), T : Temperature (K), and KT : Emprical Temkin constant related to the equilibrium binding
constant related to the maximum binding energy (L.mg-1), (L.mol-1). The adsorption data can be
analyzed according to the Eq (5). A plot of the qe versus log Ce shown in figure 4, enables the
determination of the isotherm constants KT and b shown in table 2.
2.0
qe/ mg.g
-1
1.5
1.0
Experimental
Langmuir Model
0.5
Freundlich Model
Tempkin Model
0.0
0.0
0.1
0.2
0.3
0.4
0.5
-1
Ce/ mg.L
Fig. 4: adsorption isotherms models of MB dye on the surface of attapulgite clay.
20
Table 1: Isotherm constants for various parameters adsorption isotherms for the adsorption of
MB dye onto attapulgite clay surface.
Isotherm
models
Langmuir
Freundlich
Temkin
Standard Error
Parameters
Value
qm (mg.g-1)
4.37265
0.44805
KL(L.mg-1)
1.61529
0.25069
R2
0.99484
----
F value
3240.37341
----
prob>F
3.16506E-8
---
KF
3.29878
0.02179
1/n
0.71616
0.00526
R2
0.99983
---
F value
95994.48455
---
prob>F
6.64724E-12
---
B/J.mole-1
2.25563
0.12607
KT
0.67285
0.0666
R2
0.96546
---
F value
284.29703
---
prob>F
1.34166E-5
---
4. Conclusion
The present study shows that the activated attapulgite can be used as a potential adsorbent for
the removal of methylene blue dye from aqueous solutions. The operational parameters such as
pH, adsorbent dose, and particle size were found to have an effect on the adsorption efficiency of
attapulgite clay. The adsorption was highly dependent on solution pH. The dye was optimally
adsorbed at pH 3.
The applicability of the three isotherm models for the present data follows the
order: Freundlich > Langmuir>> Temkin .
21
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Address correspondence Aseel M. Aljeboree, [email protected] ; Ayad F. Alkaim, [email protected]
25
An ecological assessment for Tigris River within Baghdad, Iraq
Fikrat M. Hassan1*
Najm A. J. AL-Zubaidi
Weeam A. A. Al- Dulaimi
1
College of Science for Woman, University of Baghdad, Baghdad, IRAQ. 2 College of PureEducation, University of Diyala , IRAQ.
* Email [email protected]
Abstract:
The present study has been conducted to assess the Tigris River within Baghdad City during
one year from October 2011 to December 2012. Four sites along the studied river were selected; the
study included measuring physiochemical factors of the Tigris River, such as: temperature (air and
water), electrical conductivity (EC), salinity (S‰), current flow, total dissolved material (TDS),
total solid suspended (TSS), total alkalinity (TA), dissolved oxygen (DO), biochemical oxygen
demand (BOD5) , total nitrogen (TN) and total phosphorus (TP). The water quality of Tigris River
was assessed using the Canadian Council of Ministry of the Environment Water Quality Index
(CCME WQI). Seven environmental factors were used to assess the WQI, they are temperature, pH,
TDS, DO, BOD5, TN and TP. The mean ranges of the studied factors were recorded: air
temperature ( 13.00-43.20 C°), water temperature (10.80-32.43 C°), current flow (0.07-0.303)
m/Sec, pH (7.94-8.62), EC ( 214.33-319.67 µS/cm), S‰ (0.130-0.191), TDS (154.50-211.83 mg/l),
TSS (1.52-1.72 mg/l), TA (44.33-80.17 mg/l), DO (6.70-13.50 mg/l), BOD5 (0.04-4.26 mg/l), TN
(14.01-19.65µg/l) and TP (0.005-0.023 µg/l ). The WQI values ranged 20.32 - 60.48. Hence, the
water quality of the Tigris River is rated as poormarginal. A statistical analysis was done using the
canonical correspondence analysis (CCA).
Keywords: Water quality index, physicochemical parameters, Tigris River, Iraq.
1. Introduction:
Tigris River is suffering from different agricultural drainages, industrial discharge
and domestic's disposal during its passing in different cities in Iraq terrain in addition to the
impact of Tharthar Lake and Diyala River (1, 2, 3, 4). Many previous researches on the Tigris
River showed a significant decline in its water quality (5, 6). Recently, few studies focused
on water quality index to fill the gap of information in this issue (7, 8 ). Less attention from
government authorities to prevent the decline in water quality of the main Iraqi water
26
resources is one of importance to use the water quality index in Iraq in order to simplify the
results of many data of water quality data. The industrialized countries developed many
criteria of water quality since 1965 when Horton (9) suggested water quality index. The
water quality index illustrates all physical and chemical features of river or an aquatic
system by simple decision whether an aquatic system is valid for different human uses or for
aquatic organism lives (10).
The present study aimed to fill the gap of information on water quality of the studied area.
2. Materials and Methods:
Four sites were chosen along the Tigris River within Baghdad City (Figure 1). Table 1 shows
the GPS values of the selected sites for the present study.
The present study was carried out from October 2011 to December 2012. Physical and
chemical properties of the river water (temperature (air and water), EC, S‰, current flow, TDS,
TSS, TA, DO, BOD5, TN and TP) were measured according to (11). The water quality was
assessed using the Canadian Council of Ministry of the Environment Water Quality Index (12).
Three measures were selected to calculate WQI (Scope, frequency, amplitude) by using the
following formula:
{
}
{
Eq1
}
{
{
∑
Eq2
}
Eq3 for values higher than Standard values
}
Eq4 for values lower than Standard values
Eq5 nse = total excursion
Eq6
27
The equations 1 and 2 for calculated Scope and frequency, respectively. While values of Amplitude
( ) was calculated by using the other equations (3, 4 and 5). The values of these three measures
were used in the following formula to calculate WQI:
√
The classification of water quality as poor, marginal, fair, good or excellent followed Al-Janabi et
al. (7). The statistic analyses were done by using correlation coefficient (r), and canonical
correspondence analysis (CCA). CCA method was conducted by using the computer program
CANOCO, version 4.5 in order to clarify the relationships between physiochemical parameters.
3. Results and Discussion:
The environmental characteristic of the water in the study area is shown in Table (2). The air
temperature reached its high value ( 43.20 c0 ) in Site 4 and its lowest value ( 13.00 c0 ) in Site 1 ,
while the water temperature ranged between 9.06 to 31 .43 in sites 1 and 4 respectively (Figure 2).
Narrow fluctuation of pH was observed during the study period, with the highest average value of
8.46 and a low of 7.8; this observation was mentioned earlier in Iraqi aquatic systems by Talling
(13). PH variation might be caused by many factors such as biotic activities, discharge of waste
water, photosynthesis and nature of river basin (14, 15). Similar results match with other studies (15,
16, 17, 18, 19, 20, 21).
High conductivity values were (319.67 µs. cm-1) at site3 in summer 2012, lower value was
(241.33 µs. cm-1) at site2 in spring 2012, while water salinity in the study area ranged (0.130 – 0.191
‰) at sites 4 and 1 in winter 2011 and summer 2012, respectively. The increasing values of
conductivity and salinity in the Tigris River during hot seasons indicated the increase of the
evaporation rate due to the discharge of agricultural and industrial wastewater (22, 23)
The total dissolved solid (TDS) represented all inorganic salts, organic material and other
soluble material in water. These constituted may be found naturally or due to discharge of
municipal, industrial and agricultural, so its values followed the trend as conductivity and salinity. It
ranged from 154.50 mg\l at site 1 spring 2012 to 211.83 mg\l at site 4 in summer 2012. Significant
differences between seasons were noticed at P<0.05. TSS values ranged (1.52 to 1.72 mg\l) at sites 2
and 4 during autumn 2011 and winter 2012, respectively (Fig.3). Many factors affecting TSS values
in water such as silting, microscopic organisms and suspended organic matter lead to catch the dust
and other materials not drawn into the water column and subsequently deposited on the bottom of
the river (21, 24). No significant differences between seasons and sites except between sites 2and 4
were noticed at P<0.05.
Total alkalinity is a function of water contents from hydroxide, bicarbonate and carbonate,
its values used to find out the validity of the water to be used in different purposes (11). Many
authors mentioned that the Iraqi water systems characterized as alkaline water due to the presence of
28
bicarbonate ions (25, 26). Its values in this study ranged (44.33- 80.17 mg/l) during autumn 2011
and summer 2012 at sites 4 and 3, respectively.
Concentration of dissolved oxygen is not recorded less than 6 mg/l in the present study. This
concentration ranged from 6.70 mg\l to 13.50 mg\l at sites 2 and 3 in autumn 2011 and spring 2012
respectively. The concentration of dissolved oxygen was affected by many factors especially
biological activities such as photosynthesis, respiration and decomposition process at the river
bottom in addition to the rainfall effects ( 27, 28, 29 ). The dissolved oxygen concentration was
found to be within the standard limited (30) in the present study. Only a significant difference
between sites 2 and 3 was observed at P<0.05.
The concentrations of BOD5 ranged from 0.04 mg\l at site 4 in autumn 2011 to 4.26 mg\l at
site1 in summer 2012. These results revealed that the study river may be suffering from pollution
(11, 26, 31). These BOD5 values may be attributed to increase in temperature during the hot months
in addition to different human activities (32).
Significant differences between sites 2 and 4 were observed at P<0.05. The water flow results
of the present study showed high flow after flooding months (early summer in Iraq), and ranged
(0.07- 0.303 m/sec at sites 3 and 1 in autumn 2011 and winter 2012 respectively. The fluctuations in
water flow are related to river channel morphology (33). Only in site 3, significant differences were
noticed among the study seasons and also significant differences recorded among sites during the
study per season except in summer 2012 at P<0.05.
Total nitrogen concentration ranged (14.01-19.65 µg/l) at sites 1 and 4 during winter and
summer 2012, respectively. The results showed no clear seasonal variation during the study period,
the source of nitrogen in aquatic systems included different chemical reactions in water, rainfall,
inputs from human activities such as; municipal, agricultural and industrial (33, 34). Significant
differences, noticed in two sites during the study seasons at level P<0.05 and no significant
differences among studied sites except in site 4, may be due to dense growth of macrophytes.
Moreover, this site is exposed to the effect of residential communities and industrial plants.
Most of phosphorus forms in environment are low solubility in water (35). Different forms
of phosphorus (inorganic and organic) found as soluble or particulate (36). The present results show
no clear variation among months that may indicate the effect of different types of discharge sources
into the river and due to rainfall that washed away soil compounds and Phosphorus agricultural
fertilized land (36, 37). TP concentrations ranged (0.005-0.023 µg/l) in sites 1 and 4 during summer
2012 and autumn 2011, respectively.
CCM WQI results revealed that the water quality of the Tigris River ranged between poormarginal. The higher value (60.48) was recorded at site 2 in summer 2012, while the lower value
was 20.32 at sit 3 in winter 2012. The WQI in all study sites ranked as marginal except in site 1 as
poor. Two studied parameters (BOD5 and TN) exceeded the standard limits.
29
CCA for water quality in the present study (Figure 4) indicated that weak positive
relationships are found between water temperature and BOD5 (r=0.101, P<0.01), while negative
relationships are found between DO and temperature ( r= -0.539, P<0.05) .Similar results appeared
between EC, TDS and temperature (r=-0.743) that may be due to evaporate effect. Positive
relationships were recorded between EC and TDS (r= 0.823), due to the relation between the
quantity and quality of dissolved salts and EC (38). Also positive relationships were found between
pH and salinity (r=0.673) because of salinity effects on solubility of CO2HCO3− and CO3= (39 ).
Positive relationships were found between total alkalinity and water flow. These results may not
agree with another study which showed negative relationships between them (40).
4. Conclusion
The present study results revealed that temperature and total nitrogen play important roles in
the water quality of Tigris River in this study. The rank of WQI for Tigris River was poor-marginal.
5. Acknowledgements
We are grateful to Department of Biology, College of Science for Women, University of
Baghdad and Department of Biology, College of Pure-Education, University of Diyala for their
support to this research.
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Changes in
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29. Amer, A. S. and Abd El-Gawad, H. A. 2012. Rapid Bio-Indi- cators Assessment of
Macrobiotic Pollution on Aquatic Environment. International Water Technology
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30. UNESCO-WHO-UNEP. 1996. Water Quality Assessment—A Guide to the Use of
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31. Adakole J.A., J.K. Balogun and A.K. Haroon. 1984. Water quality Impacts Assessment
Associated with an Urban Stream in Zaria, Nigeria . NISEB Journal , 2 (3): 195 – 203 .
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34. Lampert, W. and Sommer, U. 1997. Limnoecology: The ecology of lakes and streams.
Oxford University Press.
35. Goldman, C.R. & Horne, A.J. (1983). Limnology. McGraw-Hill Int. B. Co. 464 pp.
36. Sims, J.T & Sharpley, A. (2005). Phosphorus: Agriculture and the Environment. Publisher
by American Society of Agronomy-Crop Science Society of America-Soil Science Society
of America; illustrated Edition, 1121pp.
37. Yeoman, S.; Stephenoson, T.; Lester, J.N. & Perry, R. (1988). The Removal of
Phytoplankton during Waste Water Treatment. A review. Environ. Pollution, 49: 183- 233.
38. Weiner , E . R. (2000). Application of environmental chemistry . Lewis Puplshers , London ,
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39. Golterman, H. L. (1975). Chemistry Chapter 2. In. Witton, B.A. (Editor) River Ecology,
Studies In Ecology. Vol. 2 Blackwell Scientific Publication, pp. 39-80.
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Studies. Environ. Publ., Karad.
33
Figure (1): Map of the study area
34
Table1: GPS values of the study sites
Sites
1
2
3
4
Longitude (East)
44°20'14.57"
44°21'24.60"
44°23'0.19"
44°31'2.31"
Latitude (North)
33°23'03.37"
33°21'33.75"
33°15'54.23"
33°13'57.14"
Table2: Range (mean ±SE) of Physical and chemical properties of water in Tigris river from
Autumn 2011 to Summer 2012.
Parameters
1
2
3
4
Water temp ( C ) 28.30-11.46
(1.72±0.77)
30.06-10.80
(0.61±0.55)
31.50-12.40
(0.58±0.30)
32.43-12.76
(2.83±0.26)
pH
8.22-7.94
(0.03±0.10)
203.33-161.56
(26.82±7.65)
11.63-6.70
(1.12±0.40)
3.37-0.65
(0.50±0.40)
19.20-16.24
(1.86±0.72)
0.016-0.008
(0.01±0.002)
8.57-8.06
(0.13±0.07)
207.63-173.60
(17.06±12.88)
13.50-8.86
(0.50±0.93)
3.50-2.53
(0.37±1.33)
18.40-14.73
(0.67±0.80)
0.018-0.008
(0.015±0.002)
8.59-8.32
(0.10±0.09)
211.83-173.53
(23.34±9.24)
10.06-7.53
(0.29±0.58)
2.93-0.55
(0.53±0.03)
19.65-15.85
(1.59±1.65)
0.023-0.008
(0.02±0.002)
ᴏ
TDS (Mg/L)
DO (Mg/L)
BOD5(mg/L)
T.N(mg/L)
T.P( mg/L)
8.62-8.08
(0.17±0.02)
189.96-154.50
(11.58±2.63)
11.46-8.36
(1.24±1.80)
4.26-2.73
(2.21±1.27)
18.80-14.01
(1.68± 0.47)
0.010-0.005
(0.00±0.002)
35
40
60
40
W.T
0
0
9
350
8.5
300
PH
8
E.C
A.T
20
20
250
7.5
200
300
200
250
T.D.S
300
S‰
350
100
0
200
90
0.5
80
0.45
70
0.35
W.F
T. Alk
0.4
60
50
40
Autumn2011
Winter2012
Spring2012
Summer2012
0.3
0.25
0.2
0.15
0.1
0.05
Autumn2011
Winter2012
Spring2012
Summer2012
Figure 2: Season variation of the studied parameters ( Air temperature (AT), Water Temperature(WT), pH,
EC, S‰, TDS, Total alkalinity (T.ALK) and Water flow(W.F)) during the study period in Tigris River.
36
1.75
1.7
1.65
1.55
D.O
T.S.S
1.6
1.5
1.45
1.4
15
14
13
12
11
10
9
8
7
6
5
20
19.5
19
18.5
18
17.5
17
16.5
16
15.5
15
14.5
14
BOD5
T.N
15
14
13
12
11
10
9
8
7
6
5
Autumn2011
Winter2012
Spring2012
Summer2012
0.024
0.02
T.P
0.016
0.012
0.008
0.004
0
Autumn2011
Winter2012
Spring2012
Summer2012
Figure3: Season variation of the studied parameters (TSS, DO, BOD5, TN, TP) during the study period in Tigris
River.
37
0.6
PH
cha P
TN
pha P
Sal
WT
cha C
BOD5
EC
D O
TDS
T k
TSS
WF
AT
pha C
-0.6
TP
-0.4
0.6
Figure (4): Correlations between physical and chemical parameters according to Canoco(CCA).
38
‫‪Jornal ofthBabylon Univ. Special Issue - Proceding of 5th International Conference of Environmental Scince University of Babylon / Environmental Research Center 3-5 December 2013‬‬
‫‪5 International Conference for Environmental Researcuys-Environmental Researc‬‬
‫اىزق‪ ٌٞٞ‬اىج‪ٞ‬ئ‪ ٜ‬ىْٖش دعيخ ضَِ ٍذ‪ْٝ‬خ ثغذاد‪ ,‬اىعشاق‬
‫*‪1‬‬
‫فنشد ٍغ‪ٞ‬ذ حغِ‬
‫ّغٌ عجذ هللا عَعخ ٗئبً احَذ عي٘اُ اىذى‪َٜٞ‬‬
‫‪1‬مي‪ٞ‬خ اىعيً٘ ىيجْبد – عبٍعخ ثغذاد مي‪ٞ‬خ اىزشث‪ٞ‬خ ىيعيً٘ اىظشفخ‪ -‬عبٍعخ د‪ٝ‬بى‪ٚ‬‬
‫* ‪[email protected]‬‬
‫اىَغزخيض‪:‬‬
‫أعش‪ ٝ‬ذ اىذساعخ اىحبى‪ٞ‬خ ىزق‪ّٖ ٌٞٞ‬ش دعيخ داخو ٍذ‪ْٝ‬خ ثغذاد خاله فزشح عْخ ٗاحذح ٍِ أمز٘ثش ‪ 2011‬إى‪ ٚ‬د‪ٝ‬غَجش ‪ 2012‬رٌ اخز‪ٞ‬بس‬
‫أسثعخ ٍ٘اقع عي‪ ٚ‬ط٘ه ّٖش دعيخ ؛ ٗشَيذ اىذساعخ ق‪ٞ‬بط اىع٘اٍو اىف‪ٞ‬ض‪ٝ‬بئ‪ٞ‬خ ىْٖش دعيخ ‪ٍ ،‬ضو ‪ :‬دسعخ اىحشاسح (اىٖ٘اء ٗ اىَبء) ‪،‬‬
‫اىز٘ط‪ٞ‬و اىنٖشثبئ‪ٗ ، ) EC( ٜ‬اىَي٘حخ (‪ ، ) S‰‬عشعخ اىغش‪ٝ‬بُ ‪ٗ ،‬اىَ٘اد اىزائجخ اىني‪ٞ‬خ (‪ ، ) TDS‬اىَ٘اد اىظيجخ اىعبىقخ ( ‪)TSS‬‬
‫‪ ،‬اىقبعذ‪ٝ‬خ اىني‪ٞ‬خ ( ‪ ، ) TA‬األٗمغغ‪ ِٞ‬اىَزاة ( ‪ ، ) DO‬اىَزطيت اىح‪ ٛ٘ٞ‬ىألٗمغغ‪ ، ) BOD5 ( ِٞ‬اىْزشٗع‪ ِٞ‬اىني‪ٗ ) TN( ٜ‬‬
‫اىفغف٘س اىني‪ٗ . ) TP( ٜ‬رٌ رق‪ّ٘ ٌٞٞ‬ع‪ٞ‬خ ٍ‪ٞ‬بٓ ّٖش دعيخ ىَع‪ٞ‬شخ اإلح‪ٞ‬بء اىَبئ‪ٞ‬خ ثــــبعزعَبه دى‪ٞ‬ــــو ّ٘ع‪ٞ‬ـــخ اىَ‪ٞ‬ــــبٓ اىَعزَـــذ‬
‫عيـــ‪ ٚ‬اىَ٘د‪ٝ‬و اىنْــذ‪ٗ , ) CCM(ٛ‬اعزخذٍذ عجع ع٘اٍو ث‪ٞ‬ئ‪ٞ‬خ ىزق‪ّ٘ ٌٞٞ‬ع‪ٞ‬خ اىَ‪ٞ‬بٓ ٕٗ‪ ٜ‬دسعخ اىحشاسح ‪ ،‬األط اىٖ‪ٞ‬ذسٗع‪، ْٜٞ‬‬
‫اىَ٘اد اىظيجخ اىزائجخ ‪,‬األٗمغغ‪ ِٞ‬اىَزاة‪ ,‬اىَزطيت اىح‪ ٛ٘ٞ‬ىألٗمغغ‪ ,ِٞ‬اىْزشٗع‪ ِٞ‬اىني‪ ,ٜ‬اىفغف٘س اىني‪ . ٜ‬رشاٗحذ دسعخ حشاسح‬
‫اىٖ٘اء (‪ ، ) ° C 43.20-13‬دسعخ حشاسح اىَبء (‪ ، ) ° C 32.43-10.8‬عشعخ اىغش‪ٝ‬بُ(‪ / ً ) 0.07-0.303‬صب ‪ٗ ،‬األط‬
‫اىٖ‪ٞ‬ذسٗع‪ ، )8.62-7.94(ْٜٞ‬اىز٘ط‪ٞ‬و اىنٖشثبئ‪ٍ )319.67-241.33( ٜ‬ب‪ٝ‬نشٗعَْظ‪/‬عٌ‪ٗ ,‬اىَي٘حخ(‪)S‰ 0.191-0.130‬‬
‫‪ٗ،‬اىَ٘اد اىزائجخ اىني‪ٞ‬خ(‪ٍ)211.83-154.50‬يغٌ\ىزش ‪ٗ ،‬اىَ٘اد اىظيجخ اىعبىقخ ث‪ٍ )1.72-1.52( ِٞ‬يغٌ \ىزش ‪ٗ ،‬األٗمغغ‪ ِٞ‬اىَزاة (‬
‫‪ٍ (13.50-6.70‬يغٌ \ىزش ٗق‪ ٌٞ‬اىَزطيت اىح‪ٍ) 4.26-0.04( ٛ٘ٞ‬يغٌ \ىزش ‪ٗ،‬اىْزشٗع‪ ِٞ‬اىني‪ٍ ( 14.01 - 19.65) ٜ‬ب‪ٝ‬نشٗغشاً‪ /‬ىزش‬
‫) ٗاىفغف٘س اىني‪ٍ )0.023-0.005 (ٜ‬ب‪ٝ‬نشٗغشاً \ىزش‪ ,‬رشاٗحذ ق‪ ٌٞ‬دى‪ٞ‬و ّ٘ع‪ٞ‬خ اىَ‪ٞ‬بٓ ( ‪ٗ , )60.48- 20.32‬عي‪ ٔٞ‬رظْف ّ٘ع‪ٞ‬خ‬
‫اىَ‪ٞ‬بٓ ف‪ّٖ ٜ‬ش دعيخ ثزقذ‪ٝ‬ش حبف‪- ٜ‬فق‪ٞ‬ش‪ٗ .‬رٌ اعشاء اىزحي‪ٞ‬و اإلحظبئ‪ ٜ‬اىنّ٘ن‪ )CCA( ٜ‬ىْزبئظ اىذساعخ ‪.‬‬
‫‪39‬‬
Biodegradation of phenol- a plasmid activity in
Rhodococcus pyridinivorans GM3
Mahammed E Jabbar Al Defiery “()1”Gopal Reddy ()2
1
Local Environmental Research Center, Babylon University, Babylon, Iraq.
2
UCS, Osmania University, India.
Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the
publisher)
© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011
ABSTRACT
Biodegradation of phenol was studied by using bacterial isolate Rhodococcus pyridinivorans GM3. R.
pyridinivorans GM3 showed degradation of phenol at concentrations l.0 and 1.5 g/L within 18 and 24 hours. A
single plasmid (~11 Kb size) was isolated from R. pyridinivorans GM3 by electrophoresis separation of
plasmid. The location of phenol degrading genes was found to be on plasmid. This plasmid curing with
acridine orange and hexammine ruthenium (lll) chloride led to loss of phenol degrading ability by GM3.
KEYWORDS
Biodegradation, Rhodococcus, Plasmid, pyridinivorans.
>.> phenol-a plasmid
Introductions
Among the different toxic compounds, phenol is recognized as a pollutant and has a potential threat to human
health [1]. Phenol represents a serious ecological problem due to it’s wide spread use, toxicity and occurrence
throughout the environment; hence, it is necessary to develop efficient strategies for its waste management [2].
Genetic factors play important role in conferring biodegradation potentials on microorganisms. These factors
can affect degradation ability or metabolism of microorganisms by either preventing or stimulating growth of
the organisms and more subtly by affecting gene expression. The understanding of physiology and genetics of
such populations may prove very useful to assess and improve bioremediation, most importantly; they need to
identify general aspects in certain types of bioremediation [3]. Shimizu et al. [4] suggested that the linear
plasmid is a possible determinant responsible for propagation of the diverse degradative genes in rhodococci.
However, the plasticity of the rhodococcal genome is a feature of these bacteria and genomic rearrangements
40
Address correspondence to Mahammed, email1 [email protected]; Gopal, [email protected]
are clearly evident. Larkin et al. [5] reported that Rhodococcus has smaller circular plasmid and also harbor
many large linear plasmids that contribute to their substrate diversity and these appear to be vehicles for the
"mass storage" of numerous catabolic genes. Furthermore, Dabrock et al. [6] documented that Rhodococcus
erythropolis BD2, is able to utilize isopropylbenzene as a sole carbon and energy source and was shown to
contain a conjugative linear plasmid, the estimated size of pBD2 is 208 to 212 kb.
The effectiveness of bioremediation is rapidly advancing by bringing available molecular approaches for
examining the presence and expression of the key genes involved in microbial processes. The genes responsible
for biodegradation of pollutant and may be application of genetic engineering for constructing novel microbial
strains that have improved capacities or maximum levels of gene expression coincided with maximum phenol
degradation.
Growth medium
The mineral salts medium (MSM) consists of (g/L), 1.25 of yeast extract, 0.35 of K2HPO4, 0.35 of MgCl2.6H2O,
0.2 of Ca(NO3)2, 0.12 of FeCl2 and trace elements (0.1 mg/L ZnSO4.7H2O, 0.2 mg/L CuSO4.5H2O, 0.2 mg/L
MnSO4. 2H2O and 0.1 mg/L Na2MoO4) with phenol as the sole carbon source.
Phenol estimation
Phenol was estimated by direct photometric method [7] in portion of the medium withdrawn and
centrifuged at 5000 rpm for 10 mins to remove cell pellet and was analyzed by U.V/visible recording
spectrophotometer SHIMADZU 160A (Tokyo, Japan) at 500 nm. To the supernatant was added 4-aminoantipyrene at pH 7.9 ± 0.1 by using ammonium hydroxide (0.5N) and phosphate buffer (pH 6.8), followed by
oxidation with alkaline K3Fe(CN)6 giving a red color when phenol is present.
