International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(4), pp. 50-62, 2013 Available online at http://www.ijsrpub.com/ijsres

ISSN: 2322-4983; ©2013 IJSRPUB

50

Full Length Research Paper

Computational Prediction and Analysis of Interaction of Silver Nitrate with

Chitinase Enzyme

Shahin Gavanji1*, Hamidi Abdul Aziz

2, Behrouz Larki

1, Amin Mojiri

2

1Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

2School of Civil Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang, Malaysia

*Corresponding Author: shahin.gavanji@yahoo.com

Received 28 February 2013; Accepted 22 March 2013

Abstract. Silver nitrate is an inorganic compound with chemical formula AgNO3. Silver or silver ions have long been used in

many areas due to their strong antimicrobial activity against pathogenic microbes such as bacteria, yeast, fungi and algae.

Trichoderma spp. are considered as biocontrol and growth promoting agents for many crop plants due to their antagonistic

properties against plant pathogens. We, in this study, checked the possible interaction between silver nitrate and Chitinase

enzyme. We used Molegro virtual docker (MVD). The results obtained from docking showed us that the best pose which is

derived from MolDock score for Chitinase was -49.1418 with reranking score equal to -58. 1719. It is concluded that silver

nitrate and Chitinase have interaction. Obtained results showed that silver nitrate can be attached to Chitinase in Trichoderma

fungi cells leading to deactivation the enzymes.

Key words: Chitinase- Molecular mechanism-Silver nitrate-bioinformatics.

1. INTRODUCTION

Trichoderma viride is a filamentous Ascomycota.

Agricultural and orchard soil, which contain huge

quantities of plant roots are the best places to live for

these organisms (Samuels et al., 1999). Persoon

almost 200 years ago described the genus

Trichoderma that consists of anamorphic fungi

isolated primarily from soil and decomposing organic

matter. Isolates of Trichoderma are ubiquitous and

their isolation and culture are very easy. These

isolates can grow quickly on a wide range of

substrates and produce metabolites with demonstrable

antibiotic activity or mycoparasitic against a wide

range of pathogens. Because of these properties,

Trichoderma species have been used in biological

control of fungi including plant pathogens (Inbar and

Chet, 1992; Lumsden and Locke, 1989). Through

nutrient competition and rhizosphere competence,

mycoparasitism, enzyme, metabolism of germination

stimulants and induced defense responses in plants,

Trichoderma spp can control root foliar pathogens

(Howell, 2003; Zimand et al., 1996).

Jin and Custis (2011) also showed that

Trichoderma strains induce changes in the microbial

composition on roots, enhance nutrient uptake,

stabilize soil nutrients, promote root development, and

increase root hair formation. Since mycoparasitic and

antibiotic properties were demonstrated in 1932 and

1934 by Weindling, there have been biotechnological

applications of these fungi as biocontrol agents (Chet,

1987; Haran et al., 1996; Schirmbock et al., 1994).

Most species of the genus grow rapidly in artificial

culture and produce large numbers of small green or

white conidia from conidiogenous cells of widely

branched conidiophores which al low a relatively easy

identification of Trichoderma as a genus (Rifai,

1969). Research is in progress on multifunctional

materials containing silver nanoparticles (SNPs) in

reactive or nonreactive polymer networks for

applications as biocidal products and drug supports

(Gaidau et al., 2009).

Silver nitrate is an inorganic compound with

chemical formula AgNO3. Silver salts have antiseptic

properties. Until the development and common

adoption of antibiotics, dilute solutions of AgNO3 is

used to be dropped into newborn babies' eyes at birth

to prevent contraction of gonorrhea from the mother.

Eye infections and blindness of newborns was

reduced by this method, but incorrect dosage could

cause blindness in extreme cases. This protection was

used by Credé in 1881 for the first time (Credé, 1881).

The antimicrobial properties of silver were first

detected thousands of years ago when silver

containers have been used to store water for

preservation. Its disinfection property has been

scientifically studied for over a century. Before a

disinfectant can be efficiently used as a water

disinfectant, its inactivation kinetics must be

recognized. Kinetics generally depend on both the

dosage of disinfectant and the time of application.

Gavanji et al.

Computational Prediction and Analysis of Interaction of Silver Nitrate with Chitinase Enzyme

51

It is important to understand the kinetics so that the

minimum dosage of disinfectant can be applied for the

minimum amount of time while still effectively

inactivating any pathogens in the water. In recent

years, research has focused mostly on generated silver

ions or colloidal silver electrolytically. In addition,

silver tends to adsorb to glassware, which can lead not

only to a decrease in the silver concentration within a

given experiment but also to a release of the silver in

subsequent experiments unless measures further than

general glassware washing are taken for the removal

of silver from the glassware surface (Chambers et al.,

1962). Therefore studies must both minimize the

external factors affecting the concentration and to

measure the changes in concentration that take place

throughout the experiment. Chambers, Proctor and

Kabler established the importance of using an

effective neutralizer solution which is made of a

mixture of sodium thioglycolate and sodium

thiosulfate, rather than sodium thiosulfate alone,

which though it is effective in neutralizing other

disinfectants does not satisfactorily stop the

bactericidal action of silver nitrate. The researchers

tested the effect of pH on the kinetics, finding that a

higher pH increased the bactericidal action (Chambers

et al., 1962; Gavanji et al., 2011; Gavanji et al., 2012).

Wuhrmann and Zobrist added that at a higher

temperature, inactivation occurs faster (Wuhrmann

and Zobrist 1958). Elemental silver and silver salts

have been used as antimicrobial agents for a long time

(Gavanji et al., 2012). Silver or silver ions have long

been used in many areas due to their strong

antimicrobial activity against pathogenic microbes

such as bacteria, yeast, fungi and algae (Gavanji et al.,

2013). It may be used for controlling different plant

pathogens in a relatively safer way compared to

synthetic fungicides (Park et al., 2006). Until now,

limited studies have provided pieces of evidence of

the applicability of silver for controlling plant diseases

(Park et al., 2006).

Silver ions are very reactive, which are known to

cause the inhibition of microbial respiration and

metabolism as well as physical damage (Gavanji et

al., 2013; Bragg and Rannie, 1974; Thurman and

Gerba, 1989). Ionic silver has some disadvantages

such as its high reactivity which made it unstable and

thus easily oxidized or reduced into a metal depending

on the surrounding environment. In addition, ionic

silver causes discoloration by itself or allows other

materials to cause undesirable coloration and it does

not continuously exert antimicrobial activity. Also,

silver in the form of a metal or oxide, which is stable

in the environment, is applied in a relatively increased

amount due to its low antimicrobial activity (Park et

al., 2006). In recent years, the use of silver as a

biocide in the form of micro crystals or nanoparticles

has grown significantly, as these preparations are

useful against many resistant populations and

‘biofilms’ aggregates of microorganisms that grow on

the surfaces of bodies of water and inside water pipes

(Silver, 2003; Panyala et al., 2008; Gaidau et al.,

2009). Generally, the antimicrobial mechanism of

chemical agents depends on the specific binding with

surface and metabolism of agents into the

microorganism. Furthermore, it has been suggested

that silver ions penetrate into bacterial DNA once

entering the cell, which prevents further proliferation

of the pathogen (Woo et al., 2009).

The aim of present study was to study

bioinformatic interaction of silver nitrate and

Chitinase enzyme to determine harmful effect of

silver nitrate on the enzyme. Chitin is an example of

highly basic polysaccharides. Their unique properties

include polyxylate formation, ability to form films,

chelate metal ions and optical structural

characteristics. Like cellulose, it naturally functions as

structural polysaccharide, but differs from cellulose in

the properties. Chitin is extremely hydrophobic and is

insoluble in water. It is soluble in

hexafluoroisopropanol, hexafluoroacetone,

chloroalcohol in conjugation with aqueous solutions

of minerals acid and dimethylacetamide containing

5% lithium chloride. The nitrogen content in chitin

differs between 5 to 8% depending on the extent of

deacetylation (Yalpani et al., 1992). Acetic anhydride

can entirely acetylate chitin. Linear aliphatic N-acetyl

groups above propionyl, let rapid acetylation of

hydroxyl groups. Highly benzoylated chitin is soluble

in benzyl alcohol, dimethyl sulfoxide, formic acid and

dichloroacetic acid. The N-hexanol, N-decanoyl and

N-dodecanoyl derivatives have been obtained in

metthanesulfonic acid. Chitin is the most widespread

amino polysaccharide in nature and is estimated

annually to be produced almost as much as cellulose.