Inoculum preparation
R. pyridinivorans GM3, isolated from soil by enrichment culturing with phenol. Actively growing culture of
R. pyridinivorans GM3 was inoculated (loop full) into MSM broth with 1% glucose and 0.05% phenol and
incubated at 32°C and with agitation 200 rpm for 20 hours (approximately 109 CFU/mL).
41
Phenol Degradation
Phenol degradation was carried out in triplicate using MSM (50 mL) containing two concentrations of phenol
1.0 and 1.5 g/L in 250 mL flasks and inoculated with 1% R. pyridinivorans GM3 and incubated at 32oC, pH 8.5
and 200 rpm. The samples were collected at every 6 hours of interval from flasks containing phenol
concentration 1.0 and 1.5 g/L and phenol degradation was monitored.
Plasmid isolation
Plasmid DNA was isolated from the R. pyridinivorans GM3 by the alkaline lysis method [8] with the following
modifications. R. pyridinivorans GM3 isolates were grown for two days at 30o C in 5 mL of nutrient broth. One
milliliter of bacterial culture was centrifuged, and bacteria were resuspended in 200 mL of a freshly prepared
solution containing 0.05 M Tris-HCl (pH 8.0), 0.01 M EDTA (pH 8.0), 0.5 M NaCl, and 20% (w/v) sucrose plus 5
mg of lysozyme per mL. The bacterium was then incubated at 37 oC for two hours. Cells were then lysed by
adding 400 mL of a solution containing 2.0% (w/v) sodium dodecyl sulfate (SDS) and 0.3 M NaOH. Plasmid
DNA was precipitated with 600 uL of ice-cold isoprapanol at room temperature (30 ± 2 oC) for 10 mins and then
centrifuged for 10 mins. Samples were eluted with 40 μL sterile water. Samples of plasmid preparations were
separated along with the plasmids pET 28(a) and with 1.0 Kb DNA marker (ladder) using 0.8% agarose gels
containing 5 μg/mL ethidium brromide at approximately 5 V/cm for 2 hours [9]. Molecular weights were
observed as bands on agrose gel electrophoresis and estimated by observing the migration in the gels.
Plasmid curing
Plasmid curing is done for elimination of plasmid from its host by using curing agents and screening of the
cured cells by replica plate method. Hexammine ruthenium (lll)chloride (HRC) was obtained from Strem
Chemicals Co. U.S.A., Acridine orange (AO) was obtained from Merck Chemicals Co. Germany and sodium
dodecyl sulfate (SDS) was obtained from Sigma Chemical Co U.K. There were used to prepare standard stock
solution freshly in sterile distilled water at concentrations of 2000 µg/mL. The isolate is treated with each
curing agent in MSM broth with 1.0 g/L of phenol as per the modified method of Reddy et al. [10]. Curing
agents (HRC, AO and SDS) are diluted in MSM broth to get a concentration in the range of 6.25- 400 µg/mL.
Overnight agar slant culture suspended in 10 mL of culture (0.1 mL) was inoculated into broth and mixed
thoroughly to get uniform suspension. Culture (0.1 mL) is inoculated into 1.0 mL of test media containing
above prepared concentrations of curing agents and incubated at 32 oC overnight. Minimum inhibitory
concentration (MIC) is determined by streaking on MSM agar plates (with glucose 1.0 g/L). A sub lethal
concentration less than minimum inhibitory concentration of a curing agent is selected for curing plasmids.
R. pyridinivorans GM3 is incubated at 32oC overnight with the respective sub lethal curing concentration of
42
each curing agent that is determined from such preliminary studies. Controls are maintained without curing
agent, isolated colonies of treated and control cultures are obtained by streaking on MSM agar plates and are
picked up with sterile toothpicks and inoculated into MSM broth (50 mL) with 1.0 g/L phenol as selective
agent.
Results
Phenol Degradation
The ability of R. pyridinivorans GM3 to degrade phenol in batch culture were studied by using MSM
containing 1.0 and 1.5 g/L initial phenol concentrations with inoculum size 1%. It is clear from the results that
R. pyridinivorans GM3 showed 100% degradation within 18 and 24 hours at 1.0 and 1.5 g/L of phenol
concentrations respectively (Figure 1). The results proved that R. pyridinivorans GM3 utilized /degraded
phenol as the sole source of carbon and energy.
Plasmid isolation
Plasmid was isolated from R. pyridinivorans GM3 using gel electrophoresis and the separation resulted in
single plasmid band with bands of pET 28(a) plasmid on right (Figure 2). In Figure 3 the size of plasmid of R.
pyridinivorans GM3 was compared with 10 Kb ladder (marker) for determining the molecular size of GM3
plasmid. The results obtained showed that there was a single plasmid of ~11 Kb size in R. pyridinivorans
GM3.
Plasmid curing
Plasmid curing means elimination of plasmid from its host cell by use of a curing agent and screening of the
cured bacterial cells by replica plate method to demonstrate the presence of plasmid DNA and specific
character gene on its plasmid.
43
Plasmid curing was performed by using acridine orange (AO), hexamine ruthenium (III) chloride (HRC) and
sodium dodecyl sulfate (SDS). It was observed that growth of R. pyridinivorans GM3 was inhibited by AO
and HRC at 200 and 400 µg/mL respectively, whereas SDS was used at various concentrations did not show
any growth inhibition. Hence, sub lethal concentrations of AO (<200 µg/mL) and HRC (<400 µg/mL) were
used for curing experiments. Colonies from sub lethal concentration of these curing agents (AO and HRC)
were grown on MSM with 1.5 g/L phenol to observe the sensitivity or resistance to phenol.
Elimination of plasmid from R. pyridinivorans GM3 was 78% at 100 µg/mL by AO and 84% at 200 µg/mL by
HRC (Table 1). The results indicated that SDS at selected concentrations in this study was not effective in
plasmid curing and growth when compared to AO and HRC.
Plasmid DNA cured R. pyridinivorans GM3 cells were able to grow on MSM with glucose and agar, but failed
to grow on the MSM containing phenol as sole source of carbon, and lost ability to degrade phenol at
concentration 1.0 g/L. This indicated that the genes responsible for the degradation of phenol are present on
the plasmid DNA.
Discussion
The isolate R. pyridinivorans GM3 could degrade phenol with concentrations of 1.0 and 1.5 g/L in 18 and 24
hours respectively. The results implied that GM3 has utilized phenol as sole source of carbon in MSM. Previous
reports suggest that Rhodococcus has the ability to degrade a variety of hydrocarbon and fuel additive
compounds and could be efficiently used in bioremediation [11]. Genetic factors play an important role in
conferring biodegradation potentials to microorganisms. The functional gene was investigated to find out
whether it is present on plasmid or the chromosome. In the present study, R. pyridinivorans GM3 is found to
possess a single plasmid of ~11 Kb size (Figures 2 and 3). Kim et al.[12] reported that pulsed-field gel
electrophoresis analysis demonstrated the presence of two large megaplasmids in the Rhodococcus sp. strain
DK17 which was capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene and other
alkylbenzene isomers.
According to previous study, plasmid was found to have an important role in degrading phenol, Cho and Kim
[13] suggested that this mega plasmid could be responsible for the degradation of polycyclic aromatic
hydrocarbon by Sphingomonas sp. strain KS14. van der Geize and Dijkhuizen,[14] have shown evidence that,
these plasmids may also contribute to propagation and mobilization of genes encoding these catabolic
pathways and enzymes between rhodococci.
44
The location of phenol degradation genes was found on plasmid, this plasmid was cured with acridine
orange (AO) and hexammine ruthenium (lll)chloride (HRC) (Table 1), this observation emphasize
involvement of R. pyridinivorans GM3 plasmid in phenol degradation. The inhibition of plasmid DNA
replication was found when AO and HRC curing agents were used and further R. pyridinivorans GM3 might
lost plasmid in generation. Therefore, R. pyridinivorans GM3 was not able to grow in the presence of phenol
as it has lost the ability to degrade phenol (that is sole carbon and energy sources) due to the plasmid curing
and may lead to death of this bacterium. Chowdhuryi et al. [15] observed a similar pattern that the two
bacterial strains (Pseudomonas sp. and Staphylococcus sp.), after the plasmid curing experiment by using
ethidium bromide, lost the ability to grow in phenol, suggests the involvement of plasmids. Ajaz et al.[16]
determined the location of phenol resistant genes by acridine orange mediated plasmid curing on bacteria, all
the resistant isolates lost the characteristic (to resist phenol) after curing, thereby indicating the plasmid
genes being responsible for this property. Reddy et al. [17] reported that the 11 Kb plasmid isolated from
Bacillus cereus GMHS (degrading alpha picolin) was found to be solely responsible for degradation via
plasmid-curing studies using acridine orange and hexamine ruthenium (III) chloride.
Biodegradation of many aromatic compound have been reported to be plasmid borne; thus, in present study
the role of plasmid in phenol degradation was emphasized by R. pyridinivorans GM3, consequently it was
found that plasmid DNA may play a particularly important role in genetic adaptation and can impart novel
phenotypes, including hydrocarbon oxidizing ability to recipient organisms [18]. The degradation of many
xenobiotic and hydrocarbon compounds is known to be mediated by plasmid encoded enzymes; there is
preponderance of information showing high level of plasmid involvement in the degradation of naphthalene
and other 2- and 3-ring polycyclic aromatic hydrocarbons [19].
These observations evidently indicated that the genes responsible for the degradation of phenol are present
on the plasmid DNA. Hence, Martínková et al. [20] anticipated that Rhodococcus which carry large plasmid
can degrade the phenol into environmental friendly compounds that may be utilized in genetic engineering
for multipurpose uses and contribute to the removal of the pollutants.
45
Phenol Concentration g/L
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
6
12
Time (Hours)
1.0 g/L Phenol
18
24
1.5 g/L Phenol
Figure 1: Phenol degradation by R. pyridinivorans GM3 with initial phenol concentration of 1.0 and 1.5 g/L
Figure 2: Agarose gel separation of plasmid DNA isolated from R. pyridinivorans GM3 with (left) with pET 28(a)
(right)
Figure 3: Agarose gel electrophoresis showing the size of plasmid of R. pyridinivorans GM3 (right) with
molecular size 10 Kb ladder (left)
46
Table 1: Elimination of phenol catabolic plasmid from R. pyridinivorans GM3 using curing agent
Conc. tested
Hexammine
Acridine orange
Sodium dodecyl
(µg/mL )
ruthenium
(AO)
sulphate (SDS)
(lll)chloride (HRC)
Growth
%Curing
Growth
%Curing
Growth
%Curing
6.25
+
0.0
+
0.0
+
0.0
12.5
+
6
+
16
+
0.0
25
+
15
+
28
+
0.0
50
+
34
+
42
+
0.0
100
+
66
+
78
+
0.0
200
+
84
–
–
+
0.0
400
–
–
–
–
+
4.0
47
References
[1] Zhou, K. B.; Wang, X.; Sun, X. M.; Peng, Q.; Li, Y. D. Enhanced catalytic activity of ceria nanorods from well defined
reactive crystal planes. J. Catal. 2005, 229, 206–212.
*1+ Michałowicz, J.; Duda, W. Phenols–Sources and toxicity. Polish Journal of Environmental Studies. 2007,16,347–362.
[2]Nair, C. I.; Jayachandran, K.; Shashidhar, S. Biodegradation of phenol. African Journal of Biotechnology. 2008, 7, 4951–
4958.
[3]Watanabe, K. Microorganisms relevant to bioremediation. Current Opinion in Biotechnology. 2001, 12, 237–241.
[4] Shimizu, S.; Kobayashi, H.; Masai, E.; Fukuda, M. Characterization of the 450-kb linear plasmid in a polychlorinated
biphenyl degrader, Rhodococcus sp. strain RHA1. Applied and Environmental Microbiology. 2001, 67, 2021–2028.
[5] Larkin, M. J.; Kulakov, L. A.; Allen, C. C. R. Genomes and Plasmid in Rhodococcus. In H. M. Alvarez (ed.), Biology of
Rhodococcus. Microbiology Monographs (V:16). Springer-Verlag Berlin Heidelberg. 2010, p.73.
[6] Dabrock, B.; Kebeler, M.; Averhoff, B.; Gottschalk, G. Identification and characterization of a transmissible linear
plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism. Applied
and Environmental Microbiology. 1994, 60, 853–860.
[7] Clesceri, L. S.; Greenberg; A. E.; Eaton, A. D. Standard Methods for Examination of Water and Waste Water. 20th ed.
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Laboratories-New York.
[9]Takai, S.; Ikeda, T.; Sasaki, Y.; Watanabe, Y.; Ozawa, T.; Tsubaki, S.; Sekizaki, T. Identification of virulent Rhodococcus equi
by amplification of gene coding for 15- to 17- kilodalton antigens. Journal of Clinical Microbiology. 1995, 33, 1624–
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[10] Reddy, G.; Shridhar, P.; Polasa, H. Elimination Col E1 (pBR322 and pBR329) plasmids in Escherichia coli on treatment
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[11]Auffret, M.; Labbe, D.; Thouand, G.;. Greer, C. W; Fayolle-Guichard, F. Degradation of a mixture of hydrocarbons,
gasoline, and diesel oil additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis. Applied and
Environmental Microbiology. 2009, 75, 7774–7782.
[12] Kim, D.; Kim, Y. K.; Kim, S. K.; Kim, S. W. ; Zylstra, G. J.; Kim, Y. M.; Kim, E. Monocyclic aromatic hydrocarbon
degradation by Rhodococcus sp. strain DK17. Applied and Environmental Microbiology. 2002, 68, 3270–3278.
[13] Cho, J. C.; Kim, S. J. Detection of mega plasmid from polycyclic aromatic hydrocarbon-degrading Sphingomonas sp.
strain KS14. Journal of Molecular Microbiology and Biotechnology. 2001, 3, 503–506.
[14] van der Geize, R.; Dijkhuizen, L. Harnessing the catabolic diversity of rhodococci for environmental and
biotechnological applications. Current Opinion in Microbiology. 2004, 7, 255–261.
[15] Chowdhuryi, A. H.; Hassan, P.; Mosaddik, M. A.; Bhuyan, S. A.; Rahman, M. H.; Saha, A. K.; Hossain, M. Plasmid
mediated degradation of phenol by two bacterial strains Pseudomonas sp. and Staphylococcus sp.. Pakistan Journal of
Biological Sciences. 2000, 3, 939–942.
[16] Ajaz, M.; Noor, N.; Rasool, S. A.; Khan, S. A. Phenol resistant bacteria from soil: identification-characterization and
genetical studies. Pakistan Journal of Botany. 2004, 36, 415–424.
[17] Reddy, D. M., Paul, D.; Jogeswar, M.; Reddy, G. Biodegradation of alpha picolin – a plasmid born activity.
International Journal of Environmental Studies. 2009, 66, 737–745.
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[18] Leahy, J. G.; Colwell, R. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews. 1990, 54,
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*20+ Martínkov{, L.; Uhn{kov{, B.; P{tek, M.; Nešvera, J.; Křen, V. Biodegradation potential of the genus Rhodococcus.
Environment International. 2009, 35, 162–177.
49
Simulation Model To Predict Velocity And Pressure Distribution
Inside The Hydrocylone In Water Treatment Plant
Dr. Alaa Hussein Wadi Al-Fatlawi
Osamah Ali Hadi Al-Hashimi
Asst. Prof. / Environmental Eng. Dept.
Babylon Water Directorate
College of Engineering / Babylon University
Babylon / Iraq
Abstract - The objective of this research work is to predict the velocity and pressure distribution inside a hydrocylone
which used water as a liquid phase and inert/solid particles as a solid phase. Inside diameter of this hydrocyclone is
85mm. The proportions of each dimension proposed by Bradley are used in this work. In this study, turbulent and
swirling flow within a hydrocyclone is simulated by using commercial computational fluid dynamics (CFD) code
'FLUENT' v14.0, Gambit 2.4.6, Tecplot 360, CFD post computer software’s . The results clearly showed the contours and
diagrams of pressure and velocity inside the hydrocyclone. The pressure diagram indicates that pressure in center of
surface is less than the walls, while the velocity distribution is (7.173 m/s) which agreed with the inlet theoretical
velocity of (7.18 m/s).
Keywords: Hydrocyclone, Computational Fluid Dynamic, Fluent.
I.
W
Introduction
ith the rising of water shortage problems and decreasing of water resources due to climate changes, the need for
using all type of available water is increased, such as the re-treatment of waste water; increase the independent
on water wells, water disposed from different industries such as pulp industry … etc, usually these types of water
brings different types of suspended solids, the hydrocyclone come into existence as a sound and proved technological
alternative in separation these solids from water .Hydrocyclones have been widely used for various applications and by
Dr. Alaa Husaeen Wadie Al-Fatlawi, PhD. Environmental Engineering Department, College of Engineering, University of Babylon, Iraq.
Phone: (+964) (0) (7801595607), Email: [email protected]
Eng.Osamah Al-Hashimi, MSc. Environmental Engineering, University of Babylon, Iraq.
Phone: (+964) (0) (7700084502), Email: [email protected]
50
different industries, including the mineral, chemical petrochemical, petroleum, food and drug, pulp and paper,
[1]
environmental, and biology, among others (Rama and Udaya, 2012) .
Common hydrocyclone applications include classification of solids or removal of particulates from a liquid or a gas
stream. The use of the solid-liquid hydrocyclone has emerged as a sound alternative to conventional filtration and
other separation systems, which are bulky, require backwashing, frequent replacement of filters, chemical additives,
and have greater pressure drop, resulting in higher operating costs. The petroleum industry, for example, has utilized
the solid-liquid hydrocyclone to remove oilfield solids from produced water in order to make it suitable for down-hole
re-injection, either for reservoir water flooding or for disposal. Hydrocyclones are also an attractive solution for
[2]
offshore applications where space, efficiency, and reliability are important (Gómez, 2001) .
Different types of hydrocyclones have been used by the industry in the past to separate solid-solid (classifiers),
liquid-liquid, gas-liquid, gas-solid, and solid-liquid mixtures. This paper focuses on the application of hydrocyclone in
the field of water treatment and how the use of hydrocyclone can improve the operation of water treatment facility
and reduce the cost of operation by reducing the turbidity of water pumped into the treatment facility. In the water
industry there is a wide use to the conventional gravity based vessels which are bulky, heavy and expensive to
separate multiphase flow. The difficulty and the cost of accommodate these separation facilities has provided the
incentive for development of compact separation technology, hydrocyclones have emerged as an economical and
effective alternate for produced water and other applications, the hydrocyclone is inexpensive, simple in design with
no moving parts, easy to install and operate, and has low maintenance cost (E. Endres, et al.,2001)
II.
[3].
Objective of study
Proper hydrocyclone design is essential for achieving maximum performance and ensuring the maximum and most
reliable solids separation efficiency. However, there is still a lack of detailed understanding of hydrocyclone flow
behavior and separation mechanism that occur in hydrocyclone, thus, more researches are needed in order to achieve
these targets.
Up to date, the design of the solid liquid hydrocyclones has relied on empirical experience, and more recently on
CFD and numerical modeling, which has had some success owing to the improvement of computing power. Still, CFD
models require a large amount of computing power, and simulations are time consuming and costly (Severino, 2007)
[4]
51
So, this work aims to use the latest computer programs such as AutoCAD 3D Mechanical, Gambit 2.4.6, Ansys
Fluent V.14, TecPlot 360 and CFD Post to predict the velocity and pressure profile inside a hydrocylone.
III.
Description of Hydrocyclone Separators
The solid liquid hydrocyclone separator is a type of cyclone that facilitates the centrifugal separation of solid
particulates from a liquid stream; the hydrocyclone utilizes the energy obtained from fluid pressure to create
rotational fluid motion, yielding much larger values of the g-force that can vary from 800g to about 50,000g. This high
swirling motion is applied over a shorter residence time causing the particles suspended in the liquid to separate fast
[5]
and effectively from the liquid itself (Rushton, et al., 1996) .
The hydrocyclone is based on the principle of centrifugal force causing the separation of solids from a liquid by the
differences in density and particle size. A typical hydrocyclone consists of a cylindrical section and a conical section, as
shown in Figure 1. It does not have any internal rotating parts. An external pump is used to transport the liquid
suspension to the hydrocyclone through a tangential inlet at high velocity, which in turn generates the fluid rotation
and the necessary centrifugal force. The outlet for the bulk of the liquid is connected to a vortex finder located on the
axis of the cylindrical section of the vessel. The underflow, which carries most of the solids, leaves through an opening
[6]
(apex) at the bottom of the conical section (Wen-Ching Yang, 2003) .
(a)
(b)
Fig. 1: – Hydrocyclone (a) schematic diagram (b) flow pattern
52
IV.
Motion of suspended particle
When solid particles are fed to a hydrocyclone, they will be dispersed rapidly because of the strong turbulent
mixing at the inlet. However, there is little information about the behavior of fluid in the cylindrical part of the
hydrocyclone. This portion of the hydrocyclone is generally regarded as the preliminary separation zone, while more
thorough separation is thought to occur in the conical section. As Kelsel
[7]
proposed in 1952, "if fluid moves into the
interior, all particles that exist near the cone wall can move into the interior rapidly". Therefore, it is obvious that if the
fraction Rf of the feed liquid goes to the underflow, then the same fraction R f of all particles, regardless of their settling
rate, must also go with the liquid, together with the particles separated from the remaining fraction of the liquid (1–Rf)
[8]
leaving in the underflow. This is an important diagnostic phenomenon of the hydrocyclone (Sang Huck Park, 2003)
In the hydrocyclone interior, a particle at any point within the flow is subjected to two forces: acceleration due to
gravity and centrifugal forces, and drag imposed on particle by the flow. Only centrifugal and drag forces are taken into
account because effect of gravity is usually negligible in hydrocyclones. The movement of a particle in both the
tangential and vertical (axial) directions does not encounter resistance by any forces. Therefore, its velocity
components in those directions can be taken to be equal to the corresponding flow velocity components v t and va.
Because the centrifugal force acts in the radial direction, it prevents the particles from flowing inward to the interior of
the hydrocyclone and particles are subjected to control of "centrifugal elutriation" effect. If the centrifugal force that
acts on the particle is greater than the drag force, the particle moves rapidly to the outside; conversely, if the drag is
greater than centrifugal force, the particle moves to the inside. Because both the drag and centrifugal forces depend
on vt and va respectively for given a particle, the relative values of va and vt of all positions within the separation zone
detentions the overall performance of the hydrocyclone operation (Shojaeefard, et al, 2006)[9].
The trajectory of the discrete phase particle is obtained by integrating the force balance on the particle, which can
be written in a Lagrangian reference frame. This force balance equates, the particle inertia with the forces acting on
the particle, and can be written as in equations (1) and (2) and illustrated in Figure 2:
53
Fig. 2: Different Forces on Particle (Shojaeefard, et al, 2006)
= FD (
)+g(
Where Fx is coriolis forces, FD (
FD = (
Here,
)(
… (1)
) is the drag force per unit particle mass and,
… (2)
)
is the fluid phase velocity,
density,
) + Fx
[9]
is the particle velocity,
is the density of the particle and
is the molecular viscosity of the fluid,
s the fluid
is the particle diameter, Re is the relative Reynolds number which is
defined as:(
)
… (3)
Re =
For sub micron particles, a form of stoke’s drag law is available and in that case FD is defined as:
… (4)
FD =
Where:
=1+[
Where
(1.257 + 0.4 exp (-1.1 (
)))]
… (5)
[9]
is the mean free path (Shojaeefard, et al., 2006) ) .
54
V.
Modeling of Water Flow in Hydrocyclone
For a dilute fluid suspension, the incompressible Navier–Stokes equations supplemented by a suitable
turbulence model are appropriate for modeling the flow in a hydrocyclone. The most popular turbulence model in use
for engineering applications is the k–e model where the scalar variables k and e represent the kinetic energy of
turbulence and its dissipation rate, respectively. The standard k–e model was used to represent the turbulence in the
equipment. The model was used to predict the water flow rates in the two outlet streams for different inlet velocities
of water (Shojaeefard, 2006).
The following equations describe the transport of momentum in an inertial (non-accelerating) reference frame:-
( ⃗)
( )̿
( ⃗ ⃗ )=
Where is the static pressure,
̿
̅
̅
… (3.1)
is the stress tensor (described below), and
̅
is the gravitational body force.
̅ contains other source terms that may arise from resistances, sources, etc.
The stress tensor
̿
( ⃗
̿ is given by:
⃗ )
⃗
… (3.2)
Where μ is the molecular viscosity, I is the unit tensor, and the second term on the right-hand side is the effect
of volume dilation.
1- Boundary conditions
It was necessary to specify boundary conditions at the inlet, outlet and at the walls of hydrocyclones. Inlet velocity
was used as a boundary condition, which means that the value of the velocity is specified. A uniform velocity profile
was specified by introducing the inlet velocity and this gave the required mass flow rate. To determine the influence of
the flow rate on the velocity field and to improve the predicted axial and tangential velocity profile, a pressure
55
boundary was used to model the outlet conditions. At the walls, the default of no slip condition was applied, i.e. the
velocity equals to zero at the wall. The normal logarithmic wall function was used to specify the flow conditions in the
cells adjacent to the wall. The fluid properties at the inlet used in this study are specified in Table 1 below.
Table 1: Physical properties of water and inert particles
a-
Water -liquid (fluid)
Property
Units
Method
Value(s)
Density
kg/m
3
constant
998.2
Cp (Specific Heat)
J/kg.k
constant
4182
Thermal Conductivity
w/m.k
constant
0.6
Viscosity
kg/m.s
constant
0.001
Molecular Weight
kg/kmol
constant
18.015
b- Inert-particles
Property
Units
Method
Value(s)
Density
kg/m
3
constant
1920
Cp (Specific Heat)
J /kg.k
constant
1680
Thermal Conductivity
w/m.k
constant
0.045
The hydrocyclone in this study has a 85 mm diameter of cylindrical section as shown in Fig. 1.
56
All dimensions are in mm
Fig. 1: Hydrocyclone geometry
By using GAMBIT, pre-processing software, an unstructured triangular mesh with 1,260,881 elements has been
used for the main body of hydrocyclone. The mesh is shown in figures 3.7 and 3.8 uses unstructured triangular mesh
for the main body of the hydrocyclone. In this model the tangential inlet shown is meshed for simplicity using
triangular elements.
Figure 3.7: Unstructured triangular mesh of hydrocyclone with 100% active elements.
57
Figure 3.8: Grid elements in the (y) axis
2- Solution Steps
In addition to solving transport equations for the continuous phase, CFD allows to simulate a discrete second phase
in a Lagrangian frame of reference. This second phase consists of spherical particles dispersed in the continuous phase.
CFD computes the trajectories of these discrete phase entities, as well as heat and mass transfer to/from them. The
coupling between the phases and its impact on both the discrete phase trajectories and the continuous phase flow can
be included. We can include a discrete phase in our CFD model by defining the initial position, velocity, size of
individual particles. These initial conditions, along with our inputs defining the physical properties of the discrete
phase, are used to initiate trajectory and mass transfer calculations.
-5
For this model, a discrete phase model with a tolerance of 10 has been used. For the operation conditions, we
2
define gravitational acceleration in direction y (-9.86m/s ). After defining materials, boundary conditions and operating
conditions, the next step is to solve for CFD. A SIMPLE scheme pressure velocity coupling has been used for the
solution method. A (10,000) iterations needed to get the peak tangential velocity in the simulation. Running of this
-6
model on a dual core computer processor toke (60 hrs), with minimum accuracy of (1e ).
VI.
RESULTS AND DISCUSSION
Despite the simplicity of its construction of hydrocyclone, it displays a quite complex internal behavior,
including features as high preservation of vorticity, vortex breakdown and flow diagram. For the geometry, boundary
58
conditions, and operation conditions stated in the experimental work (chapter 3), the results of CFD modeling are
presented in figures 4.7 to 4.11:
Figure 4.7: Vertical section for pressure distribution inside the hydrocyclone
The above figure shows a half cross section of the effects of pressure on the separation. There is a positive
effect of pressure on the recovery and split flow ratio, the contours and diagrams of pressure inside the hydrocyclone.
This figure clearly indicates that pressure in center of surface is less than the walls.
Figure 4.8: Planner view for pressure distribution inside the hydrocyclone
59
Figure 4.9: Vertical section for velocity distribution inside the hydrocyclone
Figure 4.10: Planner view for velocity distribution inside the hydrocyclone
Figure 4.11: Pathlines of particles colored by time inside the hydrocyclone
60
It is notable that particles needs maximum (0.99 sec) to reach the underflow and (3.31 sec) to rise to the
overflow. This is due to the high velocity near the wall and slow velocity in the core of hydrocyclone.
An important analysis comes from the velocity profiles. As is shown in figure 4.12 below, for most heights, the
axial velocity is not a maximum in the middle of the flow, as expected. This maximum velocity is placed close to the
wall when the flow goes in direction to underflow or is close to the air core when the flow goes to direction to
overflow. This is agreed with the theory of forces inside the hydrocyclone.
Figure 4.12: Axial Velocity vs Radial Position
The tangential velocity field distribution of the multiphase system is illustrated in Figure 4.13. Maximum
positive values are noted in the regions near to the outer wall, while the minimum flow velocity is noted in the central
core of the hydrocylone. A significant increase in the tangential velocity occurs traversing from the wall inwards, at a
point interfacing with the air core, the tangential velocity starts to decrease.
Figure 4.13: Tangential Velocity vs Radial Position
Figure 4.14 shows the radial velocity inside the hydrocylone, the magnitude of radial velocity is much smaller
than that of the tangential or axial velocity which agree with what (Kelsall, 1952) proposed. However, very little
61
information is available about this velocity component. In practice, the tangential and axial velocities are usually
measured (Leeuwner and Eksteen, 2008)
[10]
.
Figure 4.14: Radial Velocity vs Radial Position
The model also gives the contour of pressure as shown in figure 4.15, The pressure is high in the upper wall of
the hydrocyclone, meanwhile inside the air-core is the lower pressure. Those results are agreed with theory.
Figure 4.15: Pressure vs Radial Position
62
VII.
Conclusions:
It can be concluded that the velocity, pressure and flow pattern within a hydrocyclone chamber can be modeled
using CFD. This will easily allow researchers studying how changes in the shape of hydrocyclone will influence its
operating performance. The ability of modern supercomputers allows the approximation of three-dimensional flow
pattern in hydrocyclones to be investigated. That will give in a near future a better understanding of hydrocyclone
performance.
VIII.
Acknowledgements
The authors wish to acknowledge the assistance of Mr. Sahir Rakim for his valuable assistance and guidance to achieve
research goal.
IX.
References:
[1] Rama Murthya, Udaya Bhaskarb, (2012), “Parametric CFD studies on hydrocyclone”, Research Development and
Technology, Tata Steel Ltd, Jamshedpur, 831007, India & ArcelorMittal Global R & D, 3001 E. Columbus Drive, East
Chicago, IN 46312, USA.
[2] Gómez, Carlos Hernán, (2001), “Oil-Water Separation in Liquid-Liquid Hydrocyclones (LLHC) - Experiment and
Modeling”, The Graduate School, University of Tulsa – USA.
[3] E. Endres, J. Dueck, Th. Neesse, (2011), “Hydrocyclone classification of particles in the micron range”, Life Sciences
Engineering Center, German Friedrich-Alexander, Universität Erlangen-Nuremberg, Branch Busan, South Korea.
[4] Severino, G. Jose, (2007), "Mechanistic Modeling of Solid-Liquid Separation in Small Diameter Hydrocyclones", The
Graduate School, University of Tulsa, USA.
[5] Rushton A., Anthony S. Ward , Richard G. Holdich, (1996), "Solid-Liquid Filtration and Separation Technology",
Wiley-VCH; 1st Edition.
[6] Wen-Ching Yang, (2003), "Handbook of Fluidization and Fluid-Particle Systems", Published March 19th 2003 by CRC
Press.
63
[7] Kelsal, D.F., 1952. A study of the motion of solid particles in a hydraulic cyclone. Transactions of the Institution of
Chemical Engineers. 30, 87– 108.
[8] Sang Huck Park, (2003), “Separation of polymer particles using a hydrocyclone”, Lehigh University.
[9] Shojaeefard M. H., Noorpoor A.R., Yarjiabadi H., Habibian M., (2006), “Particle Size Effects on Hydrocyclone
Performance”, Automotive Engineering Department, Iran University of Science and Technology. Islamic Republic of
Iran.
[10] Leeuwner M.J and Eksteen J.J., (2008), “Computational fluid dynamic modelling of two phase flow in a
hydrocyclone”, Department of Process Engineering, University of Stellenbosch.
64
‫أثرالتكنىلىجيب الحديثت في بعض االجهزة االلكترونيت والكهرببئيت‬
‫على البيئت‬
‫ً ّجشاط ٍحَذ عجذ اىشع٘ه‬.ً
‫خ اىَغزٖيل‬ٝ‫ ٍشمض ثح٘س اىغ٘ق ٗحَب‬/‫عبٍعخ ثغذاد‬
2013
‫اىخالطخ‬
ٜ‫شح اىحغٌ راد االعزعَبه اى٘اعع خظ٘طب ف‬ٞ‫خ اىظغ‬ٞ‫خ ٗاىنٖشثبئ‬ّٞٗ‫ِ ثعض االعٖضح االىنزش‬ٞ‫ اىَقبسّخ ث‬ٚ‫ٖذف اىجحش اى‬ٝ
‫ ٗثزىل ىغأ‬ٙ‫ ٍِ عٖخ ٗىَ٘امجخ اىزط٘س ٍِ عٖخ اخش‬ٜ‫ ٗاالّقطبع اىَغزَش ٗاىفغبئ‬ٜ‫بساىنٖشثبئ‬ٞ‫غخ عذً اعزقشاس اىز‬ٞ‫اىعشاق ّز‬
‫ ٍفزبػ‬, ‫خ‬ّٞٗ‫خ االىنزش‬ٞ‫خ ٗ ق٘اطع اىذٗسح اىنٖشثبئ‬ٞ‫خ ٍٗبشبثٔ رىل ( ق٘اطع اىذٗسح اىنٖشثبئ‬ٞ‫خ اىَْ ضى‬ٝ‫ اعٖضح اىحَب‬ٚ‫اىَ٘اطِ اى‬
‫ ٍٗح٘ه‬ٛٗ‫ذ‬ٝ ‫ ٍح٘ه مٖشثبء‬, ‫خ‬ٝ‫ٌ ٍع عٖبص حَب‬ٞ‫ٌ مٖشثبء ٗرقغ‬ٞ‫ رقغ‬, ‫خ‬ٝ‫ ٍع عٖبص حَب‬ٜ‫ظ ثيل) ٗ ٍفزبػ مٖشثبئ‬ٝ٘‫ (ع‬ٜ‫مٖشثبئ‬
‫خ‬ٞ‫ف‬ٞ‫ م‬ٜ‫خ) ٗاىجحش ف‬ٝ‫ ٗ عشط ثطبس‬ٛ‫ عشط عبد‬, ‫شىظ‬ٝ‫ ٗاالخش ٗا‬ٛ‫ ٍحشك ٍؤشش شبشخ اىحبع٘ة عبد‬, ٜ‫ن‬ٞ‫مٖشثبء اٗرٍ٘بر‬
‫ش ثعض‬ٝ‫ ٗرقذ‬ٜ‫بد ثشنو عش٘ائ‬ٝ‫ اىْفب‬ٜ‫ٖب ف‬ٍٞ‫ حبىخ ريفٖب ٗس‬ٜ‫ئخ ف‬ٞ‫ اىج‬ٚ‫شٕب عي‬ٞ‫ رأص‬ٙ‫جٖب ٗأعبدح اعزعَبىٖب ٍٗذ‬ٞ‫بّزٖب ٗرشم‬ٞ‫ط‬
.(AAS)ٛ‫بف االٍزظبص اىزس‬ٞ‫)عٖبص ٍط‬Fe,Ni,Co,Mg)‫اىعْبطش‬
Modern technology in some electrical and electronic appliances
on the environment
alrasool .Nibras Mohammed A
Center for Market Research and Consumer Protection
Abstract
The research aims to compare some electronic appliances and small electrical size of widespread
use, particularly in Iraq as a result of instability of electrical power interruptions continuous and
sudden hand and keep pace with the other hand and thus resorted citizen to protection devices home
and the like (breakers session electrical and breakers session electrical electronic, switch and switch
65
with protection device, a division of power and the division with the protection device, an electricity
transformer Manual and an electricity transformer automatic, engine index computer screen plain
and other Wireless, bell Plain and bell battery) and look how maintenance and installation, reuse and
its impact on environment and throw it in the waste indiscriminately and appreciation of some of
the elements by atomic absorption spectro photo meter(AAS)(Fe,Ni,Co,Mg).
Introduction
Several types of small digital equipment were also identified as important sources of precious
metals; however, mid-size information and communication technology equipment as printers, fax
machines and audio/video equipment were shown to be more important as a source of a variety of less
common metals. The physical collectability of each type of Electrical and Electronic Equipment was
roughly characterized by unit size and number of end-of-life products generated annually (1).
This situation had strayed great technological progress that has imposed itself reason to increase the
intensity of industrial hazards and its impacts on other circles, making thinking in the face of these
dangers of the biggest challenges faced by the State in general and institutions Industrial particular(2).
Waste electrical and electronic equipment has become an important target in managing material cycles
from the viewpoint of not only waste management and control of environmental pollution but also
resource conservation (3). Electrical and Electronic Equipment is developing fast and spreading over
every part of modern life. This equipment includes diverse substances that may cause serious damage
to the environment and have adverse effects on human health so it is essential to manage the waste
resulting from Electrical and Electronic Equipment in a proper way. Waste Electrical and Electronic
Equipment has been identified as a priority area to take specific measures on a European scale (4).
Waste electric and electronic equipment, or electronic waste, has been taken into consideration not
only by the government but also by the public due to their hazardous material contents (5). The aim of
this research study was firstly to determine the average concentration of four metals in this sampled
and it‘s effected on environmental.
Electronic waste, current status of the management of electronic waste and recycling technologies
for the recovery of metals from end-of-life electronic equipment. Because of the ever increasing
generation of e-waste and the hazardous nature of this waste stream, e-waste is an emerging issue.
66
Many countries have drafted legislation to improve the reuse, recycling, and other forms of recovery
of such waste. Electronic waste is significantly heterogeneous and complex in terms of the type of
components and materials. This indicates that the recovery of precious metals and copper may remain
as the major economic driver for a long time. The hierarchy of treatment of e-waste encourages the
reuse of the whole equipment first, remanufacturing, then recovery of materials by recycling
techniques, and as a last resort, disposal by incineration and land filling (5). Electronic devices may
contain up to 60 different chemical elements. However, deficiencies in the methods of combining and
technologies for recycling and disposal of waste in illegal ways mean the loss of most of these
precious resources when the arrival of the equipment to the end of life stage (7). These wastes could
have a negative impact on the environment and human health if they contaminate soil, water and air
(6).
MATERIALS AND METHODS
In this research we take 6 different part of small electrical, electronic equipments and the sample
digestion methods were found in many literatures that specializing in analysis of heavy metals differed
when work with it using one of the following concentrated acids: HCL, HNO3, HCLO4, HF or a
mixture of some of them and sometimes using H2O2 (12, 13), each time all compound of sample
crashed and subjected to acid digestion using different temperature condition (14, 15,16).
Some of the methods a 95C digestion temperature was used for 2 hr (15) others digestion at 80C for
3hr(17)and some with reflux (18,19) .In the past century the concentrated acid solution which is
consisted from three volumes of HCL mixed with one volume of HNO3 that it so called aqua regia
was commonly used for digestion, this concentrated acid solution was recently used for digestion
sample using temperature above boiling with digestion equipment (4,16,20).
Apparatus, materials and reagents:
In this work 40 ml of aqua regia has been used for digestion of sample, this acid solution was prepared
from concentrated (37%) HCL and (69%) HNO3 both obtained from Applichem-company-GmbH
Germany, the mixture was shacked for 24hr then filtered and the filtrate was further centrifuged then
the supernatant was diluted using volumetric flask by distilled deionizer water to a 100 ml.
67
A set of suitable standard solutions were prepared from 1000mg/L stock solutions of Fe,Ni,Co,Mg all
these stock were obtained from MERK company-Germany SHIMADZU AA-7000 flame atomic
absorption spectrophotometer was used for the determination of analytes. The apparatus optimum
condition for ASS is given in Table 1.
Table (1) apparatus operating parameters for ASS.
Parameters
Fe
Ni
Co
Mg
Wavelength/nm
248.3
232
240.7
285.5
Burner height/ mm
7
7
7
7
Acetylene flow rate,L/min
2.2
2.2
2.2
1.8
Slit/nm
0.2
0.2
0.2
0.5
RESULTS AND DISCUSSION
The higher concentration of Fe was found in sample 2 equal to 148.0764 ppm as in Table 1 and
the minimum concentration of Fe was found in sample 10 equal to 23.9184 ppm, for Ni was in
sample 8 equal to 119.0096 ppm the higher, and the minimum
in 3 equal to 59.7656 ppm, for Co
the minimum concentration 1.8588 ppm in sample10 and maximam was 24.9280 ppm in 12 and for
Mg the higher in sample 27.7680 ppm and lower value was 4.4732 ppm in sample 9.
Table 1 concentration of Fe,Ni,Co and Mg in ppm
Sample
1- Normal division
2- division with the protection device
3- Plain black
4- Plain black protection device
5- AC adapter manual
6- AC adapter automatic
7- Electric bell
8- ball with battery
9- Cricket breaker manual
10- Cricket breaker automatic
11- computer engine with wire
12- computer engine without wire
Fe
Ppm
42.9628
148.0764
95.8708
27.4400
105.9352
71.9948
81.7704
39.3428
31.3032
23.9184
53.5980
83.5312
Ni
ppm
107.2472
106.5820
59.7656
108.4564
85.0600
114.8972
110.7924
119.0096
118.3748
67.8996
73.9852
123.1824
Co
ppm
3.0496
2.6824
8.6496
5.0260
9.6708
5.8216
5.33412
5.5436
8.3532
1.8588
4.0896
24.9280
Mg
ppm
27.1656
24.6364
10.7212
12.9032
6.6124
10.7356
27.7680
19.5140
4.4732
9.8864
26.0400
24.4412
68
CONCLUSIONS
Heavy metal contamination in the Waste Electric and Electronic Equipment in the study was
relatively high, and the highest concentration were detected in both samples, so to reduce human
health risks we must classification waste and put this waste in special container to another reuse.
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2-European commission Environment, "Restriction of Hazardous Substances in Electrical and
Electronic Equipment", 2012.
3-X. Lu,K. Nakajima,H. Sakanakura,,K. Matsubae,H. Bai,T. Nagasaki,‖ Thermodynamic estimation
of minor element distribution between immiscible liquids in Fe–Cu-based metal phase generated in
melting treatment of municipal solid wastes‖2012,Waste management, volume 32,issue6,p11481155,(ivsl).
4-Josefina Lindblom and Luis Delgado"Implementation of Waste Electric and Electronic Equipment
Directive in EU 25" Regional Environmental Center for Central and Eastern Europe, Institute for
Prospective Technological Studies, 2006.
5-Jirang Cui, Lifeng Zhang‖ Metallurgical recovery of metals from electronic waste: A review‖,
Journal of Hazardous Materials, Volume 158, Issues 2–3, 30 October 2008, Pages 228–256,(ivsl).
6-Seong-Rin Lim,Julie M. Schoenung,‖ Human health and ecological toxicity potentials due to heavy
metal content in waste electronic devices with flat panel displays‖,2010,Journal of hazardous
materials, volume 177.issues1-3,p251-259,(ivsl).
8-Jirang Cui,Hans Jorgen Roven,‖Electronic Waste‖,2011,AHandbook of management
waste,chapter20,p281-296.(ivsl).
9-V. Coman,B. Robotin,P. Ilea,‖ Nickel recovery/removal from industrial wastes: A
review‖,2013,Resources,Conservation and Recycling. Volume 73, p229-238,(ivsl).
10- Peeranart Kiddee.Naidu, Ming,‖ Metals and polybrominated diphenyl ethers leaching from
electronic waste in simulated landfills‖2013, Journal of hazardous materials, volumes252-253, p243249, (ivsl).
‫ئخ ثشأُ ثْبء اىقذساد اىخبطخ ثبإلداسح‬ٞ‫(ىيج‬WEEE( ‫ثشّبٍظ األٌٍ اىَزحذح‬/‫ ىالرظبالد‬ٜ‫ ٗسشخ اىعَو اىَشزشمخ ىالرحبد اىذٗى‬-11
‫ فشطخ ٗاعذح‬ٚ‫خ إى‬ّٞٗ‫و اىَخيفبد اإلىنزش‬ٝ٘‫ رح‬ٚ‫ إى‬ٚ‫غع‬ٝ ‫ذ‬ٝ‫ ثشّبٍظ عذ‬,‫خ‬ّٞٗ‫خ ٗاإلىنزش‬ٞ‫ب ىَخيفبد اىَعذاد اىنٖشثبئ‬ٞ‫ئ‬ٞ‫اىَغؤٗىخ ث‬
. 2013‫و‬ٝ‫ أثش‬3 ،‫ف‬ْٞ‫ ع‬,‫خ ىيحذ ٍِ اىَخيفبد‬ٞ‫َّبرط األعَبه اىَغزذاٍخ أعبع‬
69
12-Balcerzak,M.(2002)"Sample digestion methods for the determination of traces of precious metals
by spectrometric techmiques" Analytical Sciences 18:737-750.
13-Nasralla,M.M.(1984)"Lead in jaddah urban dust"Enviro. Poll.Series B.,1984,8:133-141.
14-Lee,C. K.,Low,K.S.and Lim, C.G.(1988)"Communication V lead in Kuala Lumpur urban
dust"Pertanika11(1):157-160.
15-Yeimoglu, E.K. and Ercan,O.(2008)"Multivariate Analysis of metal contamination in street dusts
of Istanbul D-100 highway"J.Braz. Chem. Soc.19(7)1399-1404.
16-Suuzuki, K., Yabuki, T. and One, Y.(2009)"Roadside Rhododendron pulchrum leaves as
bioindicators of heavy metal pollution in traffic areas of Okayama Japan"Environ Monti Assess
149:133-141.
17-Paudyn, A.M. and Smith, R.G."Application of inductively coupled plasma atomic emission
spectrometry in occupational health"J. of Analytical Atomic Spectrometry 5:523-529,1990.
18-Bakirdere, S. and Yaman, M."Determination of lead,cadmium and copper in roadside soil and
plants in Elazig,Turkey"Enviro Monit Assess 136:401-410,2008.
19- Paudyn, A.M. and Smith, R.G."Microwave decomposition of dust,ashes,and sediment for the
determination of elements by ICP-AES"Canadian J.of Applied Spectroscopy 37(4):94-99,1992.
20-Abechi, E.S., Okunola, O.J.,Zubairu, A.A. and Apene, E."Evaluation of heavy metals in roadside
soils of major streets in Jos metropolis,Nigeria‖ J.Environ. Chem. Ecotoxical.2(6):98-102,2010.
21-Muhamad-Darus, F.,Nasir, R.A., Sumari, S.M.,Ismail, Z.S. and Otmar, N.A."Nursery
schools:Characterization of heavy metal content indoor dust "Asian J. of Environment-Behaviour
Studies 2(6):53-60,2011.
70
Effect of salt stress, application of salicylic acid and proline on
endogenous growth hormones of sweet pepper (Capsicum annum L.)
during vegetative stage.
Basheer A. AL-Alwani
Coll.of sci.
Univ. of babylon
Ali H. Jasim
Coll.of agric.
Univ. of al-qasim
Wassan M. Abu ALTimmen
Coll.of sci.
Univ. of babylon
Abstract
Factorial experiment with three factors was conducted to study the effect of salt stress on plant
hormones concentration during vegetative growth of sweet pepper (Cpsicum annuum L.) planting
individually in pots (5kg) and its interactions with exogenous application of salicylic acid and
proline. Sodium chloride (NaCl) was added to water irrigation in two concentrations (1.3 and 5
dsm/m). Three concentrations of salicylic acid (SA): 0 , 5*10-5, 10-4 M, and four concentrations of
proline : 0, 1, 5, 10 mM were sprayed exogenously on seedlings.
The results showed that salt stress was negatively affect on free IAA,GA, CK and ABA
concentrations. While increase in almost bound hormone concentrations. Spraying plants with SA
caused a decrease in free IAA,CK concentrations, while free ABA concentration was increased
significantly. In contrast, SA caused a reversible effect on bound hormones. Whereas, proline
caused a significant decrease in free IAA and ABA concentrations and an increase in bound
hormones concentrations.
Introduction
World population is increasing at an alarming rate and is expected to reach about six billion by the
end of year 2050. On the other hand food productivity is decreasing due to the effect of various abiotic
stresses; therefore minimizing these losses is a major area to concern for all nations to cope with the
increasing food requirements. Cold, salinity and drought are among the major stresses, which
adversely affect plants growth and productivity; hence it is important to develop stress tolerant crops
(Mahajan and Tuteja , 2005). The stress imposed by 25 or 50 mm NaCl reduced substantially leaf
area, dry mass, leaf chlorophyll content, stomatal conductance and net photosynthetic rate 50 days
after emergence of mustard plants (Shah,2007). Taffouo et al.,2008 showed that low concentrations
of NaCl had a negative effect on agronomic parameters and limited the growth of plants, as well as the
Na+ and K+ contents in the shoots of C. lanatus and C. moshata. Salt stress also resulted in growth
reduction, increase of Na+/K+ ratio, increase of Pro level and up-regulation of Pro synthesis genes
71
(pyrroline-5-carboxylatesynthetase, P5CS; pyrroline-5-carboxylate reductase, P5CR) as well as
accumulation of hydrogen peroxide (H2O2), increased activity of antioxidative enzymes (superoxide
dismutase, SOD; peroxidase, POX; ascorbate peroxidase, APX; catalase, CAT) and transcript upregulation of genes encoding antioxidant enzymes (Nounjan et al,2012).
Sweet pepper (capsicum annuum L.) is one of sensitive plants, (zapata et al.,2008). So, it affect
negatively when grown in saline conditions. Bethke and Drew ,1992 found that saline treatments
decreased the quality of pepper fruits and growth rate. Also, Chartzoulakis and Klapaki,2000
demonstrated that pepper plants treated to 100 or 150 moles per cubic meter NaCI had up to 85%
inhibition in photosynthetic ability.
Salicylic acid (SA) is an endogenous growth regulators of phenolic nature, which participates in the
regulation of physiological processes in plant (Ebrahimian and Bybordi,2012). It significantly
increased the fresh and dry weights of wheat plants roots and shoots under salt stress. Similarly, it
promoted the activities of antioxidative enzymes (Arfan, 2009). Salicylic acid pre-treatment
alleviated the adverse effects of salinity stress on germination percentage, length of shoot, fresh and
dry weight, photosynthetic pigments and K+ concentration (Delavari et al, 2010). Plants treated with
SA showed no recovery from excessive accumulation of Na+ in their shoot/root, under salt stress
(Mahmood et al 2010 ).
Proline (Pro) function as compatible solutes and are up regulated in plants under abiotic stress.
They play an osmoprotective role in physiological responses, enabling the plants to better tolerate
the adverse effects of abiotic stress. Exogenous application of proline considered as an important
agent to maintain osmotic potential of the plant cell (Ali, et al, 2007) and it considered as an
antioxidant agent through its role in increasing the ability of plant to tolerate salt stress (Okuma et
al, 2004).
Plant hormones are comprised of a group of structurally unrelated small molecules that regulate a
wide variety of plant processes. The hormones also act to integrate diverse environmental cues with
endogenous growth programs. So, far ten phytohormones have been identified including auxin,
abscisic acid (ABA), cytokinin (CK), gibberellin (GA), ethylene, brassinosteroids (BR), jasmonate
(JA), salicylic acid (SA), nitric oxide, and strigolactones (Davies, 1995; Browse, 2005; Vert et al.,
2005; Grun et al., 2006; Loake and Grant, 2007; Gomez-Roldan et al., 2008; Umehara et al., 2008).
Plants also utilize several peptide hormones to regulate various growth responses (Jun et al.,
2008).With the application of biochemical, genetic, and genomic approaches, many aspects of
hormone biology have been elucidated, especially in the model flowering plant Arabidopsis thaliana.
Most hormones are involved in multiple processes and impact each other through elaborate
crosstalk strategies in elucidating these hormone-signalling pathways (Santner and Estelle,2010).
The objective of the present study was to observe the effect of the individually and simultaneous
application of SA and proline as a foliar spray on the endogenous hormones of pepper plants under
saline and non-saline conditions.
72
Material and Methods
Planting method:
This experiment was conducted under saran canopy at the Department of Biology, collage of
science Babylon University. Sweet pepper (Capsicum annuum L.) seedlings of 45 days old were
obtained from gbela, Babylon. The original seeds were irrigated with water of (1.3 dsm/m). The
seedlings were planted in plastic pots containing 5 kg of soil (six pots for each treatment). Each one
supplied with 0.5 gm of NPK and granular fungicide. Seedlings were irrigated with tap water (1.3
dsm/m) for ten days twice a day before salinity treatment, followed by irrigation with salted water (5
dsm/m) every day until seedlings were reaching 70 days old.
Plants were sprayed twice with different concentrations of S.A (0 , 5*10-5,10-4 M) and proline (0 ,
1 , 5 , 10 mM). The first treatment added when the plants was 60 days old and the second treatment
after a week of the first one. The interaction between S.A and proline was applied by spraying
seedlings with proline in the concentrations mentioned above after two days of S.A application.