It is mostly found in antraphode exoskeletons, fungal

cell walls or nematode eggshells. However,

derivatives of chitin oligomers have also been

implicated as morphogenetic factors between

leguminous plants and rhizobium and even in

vertebrates, where they may be important during early

stages of embryogenesis (Merzendorfer and Zimoch,

2003).

Chitin is largely composed of alternating N-

acetylglucosamine residues, which where linked by β-

(1-4) glycosidic bonds. Since hydrolysis of chitin by

chitinase treatment leads to release of glucosamine in

addition to N- acetylglucosamine, it was concluded

that it might be the significant portion of polymer.

Chitin polymer tends to form microfibrils (also

referred as rod or crystallites) of ~3 nm in diameter

that are stabilized by hydrogen bonds formed between

the amine and carbonyl groups. Chitin micro fibrils of

International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(4), pp. 50-62, 2013

52

peritropic matrices may exceed 0.5μm in length and

frequently associate in bundles containing parallel

groups of 10 or more single microfibrils. X- ray

diffraction analysis suggested that chitin is a

polymeric substance that occurs in three different

crystalline modifications, termed α, β and γ. They

mainly differ in the degree of hydration, in size of the

unit cell and in the number of chitin chains per unit

cell. In the α form, all chains display an anti-parallel

orientation. In the β form the chains are set in a

parallel manner and in the γ form sets of two parallel

strands alternate with single anti parallel strands. The

anti parallel arrangement of chitin molecules in α

form allows tight packaging into chitin microfibrils,

consisting ~20 single chitin chains that are stabilized

by a high number of hydrogen bonds which are

formed between the molecules. This arrangement may

contribute significantly to the physicochemical

properties of cuticle such as strength and stability

(Merzendorfer and Zimoch, 2003).

In contrast, the packaging tightness and numbers of

inter-chain hydrogen bonds of the β and γ chains are

reduced, leading to an increase in the number of

hydrogen bonds with water. The high degree of

hydration and reduced packaging tightness resulted in

more flexible and soft chitinous structures, as are

found in peritropic matrices.

Fig. 1: Mechanism of Chitinases action (www.sigmaaldrich.com)

Chitinases are enzymes that catalyze the

degradation of chitin. They have been detected in

many organisms, including bacteria, fungi, plants,

invertebrates and vertebrates (Rogalski et al., 1997;

Felse and Panda, 2000). Chitinases are generally

classified into endo- and exochitinases. The

endochitinase activity is defined as the random

cleavage at internal points in the chitin chain. The

exochitinase activity is defined as the progressive

action starting at the non-reducing end of chitin with

the release of chitobiose or N-acetylglucosamine units

(figure 1). Chitobiosidase and N-acetyl-b-

glucosaminidase are considered as exochitinases

(Felse and Panda, 2000). The combination of endo-

and exochitinases results in a synergistic increase in

the chitinolytic activity (Bolar et al., 2001). In another

research the role of silver nanoparticles on growth rate

of two biocontrol agent species of Trichoderma viride

and T. harzianum was investigated by measuring the

diameter of colonies of fungi (Gavanji et al., 2012.).

So we, in this study, checked the possible interaction

between silver nitrate and Chitinase enzyme.

2. MATERIALS AND METHODS

2.1. Preparing 3 dimensional structures of silver

nitrate and Catalase and Nitrat reductase:

In the first step, amino acid sequences of Chitinase

enzyme with accession number of B6E5Q4 were

taken from NCBI website (www.ncbi.nlm.nih.gov/)

(Figure2). Then the Chitinase enzyme with the

number of 1D2K was obtained from Protein Data

Bank website (www.rcsb.com). In the next step,

Silver Nitrate with AgNo3 molecular formula

(number 22878) was provided from ChemSpider

website (www.chemispider.com) (figure3).