Plant hormones determination
Plant hormones were determined according to (Ergun et al.,2002). Either one gram fresh or dry
weight of leaves sample was taken and combined with 60 ml of methanol: chloroform: 2N ammonium
hydroxide (12:5:3 v/v/v). Each combined extract (60 ml) was kept in a bottle at -20oC in deep freeze
for further analysis. Combined extract was treated with 25 ml of distilled water. The chloroform phase
was discarded. The water-methanol phase was evaporated. The water phase was adjusted to the extract
pH value of 2.5 or 7 with 1 N HCl or 1 N NaOH respectively and 15 ml ethyl acetate was added at
each of three steps. This procedure provided the isolation of free-form IAA, GA3, ABA and zeatin
from the extraction solvent. After an incubation period of 1 hour at 70 oC, the same procedure was
used for the isolation of bound form IAA, GA3, ABA and zeatin from the extraction solvent.
Spectrophotometric assay was done using 222 nm and 280 nm wave lengths for IAA, 254 nm for
GA3, 263 nm for ABA, and 269 nm for zeatin and for all standard synthetic IAA, GA3, ABA and
zeatin and isolated samples.
Statistical analysis :
This factorial experiment included three factors (24 treatments) . Each pot was treated as one
replicate and all the treatments were repeated three times. The data were analyzed statistically with
SPSS-17 statistical software. Means were statistically compared by L.S.D test at p<5% level.
Results
Tabe (1) demonstrated that salt stress caused a significant decrease about 12.1% in free IAA
concentration. Also, the concentrations 10-4 M , 5*10-5 M of SA and the concentrations 1,5 mM of
proline caused a significant decrease in hormone concentration. Stressed plants treated with both
concentrations of SA and 1, 10 mM proline showed a significant decrease in hormone concentration.
The bilateral interaction treatments between SA and proline showed that (1 mM pro.+ 10-4 , 5*10-5 M
of SA ) and (5 mM pro.+ 5*10-5 M of SA) caused a significant decrease in free IAA concentration.
73
The triple interaction between salt,SA and proline showed that free IAA concentration was
decreased significantly in stressed plants compared to unstressed plants. Also, the same result was
observed during 1,5 mM proline treatments in unstressed plants. While free IAA concentration
increased in stressed plants. SA treatment maintain free IAA concentration in normal range
compared to control plants. All interaction treatments decreased IAA concentration.
Table (1):The effect of salt , S.A , proline and their interaction on free IAA
concentration (M) of leaves / vegetative stage.
Salt concentration
dSm/m
Proline concentration mM
S.A concentration M
0
Mean of Salt*SA
0
1
5
10
1.365
.920
.891
1.314
.710
.795
1.043
1.130
1.355
.908
.866
.889
1.054
1.218
1.120
.938
1.341
.700
.946
.639
1.032
.690
.845
.623
.931
.845
.998
-4
1.3
10
-5
5*10
0
5
-4
10
-5
5*10
Mean of proline
L.S.D0.05
Salt * Proline
1.115
salt * SA * proline =0.4
proline=0.164
1.3
5
L.S.D0.05
salt * SA =0.2
1.122
.920
1.062
1.025
.906
.798
Mean of salt
1.143
.874
.933
1.188
1.035
1.087
salt * proline = 0.23
.815
.929
salt =0.12
.807
.910
Mean of S.A
1.127
.987
1.055
1.126
1.074
1.026
.748
.994
.885
.913
1.194
.799
0
S.A * Proline
-4
10
-5
5*10
L.S.D0.05
.744
.983
SA * proline = 0.283
.930
SA=0.14
Table (2) showed no significant effect in bound IAA concentration between unstressed and
stressed plants neither treated with SA and proline nor untreated plants. While, bound IAA
concentration increased significantly during SA 5*10-5 M treatment of unstressed plants. But it
decreased in stressed plants. Whereas, proline had no effect on hormone concentration both in
stressed and unstressed plants. The combination (10mM pro.+ 5*10-5 M SA) showed a significant
increase in bound IAA concentration compared with control plants. The same results were observed
at the combinations (5,10mM pro.+ 5*10-5 M SA) and (1,5 mM pro.+ 10-4 M SA) of stressful and
unstressed plants.
74
Table (3) clarify a non significant decrease in free GA concentration in stressful plants. Also,
neither SA nor proline treatments lonely or in combination had significant effect on the plants.
Table (2):The effect of salt , S.A , proline and their interaction on bound IAA concentration (M) of leaves /
vegetative stage.
Salt concentration
dSm/m
0
1.3
10
-4
5*10
-5
0
10-4
5
5*10
-5
Mean of proline
L.S.D0.05
0
1
5
10
Mean of
Salt*SA
.5085
.5943
.2867
.6581
.512
.5147
.3886
.6397
.7269
.567
.7105
.5555
.7747
.8475
.722
.6417
.6101
.5785
.8122
.661
.6190
.7804
.8078
.4026
.652
.6467
.5454
.5350
.6548
.595
.607
.607
.604
.684
S.A concentration M
proline=0.095
salt * SA =0.12
Mean of salt
1.3
.578
.513
.567
.744
.600
5
.636
.645
.640
.623
.636
Salt * Proline
L.S.D0.05
salt * proline =0.14
0
10-4
S.A * Proline
5*10
-5
salt =0.07
.575
.602
.433
.735
.586
.567
.584
.724
.565
.610
.679
.550
.655
.751
.659
SA * proline =0.17
L.S.D0.05
Mean of S.A
SA=0.08
Table (3):The effect of salt , S.A , proline and their interaction on free GA concentration
(M) of leaves / vegetative stage.
Salt concentration
dSm/m
S.A concentration
M
Salt * Proline
1
5
10
0
28.0209
24.2453
22.1858
34.4385
27.223
10
19.6583
24.7445
29.5291
31.8070
26.435
5*10-5
35.6918
28.2289
26.1643
29.8619
29.987
0
23.8708
27.3656
30.8604
26.4919
27.147
10-4
27.1680
19.5439
30.6420
18.5350
23.972
5*10-5
26.5647
22.2014
16.2259
26.6791
29.987
26.829
24.388
25.935
27.969
Mean of proline
L.S.D0.05
Mean of Salt*SA
0
-4
1.3
5
27.790
25.740
25.960
32.036
27.881
5
25.868
23.037
25.909
23.902
24.679
salt =3.47
Mean of S.A
25.946
25.805
26.523
30.465
27.185
10-4
23.413
22.144
30.086
25.171
25.203
5*10-5
31.128
25.215
21.195
28.271
26.452
0
L.S.D0.05
Mean of salt
1.3
L.S.D0.05
S.A * Proline
salt * SA =6.01 proline=4.9
SA * proline =8.5
SA=4.25
75
Table (4) demonstrated a significant decrease in bound GA concentration in stressful plants.
Alternatively, SA and proline had no significant effect on hormone concentration. Meanwhile, 5*105
M SA caused a significant increase in bound GA concentration in unstressed plants, while it
caused a decrease in GA concentration in stressful plants. But, proline at 5 mM caused an increase
in hormone concentration compared with 0 mM.
The combination between SA and proline showed that ( 1 mM pro.+ 0, 10-4 M SA) caused an
increase in GA concentration.
The triple combination between salt ,SA and proline showed that 5*10-5 M SA caused an
increase in GA concentration in unstressed plants. Whereas no significant effect appeared neither in
stressful plants nor in the combination treatments.
Table (4):The effect of salt , S.A , proline and their interaction on bound GA
Salt concentration
dSm/m
1.3
S.A
concentration
M
0
0
1
5
10
11.3347
13.2668
9.0257
11.4361
11.9250
12.1772
10.5676
12.1070
14.7125
11.1709
12.6921
9.0153
10.5221
12.0290
12.7857
12.1226
7.2315
12.0550
11.5610
5.7935
10.7341
10.826
8.6122
11.301
10.5468
11.070
10.9681
10.869
-4
10
5*10
-5
0
5
-4
10
5*10
-5
Mean of proline
L.S.D0.05
Salt * Proline
salt * SA * proline = 2.41
proline=0.99
1.3
12.657
5
L.S.D0.05
0
S.A * Proline
11.266
11.694
12.840
10.922
9.160
10.215
Mean of
salt
10.762
12.110
11.933
8.994
10.396
11.379
salt =0.7
9.628
10.099
Mean of S.A
10.175
11.894
10.527
11.779
11.094
9.578
12.116
11.064
8.950
10.427
11.619
11.877
SA=0.85
11.528
-4
10
5*10
L.S.D0.05
12.205
salt * SA =1.21
Mean of
Salt*SA
-5
12.723
9.892
SA * proline =1.71
The results in table (5) showed that salt stress caused a significant decrease in free CK
concentration about 30.5%. Also, we demonstrated that 10-4 M SA caused a significant decrease in
free CK concentration compared with control plants. But, 5*10-5 M SA maintain hormone
concentration in the leaves. Whereas, proline caused hormone concentration decrease.
76
The combination between salt and SA had no effect on free CK concentration in unstressed and
stressful plants which their hormone concentration still lower than control plants. The same results
observed in proline treated plants.
The combination between SA and proline showed that (5,10 mM pro.+ 10-4 M SA) caused a
significant decrease in free CK concentration, whereas10-4 M SA alone increased it.
The triple interaction between salt ,SA and proline showed that both concentrations of SA and
the interaction (10 mM pro.+ 5*10-5 M SA) caused an increase in free CK concentration in
unstressed plants, but salt stress cancelled the positive effect of SA.
Table (5):The effect of salt , S.A , proline and their interaction on free CK
Salt concentration
dSm/m
S.A concentration
M
1.3
0
0
1
5
10
8.7112
10.2875
8.9020
9.6376
11.6121
9.9349
5.6802
6.2942
11.5596
10.1838
7.3755
7.6520
6.5140
5.3760
11.5319
7.4764
9.2712
6.0508
3.5467
3.6545
7.0685
9.312
6.8445
8.303
7.5331
6.402
6.6910
7.548
-4
10
5*10
-5
0
5
-4
10
5*10
-5
Mean of proline
L.S.D0.05
Salt * Proline
salt * SA * proline = 2.54
proline=1.04
1.3
5
S.A * Proline
10.1627
6.7546
5.6308
7.0343
7.319
9.155
9.309
6.470
5.485
salt =0.73
5.941
6.473
Mean of S.A
8.182
8.401
7.139
8.557
8.070
10.442
7.993
4.613
4.974
7.006
7.454
9.111
SA=0.9
8.598
10.135
-4
10
5*10
L.S.D0.05
8.3803
Mean of salt
7.997
0
9.3845
salt * SA =1.27
10.628
L.S.D0.05
Mean of
Salt*SA
-5
9.314
8.514
SA * proline =1.8
Table (6) showed that bound CK increased during salt stress treatment about 24.2%. The same
results observed at 10-4 M SA treatment. Whereas, proline had no significant effect on bound CK
concentration.
The interaction between salt and SA showed that untreated plants and treated plants with 10-4 M
SA caused an increase in hormone concentration. The same results were observed in plants treated
with 1,5 mM proline in stressful plants when compared with unstressed plants.
The table also showed disappearance of significant differences in bound CK concentration in
unstressed plants except of (0, 10 mM pro.+ 5*10-5 M SA) which increase hormone concentration.
77
The same results were found in stressful plants treated with the interaction (1,5 mM pro.+ 5*10 -5 M
SA) and 10-4 M SA alone when compared with control plants.
Table (6):The effect of salt , S.A , proline and their interaction on bound CK
Salt concentration
dSm/m
S.A concentration
M
1.3
0
10
1.3
5
6.7311
8.4830
6.9219
6.7062
6.4255
4.8464
5.6885
9.8519
4.6155
6.7449
8.7637
9.7766
10.7894
10.6719
8.6185
11.6149
7.0713
6.8528
8.4623
6.8058
8.229
9.2504
7.312
8.8978
7.769
6.5804
7.824
10
salt * SA =1.58
7.397
proline=1.3
Mean of
Salt*SA
6.9423
5.9167
7.9711
9.4870
8.5003
7.8836
Mean of salt
5.924
6.691
7.761
6.943
8.699
8.847
salt =0.91
7.887
8.624
Mean of S.A
7.198
8.254
9.636
7.770
6.943
9.161
6.748
5.850
7.075
8.624
7.821
8.626
SA=1.12
6.943
9.061
0
-4
5*10
L.S.D0.05
5.6332
L.S.D 0.05
S.A * Proline
10
-5
Mean of proline
Salt * Proline
5
-4
5*10
L.S.D 0.05
1
-5
0
10
0
-4
5*10
5
-5
8.329
6.933
SA * proline =2.23
Table (7) showed that salt stress caused a significant decrease in free ABA concentration about
10.7%.When the plants were treated with SA, the significant effects were disappeared. Whereas,
proline treatments(1,10) mM caused decrease hormone concentration.
The combination between salt and SA or proline clarify a significant decrease in free ABA
concentration when unstressed plants were treated with 10-4 M SA ,whereas, the concentrations 0,
5*10-5 M SA and 1,5 mM proline caused the same results in stressful plants when compared with
control once.
The combination between SA and proline had no significant effect on free ABA concentration
except the interaction (5 mM pro.+ 5*10-5 M SA) which cause free ABA decrease. The triple
interaction between salt, SA and proline showed no significant effect of almost treatments in
stressful plants.
78
Table (7):The effect of salt , S.A , proline and their interaction on free ABA
Salt concentration
dSm/m
1.3
S.A
concentration
M
0
0
1
5
10
387.6787
316.9242
294.4055
442.0561
248.9844
326.1363
277.7085
351.9816
458.2414
366.6955
339.5068
313.7895
282.6024
251.4154
381.5373
342.5775
460.6084
256.7892
391.5171
248.3447
351.5977
370.150
280.7792
304.988
213.7991
294.725
348.2711
352.461
-4
10
-5
5*10
0
5
-4
10
-5
5*10
Mean of proline
L.S.D0.05
Salt * Proline
1.3
364.968
5
336.585
375.332
273.390
L.S.D0.05
0
S.A * Proline
salt * SA =46.9
proline=38.33
Mean of
Salt*SA
360.2661
301.2027
386.4952
297.5962
339.3148
298.6118
Mean of salt
303.874
391.858
349.321
285.577
salt =27.11
313.064
311.841
Mean of S.A
350.734
299.763
272.910
392.317
328.931
354.796
291.463
334.613
300.163
320.259
364.904
SA=33.2
342.553
-4
10
-5
5*10
L.S.D0.05
404.920
323.737
276.653
SA * proline =66.4
Table (8) clarify a significant decrease about 18.9% in bound ABA concentration during stress
treatment. Also, we observed a decrease in hormone concentration in plants treated with 10-4 M SA
and its increase in plants treated with 5*10-5 M SA. This is observed increase the concentration of
the bound hormone when spraying with the concentrations(1 and 10) mM proline. While, 5 mM
caused hormone decrease significantly.
Bilateral interactions showed increase free ABA concentration significantly in stressful plants.
The same result were observed in unstressed plants treated with 5*10-5 M SA. While, it decreased in
stressful plants treated with (0,5.10) mM proline.
Sequential spraying with SA and proline showed that bound ABA concentration decreased in
plants treated with (0,5,10 mM pro.+ 10-4 M SA).
The triple interaction clarify a significant increase in bound ABA concentration when exposing to
salt stress without any treatment. But its concentration decreased when stressful plants sprayed with
(0,1,5,10 mM proline +5*10-5 M SA),while there were no effect in plants treated with 10-4 M SA.
79
Table (8):The effect of salt , S.A , proline and their interaction on bound ABA
concentration (M) of leaves /vegetative stage.
Salt concentration
dSm/m
S.A
concentration
M
0
0
1
5
10
237.5012
276.8128
181.9403
249.1763
227.8092
246.6174
225.9220
271.8869
398.2343
375.3319
365.2880
351.5977
289.9914
228.3850
515.3056
275.2135
180.5969
274.8297
261.3953
168.5699
183.9235
263.277
261.9710
287.592
173.6877
239.436
246.6174
287.795
-4
1.3
10
-5
5*10
0
5
-4
10
-5
5*10
Mean of proline
L.S.D0.05
Salt * Proline
proline=23.12
1.3
287.848
5
L.S.D0.05
0
S.A * Proline
salt * SA =28.3
Mean of
Salt*SA
236.3577
243.0589
413.5400
286.2969
221.3479
216.5499
Mean of salt
299.587
257.717
345.456
297.652
238.706
275.597
221.156
salt =16.4
230.134
241.398
Mean of S.A
294.549
283.402
205.163
262.195
261.327
204.203
260.724
243.659
220.228
232.203
291.079
318.651
269.488
380.962
SA * proline = 40.04
SA=20.02
315.045
-4
10
-5
5*10
L.S.D0.05
Discussion
Plant hormones auxin (indole-3-acetic acid), gibberellins, cytokinins, and abscisic acid are central
to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the
hormone concentration can reveal different plant strategies to cope with the stress, e.g., suppression
of growth or mobilization of plant metabolism.
As mentioned in our previous research the growth parameters were decreased significantly (Jasim
et al,2012), This is may be related to the lack of cell division resulting from a lack of free auxin and
free CK table (1 and5). This is compatible with (Yew et al., 2010) who found that CK is one of the
hormones necessary to stimulate the elongation of the shoot. In addition, (Vernoux et al., 2010)
mentioned that free auxin plays an important role in meristimatic cell division of the shoot to
configure the parts of the plant, or due to the inability of GA concentration to cope with the adverse
effect of salt stress even though it does not differ significantly from its concentration in the
treatment of control plant (table 3). In addition our results regarding free and bound ABA decreases
in the leaves were in agreement with the decrease wet weight of the leaves due to decrease hormone
efficiency in controlling stomata closure.
Spraying pepper plant with SA caused decrease free IAA and CK concentrations table(1 and 5)
because SA might interfere with auxin responses resulting in stabilization of the Aux/IAA repressor
proteins and inhibition of auxin responses (Wang et al., 2007). Or, due to its ability to inhibit CK
signaling because SA negatively regulates cytokinin signaling creates a sort of balance helps the plant
to withstand stress (Argueso et al.,2012;Choi et al.,2011).
80
Exogenous proline supply showed a negative role in maintaining cell hormone system during
vegetative stage of plant growth table (1,5,7) due to its poisoning effect causing mitochondria and
chloroplast break down and accelerating programmed cell death (Bonner et al., 1996; Hellmann et al.,
2000; Hare et al., 2002). This result was compatible with (Mattoli et al., 2009) who demonstrated that
exogenous proline cause excessive increase of endogenous proline in shoot and root leading to cell
damage. Or this decrease in hormones concentration as a result of an attempt of plant to cope against
the stress conditions to reduce water loss.
Many studies had noted to the role of bound IAA with increased susceptibility of the plant to
stress tolerance (Muller, 2011). Junghans et al., 2006 isolated enzymes liberated for auxin (Axin conjugate hydrolase) from tissues exposed to stress in the poplar plant, also he found that the IBA glucose has a role in the response of the plant to saline stress (Tognetti et al., 2010), and found that
bound CK had an importance in plant development because its ability to organize active CK
concentration and its transmission and inhibition (Auer, 1997) and this is evidenced by the results of
this study table (2,6). In addition, bound ABA had an active role in plant stress tolerance which is
associated with an increased in endogenous proline content table (8). Dietz et al.,2000 demonstrated
that bound ABA concentration decreased when exposed to saline stress due to increasing the
effectiveness of certain enzymes liberated bound hormone and the formation of active form of the
hormone and the most important of these enzymes is B - glucosidase which rises effectiveness during
plant exposure to salt stress. It is believed that the external proline stimulates the effectiveness of this
enzyme. The accumulation of endogenous proline needs to change the sensitivity of the cells to the
ABA by affecting the metabolism or association of the hormone, and that many of the metabolic
pathways of the hormone was considered within the mechanical mechanism of hormonal imbalance
that prevents ABA accumulation to maintain a certain concentration of the hormone that fits the type
of stress )Verslues and Bray,2006 ( .
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83
The relationship between the levels of Cardiac troponin I (cTnI) and
criteria for the metabolic syndrome in patients with type II diabetes.
Amira kamal Mohammed1().
1
, Iraq Biology Department, Environmental Research \ Babylon University \, Babil University -51002
Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher)
© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011
ABSTRACT
The aim of this study was to find out the type and extent of the relationship between cardiac troponin I and
criteria for the metabolic syndrome among patients with type II diabetes as defined by the World Health
Organization (WHO) in the diagnosis of the metabolic syndrome criteria. This study was conducted on
patients with diabetes during their review in the laboratory of Murjan Hospital Educational Specialist/ Babel
province for the period from February / March 2013 .This study included 43 patients with type II diabetes 18
(41.86%) were male, 25 (58.14%) of females and the average age (Mean56.35 ± standard error 1.04 years) for
age groups ranging from 42-65 years and the average length of injury (Mean 6.61 ± standard error 0.74 years).
and rates level of cardiac troponin I (Mean 0.023 ± standard error 0.003 μg/L). The results showed no
significant correlation p => 0.05 between levels Troponin I cardiac and the metabolic syndrome criteria.
KEYWORDS
Type keywords : metabolic syndrome (MetS) , troponin I , dyslipidemia, hypertension, insulin resistance, BMI.
‫الخالصة‬
‫القلبي ومعيايير مر يى متال مية ا ييم الم يابي بيدا سيرر النيوع‬
‫هيذه الدراسية عليى المر يى دا سيرر النيوع‬
. 2013 ‫ آذار‬/‫م يباط‬
‫تهدف هذه الدراسة على إيجاد نوع ومدى العالقة بيي تربيوني‬
‫ أجريي‬. ‫الثان حسب تعريف منظمة ال حة العالميية يي تيي يع معيايير متال مية ا ييم‬
‫ مرر محايظة بابل للمدة‬/
‫الثان أثنا مراجعتهم م تبر السرر ي مستيفى مرجا التعليم الت‬
±56,35 ‫) م اإلناث ومتوسيط عمير (معيدل‬٪58,14( 25 ،‫) ذرور‬٪41,86( 18 ‫النوع الثان‬
.)‫ سينة‬0,74
‫ ال طيأ المعييار‬± 6,61 ‫ سنة ومتوسط ميدة إ يابة (معيدل‬65-42 ‫م‬
‫ مري ا بدا سرر‬43 ‫هذه الدراسة‬
‫العمرية تراوح‬
‫يمل‬
‫ سنه) للفئا‬1,04 ‫ال طأ المعيار‬
‫ أظهير النتيائم عيدم وجيود عالقية ذا د لية‬.)‫ لتير‬/ ‫ ميرروغيرام‬0,003 ‫ ال طأ المعيار‬±0,023 ‫ومتوسط مستوى تربوني القلب (معدل‬
.‫وبيييييييييييييييييييي معيييييييييييييييييييايير متال مييييييييييييييييييية ا ييييييييييييييييييييم‬
‫معنويييييييييييييييييييية بيييييييييييييييييييي مسيييييييييييييييييييتويا التربيييييييييييييييييييوني القلبييييييييييييييييييي‬
84
Special Issue - Proceding of 5th International Conference of Environmental Scince University of Babylon / vironmental Research Center 3-5
December 2013
1introduction
The metabolic syndrome (MetS) is a clustering of metabolism-related disorders and conditions that may
share common pathophysiological: including glucose intolerance/insulin resistance, abdominal obesity,
dyslipidemia, and high blood pressure—hypertension (HTN) Insulin resistance and obesity play the
central role in the pathophysiology of the MetS. [1,2]. The first set of criteria for the diagnosis of metabolic
syndrome was proposed in 1999 by WHO [3].The WHO diagnosis required proof of insulin resistance,
either through the existence of glucoregulation issues (altered fasting plasma glucose, altered tolerance
to glucose or type 2 diabetes mellitus), either through the use of a hyperinsulinemic euglycemic clamp,
which requires a level of glucose binding under the lowest quartile for the studied population. Together
with insulin resistance, at least two of the following parameters need to be associated: 1) arterial
hypertension, BP ≥ 140/90 mmHg; 2) increased triglycerides concentration ≥150 mg/ dl (1.7 mmol/l)
and/or decreased concentration of HDL-cholesterol - men < 35 mg/dl (0.9 mmol/l), women < 39 mg/dl (1
mmol/l); 3) obesity – waist-to-hip ration: men >0.9, women > 0.85 and/or BMI > 30 kg/m2;4)
microalbuminuria(urinary albumin excretion ≥ 20 μg/min or albumin/creatinine ≥ 30 mg/g). *4+ The MetS
increases risk of myocardial infarction (heart attack) and stroke; it is also associated with an increased
cardiovascular disease mortality and all-cause mortality [5,6]. Cardiac troponin T (cTnT) and cTnI are
currently used as sensitive and specific laboratory markers to assess the presence of myocardial damage
or myocardial necrosis and used in the diagnosis of acute myocardial infarction and for risk stratification
of patients with acute coronary symptoms [7,8].Cardiac troponin I (cTnI) analysis is a highly sensitive
and specific method for the detection of myocyte injury which has been used to diagnose myocardial
damage in many mammalian species including humans, dogs and cats [9- 11].
Materials and method
The materials that were used in this research is the Estimate bar the level Microalbuminuria, Kit to measure
the level of Troponin (cTnI) Equipped from Company BioMérieux , Kit to measure the lipid profile of the
company BIOLABO and the company Rondo and , (Kit ) to measure a patient's blood sugar level is equipped
with a Company Audit. Was conducted practical part of the research in the laboratory of Murjan Hospital
Educational Specialist, as samples were collected (blood and urine) for people with diabetes type II during
fasting and before breakfast (8- 12 hours) and randomly, Where the level of glucose were measured in
fasting serum after 12 hours of fasting according to the principle of enzymatic oxidation presence of an
enzyme Glucose _Oxidase (GOD) [12]. Measuring the level of triglycerides TG in fasting serum after 12
hours of fasting according to the principle of
enzymatic hydrolysis [13,14]. Measuring the level of high-density lipoprotein HDL in the blood serum of
human according to the principle that the low-density lipoprotein LDL and lipoprotein very low density
VLDL and chylomicrons resides within the blood serum were separated and deposited mediated Add
solution phosphotungstic acid-containing ions of magnesium and Concentration was measured after a
process of centrifugation The wavelength of 500 Nanomitra in Temperature (21) ° C. [15]. Estimate
Microalbuminuria
levels
mediated
by
dipping
Microalbuminuria
examination
bar
measuring
Microalbuminuria in lactation cursor to the middle of the mark on the tape for 5 seconds and then taken out
and left for a minute, compared to the color on the color ribbon on the box and read the result Balmelgram
Address correspondence to Amira kamal: [email protected].
85
December 2013
per liter. Calculate body mass index (BMI) by measuring the weight (kg) and height (m) for each person was
measured body mass index according to the following equation: BMI=Wt/(Ht)² Where: weight = weight (kg),
(Ht)²= box height (m)² [16]. Measuring levels of systolic blood pressure and diastolic mediated by a Mercury
sphygmomanometer where readings were taken after 10 minutes from the first reading [17]. Measurement of
troponin in the blood serum of patients immune-mediated way, Which works according to the principle of
sandwich Immunoassy. It is an automated quantitative test which is used for the determination of human
cardiac troponin I in human serum by using an enzyme-linked fluorescent assay technique( ALFA). The
measurement values of the VIDAS Troponin I Ultra kit range from 0.01to 30 μg/L. A value of more than 0.11
μg/L is considered to be significant *18+.
Statistical Analysis
The statistical analysis of this study was made by using SPSS program (Version 15.0) and the statistical
processes used here were Means, Standard Error, One –sample T Test , Correlation coefficient and Linear
regression[19].
Results
The results showed The average metabolic syndrome criteria (as defined by the World Health
Organization) were as follows: Average high level of
Fasting blood glucose (mmol/l.) has reached
11.28(mmol/l.), Average level of Systolic B. P. 15.11 (mmHg), : Average high level of Diastolic B. P. 8.981
(mmHg),
Average
high
body
mass
index
31.97(Kg/m2),
Average
high
level
of
Microalbuminuria37.91(mg/l), Average high level Triglycrides 2.06 (mmol/l) Or the low level of highdensity lipoprotein 1.058(mmol/l) For age groups ranging between (42.00-65.00 years) and the average
age was 56.35 years and the duration of injury by diabetes type II ranged between (1.00-2.00Year) while
the average levels of cardiac troponin in the blood serum of The Following patients were within normal
limits, reaching 0.023 (μg / L)to range between 0.01-0.10. See table 1. Results showed no linear
relationship significant (p> 0.05) between the levels of fasting blood glucose FBG(mmol/L) and levels
Cardiac troponin I (cTnI) (μg / L) In the blood serum of the male and the female If the linear equation Y =
0.02 + 0.00 * X and correlation coefficient (r) is equal to(0.01). See Figure (1): a, and no linear relationship
significant (p> 0.05) between the levels of High density lipoproteins HDL (mmol/L) and levels Cardiac
troponin I (cTnI) (μg / L) In the blood serum of the male and the female If the linear equation Y = 0.03 + 0.01 * X and correlation coefficient (r) is equal to(0. 1) See Figure (1): b, and no linear relationship
significant (p> 0.05) between the levels of Triglycrides(mmol/L) and levels Cardiac troponin I (cTnI) (μg /
L) In the blood serum of the male and the female If the linear equation Y = 0.02 + -0.00 * X and correlation
coefficient (r) is equal to(0.01). See Figure (1): c, and no linear relationship significant (p> 0.05) between
the levels of Microalbuminuria (mg/L) and levels Cardiac troponin I (cTnI) (μg / L) In the blood serum of
the male and the female If the linear equation Y = 0.03 + -0.00 * X and correlation coefficient (r) is equal
to(-0.3) See Figure (1): d, and no linear relationship significant (p> 0.05) between the levels of Systolic
86
December 2013
blood pressure (mmHg) ) and levels Cardiac troponin I (cTnI) (μg / L) In the blood serum of the male and
the female. . If the linear equation Y = -0.00 + 0.00 * X and correlation coefficient (r) is equal to(0.11) See
Figure (1): e, and no linear relationship significant (p> 0.05) between the levels of Diastolic blood pressure
(mmHg) ) And levels Cardiac troponin I (cTnI) (μg / L) In the blood serum of the male and the female. . If
the linear equation Y = 0.05 + -0.00 * X and correlation coefficient (r) is equal to(- 0.2) See Figure (1): f, and
no linear relationship significant (p> 0.05) between the levels of Body max index(Kg/m2) and levels
Cardiac troponin I (cTnI) (μg / L) In the blood serum of the male and the female If the linear equation Y=
0.05 + -0.00 * X and correlation coefficient (r) is equal to(-0.22) See Figure (1): g.
Discussion
The results showed that the average cardiac troponin levels were within normal levels in samples of
patients with metabolic syndrome. Results also showed no significant relationship between the average
levels of the metabolic syndrome criteria and the levels of cardiac troponin See Table 1, Figure 2. The
Cardiac troponins play a central role in diagnosis and risk stratification in acute coronary syndromes ,
acute myocardial infarction(AMI) but troponins are recognized as markers of cardiac myocyte injury, not
of the etiology of injury.Awide range of clinical conditions has been associated with increased [20].
The elevated troponin levels could reflect silent myocardial necrosis especially in patients with a history
of ischemic heart disease[21,22]. left ventricular hypertrophy associated with perfusion defects or
abnormal coronary vasomotion[23,24]. left ventricular systolic dysfunction[25]. increased cardiac preload
with myocardial stretch [26,27]. microvascular disease, especially in diabetic patients[26,28]. endothelial
dysfunction secondary to oxidative stress and inflammation[29,30]. episodes of hypotension during
dialysis or cardiac injury secondary to calcium and oxalate deposition[31]. Many studies show that in
addition to the main components of MS, there are other components playing an equally important role in
the development of ischemic heart disease ( IHD). Such components are age, smoking, lifestyle
peculiarities, gender, genetic factors, low-density lipoprotein cholesterol β phenotype, reduction of
endothelial function, increased activity of C-reactive protein, fibrinogen, lipoprotein (α), homocysteine,
lowdensity lipoprotein cholesterol, uric acid, VII factor, interleukin-6, and plasminogen activator
inhibitor and they are associated with pathogenesis of MS componentsThe assessment of acute
myocardial infarction (AMI) as formally established by the World Health Organization(WHO) requires at
least 2 of the following criteria: a history of chest pain, an ST-segment elevation on the electrocardiogram,
and increased cardiac enzymes, such as creatine kinase (CK), its MB (CK-MB) isoenzyme and troponin
[32]. The early release of troponin is similar to those of total CK and CK-MB, with a rise at 4 to 6 hours
after AMI. troponin levels remain elevated, and may be detected for 3 to 7 days following AMI The
absence of a significant relationship between the average levels of the metabolic syndrome criteria and
between the levels of cardiac troponin may be due to several reasons, including hyperglycaemia and
elevated troponin concentrations are less well established, Acute coronary syndromes (ACS) include a
spectrum of subjects who present with chest discomfort, with or without other clinical features, caused by
87
December 2013
myocardial ischaemia. the presence of hyperglycaemia did not affect the presenting absolute troponin I
concentration. These findings are supported by other studies[33,34]. Or because of the absence of the
main factors leading to higher average rates of troponin samples within the current study, including
:cardiac dysfunction, left ventricular hypertrophy, as well as subclinical myocardial infarction and is
associated with an increased risk of morbidity and mortality in those patients. [35,36]. Or because A
series of risk that influence e.g. gender and age relate to the development of atherosclerosis and the risk
of presenting with ACS such as Post menopause, the risks myocardial infarction increase for women but
remain lower than for men of corresponding age [37]. Or because of not taking into consideration the
family history of heart disease for each patient and its relationship to the level of cardiac troponin in the
study sample[32]. Or the right type of treatment which takes him with metabolic syndrome and taking
into account the lack of exposure to other serious complications such as heart disease [38]. Or due to lack
of patients suffering with chest pain, in whom unstable coronary disease is possible but not overt, are at
higher risk of cardiac events if troponin is elevated [39].
Acknowledgements
I thank all diabetic patients who have collaborated with me to give a sample of blood, urine and provide
me with information about their health status also thank Prof. Dr. Mushtaq Talib Wtwt from Medicine
College, Babylon University, which paved me the idea of the study.
88
December 2013
.
Table 1: Clinical characteristics of patients with metabolic syndrome and Cardiac troponin I rate among the patients
* Standard Error
Variable
Mean (SE*)
Range
Fasting blood glucose
(mmol/l.)
11.28(0.61)
6.20-19.40
Systolic B. P. (mmHg)
15.11(0.29)
8.00-17.60
Diastolic B. P. (mmHg)
8.981(0.15)
6.90-11.70
Elevated Microalbuminuria
(mg/l)
37.91(4.62)
0.00-90.00
Reduced High Density
Lipoprotein .(mmol/l)
1.058(0.045)
0.60-2.00
Elevated Triglycerides
.(mmol/l)
2.06 (0.11)
Increased Body Mass
Index(Kg/m2)
31.97(0.82)
22.70-45.90
Duration of Diabetes
Mellitus Type 2 (years)
6.61(0.74)
1.00-2.00
Age (years)
56.35(1.04)
42.00-65.00
Cardiac troponin I (cTnI)
(μg/L)
0.023(.003)
0.01-0.10
Figure 1 : the relationship between the metabolic
0.70-4.60
syndrome criteria as defined by the World Health
Organization: (a) Fasting blood glucose (mmol/L),(b)
High
density
lipoproteins
HDL(mmol/L),(c)
Triglycrides(mmol/L),(d)Microalbuminuria
(mg/L),(e) Systolic blood pressure (mmHg), (f)
Diastolic blood pressure (mmHg),(g) Body max
index(Kg/m2), and the levels of Cardiac troponin I
(cTnI)( μg/L) In the blood serum of the male and the
female.
b
0.10
0.10
0.08
0.08
0.06
0.06
0.04
Y = 0.02 + 0.00 * X
r = 0.01
0.02
p =>0.05
7.50 10.00 12.50 15.00 17.50
Fasting blood glucose (mmol/L)
n = 43
troponin I (cTnI) )( μg/L)
a
Y = 0.03 + -0.01 * X
r = - 0.1
0.04
p =>0.05
0.02
n = 43
1.00
1.50
2.00
High density lipoproteins HDL (mmol/L)
89
December 2013
d
c
0.10
0.10
0.08
Y = 0.03 + -0.00 * X
Y = 0.02 + -0.00 * X
0.06
r = -0.01
0.04
p =>0.05
0.02
n = 43
1.00
2.00
3.00
0.08
0.06
r = -0.3
p =>0.05
0.04
n = 43
0.02
0.00
25.00
50.00
75.00
Microalbuminuria (mg/L)
4.00
Triglycrides(mmol/L)
f
e
0.10
0.10
Y = -0.00 + 0.00 * X
r =0.11
0.06
p =>0.05
0.04
n = 43
0.02
8.00
10.00 12.00
16.00
Systolic blood pressure (mmHg)
0.08
0.08
Y = 0.05 + -0.00 * X
0.06
r =- 0.2
p =>0.05
0.04
n = 43
0.02
7.00
8.00
9.00
10.00
11.00
Diastolic blood pressure (mmHg)
g
0.10
Y= 0.05 + -0.00 * X
0.08
r =-0.22
0.06
p =>0.05
0.04
n = 43
0.02
25.00 30.00 35.00 40.00 45.00
Body max index(Kg/m2)
90
December 2013
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[24 ] Porter, GA.; Norton, TL.; Lindsley, J. Relationship between elevated serum troponin values in end-stage renal disease
patients and abnormal isotopic cardiac scans following stress. Ren Fail. 2003,25,:55-65.
[25] Mallamaci, F.; Zoccali, C.; Tripepi, G.; Benedetto, FA.; Parlongo, S. Diagnostic value of troponin T for alterations in left
ventricular mass and function in dialysis patients. Kidney Int. 2002,62,1884-90.
[26] Iliou, MC.; Fumeron, C.;Benoit, MO. Tuppin, P.; Calonge, VM.;Moatti, N.; Buisson, C.; Jacquot, C. Factors associated with
increased serum levels of cardiac troponins T and I in chronic haemodialysis patients: Chronic Haemodialysis And New
Cardiac Markers Evaluation (CHANCE) study. Nephrol
Dial Transplant. 2001,16,1452-8.
[27] Wayand, D.; Baum, H.; Schatzle ,G.; Schärf, J.; Neumeier, D. Cardiac troponin T and I in end-stage renal failure. Clin Chem.
2000,46,1345-50.
[28] Freda, BJ.; Tang, WH.; Van Lente, F. Peacock, WF.; Francis, GS. Cardiac troponins in renal insufficiency: review and
clinical implications. J Am Coll Cardiol. 2002,40,2065-71.
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MRCPAbnormalities of endothelial function in patients with predialysis renal failure. Heart. 2000,83,205-9.
[30] Scott, B.; Deman, A.;Peeters, P. CVan den Branden, C .;
Stolear,J.C.; Van Camp,G and Verbeelen,D . Cardiac troponin T and malondialdehyde modified plasma lipids in
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Vasavada, C.; Terrence, MD.; Sacchi, J. MD. Prognostic value of serum cardiac troponin I in ambulatory patients with
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December 2013
Concentration of some heavy metals in C.carpio fish collected from
Main outfall drain and its concentration in blood of local fishermen
Basim Y. ALKhafaji () A frah A. Maktoof
‫٭‬Biology Department –Science Collage-Thi-Qar University
ABSTRACT
The present study was carried out to assess the levels of six metals Pb, Cd, Ni, Mn, Co and
Cu in different tissues of C.carpio from main outfall drain also Cd and Pb in blood samples of
local fishermen. The levels of heavy metals were analyzed by flam atomic absorption
spectrophotometer(FAAS). Total levels in muscle, gill, liver and kidney tissues of the fish
showed a wide range. The order of heavy metals concentration in muscle Mn> Cu> Ni> Co> Pb>
Cd , in gills Mn> Pb> Ni> Cu> Co> Cd, in liver Mn> Co> Cu> Pb> Ni> Cd and in kidney Mn>
Pb> Cd> Cu> Co> Ni. The present study showed differences in metal concentrations in different
tissues. This due to the nature and the function of the tissue and ability of fish on regulating the
level of the metals in their bodies during the uptake and elimination processes.
The range of metals concentration µg/g in blood were (0.003-0.014)Cd and ( 1.6- 2.3) Pb.
The estimated values of all metals concentration in muscle and blood human in this study
were below the established values.
1: Introduction
The pollution of the aquatic environment with heavy metals has become a world wide
problem during recent years because they are indestructible and most of them have toxic effects
on organisms [1]. Among environmental pollutants toxic effect and ability to bioaccumulation
aquatic ecosystems [2]. The presence of heavy metals in aquatic ecosystem is the result of two
main sources of contamination; natural processes and natural occurring deposits and
anthropogenic activities. The main sources of heavy metal pollution to life forms are invariably
the result of anthropogenic activities [3, 4]. In the fresh water environment, toxic metals are
potentially accumulated in sediments and marine organisms and subsequently transferred to
Email :Basim_Y_ d @yahoo.com
94
December 2013
man through the food chain. Therefore, heavy metals can be bioaccumulated and biomagnified
via the food chain and finally assimilated by human consumers resulting in health risks [5]. As
a consequence, fish are often used as indicators of heavy metals contamination in the aquatic
ecosystem because they occupy high trophic levels and are important food source [6, 5].
Heavy metals including both essential and non essential metals have a particular significance
in ecotoxicology, since they are highly persistent and all have the potential to be toxic to living
organisms [7]. Studies have been done on heavy metals in rivers and fish [8, 9, 10, 11].
Bioaccumulation and magnification is capable of leading toxic level of these metals in fish
even when the exposure is low. The presence of metal pollutant in fresh water is known to
disturb the delicate balance of the aquatic systems. Fishes are notorious for the ability to
concentrate heavy metals in their muscles and since the play an important role in human
nutrition, they need to be carefully screened to ensure that unnecessary high level of some toxic
trace metals are not being transferred to man through fish consumption [12].
In recent years, awareness that heavy metals play a very important role, either beneficial or
harmful, in human health has increased .
Fish tissue accumulate heavy metals from their environment. They can act as indicators for
the levels in aquatic environment. It can be a source of food, leading to the possibility of
bioaccumulation metals in higher trophic levels of the human food chain. Humans may be
unwittingly exposed to heavy metals contaminated food, water and air [13]. The level of
Cadmium and lead in blood can be taken as a representative of dose/ exposure [14].
Consequently, the objectives of the present study were to determine the total level of six
heavy metals (Pb, Cd, Cu, Co, Ni and Mn) in the liver, gills, muscles and kidney of C. carpio, and
to assess whether edible part are safe for human consumption to evaluate relationship between
the accumulation metal in level in the edible part tissue and human blood sample obtained
from local fishermen.
95
December 2013
2: Materials and Methods:
2-1: Study area :
Main outfall drain is a river use to discharge the effluents of agriculture activities from its
both side. It is extended from Al-Shaklawiya near Baghdad north until Al-Basrah at the south
with length about 565 km [15]. It is dividing into three sectors (North, Mid and South), the
south sector (which the present study area is a part of its) extended from the end of the mid
sector until Shatt Al-Basarah in the south, with length about 165 km. The discharge of water is
220m3/sec in this sector [16]. New branch was opened in this sector with length 7 km, use to
transform the water to the marshes south Al-Nassiriya city.
Three stations were selected in the south sector of this river to implemented, the present
study, these are station 1(St.1) was near Al-Holandee bridge and the general caragge in the
center of Al-Nassiriya city, St.2 was 20 km far from the first station, while St.3 was in the
beginning of the new branch (Fig.1).
2-2: Materials and Methods :
Fish samples were captured from the study area by using gill nets 25*25 mm mesh size. The
captured fish were then placed in polyethylene bags and frozen immediately and transferred to
laboratory. In the laboratory, the fish washed with deionized water, standard length and weight
were measured to the nearest mm. and mg. respectively, then the abdominal cavity of each
specimen was opened and the organs gills, liver and kidney were separated, whereas muscle
was taken from the left posterior side of each fish, tissues were then dried under 105° for 24 hr.
The determination of metals in fish sample was done according to the following procedure
described by [17]. For two groups of the study samples were chosen on human blood. The first
group consisted of local fishermen (15 persons), while the second group, represented the control
sample, which consisted of (15 persons) away from the study area. The blood samples have
been taken from different individual, considering their age and sex, whether they are smokers
or not. Following the traditional method of blood withdrawn (5 cc)of blood is taken from each
96
December 2013
donor and placed in the tubes containing EDTA. The tubes contain all the samples to be
transported directly to the educational laboratories of the hospital near the study area .
Blood samples were taken from selected persons in Thi-Qar Province by sterile plastic
disposable injections (5) ml from every donor. The pipes that contained blood samples were
then transferred to centrifuge with a speed of 3000 rpm for 15 minutes. These tubes are sterile
and have good plastic covers. The serum is separated from all samples. The concentrations of
most other metals should be measured. The separated serum were diluted using micro-pipette
to separate the serum from the sediment of the blood .
Measuring samples was done by flame atomic absorption
spectrophotometer (flame
atomic absorption). The flame atomic absorption is the best and the simplest one used to
measure the concentrations of the some trace metals in the samples.
2-3: Statistical analysis:
The data of this study was used to analyze the variance, T test, mean, standard error and
standard deviation to test the significance of mean difference. Data has been analyzed by using
statistical analysis system [18].
3: Results:
The mean concentration of heavy metals in different fish organs are presented in Table (3),
Figure(2,3,4,5,6,7). The average concentration of Pb in different fish organs (liver, gills, muscles
and kidney) was 12.53, 19.71, 6.01 and 20.75 µg/g dry weight, respectively. The data was
showed that kidney accumulated the highest concentration, while muscles accumulated the
lowest.
The mean Cd concentrations were (0.08 µg/g in liver, 0.12 µg/g in gills, 0.04 µg/g in muscles
and 1.31 µg/g kidney respectively. The data revealed that, kidney accumulated the highest
concentration of Cd, while muscle accumulated the lowest concentration.
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December 2013
Nickel showed high concentration in gills and lowest concentration in liver. The average
concentration of manganese were 38.21 µg/g, 40.44 µg/g, 34.79 µg/g, 30.12 µg/g , in liver, gills,
muscles and kidney respectively. This mean that gills accumulated the highest concentration of
Mn, whereas kidney accumulated the lowest concentration. The highest concentrations of Co
and Cu was recorded in liver, while gills accumulated the lowest.
Heavy metals in human blood samples
Fifteen fishermen and fifteen males as control were investigated. Their ages ranged from (24
to 30) years for both fishermen and controls.
The concentration of trace elements in the serum were recorded higher values in fishermen
compared with control group (Table 2). However, the differences between mean Pb blood
content of both fishermen and control were found significant (t = 18.73 ; P<0.001). The statistical
analysis showed that there were significant differences (t = 8.36 ; P<0.001) in Cd mean among
the two studied samples.
4: Discussion :
The contamination of soils, sediments, water resources and biota by heavy metal is of major
concern especially in many industrialized countries because of their toxicity, persistence and
bioaccumulative nature [19]. Heavy metal contamination in sediment can affect the water
quality and bioaccumulation of metals in aquatic organisms, resulting in potential long-term
implication on human health and ecosystem [20]. The results of the present study showed that
the concentration of heavy metals varied among the organs of the studied species, this may be
due to the species-specific mechanisms. [21] indicated that the differences in accumulation
metal interdependency of the uptake and elimination rates of metals. The results obtained for C.
carpio showed that, liver accumulate and concentrate highest concentrations of Cu and Co. [22]
reported that the liver were the major site for Cu and Zn accumulation. High accumulation in
the kidney of Cd and Pb corroborated the results obtained by [23]. The results of the present
study disagree with those found by other researchers [24]. The results of this study showed that
the muscle tissue of these fish is not an active organ in accumulating heavy metals. Metal levels
98
December 2013
in liver reflect the high metal storage capacity of the organ. Liver stores heavy metals by
producing metallothionens which appears as a metal detoxification mechanism within the body
[25]. Metallothionens are a family of low molecular weight cystein rich proteins that occur in the
animals. Their synthesis can be induced by a wide variety of metal ions including Cd, Cu, Zn.
The accumulation of metal in liver and gills of food fish do not directly affected human health
because these are not edible parts. Nevertheless, the predatory animals such as birds whom
consume the whole fish from the river are at risk of excess metal contamination.
Muscles are the main edible part of fish and can directly influence human health. Therefore,
most governorates have established toxicological limits for heavy metals in seafood [5]. Heavy
metals tend to accumulate in different body organs. These metals are dangerous for fish in turn
they lead to serious problems in both man and animals. The difference between blood lead
levels in controls and fishermen leaves little doubt that regular exposure in high traffic areas
and diet type is associated with increased blood cadmium and lead concentrations [26].
The results obtained for the blood Pb and Cd levels for fishermen were higher than controls.
This can probably be attributed to various factors including in general, the type of food eaten by
individual donors, as there is an inverse relationship between concentration of trace metals in
the body and healthy diet. Comparing the concentrations in blood of fishermen obtained from
this study with the [27] guideline value for cadmium in blood, had dectable sample and lower
concentration than the permissible levels stipulated i.e. (0.0003-0.0012) ppm [28].
The values in the amount of less than 2 ppm border within the limits of this nature of lead
in blood [29, 30].
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December 2013
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Kennish, M.J. Ecology of Estuaries: Anthropogenic effects. CRC, Press, Boca Raton, (1992) Fl pp: 494.
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[6]
Blasco, J.; Rubio, J.; Forja, J.; Gomez-Parra, A. and Establier, R. Heavy metals in some fishes of the muglidae
family from salt-pounds of Codiz Bay SW Spain. Ecotox. Environ. Res. (1998) 1: 71-77.
[7]
Storelli, M.M.; Storelli, A.; D΄ddaabbo, R.; Morano, C.; Bruno, R. and Marcotrigiano, G. Trace elements in
loggerhead turtles (Coretta coretta) from the Eastern Mediterranean: Overview and Evaluation. Environ.
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[8] Al-Taee, M.M. Some metals in water, sediments, fishes and plants of Shatt Al-Hilla river, Ph.D. Thesis. Univ. of
Babylon. Iraq. (1999) 128 pp.
[9]
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Begum, A.; Amin, M,N.; Kaneco, S. and Ohta, K. Selected elemental composition of fish, Cirrhina mrigala and
Clarius batrachus from the fresh water Dhanmondi lake in Bangladesh. Food Chem., (2005) 93: 439-443.
[10] Saeed, S.B. and Shaker, S. K. Trace metal distribution in tissues of Cichlids (Oreochromis niloticus) collected
from River Nile. Egypt. Bull. NRC, Egypt (2008) 19 (3): 225-233.
[11]
Al-Khafaji, B.Y. Distribution Pattern of Selected Heavy metals in Water, Sediments and two species of fish
th
from Al-Hammar Marsh south of Iraq. The 5 Scientific Conference 2011-college of Science- Babylon Univ.,
(2010) 5: 115-124
[12] Adeniyi, A.A. and Yusuf, K.A. Determination of heavy metals in fish tissues, water and bottom sediments from
Epe and Badagry lagoons, Lagos, Nigeria. Environ. Monitor. Assess., (2007) 37: 451-458.
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[13] Wu, T.N.; Yang, G.Y.; Shen C.Y. and Liou, L.S.H. (1995): Lead contamination of candy: an example of crisis
management in public health. Lancet, 346 : 1437-1442.
*14+ Baldwin, D. and Marshall, W. Heavy metal poisoning and it’s laboratory investigation. Ann. Clin. Biochem.,
(1999) 36: 267-315.
[15] Al-Kubaysi, A.Al.R. The ecological study of suddam river. Ph.D. Thesis. Univ. of Baghdad. (1996) 143 pp.
[16] Mohammed, S. S. Evaluation the quality of water in Al-Masab Alamm river in Thi-Qar Governorate.
M.Sc.
Thesis. College of Science, Univ. of Thi-Qar. (2010) 106 p.
[17]
Strugen, R.E.; Desaulnicrs, J.A.; Berman, S.S. and Russell, D.S. Determination of trace metals in estuarine
sediment by graphite furnace atomic absorption spectrophotometer. Anal. Chem. Act. (1982) 134: 288-291.
th
[18] SAS, Statistical Analysis System, User's Guide. Statistical. Version 7 ed. SAS. Inst. Inc. Cary. N.C. U.S.A.
(2004).
[19]
Ikem, A.; Egiebor, N.O. and Nyavor, K. Trace elements in water, fish and sediment from Tuskegee lake,
southern U.S.A. Water, Air and Soil Pollution, (2003) 149: 51-75.
[20] Fernandes, C.; Fontainhas-Fernandes, A.; Peixoto, F. and Salgado, MR. Bioaccumulation of heavy metals in
Liza saliens from the Esomriz-Paramos coastal lagoon, Portugal. Ecotox. Environ. Saf., (2007) 66: 426-431.
[21] Cross, F.A.; Hardy, L.H.; Jones, N.Y. and Barber, R.T. J.Fish.Res..Bd.Con., (1973) 30: 1287-1291.
[22] Al-Khafaji, B.Y.; Mohammed, A.W. and Maqtoof, A.A. Distribution of some heavy metals in water, sediments
and fish Cyprinus carpio in Euphrates river Near Al-Nassiriya city center south Iraq. J. Thi-Qar Science, (2011)
8 (1):552-560.
[23] Abida, B.; Ramaiah, M.; Harikrishna, I.K. and Infanulla, K. Analysis of heavy metals in water, sediments and
fish sample of Madivala lakes of Bangalore, Karnataka. Int. J. Chem. Tech. Res., (2009) 1(2): 246-249.
[24]
Malik, N.; Biswas, A.K.; Qureeshi, T.; Borana, K. and Virha, R. Bioaccumulation of heavy metals in fish tissues
of a freshwater lake of Bhopal. Environ. Monit. Assess., (2010) 160: 267-276.
[25] Roesijadi, G.; and Robinson, W.E. Metal regulation in aquatic animals: mechanism of uptake, accumulation
and release In: Aquatic toxicology (Molecular, biochemical and cellular perspectives) (eds. D.C. Malins and
G.K. Ostrander), Lewis publishers, London (1994).
[26]
Manser, W.; Lalani, R.; Haider, S. and Khan, M.A. Trace element studies on Karachi Population. Part V. Blood
lead levels in normal healthy adults and Grammar School children. J Pak Med Assoc; (1990) 40:150-4.
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nd
[27] WHO (World Health Organization) Guideline for Drinking Water Quality 2 ed. (1996 b). Vol. 2: 940-951.