MLGFLGKSVALLAALQATLTSATPVSTNDVSVEKRASGYTNAVYFTNWGIYGRNFQPQDLVASDITHVIYSFMNFQADGTVVSGDAYAD

YQKHYSDDSWNDVGNNAYGCAKQLFKLKKANRNLKVMLSIGGWTWSTNFPSAASTDANRKNFAKTAITFMRDWGFDGIDVDWEYPAD

DTQATNMVLLLKEIRSQLDAYAAQYAPGYHFLLSIAAPAGPEHYSALHMADLGQVLDYVNLMAYDYAGSWSSYSGHDANLFANPSNPN

FSPYNTDQATKAYINGGVPASKIVLGMPIYGRSFESTNGIGQTYNGIGSGSWENGIWDYKVLPKAGATVQYDSVAQAYYSYDSSSKELISF

DTPDMVSKKVSYLKNLGLGGSMFWEASADKTGSDSLIGTSHRALGSLDSTQNLLSYPNSQYDNIRSGLN

Fig. 2: Amino acid sequence of Chitinase (Trichoderma harzianum-Hypocrea lixii)

Gavanji et al.

Computational Prediction and Analysis of Interaction of Silver Nitrate with Chitinase Enzyme

53

2.2. Molecular docking study

Molegro virtual docker (MVD) 2011.4.3.0 is used for

computer simulated docking study. Before initiation

the docking operation, protein and ligand structures

were prepared using MVD. For this purpose, charges

assigned to the model of protein and ligands structures

and flexible torsions in ligands were detected by this

software (Gavanji et al., 2013).

Fig. 3: A: Structure of silver nitrate. B and C: structure of Chitinase enzyme.

3. RESULTS AND DISCUSSION

3.1. Chitinase Structure Analysis

The Chitinase consists of 424 amino acids, and its

molecular weight is 46293 Da. The red parts in figure

4 show the hydrophobic regions and the blue parts

show the negative charge (figure 4).

3.2. Finding ligand binding sites:

3DLigand Site server (http://www.sbg.bio.ic.ac.uk)

was used for prediction of potentially binding sites of

model. In the server output TRP, PHE, TRP, THR,

ASP, TRP, GLU TRP were predicted as present in

binding site(table 1). The position and the percentage

of each amino acides is shown in table 4. Also as an

alternative approach, MVD is used for finding cavities

of model. For this purpose, probe size was 1.2, max

number of ray checks was 16, minimum number of

ray hits was 12 and Grid Resolution was 0.8. Five

cavities were found by MVD (figure 5).

Fig. 4: The hydrophobic regions and negative charge.

3.3. Finding ligand binding sites with Site Hound-

web server:

Site Hound-web identified ligand binding sites by

computing interactions between a chemical probe and

a protein structure. By using web server, 10 regions

with different levels of energy had been recognized

(figure 9). The regions are divided into A to J (table

2). The highest energy is related to ligand bound to

enzyme at A position which is equal to -11112.50 and

the lowest one at A position is predicted to be -465.75

(table 2 and 3).

International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(4), pp. 50-62, 2013

54

Table 1: ligand binding sites by computing interactions with 3D Ligand Site server.

Fig. 5: Cavities of Chitinase predicted model. MVD used for cavity detection. Detecting parameters: probe size 1.2, max

number of ray checks was 16, minimum number of ray hits 12 and Grid Resolution 0.8

Table 2: ligand binding sites by computing interactions with Site Hound-web server.

Bioinformatic checking of ligand bound to amino

acids showed that the best position for interaction

between ligand and amino acids is A position in

which the ligand (X:66,Y:21, Z39) is bounded to

amino acids existed in this position(Figure6 and 7).

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Computational Prediction and Analysis of Interaction of Silver Nitrate with Chitinase Enzyme

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Table 3: Amino acides involved in interaction with different groups.

Fig. 6: features of ligand bound to amino acides presented in A position.

International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(4), pp. 50-62, 2013

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Fig. 7: Features of ligand bound to amino acides presented in A position.

3.4. Molecular docking study with Molegro virtual

docker (MVD):

MolDock score (Thomsen and Christensen, 2006)

with a grid resolution of 0.30 Â was used as scoring

function for docking (Figure 8). Internal electrostatic

interaction and hydrogen bond between ligand and

protein were permitted. MolDock SE was used as the

docking algorithm and ten runs for ligands were

carried out. After docking, energy minimization and

Gavanji et al.