[28] WHO World Health Organization Trace elements in human nutrition and health. International atomic energy
agency. WHO Library publication data. Geneva.: (1996 a) 194-215, 256-259.
[29] Waldbott, G.l. Health effects of Environmental pollution, MOS by com. U.S.A. (1978).
th
[30] Bomford, G.l. Hutchisous clinical methods 6 edition. London, (1975)123p.
102
December 2013
Antibacterial activity of agent produced by Streptomyces spp. isolated
from soil samples
Samer M.Al-Hulu
Babylon University, College of Science
Abstract:
Twenty-Four soil samples were collected from Hilla city. Nine isolates of Actinomycetes were recovered.
Five isolates were identified as Streptomyces spp. according to morphological and biochemical test. The
cultural characteristics of Streptomyces spp. isolates were grey aerial mycelium, yellow-brown substrate
mycelium when cultured on yeast malt extract agar. Antibacterial activity of Streptomyces spp. isolates
against S.aureus were tested. The results showed that Streptomyces spp.2 was most active against
S.aureus with 18 mm inhibition zone compared with other isolates with inhibition zone (10,12,15,16mm)
subsequently. Streptomyces spp.2 was selected for extraction of antimetabolite agent. The antibacterial
activity of agent was tested by well diffusion method. The results showed that the antimetabolite agent
had inhibition zone was 20 mm against S.aureus , 16 mm against E.coli and 10mm against P.aeruginosa.
:‫ال ال ة‬
‫ميي‬
‫مسيية عيي‬
‫يي‬
‫ي‬.
‫ميي ا رتينومايسييتا‬
‫تمتليا مايسيليم هيوائ ذو ليو ر ا ي‬
‫ارتيييف تسييعة عيي‬.‫جمعيي اربييش وعيييرو نمييوذن تربيية ميي مدينيية الحلة‬
‫ائع ال رعيية للعي‬
‫ وجد ا ال‬.‫اليرلية والبايوريمياوية‬
‫الستربتومايسس حسب ال فا‬
‫ يح يي الفعالييية الم ييادة لعيي‬.‫ييفرا الييى بنييية اللييو عنييدما رعيي علييى وسييط مسييت لع ال ميييرة واليييعير‬
‫د البرترييا العنقوديية ميش قطير تثبييط‬
‫ ارثر يعالية‬2 ‫النتائم ا الع لة الستربتومايسس‬
‫ سيت الع العاميل‬2 ‫الع لية الستربتومايسيس‬
‫ اظهر‬. ‫د البرتريا العنقودية‬
‫ ا تيير‬.‫مليم‬10,12,15,16 ‫اقطيار تثبييط‬
‫البقية الت اظهر‬
‫أظهير النتيائم إ العاميل الم ياد أعطيى قطير تثبييط‬.‫د البرتريا بواسطة طريقة ا نتيار م الحفر‬
.‫د برتريا ال ائفة ال نجارية‬
‫ ملم‬10 ‫د برتريا ا يريييا القولو و‬
‫ومييادة اسيياس‬
‫الستربتومايسس‬
‫ملم مقارنة مش الع‬18
‫الفعالية للم اد‬
‫ يح‬. ‫الم اد‬
‫ ملم‬16 ‫د البرتريا العنقودية و‬
‫ ملم‬20
103
December 2013
Introduction:
Streptomycetes are members of the actinomycetes, which live in natural environment (Augustine et al
2004).They are saprophytic organisms which spend the majority of their life cycles as semidormant
spores. During the life cycle, Streptomycetes spores germinate to produce substrate mycelium, which
during maturation fragments into chains of spores(Flärdh, 2003). Generally, synthesize a sizeable
number of diverse natural secondary metabolites, the best known of which are antibiotics currently
used worldwide as veterinary and pharmaceutical industry (Saadoun and Gharaibeh, 2003).
Streptomyces spp. are ubiquitous in soil microbial communities, and plant materials, waters and marine
sediments (Zaitlin, et al 2003). The substrate mycelium uses extracellular hydrolytic enzymes to gain
nutrition from organic compounds that resist degradation by many other microbial groups, e.g. plant
and fungal cell wall polysaccharides and insect exoskeletons. The members of Streptomyces are
distinguished by their ability to produce an array of secondary metabolites (Berdy 2005, Jensen et al
2005).
Many antimicrobial substances were isolated from Streptomyces spp. which including aminoglycosides,
anthracyclins,glycopeptides,
-lactams,
macrolides,
nucleosides,
peptides,polyenes,
polyester,
polyketides, actinomycins and tetracyclines. Most of the antibiotics are extracellular-secondary
metabolites which are normally secreted in culture media and served as intermediates from primary
metabolisms as precursors for their biosynthetic process(Mellouh,2003).
Streptomyces spp. are gram positive filamentous bacteria that produce and secrete a wide array
of biologically active compounds including antibiotics, hydrolytic enzymes and enzyme
inhibitors (Compant et al., 2005; Fravel et al., 2005; Shantikumar Singh et al., 2006).
This study aimed to isolate of a new Streptomyces from localy soil with
antibacterial activity and
extraction of antibacterial agent.
104
December 2013
Sample collection:
Twenty-Four soil samples were collected from Hilla city, during the period from June 2013 to July 2013.
Isolation of Actinomycetes:
Soil samples collected from the local soils were pretreated with calcium carbonate and dried in hot air
oven at 45oC for 1 hr. in order to reduce the incidence of bacteria and molds .Soil dilution plate
technique was employed to isolate the actinomycete isolates on yeast malt dextrose (YMD) agar
medium and pH adjusted to 7.2 and the plates were incubated at 30oC for 10 days (Shirling, and Gottlieb
1966,Williams et al 1971).
Characteristics of Actinomycetes isolates:
Cultural characteristics of actinomycetes were recorded on YMD agar which includes color of aerial
mycelium, color of substrate mycelium and pigmentation of the selected actinomycete isolates medium.
The morphological characteristics of actinomycete isolates were examined by slide culture method
(Shirling, and Gottlieb 1966). Utilization of carbon sources by the strains was carried out according to
the methods of Gottlieb 1961, Berges, 2000).
Isolation of antibacterial metabolites:
o
Culture medium was inoculated by Streptomyces and incubated at 30 C for 7 days in a incubator. After
the incubation period, the culture filtrate was separated from the mycelial cake using filter paper, After
that traces of fermentation broth was separated from broth by centrifuged at 5000 rpm for 15 min . The
solvent was added to the supernatant in 1 : 1 proportion (Augustine et al., 2005).
Extraction of the antibacterial agent :
Antibacterial compounds were recovered from the filtrate by solvent extraction with ethyl acetate in the
ratio 1:1 (v/v) and shaken well for 1 hr. Ethyl acetate phase was separated and evaporated to dryness in
water bath at 80 - 90°C. Residue was weighed and redissolved with little ethyl acetate (Dharmaraj et al.,
2010).
105
December 2013
Antibacterial activity of antibacterial agent:
The antibacterial agent were screened for antibacterial activity against Escherichia coli Stapyllococous
aureus, and Pseudomonas aeruginosa by using well diffusion method. The isolates were grown in a
production broth until adequate turbidity was achieved. 100 µl of the actinomycetes broth culture was
placed in wells made on Muller Hinton agar plates seeded with the test bacterial pathogen cultures. The
plates were incubated at 37°C and observed for inhibition zone after 24 h. (NCCLS) (2003).
Results and discussion:
Isolation of Actinomycetes:
A total Twenty-four soil samples were collected from different places in Hilla city. Nine isolates
were identified as actinomycetes spp. Out of these five Streptomyces isolates were detected when
cultured on yeast malt extract agar.
Streptomyces are aerobic, gram positive actinomycetes that form an extensively branched, substrate
mycelium, aerial hyphae which typically differentiate into chains of spores, have LL-diaminopimelic acid
but lack major characteristic sugars in whole-organism hydrolysates (Lechevalier and Lechevalier, 1970)
and contain DNA rich in guanine plus cytosine (Williams et al., 1989; Manfio et al., 1995).
Morphological and biochemical characteristics of Streptomyces isolates:
Cultural characteristics such as color of aerial mycelium, color of substrate
mycelium of
Streptomyces spp isolates were recorded on Yeast Malt Dextrose agar medium. Morphological and
biochemical test was recorded (Shirling, and Gottlieb 1966,Berges, 2000). Streptomyces isolates were
earthy odor, gram positive , grey aerial mycelium and yellow – brown substrate mycelium. Biochemical
tests such as catalase, oxidase , and utilization of carbon sources were maked (Table 1).
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December 2013
Table(1) Morphological and biochemical test of Streptomyces spp. isolates.
Test
Gram stain
Aerial mycelium
Substrate
mycelium
Earthy odor
catalase
oxidase
Utilization of
carbon source:
glucose
sucrose
xylose
mannitol
Strep.spp.1
+
grey
yellowbrown
+
+
+
+
+
-
Streptomyces spp. isolates
Strep.spp.2
Strep.spp.3
Strep.spp.4
+
+
+
grey
grey
grey
yellowyellowyellow-brown
brown
brown
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
-
Strep.spp.5
+
grey
yellowbrown
+
+
_
+
+
+
+
Antibacterial activity of Streptomyces isolates :
Antibacterial activities of the culture filtrate for five Streptomyces isolates against S.aureus were
recorded. The culture filtrate of Streptomyces isolates were active against S.aureus with different
inhibition zone (Table 2). Streptomyces spp.2 was most active against S.aureus with 18mm inhibition
zone. This isolate was selected for extraction of agent.
Our results agreed with result obtained by El-Naggar
et al., (2001) showing the highest
antimicrobial activity produced from Streptomyces violatus. Several species of Streptomyces from
different soils and water samples are a virtually unlimited source of natural secondary metabolites and
many kinds of which are used as pharmaceutical and agrochemical products(Ben-Fguira et al 2005,
Pamboukain et al , 2004, Ruiz et al 2010, Zheng et al 2012).Streptomyces are prolific sources of novel
secondary metabolites with a range of biological activities that may ultimately find application as anti–
infective and –cancer agents or other pharmaceutically useful compounds (Bibb, 2005).
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December 2013
Table(2) Antibacterial activity of culture filtrate for five Streptomyces isolates against
S.aureus
Streptomyces isolates
Streptomyces spp.1
Streptomyces spp.2
Streptomyces spp.3
Streptomyces spp.4
Streptomyces spp.5
Inhibition zone (mm)
10
18
12
15
16
Antibacterial activity of agent produced by Streptomyces spp.2 :
Antibacterial activity of agent was studied. The results showed that the agent was active against test
pathogens. Highest inhibition zone of gent produced by Streptomyces spp.2 against S.aureus with 20
mm and 16 mm against E.coli, and lowest inhibition zone against P.aeruginosa with 10mm (Figure 1).
Our results agreed with results obtained by Oskey et al (2004), who found that actinomycetes
isolated from soil samples were active against S.areus and E.coli with inhibition zone 16,15 mm
subsequently.Wide range of molecules with broad spectrum activities, that is, anti-bacterial, anti-fungal,
- anti tumour, anti -parasitic and – anti -viral
metabolites was produced by Streptomyces spp.
(Naeimpoor and Mavituna, 2000,Atta and Ahmad, 2009 and Lucas et al., 2013).
Actinomycetes, and more specifically, Streptomycetes that produce various bioactive natural products
including antibiotics, are being used as pharmaceuticals and agrochemicals (Lazzarini et al., 2001; Watve
et al., 2001; Sajid et al., 2008).
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December 2013
20
10
Inhibition zone (mm)
30
0
S.aureus
E.coli
P.aeruginosa
Test pathogens
Figure (1) Antibacterial activity of agent produced by Streptomyces spp.2
References:
Atta HM, Ahmad MS (2009).Antimycin-A Antibiotic Biosynthesis Produced by Streptomyces Sp. AZ-AR262: Taxonomy, Fermentation, Purification and Biological Activities. Aus. J. Basic, Appl. Sci. 3(1): 126135.
Augustine, S.K., Bhavsar, S.P., Baserisalehi, M. and Kapadnis, B. P. (2005).A non-polyene antifungal
antibiotic from Streptomyces albidoflavus PU 23 ; Indian. J. Exp. Biol,42:928–932.
Augustine S K, Bhavsar S P, Baserisalehi M and Kapadnis B P (2004). Isolation, characterization and
optimization of antifungal activity of an actinomycete of soil origin; Indian J. Exp. Biol.42 928–932
Ben-Fguira, L.F., Fosto, S, Mehdi, R.B, Mellouli L, and Laatsch H (2005).Purification and structure
elucidation of antifungal and antibacterial activities of newly isolated Streptomyces sp. strain US80.
Microbiol.Res., 156: 341-347.
view. J Antibiot, 58:1–26
Berdy J (2005). Bioactive microbial metabolites: a personal
Bergey’s manual of determinative bacteriology (2000). Actinomycetales, 9th
edition.
Bibb MJ (2005). Regulation of secondary metabolism in Streptomycetes. Curr. Opin. Microbiol. 8: 208215.
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December 2013
Compant S, Duffy B, Nowak J, Clement C, Ait BE (2005). Mini review - Use of plant growthpromoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future
prospects.; Appl. Environ. Microbiol., 71(9): 4951-4959.
Dharmaraj S B. Ashokkumar and K. Dhevendaran (2010). Isolation of marine Streptomyces and the
evaluation of its bioactive potential. Afr. J.Microbiol. Res., 4 (4), pp. 240-248.
Flärdh, K. (2003). Growth polarity and cell division in Streptomyces. Curr. Opin. Microbiol. 6: 564–571.
Fravel DR, Deahl KL, Stomme JR (2005). Compatibility of the biocontrol fungus Fusarium oxysporum
strain CS-20 with selected fungicides.; Biol. Control 34: 165-169.
Jensen PR, Mincer TJ, Williams PG,and Fenical W (2005) Marine actinomycetes diversity and natural
product discovery. Antonie Van Leeuwenhoek, 87:43–48
Lazzarini A, Cavaletti L, Toppo G, Marinelli F (2001). Rare genera of actinomycetes as potential sources
of new antibiotics. Anton. Van Leeuwen, 78: 399-405.
Lechevalier MP, Lechevalier H (1970). Chemical composition as acriterion in the classification of aerobic
actinomycetes. Int. J. Syst.Bacteriol., 20: 435-443.
Lucas, X, Senger C, Erxleben, A, Gruning, B A.Doring, K,Mosch J, Flemming, S. and Gunther, S.
(2013).StreptomeDB: a resource for natural compounds isolated from Streptomyces species.Nucleic
Acids Research, , Vol. 41
Manfio GP, Zakrzewska-Czerwinska J, Atalan E, Goodfellow M (1995).Towards minimal standards for
the description of Streptomyces species. Biotechnologia, 7-8: 242-253.
Mellouli, L., Ameur-Mehdi R.B., Sioud, S, Salem M, and Bejar S (2003).Isolation, purification and partial
characterization of antibacterial activities produced by a newly isolated Streptomyces sp. US24
strain.Res. Microbiol. 154: 345-352.
National Committe for Clinical Laboratory Standards (NCCLS) (2003). Performance standards for
antimicrobial susceptibility testing:Approved standard. M2-A8. National Committee for Clinical
Laboratory Standards. Wayne, Pa.
Naeimpoor F, Mavituna F (2000). Metabolic Flux Analysis in Streptomyces coelicolor under Various
Nutrient Limitations. Metab.Eng. 2(2): 140-148.
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Oskay M (2009). Antifungal and antibacterial compounds from Streptomyces strains. Afr. J. Biotechnol.,
8: 3007-3017.
Oskay M, Tamer A, and Azeri C (2004). Antibacterial activity of some actinomycetes isolated from
farming soils of Turkey. Afr. J.Biotechnol. 3(9): 441-446.
Pamboukain CRD, Facciotti MCR (2004). Production of the antitumeral retamycin during continuous
fermentations of Streptomyces olindensis. Process Biochem. 39: 2249-2255.
Ruiz, B., A. Chávez, A. Forero, Y. García-Huante, A. Romero, M. Sánchez, D. Rocha, B. Sánchez, R.
Rodríguez-Sanoja, S. Sánchez and E. Langley, 2010. Production of microbial secondary metabolites:
Regulation by the carbon source. Critical Rev. Microbiol., 36: 146– 167.
Saadoun, I. and R. Gharaibeh, (2003).The Streptomyces flora of Badia region of Jordan and its potential
as a source of antibiotics active against antibiotic- resistant bacteria. J. Arid Environ. 53: 365 - 371.
Sajid I, Fotso Fondja Yao CB, Shaaban KA, Hasnain S, Laatsch H (2008). Antifungal and antibacterial
activities of indigenous Streptomyces isolates from saline farmlands: prescreening, ribotyping and
metabolic diversity. World J. Microbiol. Biotechnol. DOI: 10.1007/s11274-008-9928-7
Shirling EB, and Gottlieb D (1966). Methods for characterization of Streptomyces species. Int. J. Syst.
Baceriol., 16: 313-340.
Shantikumar SL, Baruah I, Bora TC (2006). Actinomycetes of Loktak Habitat: Isolation and screening for
antimicrobial activities.; Biotechnol., 5(2): 217 -221.
Watve MG, Tickoo R, Maithili M, Jog B, Bhole D (2001). How many antibiotics
are produced by the genus Streptomyces? Arch. Microbiol. 176: 386-390.
Williams ST, Cross T (1971). Isolation, purification, cultivation and preservation of actinomycetes.
Methods Microbiol. 4: 295-334.
Williams ST, Goodfellow M, Alderson G (1989). Genus Streptomyces Waksman and Henrici, 1943,
339AL. In: Williams ST, Sharpe ME, Holt JG (eds.), Bergey’s Manual of Systematic Bacteriology, Williams
and Wilkins, Baltimore, MD, 4: 2452-2492.
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Zaitlin B, Watson SB, Ridal J, Satchwill T,Parkinson D (2003). Actinomycetes in Lake Ontario: Habitants
and Geosmin and MIB production. Am. Water Works Assoc. J. 95(2): 113-118.
Zheng D , Han L, Li Y, Li
J, Rong H, Leng, Q, Jiang Y, Zhao, L and Huang, X (2012)
Isostreptazolin and Sannaphenol, Two New Metabolites from Streptomyces sannanensis.Molecules, 17,
836-842
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December 2013
VENTILATION EFFECT ON SOLAR PANEL PERFORMANCE
Isam Azeez Hassoon , Emad Jaleel Mahdy, Emad Mutab Muhsen
ِ‫ عَبد ٍزعت ٍحغ‬, ٛ‫و ٍٖذ‬ٞ‫ عَبد عي‬, ُ٘‫ض حغ‬ٝ‫عظبً عض‬
Ministry of Science & Technology , Renewable Energy Directorate
Abstract
In this study, analysis of the benefit of a monument solar panels so that it is
leaving a vacuum between the panel and the surface Thread him and was also the
comparison between the two panels, one adjacent to the surface and another was
left a vacuum of 10 cm between the panel and the surface topic it was reached
leaving space between the panel and the surface leads to increased in the panel
conversion efficiency of energy depending on "the low temperature of the solar
panel was found that the high temperature of the solar panel 5 degrees Celsius
leads to a decrease in energy conversion efficiency of the board by approximately
0.4%."
Keywords: Photovoltaic, ventilation gap, roof installation.
‫الخالصة‬
‫تم في ىذه الدراسة تحميل فائدة نصب االلواح الشمسية بحيث يتم ترك فراغ بين الموح والسطح الموضوع عميو‬
‫ سم بين الموح والسطح‬01 ‫وكذلك تم المقارنة بين لوحين احدىما مالصق لمسطح واخر تم ترك فراغ قدره‬
‫الموضوع عميو وتم التوصل الى ان ترك فراغ بين الموح والسطح يؤدي الى زيادة في كفاءة تحويل الموح‬
‫ درجات‬5 ‫لمطاقة تبعا" النخفاض درجة ح اررة الموح الشمسي حيث تبين ان ارتفاع درجة ح اررة الموح الشمسي‬
."‫ تقريبا‬0.4% ‫مئوية يؤدي الى انخفاض في كفاءة تحويل الطاقة لموح بمقدار‬
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Introduction
Solar energy has become a real alternative to replace some fossil fuel consumption.
One important application of solar energy is the integration of PV panels on
buildings. Today there are many options to integrate PV panels in buildings new
buildings, on the façade, roof, windows, as shadow elements... But in old buildings
the most used option is the integration on the roof.
Temperature is an important factor to consider when a PV system is designed. We
could make the mistake of thinking that the higher temperature we have the better
efficiency we‘ll get. The real effect of temperature is just the opposite. The
conductivity of a semiconductor increases when the temperature is higher,
therefore it is easier for electrons to holes elsewhere in the material to fill, being
the electrical balance in the cell increases, the electric field falls away to the
boundary, so the load can no longer remain well separated. The result is a
decreasing tension between the two layers. This effect is illustrated in the figure
below:[1],[2]
Fig. (1). Effect of temperature on voltage
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December 2013
On a polycrystalline panel, the effect of temperature on efficiency can be around
0,45%/ºC, while in a monocrystalline panel the effect is a bit lower. In terms of
watts, in a polycrystalline panel of 135 Watts, with an efficiency of 14% at 25ºC,
we could lose around 12 Watts per panel at 45ºC and 24 Watts per panel at 65ºC.[3]
We should try to keep the panel as cool as possible by applying passive measures
such as a natural ventilation system. In the following parts of this report we‘ll
analyze the real effect of having ventilation between the panel and the roof for
different climate conditions.
We can define the efficiency power conversion of solar panel it‘s the ratio between
power emerging from solar panel and power from solar radiation incident see eq.
(1).
Where η : is the efficiency of power conversion
Pout : represents the value of power emerging from the solar panels
Pin : represents the value of solar radiation incident on the solar panels
A : represents an area of the solar panel ,its 0.34 m2
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December 2013
Practical part
To demonstrate the effect of high temperature on the performance of the solar
panel was erected tow solar panel with power 50W southward geographical city of
Baghdad and tilt angle (300) which angle are used when integrating solar panels
with the buildings where they were raised, one for the panel leave space 10cm
between the surface Bracket and panel while the other is adjacent to the surface as
shown in Fig (2).
Fig.(2) Experimental structure
Were examined performance solar panels in each case by (PV-Analyzer device)
where gives the full report on the electrical properties and the amount of
performance in relation to the amount of solar radiation falling and the temperature
of the solar panel, which is measured "by the device shown in Figure (3).
Fig. (3) PV-Analyzer device
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December 2013
When we tasting the panel by PV-Analyzer without ventilation we found the
electrical characteristics of the readings as well as electrical characteristics curve
see fig.(4)
Fig.(4) represents electrical characteristics of the readings as well as electrical
characteristics curve for panel without ventilation
We can calculate the efficiency of power conversion by eq. (1) found it was 8.9%
And the solar panel temperature was 50.6 C0
But when we tasting the panel with ventilation we found electrical characteristics
of the readings as well as electrical characteristics curve see fig.(5)
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December 2013
Fig.(5) represents electrical characteristics of the readings as well as electrical
characteristics curve for panel with ventilation
In this case the efficiency of power conversion was 9.3%
And the solar panel temperature was 46.1 C0.
Conclusion
We conclude from the research that the ventilation solar panels and so leave space
between the solar panel and the Bracket has an impact on the high temperature
solar panel and thus on his performance in energy conversion it was found that
when put panels horizontally at tilt angle 300, the effect of ventilation in the high
temperature of the panels and clear where he was the difference in the percentage
between the efficiency of power conversion of the tablets (0.4%)
In the end we can say when the temperature of solar panel increase about 5 C 0 the
the efficiency of power conversion decrease around 0.4 % .
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December 2013
References
[1]-P. Jinliang Yuan, EFFECT OF VENTILATION IN A PHOTOVOLTAIC
ROOF Department of Energy Sciences, Lund Institute of Technology ,2011.
[2]- David L. King, Jay A. Kratochvil, and William E. Boyson, Temperature
Coefficients for PV Modules and Arrays: Measurement Methods, Difficulties, and
Results, Presented at the 26th IEEE Photovoltaic Specialists Conference,
September 29- October 3, 1997, Anaheim, California.
[3]- Dominguez, Anthony. Modeling of roof heat transfer under solar photovoltaic
panels, 2010 .
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December 2013
Effect of extracellular synthesis silver nanoparticles produced by
Morganella morganii on some immunological parameters.
Frial Gemeel Abd, Ali Jabbar Abdul hussain Al-kawaz
Babylon University, Science Faculty- Biology Department, Iraq.
Abstract:
One hundred eighty eight mid-stream urine samples were collected from patients suffering from
Catheter Associated Urinary Tract Infections, (CAUTI), who visit urological consultant clinic of Hillah
Teaching Hospital in Hillah, Babylon province- Iraq during a period from November 2012 to January
2013. All urine samples were subjected for standard bacteriological procedures to check bacteriuria. The
results show that 9/159 (5.66%) give positive culture for Morganella morganii . For detection the ability
of this bacteria to produce silver nanoparticles we are used the XRD the result of XRD for the control
shows amorphous phase but after loading the silver nitrate (AgNO3) on the surface of the isolates we get
new peacks (111,200,220,311) for isolates number 2, 3, 4,5,6,7 and the isolates and the isolate number
1,8 and 9 show negative result (the amorphous phase). These peaks approved that silver ions doping on
the surface and this confirmed by using TEM. The UV-VIS used to study the behavior of silver
nanoparticles this shows it is clear at zero hour there is no reaction of her and at 24 ,72 and 120 the
increased in intensity will be observed in the range of (400 & 500)nm. 40 µg /dl of AgNPs was used to
immunization three rabbit ,Phagocytic activity , E-rosette and skin test were studied and found there is
no differences between test group and control in phagocytic activity(59.84,59.003)respectively while, Erosette (48.06,15.72 )and the AgNPs induce delayed type hypersensitivity.IL-2and IL-8 concentrations
were detected by enzyme linked immune sorbent assay and found that IL-8 was decreased while IL-2
increased in test group and control respectively.
Introduction:
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection,
accounting for more than 1 million cases each year in the US hospitals and nursing homes. The
significant number of infections and dissemination of resistant bacteria in hospitals make it important to
find ways to decrease their incidence [1].
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December 2013
Morganella morganii is a gram-negative rod commonly found in the environment and in the intestinal
tracts of humans, mammals, and reptiles as normal flora. Despite its wide distribution, it is an
uncommon cause of community-acquired infection and most often encountered in postoperative,
catheter-associated bacteriuria and other nosocomial settings [2].In the human urinary tract, the ability
of Morganella morganii to hydrolyze urea has been linked to formation of xanthine calculi as well as the
more typical struvite and carbonite apatite stones [3].
Morganella morganii possesses many virulence associated factors especially the production of urease
as well as hemolysin, protease ,and some adhesive factors which play a major role in colonization of
bacteria to human tissues [4].Risk factor for the development of Morganella morganii infection includes
surgical or non-surgical trauma, prior exposure to B-lactam antibiotics, diabetes mellitus, malnutrition,
debilitation, alcoholism, and certain snake bites [5]. However, Morganella morganii infections respond
well to appropriate antibiotic therapy; however, its natural resistance to many beta-lactam antibiotics
and cephalosporin may lead to delays in proper treatment [6].
As nanotechnology is emerging as an interdisciplinary field with potential to influence various aspects
of human life through a myriad of applications, biological synthesis of nanomaterials is gaining particular
attention as a rapidly growing discipline of Bionanotechnology with an enormous application potential
in the coming future [7].
There has been a strong interest in developing environmentally benign protocols for biological
synthesis of nanomaterials that do not involve toxic chemicals in synthesi process. Biological synthesis of
various metal nanoparticles by using prokaryotic as well as eukaryotic organisms including bacteria,
fungi, plants [8]. However, among various organisms studied until to date, prokaryotes remain the
choice of organism for biological synthesis of nanomaterials . This is predominantly because prokaryotes
offer well-defined advantages over eukaryotic organisms such as easy handling, ease of downstream
processing and ease of genetic manipulation [9].
Bacterial synthesis of silver nanoparticles (AgNPs) is particularly attractive from microbiology
perspective due to existence of well-known silver resistance machinery in few silver resistant bacterial
species, thus making their study significantly important for biomedical applications [10]. Moreover,
silver nanoparticles have remained an attractive choice of nanomaterial because of their ability of
encompassing broad application area from electronics to medicine to food technology [11].
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December 2013
Most of the natural processes also take place in the nanometer scale regime. Therefore, a confluence
of nanotechnology and biology can address several biomedical problems, and can revolutionize the field
of health and medicine nanotechnology is currently employed as a tool to explore the darkest avenues
of medical sciences in several ways like imaging sensing targeted drug delivery and gene delivery
systems and artificial implants . The new age drugs are nanoparticles of polymers, metals or ceramics,
which can combat conditions like cancer and fight human pathogens like bacteria [12]
The development of new resistant strains of bacteria to current antibiotics has become a serious
problem in public health; therefore, there is a strong incentive to develop new bactericides [13].
Bacteria have different membrane structures which allow a general classification of them as Gramnegative or Gram positive. The structural differences lie in the organization of a key component of the
membrane, peptidoglycan. Gram negative bacteria exhibit only a thin peptidoglycan layer (~2–3 nm)
between the cytoplasmic membrane and the outer membrane [14]. In contrast, Gram-positive bacteria
lack the outer membrane but have a peptidoglycan layer of about 30 nm thick [15].
Silver has long been known to exhibit a strong toxicity to a wide range of micro-organisms for this
reason silver-based compounds have been used extensively in many bactericidal applications , Silver
compounds have also been used in the medical field to treat burns and a variety of infections[16].
Commendable efforts have been made to explore this property using electron microscopy, which has
revealed size dependent interaction of silver nanoparticles with bacteria [17]. Nanoparticles of silver
have thus been studied as a medium for antibiotic delivery [18]. The synthesize composites for use as
disinfecting filters [19] and coating materials [20] However, the bactericidal property of these
nanoparticles depends on their stability in the growth medium, since this imparts greater retention time
for bacterium– nanoparticle interaction. There lies a strong challenge in preparing nanoparticles of silver
stable enough to significantly restrict bacterial growth. Studies were carried out on both antibiotic
resistant (ampicillin- resistant) and nonresistant strains of gram-negative (Escherichia coli) and a nonresistant strain of gram-positive bacteria (Staphylococcus aureus). A multi-drug resistant strain of gramnegative (Salmonella typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was
also subjected to analysis to examine the antibacterial effect of the nanoparticles [21]. Efforts have been
made to understand the underlying molecular mechanism of such antimicrobial actions. The effect of
the nanoparticles was found to be significantly more pronounced on the gram-negative strains,
irrespective of whether the strains were resistant or not, than on the gram-positive organisms,
antibacterial effect of the nanoparticles depend on their stability in the medium as a colloid, which
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December 2013
modulates the phosphotyrosine profile of the bacterial proteins and arrests bacterial growth. The
bactericidal effect of silver ions on micro-organisms is very well known; however, the bactericidal
mechanism is only partially understood. It has been proposed that ionic silver strongly interacts with
thiol groups of vital enzymes and inactivates them [22]. Experimental evidence suggests that DNA loses
its replication ability once the bacteria have been treated with silver ions . Other studies have shown
evidence of structural changes in the cell membrane [23]. Silver ions have been demonstrated to be
useful and effective in bactericidal applications, but due to the unique properties of nanoparticles
nanotechnology presents a reasonable alternative for development of new bactericides. Metal particles
in the nanometer size range exhibit physical properties that are different from both the ion and the bulk
material. This makes them exhibit remarkable properties such as increased catalytic activity due to
morphologies with highly active facets [24].
The current study aimed to phenotypic and genotypic investigation of silver nanoparticles among
Morganella morganii isolates recovered from catheter-associated urinary tract infection (CAUTI).
Materials and Methods:
Patient and samples:
One hundred eighty eight mid-stream urine sample were collected from patients suffering from (CAUTI)
who visit urological consultant clinic of Hillah Teaching Hospital in Hillah, Babylon province- Iraq during a
period from November 2012 to January 2013. all samples were subjected to standard bacteriological
procedure including culturing on blood and MacConkey's agar plates for isolation and incubated for 24-48
hours at 37C0[25].
All suspected Gram negative isolates were screening by traditionally tests and then confirmed
By Viteck 2 compact system (Biomérieux).
DNA extraction, primer designing and PCR conditions:
All Morganella morganii isolates were subjected for DNA extraction according to the protocol provided
by manufacturer (Geneaid/Taiwan). The primer pair used to investigate silE gene was designed in this
study
using
Workbench
-
Primer
3
software.
The
primer
sequence
was
DAKW
F:5-
GTGAATATCCATGAGCGGGT-3 ; DAKW R: 5-CAACTGCAGCTCTTTCATGC-3. The PCR product size was 280
bp.
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December 2013
The PCR conditions indicated in this study was Simple 3-step PCR protocol as shown in table (1).
Table (1) The cycling conditions of phylogeny groups
Steps
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Hold
Temperature
95 C°
95 C°
59 C°
72 C°
72 C°
4C°
Time
2 min
30 sec
30 sec
30 sec
5 min
10
No. of cycles
1
30
1
Table (2) The 30 µl PCR mix
Item
Master mix
Target DNA
Forward Primer (10pm/ µl)
Reverse Primer (10pm/ µl)
Nuclease free water
Total volume
Volume
15 µl
5 µl
2.5 µl
2.5 µl
5 µl
30 µl
Method of Biosynthesis and phenotypic investigation of silver nanoparticles:
The method of silver nanoparticles production by Morganella morganii was achieved as indicated by
Song et al.,(2009)26 as follow: the Morganella morganii isolates were initially grown at 37 C for 24 h in a
500-mL Erlenmeyer flask that contained LB broth (100 mL) in a shaker incubator set at 200 rpm and then
The bacterial growth were incubated with aqueous 5 mM solutions of AgNO3 at 37C in a shaker
incubator at 200 rpm in the dark, and the reactions were carried out for up to 120 h (5 days). The
extracellular synthesis of AgNPs was initially detected by visual inspection of the culture flask for a
change in color of culture medium from clear light-yellow to brown/green. The separations of AgNPs
from bacterial cells were performed by centrifugation of aliquots of culture supernatants (1.5 mL) at
3000 rpm for 6 min at 25C.
The UV-vis analysis was done as follow, the AgNPs suspensions were diluted 10 times using MilliQ
deionized water at every time point and UV-vis spectra were obtained. For X-Ray Diffraction (XRD)
analysis, the samples were prepared by precipitating AgNPs obtained after 20 h of biosynthesis at
13,000 rpm for 20 min, followed by four washings with MilliQ deionized water, and drop casting the
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December 2013
samples onto a glass substrate. For TEM analysis, AgNPs samples obtained after 20 h of reaction were
prepared by drop casting the colloidal suspensions of AgNPs onto carbon-coated Cu grids followed by
drying under air for 24 hours [26].
Immunological study
Rabbit groups
Six Laboratory rabbits were left for two weeks for adapted conditions and kept at libitum
conditions of food and drinks and divided into two groups. First one were injected with silver
nanoparticles while the second group was injected with normal saline and considered as control group.
Immunization protocols
Sex mature rabbits were adapted in the laboratory animals .Each rabbit was about 2 Kg of weight. The
animals were immunized intramuscularly and subcutaneously with AgNPs produced by M.morganii (40
mg/kg). This dose were administrated in five injection for ten days between each dose one day. After
this period the animals was anesthetized by chloroform. Blood sampling were done according to the
following:
Blood sampling
A total of 6 blood samples were collected from 3 immunized rabbits and 3 non-immunized
rabbits as a control groups. From each rabbit , a blood sample of 8 -10 ml was drawn aseptically by heart
puncture with a disposable syringe. Half of the blood samples were left at room temperature till being
clotted, and then were centrifuged at 3000 rpm for 5 min. the sera were aspirated from the whole blood
, then divided into 0.5 ml small tubes , and stored at – 200c till testing time . Each tube was used once
to avoid repeated freezing and thawing. The second half of tube samples were placed in EDTA tube as
anticoagulant. The samples were processed for assessment phagocytic activity and for separation Tlymphocyte to detect E-rosette formation.
Nitrobluetetrazolium (NBT) dye reduction test:
This test is used to detect phagocytic activity of phagocytic cells [31].
E-rosette forming test
The E-rosette test was done according to the method of [33].
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December 2013
Delayed type hypersensitivity (DTH)
This test was done for M.morganii silver nanoparticles primed rabbit by injection intradermally 0.1 ml
of AgNPs(40 mg/kg) in all tested animals and recording the observed skin change by measuring the
diameters of the erythema , edema , and necrosis by ruler in comparison with control animals [37].
Interleukin-2 and Interleukin-8
The concentrations of IL-2 ,IL-8 were done according the commercial kits(Biomérieux).
Results and Discussion:
One hundred eight eighty samples were collected during the period from November 2012 to January
2013 patients who were suffering from (CAUTI) patients in Hilla Teaching Hospital in Hilla Province, a
mid-stream urine the sample were primarily grown on to blood and MacConkey's agar plates for
isolation and incubated for
24-48 hours at 37C0[27].All samples culturing on traditional and
conventional media. As showed in figure (1), a total of 159 positive cultures, just nine showed positive
for Morganella morganii, all from male. The identification of these isolates depends on the main
characteristics of these bacteria according to [28].
Figure (1) percentage of M. morganii in CAUTI patients
API 20E was used to confirm the results of identification and the results of API 20E come with those
obtained in traditional tests. Also the system two Vitek was used to confirm the results of identification
and the results of API 20E there was different between species but the result probability between (95-
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99%)are show in table (3). And the result As follows, catalase positive, oxidase negative, urease positive
and negative to gelatinase and phenylalanine deaminase positive . Also, the isolates were found to be
unable to ferment the sugars: Lactose, Mannitol, Sorbitol, Sucrose, Arabinose, Inositol, Rhamnose,
Melibiose, but able to ferment Glucose only with gas.
Table (3) Viteck 2 compact system results
This study was performed to determine whether extracellular silver nanoparticles (AgNPs) production by
Morganella morganii. Morganella morganii isolate were able to synthesize extracellular Ag nanoparticles.
To undertake this study Morganella isolates were exposed to 5 mM colorless AgNO3 solutions Morganella
morganii formed dark brown coloured solutions within 20 h of reaction figure (2) , and the color of the
solutions did not significantly change from that point onward , even after continuing the reaction for up to
5 days. To understand the nature of nanoparticles, detailed physico-chemical characterization of
extracellular AgNPs formed by all Morganella morganii was carried out using UV-Vis absorbance
spectroscopy, and X-ray diffraction (XRD).
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December 2013
Figure (2) medium with AgNO3 (5mM) and controlled sets (negative and positive) at room temperature.
Figure (3) shows the UV-vis absorbance spectra of colloidal solutions obtained after reaction of all
Morganella isolates with 5 mM AgNO3 for zero, 24,72 and 120 –h . The presence of a characteristic Ag
surface Plasmon resonance (SPR) between 400 and 500 nm is clearly evident in all the samples, thus
confirming the formation of extracellular AgNPs by all Morganella morganii[29]. The differences in the
position of absorbance of SPR features of AgNPs synthesized by different isolates of Morganella morganii
was notable, which was most likely due to the difference in the size and/or shape of Ag nanocrystals
synthesized by these bacteria [ 30]. It is also interesting to note as the result showed that the Morganella
morganii started synthesizing AgNPs as early as within 1 h of reaction and the yield of AgNPs increased as
the reaction progresses over a period of time.
Figure (3) UV-VIS absorbance spectroscopy for AgNPs from Morganella morganii
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December 2013
However, the amount of AgNPs produced by these bacteria reached to a saturation state somewhere
between 20 h and 120 h of reaction. This suggests that although all Morganella isolates have the capability
to reduce (Ag+) ions to form AgNPs (Ag0), the rate of AgNPs formation may vary among them. The
maximum absorbance intensity of the Ag (SPR) feature of different isolates of Morganella morganii differ
with the time of reaction. It is also interesting from the result note that AgNPs production by Morganella
reached to a saturation state within 20 h of reaction, after which no further increase in AgNPs synthesis
was observed and this noted from the result of x-ray diffraction (XRD).
During this study In my opinion, 20 h time point provides a better representation than 120 h time point
for comparison between AgNPs synthesized by Morganella isolates, because at 20 h time point AgNPs
biosynthesis is in its log (growth) phase, which enables to capture the state of as formed particles, rather
than a possibility of their further modification while AgNPs stay in the bacterial growth media up to 120 h.
And during this study I noted the rate of AgNPs formation was found to be maximum at 5 mM AgNO3
concentration, and was reduced by increasing the precursor concentration and over the 5 mM AgNO3 the
bacteria was die and this result agree with [29].
The crystallography of AgNPs formed by different isolates of Morganella after 20 and 72 and 120 h of
reaction was investigated by XRD. As is evident from XRD patterns in Figure(4) , extracellular AgNPs
synthesized by Morganella morganii are highly crystalline in nature, that could be perfectly indexed to
the {111}, {200}, {220} and {311} Bragg reflections of the face centered cubic (fcc) form of crystalline
silver so this result agree with this [30].
XRD analysis thus provided a clear indication of formation of high quality crystalline AgNPs using a
Morganella morganii mediated biosynthesis process..The UV-vis, XRD results presented in this study
clearly demonstrate that formation of AgNPs is a genus-wide characteristic phenotype of Morganella
morganii. Further experiments were performed to explore whether AgNPs formation is a characteristic
phenotype restricted to genus Morganella, or whether other taxonomically related genera of
Enterobacteriaceae family also show this feature.to obtain this I do comparative analysis of AgNPs
synthesis using laboratory strains of Escherichia coli, Serratia marcescens, Kelebisella
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December 2013
pneumonia and Aeromonas hydrophila was performed in the presence of 5 mM AgNO3, no AgNPs
formation was observed in any of these closely related organisms. This strongly suggests that AgNPs
synthesis in the presence of Ag+ ions is a phenotypic character that is uniquely associated with
Morganella morganii.
Figure (3-4) X-ray diffraction results for Morganella morganii different time of reaction compared with
control, XRD patterns recorded showing 4 sharp peaks corresponding to the diffraction from 111, 200,
220 and 311 planes of silver.
Figure (5) show the results of TEM analysis, AgNPs samples obtained after 20 h of reaction were
prepared by drop casting the colloidal suspensions of AgNPs onto carbon-coated Cu grids followed by
drying under air for 24 hours this test was performed at university of Technology in Baghdad.
Figure (5) Show Transmission electron microscopy (TEM) images of extracellular AgNPs formed by
Morganella morganii (36.9nm).
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December 2013
Concerning the investigation of the silver nanoparticles gene (silE) the results reveald that 6/9 (66.7%)
of isolates have silE as shown in figure (6).
Fig.(6) 2% agarose gel electrophoresis of 280bp PCR products of silE gene . The lane M represent
100bp Molecular marker , lane 2,3,4,5,6,7 represent sample no. of positive results while lane 1, 8
and 9 represent sample no. of negative results. Lane control represents negative control for silE
gene.
Nitrobluetetrazolium reduction test was used for neutrophil phagocyte assessment. The
reduction was graded on the basis of the number of neutrophils with intracellular deposit of formazan
stippling per 100 neutrophils counted.this test was used to analyze the possible differences in
phagocytic function of the PMN cells in rabbits immunized with silver nanoparticles produced by
Morganella morganii and control with normal saline only. The result showed a systemic increase in NBT
activity of rabbit immunized with AgNPs (40 µg/kg) reached 59.003%. Statistical analysis shows that
AgNPs had the same effect on phagocytosis of polymorphnuclear cells (PMNs) in peripheral blood such
as other types of antigens. While the control group show significant decrease (P>0.05)in phagocytic
activity and the mean value was 59.84%.Table(4)
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Table (4): NBT reduction test in rabbits immunized with AgNPs
Antigen
Phagocytic activity (%) Mean ± Standard deviation
Normal saline
59.84 ± 4.953
Silver nanoparticles
59.003 ± 4.744
the result showed a systemic increase in phagocytic activity of rabbit immunized with AgNP s this
antigen taken by neutrophils leading to cell activation and subsequent enhancement of the phagocytic
ability of these cells. This result almost comparable with [32] study the were found that the phagocytic
activity was decrease at mucosal and systemic levels after immunization of rabbits with NPs.
E-rosette was decreased when the rabbits were immunized with AgNPs(40µ g/kg) and the mean
values were 34.94% ,while the E-rosette in rabbits injected with normal saline as control group was
70.216% table ( 5 ) . There are significant increase in mean values of E-rosste for AgNPs In comparison
with control group (P >0.05).
Table (5) the percentage of systemic E- rosette forming T- lymphocytes in rabbits immunized with
AgNPs
Antigen
E- rosette forming T- lymphocytes (%)Mean± Standard deviation
Normal saline
15.7233 ± 6.74
Silver nanoparticles 48.06 ± 0.03
The major function of T cells can be divided into two categories: the first(cytotoxicity) is to lyse
cells expressing specific antigens ;the second (delayed hypersensitivity ) is to release cytokines , thereby
triggering an inflammatory response [34]. CD2 is a lymphocyte surface glycoprotein of 50-58 KD that
expressed on all thymocytes, T cells and NK cells, the heterotypic interaction (Rosetting) between CD2
and its major ligand leukocyte function antigen-3(LFA-3 , CD58) which expressed by most nucleated cells
as well as by erythrocytes enhances T cells antigen recognition. CD2 engagement by LFA-3 expressed on
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an APC stimulates T cell proliferation and differentiation, in addition to its role in stabilizing cell-cell
contact the interaction between CD2 and CD58 delivers an activating signal to the T lymphocyte[35].
This result agree with [36] who found that concentration of lymphocytes increasing with the time also
the toxicity rang increase after exposed to different dilution of AgNPs with different time periods.
Skin test of rabbits primed with AgNPs revealed erythema, pus cell, induration and necrosis. Skin
reactions started after 4 hour with erythema the mean value was 1 mm. The maximum mean value of
induration, pus cell and necrosis diameter was 9.5 mm after 24 hour and increase to 10 after 48 hour
and after 72 hour it decreased to 8 mm.
Table (6):Delayed type hypersensitivity(DTH) in rabbits immunized with AgNP s
Reaction(mm)mean ± S.D
After 4
hour
Normal
0
After 24 hour
After 48 hour
After72 hour
0
0
0
Erythema,
Erythema,
induration,necrosis,
induration,necrosis,
pus cell
pus cell
9.5 ±2.5
10 ± 0.577
saline
Erythema
AgNPs
1±0.4
Erythema,
induration,necrosis
8±1
*S.D: Standard Deviation
DTH response require prior immunological sensitization to a specific antigen and thus are
categorized as recall, or memory T cell response , they can identify specific antigens to which the host
has already made an immune response and they provide an index of the current T cell reactivity to
specific recall antigens and the rea of redness and sometime the degree of swelling that can be
measured to provide an index of DTH reactivity [38].
In delayed type hypersensitivity (DTH) in humans , neutrophils are the first responders, following
by a mix of mononuclear cells composed of T cells and macrophages while in mice the murine response
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to antigens in DTH in riche in neutrophils[39]. Interstitial cellular infiltrates contain a predominance of
CD8+ T and CD4+ T cells and macrophages CD8 cells likely mediate cytotoxic reactions to foreign MHCexpressing cells, while CD4 T cells produce gamma interferon, while promotes a cytokine cascade that
activates local vascular endothelial cells , this promotes the rapid recruitment of neutrophils and
subsequently additional macrophages and T cells cytokines and induce delayed- type hypersensitivity
(DTH) reactions,Th1 cells also secrete IL-2 in addition to gamma IFN, IL-2 and gamma INF induce the
development of TH1 cells
Systemic IL-2 and IL-8 mean values showed significant differences between rabbits groups which
immunized with normal saline and and those with silver nanoparticles and the mean values were
(53.2333 ± 12.62630,39.1000 ± 4.63573)for IL-8 and (135.2667± 5.65007, 152.1667 ± 5.56007)for IL-2
respectively table (6).
Table (7) concentrations of IL-2 and IL-8 (pg/ml) in rabbits immunized with AgNPs.
Antigen
IL_8(pg/ml) mean ± S.D
IL_2(pg/ml) mean ± S.D
Normal saline
53.2333 ± 12.62630
135.2667± 5.65007
AgNPs
39.1000 ± 4.63573
152.1667 ± 5.56007
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Role of camel's milk against lead induced hematoxocitiy in male rats
Khalid G. Al-Fartosi 1, Mohammed A. Ouda2, and Hanan B. S. Al-Jabery 1
1
Department of Biology, College of Science, University of Thi-Qar,Iraq.
2
Department of Chemistry, College of Science, University of Thi-Qar, Iraq
________________________________________________________________
Abstract
Lead is a heavy metal that has been known for its adverse effects on many body organs and thus
their function. In this study, the toxic effect of lead on blood was investigated, and camel's milk (a well
known nutritious, antioxidant, and medicinal milk) was administered orally to prevent lead's toxicity.
Thirty six mature male rats were used in this study, they were divided randomly into 6 equal groups, the
first group injected I.P with (0.25 ml) distal water, while the second group administered with (1 ml) of
camel's milk, the third and the fourth groups injected I.P with lead acetate (1, 2 mg/kg) respectively, the
fifth and the sixth groups injected I.P with lead acetate (1, 2 mg/kg) respectively, then they administered
with (1 ml) of camel's milk. The duration of treatment was thirty five days. The results indicated a
significant decrease (P
) in red blood cells (RBCs) count, hemoglobin concentration (Hb) and
packed cell volume (PCV). In addition there was leukocytosis, lymphocytosis and eosinopenia which
were prominent at higher dosage of lead acetate administration. The administration of Camel’s milk for
male rats treated with lead acetate improved the changes in the previous parameters.
Key words: lead acetate, anemia, leukocytosis, Hemoglobin concentration.
Introduction
Lead and other heavy metals create reactive radicals which damage cell structure including DNA
and cell membrane (1). Lead poisoning can cause a variety of symptoms and signs which vary depending
on the individual and the duration of lead exposure (2,3). The amount of lead in blood and tissues, as
well as the time course of exposure, determines the level of toxicity (4). Blood often shows pathological
changes before the external signs of poisoning become apparent.
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Lead is absorbed through digestive and respiratory tracts, and skin. After absorption into the
blood, 99% of erythrocytes and the remaining 1 percentage stay in plasma to be carried to other tissues.
The major biochemical effect of lead is its interference with heme synthesis which lead to hematological
damage (5).
Camel milk different from other ruminant as it is low in cholesterol, sugar and protein but high
in mineral (potassium, sodium, iron, copper, zinc and magnesium), vitamin A, B2, C, E, and contain a high
concentration of insulin and immunoglobulins (6,7), It has no allergic properties and it can be consumed
by lactase deficient persons and those with weakend immune systems(8,9). This milk is considered by to
have medicinal properties. In Sahara, fresh butter is often used as a base for medicines. The products
developed also include cosmetics or pharmaceuticals. A series of metabolic and autoimmune diseases
are successfully being treated with camel's milk. In India, camel's milk is used therapeutically against
dropsy, jaundice, problems of the spleen, tuberculosis, asthma, anemia, piles and diabetes (10),
Beneficial role of raw camel's milk in chronic pulmonary tuberculosis patients has been Observed (11). In
repeated trials, it was observed that there was 30-35% reduction in daily doses of inulin in patients of
type 1 diabetes receiving raw camel's milk (12). In our present study, we didn't find any study dealt with
the therapeutic effect of Camel's milk against the bad effect of lead acetate, therefore we tested the
role of Camel's milk against toxicity of lead acetate in male rats.
Material and methods
-Camel's milk samples
Daily milk samples were collected early in the morning from camel farm in the Al-Nassiriya city,
Thi-Qar Province, Iraq. Milk was collected from camel by hand milking as normally practiced by the
framers. The samples were collected in sterile screw bottles and kept in cool boxes transported to
laboratory.
-Lead acetate solution preparation
Lead acetate is supplied by BDH (England) which dissolved in distal water (0.25 ml/animal/day).
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-Animal preparation
Male Wistar rats (3 months old, 200-250g, are obtained from animal house of biology
department/college of science/university of Thi-Qar/Iraq). They are housed in a room at constant
temperature of (20-22 ) with 12 h light/dark cycles and fed a standard laboratory rat diet and water ad
lbitum.
-Animals division system
1. The first group (control) (n=6) is treated with distal water for 5 weeks.
2.
The second group (milk) (n=6) is treated orally with camel's milk (1 ml/animal/day).
3. The third group (n=6) is treated with (1 mg.kg-1) from lead acetate.
4. The fourth group (n=6) is treated with (2 mg.kg-1) from lead acetate.
5. The fifth group (n=6) is injected with lead acetate (1 mg.kg-1), then treated orally with camel's
milk at doses (1 ml/animal/day).
6. The sixth group (n=6) is injected with lead acetate (2 mg.kg-1), then treated orally with camel's
milk at doses (1 ml/animal/day).
The experiment continues for (35 day), at the end of the experimental period the animals are
sacrificed, the blood samples are collected, two milliliter of blood collected into EDTA tubes were
analyzed for the levels of hematological parameters such as total red blood cells (RBC), hemoglobin
(Hb), packed cell volume (PCV), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean
corpuscular hemoglobin concentration (MCHC), total white blood cell (WBC) using an automatic
hematological assay analyzer, (Nihon Kohden Corporation, Japan). Blood smears were also stained with
Giemsa for differential WBC count (13).
Statistical analysis
In this study, ANOVA analysis and LSD tests are used according to (SPSS version 14) program at
the (P 0.05) to find the mean between all treatments.
Results
The hematological parameters of rats exposed to two doses of lead acetate are presented in
(table 1), there was significant reduction (P 0.05) in the packed cell volume (PCV), hemoglobin (HB), red
blood corpuscular (RBC) and mean corpuscular hemoglobin concentration (MCHC), while mean
corpuscular volume (MCV) showed significant increase (P 0.05) in male rats treated with lead acetate
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compared with control group, while the orally administration of Camel’s milk for male rats treated with
lead acetate improved the changes in the previous parameters.
Table (1): Effect of camel milk on erythrocytes of male rats treated with lead acetate.