Computational Prediction and Analysis of Interaction of Silver Nitrate with Chitinase Enzyme

57

optimization of hydrogen bonds were performed. The

energy threshold was 100.00 and similar poses were

ignored. Docking results are evaluated based on

MolDock and reranking score. Rerank score is

estimated for interaction. For the defined docking

radius in Catalase-peroxidase and Nitrate reductase

enzyme, the best pose which is derived from

MolDock score for Chitinase enzyme was -49.1418

with Reranking score equal to -58.1719 (Table 4).

Table 4: Binding energy level of top five poses of silver nitrate to Chitinase.

Fig. 8: interaction of silver nitrate with Chitinase enzyme

The microscopic study of interaction between T.

harzianum and Rhizoctonia solani showed that as the

result of T. harzianum activity, the cell wall of fungi

will be affected. Studies showed that at the very

beginning of Mycoparasitic activity of fungi, the

Myceliums of Trichoderma hoop around the

Myceliums of the host and then penetrate into it and

make changes in its cell wall and finally disturb the

cytoplasm structure. With regard to purification and

study the extracellular enzymes through

Mycoparasitic activity of T. harzianum, it was

revealed that the major part of changes in cell wall of

the host are the result of direct action of Hydrolytic

enzymes of Trichoderma (Artigues and Vavet, 1984;

Elad et al., 1985) which are glucanase, protease and

Chitinase(Ridout et al., 1988) that by their synergistic

activitis cause disturbance in cell wall(Haran et al.,

1996; Elad et al., 1985; Lorito et al., 1994). Indeed,

the presence of Chitinase enzymes is very effective in

biocontrol activitis. In different species of

Trichoderma, there have been some exo and endo

chitinases by which the glycosidic bounds will be

broken leading to destruction of cell wall (Haran et

al., 1996).

International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(4), pp. 50-62, 2013

58

Fig. 9: Ligand bound to amino acides presented in A, B, C, D, E and F position.

Studies about chitinase enzymes in T. harzianum

showed that there have been some types of the

enzymes existed in the fungi. The experiments

revealed four enzymes in the fungi which are

CHIT52, CHIT42, CHIT33 and CHIT31 respectively

(Haran et al., 1995). The studies suggested that

CHIT52, with molecullar weight of 52 kDa, is very

sensitive to heat. The appropriate temperature and pH

for enzyme activity are 40 °C and 4 respectively.

Studies about the effect of metal ions on enzyme

activity reported that the metals show no considerable

role in chitinase enzymes activity (Harman et al.,

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Computational Prediction and Analysis of Interaction of Silver Nitrate with Chitinase Enzyme

59

1993, Ulhoa and Peberdy, 1992). Experiments on the

effect of different chitinases on the cell wall of host

fungi showed that each of these enzymes affects the

cell wall exclusively. In a study about the direct effect

of CHIT42, CHIT33 and CHIT31 on the cell wall of

Botrytis cinerae revealed that only CHIT42 enzyme

can have Hydrolytic affect and two other enzymes

(CHIT33 and CHIT31) increase the hydrolize effect

of CHIT42 and they cannot make any destruction to

the cell wall by themselves (De la Cruz et al., 1993;

De Marco et al., 2000). Although some kinds of

chitinase enzymes have been purified in different

species of Trichoderma, but study about their features

at the level of DNA is basically refered to 3

endochitinases CHIT31, CHIT33 and CHIT42(Limon

et al., 1995; Zeilinger et al., 1999).

Although the antimicrobial properties of silver

have been known for centuries, recently we have only

begun to understand the mechanisms by which silver

inhibits bacterial growth. It is proposed that silver

atoms bind to thiol groups (-SH) in enzymes and as a

result cause the deactivation of enzymes. Silver forms

stable S-Ag bonds with thiol-containing compounds in

the cell membrane that are involved in transmembrane

energy production and ion transportation (Klueh et al.,

2000). It is also believed that silver can join in

catalytic oxidation reactions that result in the

construction of disulfide bonds (R-S-S-R). Silver

performs this process via catalyzing the reaction

between oxygen molecules in the cell and hydrogen

atoms of thiol groups so that water is subsequently

released as a product and two thiol groups become

covalently bonded to one another through a disulfide

bond (Davies and Etris, 1997).

The silver-catalyzed formation of disulfide bonds

could possibly modify the shape of cellular enzymes

and consequently affect their function. However, these

effective biocidal properties have the potential to

adversely affect beneficial bacteria in the environment

especially those existed in the soil and water.

Exploration is increasing into the potential toxic

mechanisms and long-standing effects by which these

nanomaterials could cause environmental dangers

throughout broad production and use. Extensively

used nanoparticles, such as silver nanoparticles will

most likely enter the environment and may produce a

physiological response in certain organisms, possibly

altering their fitness and finally might affect their

populations or community densities. Although there

have been studies discovering the toxicity of metal

NPs and despite their widespread application,

currently there is inadequate toxicity data necessary to

fill the gap for the ‘‘source pathway receptor-impact’’

framework necessary for correct risk measurement of

Ag NPs (Laban et al., 2010). These results suggest the

possibility using of silver nanoparticles to eliminate

phytopathogens. Several parameters will require

estimation proceeding to practical application,

including phytotoxicity and antimicrobial effects in

situ and development of systems for delivering

particles into host tissues that have been colonized by

phytopathogens.

It is believed that nanometer-sized silver particles

have different physical and chemical properties from

their macro scale counterparts which alter their

interaction with biological structures. Research has

been focused on antibacterial material containing

various natural and. Among them, silver or silver ions

have long been known to have potential inhibitory and

bactericidal effects as well as a broad spectrum of

antimicrobial activities (Kandile et al., 2010). Because

of its antimicrobial properties, silver has also been

used in filters to purify drinking water and to clean

swimming pool water (Wijnhoven et al., 2009). The

nanosilver products make broad claims concerning the

power of their nanosilver components such as:

elimination of 99% of bacteria, rendering material

permanently and killing approximately 650 kinds of

harmful germs and it was 2 to 5 times faster than other

forms of silver (Gavanji et al., 2013). Some studies

have reported that the positive charge on the Ag ion is

necessarily essential for its antimicrobial activity

through the electrostatic attraction between negative

charged cell membrane of microorganism and positive

charged nanoparticles.

4. CONCLUSION

Based on obtained results using Bioinformatic

softwares, it is clear that silver nitrate can cause

harmful effect to Chitinase enzyme leading to

deactivation of the enzyme. Hence, it is suggested to

use silver nitrate with care in environment.

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62

Shahin Gavanji graduated in Biotechnology at MSc at the Department of Biotechnology, Faculty of

Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran. He has over 10 international

medals in invention. Shahin Gavanji's research has focused on Pharmacy and Pharmacology, Nano

Biotechnology, Bioinformatics, Biotechnology - Medical Biotechnology. He is editor in chief of

International Journal of Scientific Research in Inventions and New Ideas.

Dr Aziz is a Professor in environmental engineering at the School of Civil Engineering, Universiti Sains

Malaysia. Dr. Aziz received his Ph.D in civil engineering (environmental engineering) from University of

Strathclyde, Scotland in 1992. He has published over 200 refereed articles in professional

journals/proceedings and currently sits as the Editorial Board Member for 8 International journals. Dr

Aziz's research has focused on alleviating problems associated with water pollution issues from industrial

wastewater discharge and solid waste management via landfilling, especially on leachate pollution. He

also interests in biodegradation and bioremediation of oil spills.

Behrouz Larki is MSc student at Isfahan (Khorasgan) Branch, Islamic Azad University. Behrouz Larki 's

research has focused on Biology, Pharmacy, Linguistic and Translation. He is managing editor of

International Journal of Scientific Research in Inventions and New Ideas.

Amin Mojiri is a PhD candidate in Environmental Engineering, School of Civil Engineering, Universiti

Sains Malaysia (USM), Pulau Pinang. He is fellowship holder and research assistant at the School of Civil

Engineering (USM). He has more than 18 articles in the international journals. He is editor and reviewer of

some international journals. His area of specialization is waste management, waste recycling, wastewater

treatment, wastewater recycling, and soil pollutions.