Animal
RBCs
Hb
PCV
MCV
MCH
MCHC
groups
(106/mm3)
(g/dl)
(%)
(m3)
(pg)
(g/dl)
7.15 a
14.36 a
39.33 a b c
54.33 b c
18.76 a b
35.43 a
± 0.13
± 1.44
± 0.27
± 1.20
± 0.29
± 0.45
7.70 a
17.51 a
41.18 a
53.16 c
19.96 a
36.33 a
± 0.18
± 2.74
± 0.41
± 1.10
± 1.11
± 0.62
6.90 a
12.26 b c
38.16 b c
57.83 ab
17.68 b
30.80 d e
± 0.26
± 0.36
± 0.39
± 1.57
± 0.29
± 0.83
5.90 b
10.60 c
36.56 c
61.50 a
17.55 b
28.90 e
± 0.42
± 0.43
± 1.45
± 1.47
± 0.38
± 0.17
7.11 a
12.72 b c
41.00 a b
55.83 b c
17.93 b
32.21 c d
± 0.35
± 0.21
± 1.30
± 1.53
± 0.50
± 0.42
13.05 b c
39.70 a b
54.66 b c
18.11 b
33.06 b c
± 0.22
± 0.39
± 1.49
± 2.34
± 0.44
± 1.36
0.81
3.67
2.9
4.6
1.7
2.16
G1
G2
G3
G4
G5
7.2
a
G6
LSD
Values are means ± S.E.
Different letters refer to significant differences (P ≤ 0.05)
Same letters refer to no significant differences (P ≤ 0.05)
Also the results showed non significant differences ( P ≤ 0.05 ) in monocytes, basophiles and
neutrophiles count in the male rats treated with lead acetate, and the male rats treatment with lead
acetate and camel's milk compared with control group. There was significant increase ( P ≤ 0.05 ) in
platelets count, WBCs and lymphocytes count in male rats treated with ( 1, 2 mg/ kg ) of lead acetate
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December 2013
compared with control group, while acidophils count showed significant decrease ( P ≤ 0.05 ) in male
rats treated with 2 mg/ kg of lead acetate compared with control group. However, a more pronounced
improves of the above mentioned parameters of male rats injected with lead acetate when treated with
camel milk (table 2).
Table (2): Effect of camel milk on leucocytes of male rats treated with lead acetate.
Animal
WBC
Lym.
Mon.
Acido.
Baso.
Neutro.
Groups
(103/mm3)
(%)
(%)
(%)
(%)
(%)
7.75 c
69.16 b
8.62 a
4.26 a
0.00 a
17.30 a
± 0.30
± 0.83
± 0.39
± 0.67
± 0.00
± 0.82
7.50 c
69.00 b
8.31 a
4.56 a
0.20 a
16.66 a
± 0.57
± 3.21
± 0.56
± 0.47
± 0.16
± 0.62
10.56 a
69.33 b
9.48 a
4.06 a
0.33 a
17.28 a
± 1.32
± 1.97
± 0.77
± 0.66
± 0.33
± 0.47
12.33 a
79.00 a
9.80 a
2.43 b
0.50 a
18.15 a
± 0.59
± 3.48
± 1.36
± 0.52
± 0.50
± 0.79
7.36 c
69.00 b
8.81 a
4.30 a
0.25 a
16.86 a
± 0.56
± 2.28
± 0.56
± 0.38
± 0.17
± 1.06
8.03 b c
67.83 b
8.81 a
4.65 a
0.16 a
17.06 a
± 1.14
± 2.32
± 0.86
± 0.57
± 0.16
± 0.51
2.4
7.24
2.4
1.6
0.8
2.12
G1
G2
G3
G4
G5
G6
LSD
Values are means ± S.E.
Different letters refer to significant differences (P ≤ 0.05).
Same letters refer to no significant differences (P ≤ 0.05).
142
December 2013
_Discussion
The results of the present study demonstrated that lead acetate administration to male rats result in
significant decrease in PCV, Hb, RBCs count , MCHC and a significant increase in lymphocytes and WBCs
count, MCV, platelets count compared with control group. This finding therefore corroborates similar
findings reported by ( 14, 15, 16 ) in mice, whom observed a significant decrease erythrocytes number,
hemoglobin concentration in rats that injected with lead acetate. Anemia is a one of the early
manifestations of lead poisoning, it result from reduction of the life span of circulation erythrocyte as
well as by inhibition the body's ability to make hemoglobin by interfering with several enzymatic steps in
hem pathway (17, 14). Ferrochelatase, which catalyze the insertion of iron into protoporphyrin IX, is
quite sensitive to lead. Erythrocyte Na-K-ATPase is somewhat inhibited by lead suggesting a loss of cell
membrane integrity this may account for the shortened lifespan of erythrocytes (17), also, lead can
cause damage in the erythrocytes originated defective cells that are eliminated by spleen and their
hemolysis (14).
In the present study the increase in the mean corpuscular volume and the decrease in the mean
corpuscular hemoglobin concentration of male rats treated with lead acetate compared with control
group may result from the toxic effect of lead acetate that affect on red blood cells count and
hemoglobin concentration, because the validity of these indexes is influenced by the value of red cell
count, hemoglobin concentration and packed cell volume (18), but the changes in these hematological
parameters are improved to near normal levels after five weeks treatment of camel's milk which
indicate that it offered protection by preserving the structural integrity of erythrocytes membrane
against lead acetate.
The present study shows that the oral administration of lead acetate caused significant increase in
the platelets count, white blood cell count and lymphocyte percentage in the other hand treatment of
male rats with lead acetate produced significant decrease in the acidophilus percentage. In the present
study, total leukocyte count had increased mainly due to an increase in lymphocyte count. There was
also an increase in basophiles, neutrophil and monocytes percentage which wasn't statistically
significant ( P ≤ 0.05 ). In some reports, leukocytosis has been attributed to the lead- induced
inflammation (19). These finding corroborates with (20) whom find a three- fold increase in neutrophil
and monocyte count along with severe leukocytosis in the young rats that were exposed to lead.
Controversies exist about monocytes; since in some studies lead- induced monocytopenia (21, 22) and
in others significant increases in monocyte count have been reported (19, 20). The reason for such
143
December 2013
difference is probably due to the extent of lead- induced inflammation. Consistent with other reports
severe eosinopenia were observed in this study (22). Platelets count showed considerable increase
compared to the control group. In the previous studies , some cases of thrombocytopenia after lead
intoxication followed by thrombocytosis have been reported (19, 23), which is consistent with the
findings of this study which was conducted over along period of 12 weeks, but there was improved in
the previous parameters after treatment with camel's milk due to antioxidant activity and it may
possible have chelating effect on toxicants (24).
The protective effect of camel's milk could be attributed to it's antioxidant activity and it may
possibly have chelating effects on zinc. It has been reported that camel's milk contains high levels of
vitamins A, B2, C and E and very rich in magnesium (Mg) and other trace elements (25). These vitamins
are antioxidants that have been found to be useful in preventing tissue injury caused by toxic agents. Mg
protects cells from heavy metals such as aluminum, mercury, lead, cadmium, beryllium and nickel,
which explains why remineralization is so essential for heavy metal detoxification and Chelating. In fact,
Mg deficiency has been associated with production of ROS (26). Additionally, Mg protects cells against
oxyradical damage and assists in the absorption and metabolism of vitamins B, C and E which are
antioxidants important in cellular protection. Recent evidence suggests that vitamin E enhances
glutathione levels and may play a protective role in Mg deficiency induced cardiac lesions. Magnesium
protects the cell against oxy radical damage and assists in the absorption and metabolism of B vitamin, C
and E vitamins, which are enhances glutathione levels and may play a protective role in magnesium
deficiency- induced cardiac lesions (27). Also, it has been reported that magnesium is very essential for
biosynthesis of glutathione, because the enzyme glutathione synthetase requires γ- glutamyl cysteine,
glycine, and ATP and magnesium ions to form glutathione (28). Vitamin C is a strong antioxidant (10, 29).
The detoxification effect of vitamin C is manifested by the removal or minimization of free radicals
produced by the heavy metals (30, 31). Also, vitamin C protects DNA from oxidative damage (32, 33),
reduces DNA damage exerted by irradiation (34) and also reduces micronucleus (MN) frequencies in
polychromatic erythrocytes of bone marrow in rodents exposed to heavy metals and radiation ( 35) .
Furthermore, milk exhibits a range of biological activities. These biological activities are mainly due to
peptides and proteins in milk. Bioactive peptides are produced during the digestion of milk in the
gastrointestinal tract (36). The beneficial health effects of milk proteins can be classified as
antimicrobial, antioxidative, antithrombotic, antihypertensive or immuno-modulatory ( 37).
144
December 2013
References
1- Flora, S.; Mittal, M. and Mehta, A. (2008). Heavy metal induced oxidative stress & its possible
reversal by chelation therapy. The Indian Journal of Medical Research ,128 (4): 501–23 .
2- Kosnett, M.J. (2005). Lead. In Brent, J. Critical Care Toxicology: Diagnosis and Management of the
Critically Poisoned Patient. Gulf Professional Publishing.
3- Karri, S.k.; Saper, R. and Kales, S. (2008). Lead encephalopathy due to traditional medicines. Current
Drug Safety 3 (1): 54–9.
4- Pearson, H.A. and Schonfeld, D.J. (2003). Lead. In Rudolph, C.D..Rudolph'sPediatrics, 21st edition.
McGraw-Hill Professional.
5- Awad, M. and William, J. (1997). Textbook of biochemistry with clinical correlation. John Wiley and
Sons, INC, New York.
6- Kamal, A.M.; Salama, O.A. and El-Saied, K.M. (2007). Changes in amino acids profile of camel
milk protein during the early lactation. Int. J. Dairy. Sci., 2: 226-234.
7-Al-Hashem, F. (2009). Camel milk protects against aluminum chloride-induced toxicity in the liver
and kidney of white albino rats. Am. J. Biochem. Biotech., 5: 98-108.
8-Inayat, S.; Akbar, A.; Khaskheli, M. and Hussain, M. (2003).Study of the effect of processing on the
chemical quality of soft unripened cheese made from camel milk. Pak. J. Nutr., 2: 102-105.
9- Yateem, A.; Balba, M.; Al-Surrayai, T.; Al-Mutairi, B. and Al-Daher, R. (2008).Isolation of lactic
acid bacteria with perobioticprotential from camel milk.Int. J. Dairy Sci., 3: 194-199.
10-Rao, M.; Gupta, R. and Dastur, N. (1970).Camels‘ milk and milk products.Indian J. Dairy Sci. 23:
71-78.
11-Mal, G.; Sena, D.; Jain, V. and Sahani, M. (2001).Therapeutic utility of camel milk as nutritional
supplement in chronic pulmonary tuberculosis.Livest. Int., 7: 4-8.
12-Agarwal, R.; Swami, S.; Beniwal, R.; Kocher, D. and Kothari, R. (2002). Effects of camel milk on
glycemic control, risk factors and diabetes quality of life in type-I diabetes: A randomized
prospective controlled study. Int. J. Diabetes Dev. Countries, 22: 70-74.
13- Dacie, J. and Lewis, S. (1984).Practical hematology. ch3. 6th edition.Churehlell.Living stone. Edinburgh.
London, Melbourne and newyourk.Pp: 28-31.
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14- Teijon, C.; Delsocorro, J.; Martin, J.; Lozano, M.; Bernardo, V. and Blaco, D. (2000). Lead
accumulation in rats at non acute doses and short periods of time:Hepatic, renal and
hematological effects. Ecotoxico.Environm.Restor., 3: 36-41.
15- Mugahi, M.H. Heidari Z. Sagheb HM. Barbarestani M. Effect ofchronic lead acetate intoxication on
blood indices of male adult rats.Daru 2003:11(4) 147-151.
16- Khan, M.S.H.; Mostofa, M.; Jahan, M.S.; Sayed, M.A. and Hossain, M.A. (2008). Effect of garlic
and vitamin B-complex in lead acetate induced toxicities in mice.Bangl. J Vet Med. 6(2):203210..
17- Doull, J.; Klaassen, C.D.; Amdur, M.O.; Casaratt and Doulls. (1980). Toxicology. 2nd ed. united
state, Macmillan publishing co., Pp: 415-421.
18- Jain, N.C. (1986).Schalms veterinary hematology.USA, Lea and Fibiger.Pp: 276-82.
19-Yagminas, A.; Franklin, C.; Villeneuve, D.; Gilman, A.P.; Little, P.B. and Valli, V.E.
(1990).Subchronic oral toxicity of triethyllead in the male weanling rat. Clinical, biochemical,
hematological, and histopathological effects.Fundam. Appl. Toxicol., 15: 580-596.
20- Hogan, G.R. and Adams, D.P. (2004).Lead-induced leukocytosis in female mice.Archives of
Toxicology, 41: 295-300.
21- Zook, B.C. (1972). Lead poisoning in dogs. Am. J. Vet. Res. 33: 981-902.
22-Xintaras, C. (1992).Impact of lead-contaminated soil on public health. Analysis paper,U.S.
Department of health and human services. Public Health Service. ATSDR (Agency for Toxic
Substances and Disease Registry), Atlanta, Georgia.
23- Sudakova, A.; Shevchenko, Z. and Nosova, L. (1983). Peripheral blood and bone marrow cell
status of white rats with long-term lead exposure. Tsitol.Genet., 17: 3-7.
24- Al-Humaid, A.; Mousa, H.; El-Mergawi, R. and Abdel-Salam, A.(2010).Chemical composition
and antioxidant activity of dates and dates-camel-milk mixtures as a protective meal against
lipid peroxidation in rats.Am. J. Food Technol., 5: 22-30.
25- Knoess, K. (1979).Milk production of the dromedary. Proceedings of the 1st International
Symposium on Camels, Sudan, Pp: 201-214.
26- Martin, H.; Richert, L. and Berthelot, A. (2003).Magnesium deficiencyinduces apoptosis in
primary cultures of rat hepatocytes. J. Nutr., 133: 2505-2511.
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27- Barbagallo, M. (1999). Effects of vitamin E and glutathione on glucose metabolism: role of
magnesium. Hypertension, 34: 1002-1006.
28- Varginia, M.; Smith, M.; Brauner, M. J. and Majerus, W.P. (1971). Glutathione biosynthesis in
human erythrocytes. J. Clin. Invest. 50: 507-513.
29- Sato, M. and Bremner, I. (1993). Oxygen free radical and melatothionein. Free Radic. Biol. Med., 14:
325- 327.
30- Gebhart, E. (1984). The action of anticlastogens on chemically induced SEC. In: R.R. Tice and A.
Hollaender, Editors, Sister Chromatid Exchange – 25 years of Experimental Research- Part A and
B, Plenum Press, New York, pp. 319- 332.
31- Herbaczyńska, C. K.; Ktosiewicz, W.B.; Cedro, K.; Wasek, W.; Panczenko, K.B. and Wartanowicz,
M. (1995). Supple- mention with vitamins C and E suppresses leukocyte oxygen free radical
production in patients with myocardial infarction, Eur. Health J. 16: 1044- 1049.
32- Eylar, E.; Baez, I.; Navas, J. and Mercado, C.(1996). Sustained levels of ascorbic acids are toxic and
immune-suppressive for human T cells, Proc. Royal Health Sci. J. 15: 21-26.
33- Antunes, L. M. and Takahashi, C.S. (1999). Protective and induction of chromosomaldamage by
vitamin C in human lymphocyte cultures, Terato.Carcino.Mutagen. 19: 53- 59.
34- Green, M. H.; Lowe, J. E.; Waugh, A. P.; Aldridge, K. E.; Cole, J. and Arlett, C. F. (1994). Effect of
diet and vitamin C on DNA strand breakage in freshly- isolated human white blood cells, Mutat.
Res. 316: 91-102.
35- Konopaka, M.; Widel, M. and Rzeszowska, W. J. (1998). Modifying effect of vitamins C, E and betacarotene against gamma-ray-induced Dna damage in mouse cells, Mutat. Res. 417: 85-94.
36- Korhonen, H. and Pihlanto, A. (2001). Food- derived bioactive peptides- opportunities for designing
future foods, Curr. Pharm. Des. 9 (2001), pp. 1297- 1308.
37- FitzGerald, R. J. and Meisel, H. (2000). Milk protein derived inhibitors of angiotensin- I- converting
enzyme, Br. J. Nutr. 84: 33-37.
147
December 2013
Green Industrial Aspects of Jaleel’s Process in Providing Free Carbon-Iron
Industry
Jaleel K. Ahmed* and Alaa K. H. Al-Khalaf**
*
Babylon University, P.O. Box 4 (mail of Babylon University), Hilla, 00964IQ, Babylon/ IRAQ
**
Green Al-Qasim University/ College of Environmental Sciences/ Hilla, 00964IQ, Babylon/ IRAQ
E-mail address: [email protected]; [email protected]
Abstract:
According to the study of the principles of green chemistry and calculations of environmental
efficiency parameters of Jaleel (Iraq) process1, this process can be considered as a greener
approach to the sustainability development in the Iron industry worldwide.
Water hydrogen
3H 2 + 3/2 O2
(Electrolysis) DC
3H2 O
Room temp.
Recycling
Reduction by pure
molecular hydrogen
Direct reduction
3H 2 + Fe2 O3
3H 2 O
+
2Fe
Free-carbon DRI
In this paper, we show a green source of free carbon-reducing gas that is water hydrogen as pure
H2 >99% obtained from electrolysis process of alkaline water which is also produced ~98.5%
oxygen gas as well as heavy water residue. The water hydrogen (>99% H2) used in the
production of directed reduced Iron (DRI) instead of reduced gas that produced from natural gas
and coke, where the percentage (H2 ≈75%, and CO ≈14%) and (H2 ≈50%, CO ≈48%)
respectively.
The annual world DRI production is about 73.3 million tons that is contributed by most common
technologies such as Midrex (USA) process: 60.5% of global gas based DRI production, along
with HYL (Mexico) process: 15.9% and others that is coal based: 23.6%.
In the case of water source for reducing gas, no polluted gas is discharged to the atmosphere,
while in the case of two traditional sources (natural gas and coke); huge quantity of carbon
dioxide is produced (3×105 and 6×106 ton respectively) as a greenhouse gas contributing to
climatic change. These parameters such as atom economy, yield, environmental factor
(E-Factor), environmental quotient (EQ), and effective mass yield (EMY) have been calculated.
148
December 2013
Introduction:
Green chemistry is a group of several modern techniques and industrial processes for chemical
reactions that distinguished with efficient energy. It can serve to reduce or prevent formation of
not desired by-products, prevent using reagents or toxic solvents, use renewable resources and
avoid use of these non renewable materials before their depletion in the nearest future, e.g. oil
and coke.2
The 12th Applied principles of green chemistry are aimed to make the chemical reactions safer
and not harmful, eco-friendly for environment and human, more efficiently and cleanly,
3
and
reduce or prevent use or produce the toxic and dangerous materials on these steps: design,
industry, applied of chemicals and ability of its degradation.4
Measurements of environmental efficiency parameters:
There are some important principles of green chemistry such as:
1. Atom Economy: this concept is considered as a design tool for the chemical reactions that
refers to numbers of reactant atoms that corporate to form the desired product and the numbers of
other reactant atoms to form by-product as the following equation depending on the original
chemical equation and the same regarding with parameters of selectivity and yielding of product:
Atom Economy = Molecular mass of Product
x 100%
Molecular mass of all Reagents
2. Selectivity = Yield of desired product
x 100%
Amount of substrate converted
3. Yield = Actual quantity of products achieved
x 100%
Theoretical quantity of products achievable
4. E-Factor: this concept can be defined as the ratio between the waste amounts and the amounts
of the desired product as the following equation:
E-factor = [Raw Materials (Total input)-Product]/Product
149
December 2013
This parameter is a good tool used to show the dilemma of the waste that accompanied with
production of desired material. However, all the amounts of used chemicals in the chemical
reaction from the starting to the ending including the water used in washing and purification
processes. The consumption of large amounts of these benign waste such water, diluted ethanol,
acetic acid, and low concentrations of inorganic salts will make the environmental factor seem to
be worse than the truth. Table (1) showed the annual production, total waste, and values of
E-factor for the industry segments.5
Table 1. Values of E-factor for the different types of industry.
Type of industry
Annual production (ton)
E-factor
Total waste
(ton; approx.)
Pharmaceuticals
10-103
25->100
103
Fine chemicals
102-104
5->50
104
Bulk chemicals
104-106
<1-5
105
Oil refining
106-108
0.1
106
5. Environmental Quotient: is multiple of E-factor by the factor of non desired by-products
(Q-factor). E.g. Q equal to (100) for salts of heavy metal and equal (1) for NaCl as the following
equation:
EQ = E-factor × Q-factor
6. Effective Mass Yield (EMY): is the ratio between the amount of desired product and amount of
non-benign material used. However, this parameter treats the problem of calculating the benign
waste in law of E-factor. The environmental benign waste like water will be taken in account as
in below:
EMY = Mass of Product
x 100%
Mass of non-benign material used
Results and Discussion:
150
December 2013
From data of Jaleel‘s process1 which used in measurements of the following parameters as in
Table (2), depending on the chemical equation of water electrolysis for production of water
hydrogen:
H2O
DC
Room temp.
H2 + 1/2 O2
Table 2. Parameters of the environmental efficiency for the electrolysis process of water. *
Parameters of environ. Efficiency
Electrolysis process of water
‘Greenness’
H2
O2
Atom economy
11.11
88.88
Quite good
Yield (%)
99.5
98.5
OK
Selectivity (%)
11.10
88.9
Quite good
E-Factor
8.0
0.12
Quite good
E-quotient
8.0
0.12
Quite good
EMY (%)
Maximum value
Maximum value
Excellent
* Water is considered as a benign waste; Q equal 1.
The above table shows the values of environmental efficiency parameters for the reaction of
water electrolysis to produce pure hydrogen, pure oxygen (high molecular mass than hydrogen),
and heavy water residue (in ratio of hydrogen: deuterium 5000:1 part). The atom economy (%)
of the hydrogen in the traditional two sources of reducing gas (natural gas and coke) are 6.66 and
19.88 respectively and other percentage for carbon mono CO and dioxide CO2. So, electrolysis
process of water is clean, economic, efficient, and eco-friendly process.
Pure hydrogen, pure oxygen, and residue of deuterium oxide (heavy water D2O) are produced
from hydrolysis process of water. All these products have great benefit and use in several
important industries and purposes. By other hand, using pure reducing gas at high temperature
leads to clean production of high grade sponge iron and clean manufacturing of steel without
impurities like sulphur and phosphorous because these fines are corrosive, polluting, and
contaminates the product resulting in iron of poor mechanical properties.
However, pure hydrogen will take place in the reduction process alone without any other
reducing gas like carbon monoxide (CO) will avoid many serious problems1 that affected the
151
December 2013
production of DRI such as: re-oxidation of iron occurs when the pressure of carbon dioxide
increases by the following reversible reaction:
Fe2O3 + 3CO
2Fe + 3CO2
The final stage in the production of DRI according to Midrex and HYL technologies is called
‗cooling and carburizing process‘ which inhibits the reduction of the iron oxide because the
carbon is deposited as iron carbide6 which is easily hydrolysed by water to give a variety of
hydrocarbons, free carbon (graphite or carbon black), hydrogen (explosive gas which is
dangerous in transportation of DRI) 7 and iron oxide (wustite form FeO as residual unreduced
iron oxide in DRI).6, 8, 9 While in case of pure water hydrogen (Jaleel‘s process) as reducing gas
acts as only cooling and reducing any residual iron oxide ore.1
1998,10 Qatar steel company lted (QASCO) using electric arc furnaces (EAF) for production of
DRI had mentioned that storage of DRI for long periods of time is affecting its metallization
gradually by losing about 1 % after 6 months of storage in the open yard due to surface
deoxidation.
The deposited carbon leads to sealing of the porosity of both the iron pellets (preventing the
reduction step leads to wasteful consumption of reducing gases and requires more time for
reduction) and sponge iron leads to creating of ―Hot spot‖ which are causing fire hazards due to
high energy liberated by exothermic reaction between carbon dioxide and deposited carbon.1
C + CO2
2CO
While through Jaleel‘s process, the only reaction would be happened to reduce the iron ore to
give the product of sponge iron as clean target (free carbon-DRI) with water as recycled
by-product only. The following reaction is one direction reaction efficient, economic, and clean
with no chance to be reversible reaction after continuing removing of water vapour by
dehumidifiers units for each step:
Fe2O3 + 3H2
2Fe + 3H2O
As a comparison, table (3) shows the typical quality of DRI (sponge iron) produced from three
technologies: Midrex process plant at QASCO‘S EAF, HYL process, and free carbon-Jaleel‘s
process (the last two process using the same ore and time of reduction):
152
December 2013
Table 3. Chemical analysis for three technologies: Midrex, HYL, and free carbon Jaleel‘s
process.
Specification
Midrex process
HYL process
Free carbon-Jaleel’s process
Metallization
95.00
85.00
92.00
Total iron
92.50
87.9
91.00
Metallic Fe (free iron)
87.90
74.90
83.90
Carbon
1.54
2.2
Free
Gangue
4.70
3.8
4.2
Lime
Not checked
2.3
2.6
Oxygen
Not checked
3.8
2.09
Sulphur
0.015
0.014
0.002
Phosphorous
0.035
0.045
0.004
Copper
Not checked
0.000
0.000
Tin
Not checked
0.000
0.000
Nickel
Not checked
0.020
0.025
Chromium
Not checked
0.000
0.000
Table (3) clearly reflects that Jaleel‘s process has free carbon content because it uses clean and
pure water hydrogen with no carbon content (CO gas). However, the metallization percentage (is
the free iron over total iron) was 95, 92, and 85 which is high in Midrex, Jaleel, and HYL
respectively. While the amounts of combined iron that refers to oxygen content (the difference
between total iron and free iron) present as residual iron oxide (FeO; g) was 0.046, 0.071, and
0.13 for the Midrex, Jaleel, HYL respectively.
References:
1. Jaleel K. Ahmed, J. Adv. Oxid. , 2010, 13, (1), 1-6.
2. Mike Lancaster, ‘Green Chemistry: An Introductory Text’, RSC Paperbacks, University of
York, 2002.
3. Keith Smith, Alaa K. H. Al-Khalaf, Gamal A. El-Hiti and Samuel Pattisson, Green Chem.,
2012, 14, 1103-1110.
153
December 2013
4. P. T. Anastas and J. C. Warner, ‘Green Chemistry: Theory and Practice’, Oxford University
Press, Oxford 1998.
5. R. A. Sheldon, Chem. & Ind., 1992, 1, 903.
6. W. Pietisch, Directed Reduced Iron; British Foundary-man, 1978, p. 71.
7. F. A. Cotton, and G. Wilkinson, Advanced Inorganic Chemistry, Interscience Publishers,
1962, 225.
8. J. K. Ahmed, In Proceeding of 7th International Conference and Exhibition on Iron and Steel
Industry; Arab Steel, (Beirut), 2004; p. 35.
9. J. K. Ahmed, In Proceeding of 1st International Symposium and Exhibition on Arab Steel
Industry; Arab Steel, (Abu Dhabi), 2006.
10. Nabil D. Takla, AISU’S 2nd Electric Furnace Symposium in Damascus, Syria, October 18-20,
1998; Direct From Midrex 2nd Quarter1999; Direct From Midrex 3rd Quarter 2012.
154

Detection of β- lactam Genes in Pseudomonas aeruginosa Isolates

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