Biodegradation of malathion pesticide by silver bio-nanoparticles of Bacillus licheniformis extracts

Abstract

This study investigated the silver nanoparticles synthesis by biological method using Bacillus licheniformis extracts was successfully applied for the determination of quantitative biodegradation of malathion pesticide, which found in diverse water samples. Structural and morphological analysis of silver bio nanoparticles studied following Zeta potential, AFM, FTIR and HPLC analysis. The silver bio nanoparticles were stable in zeta potential which recorded of -32.22mV. The size and diameter of the nanoparticles were evaluated in average 144nm as displayed via Atomic Force Microscope. The Transform Infrared Spectroscopy (FTIR) was conducted to determine the various functional groups in the silver bio nanoparticles. The biodegradation of malathion by using silver bio nanoparticles was determined quantitatively using HPLC techniques, which began during seven days. We consider that this strategy has technological potential in the biodegradation of malathion pesticide contaminated water through green biosynthesis.

Full text

Biodegradution of malathion pesticide by using Silver bionanoparticles of
Bacillus licheniformis extracts
Marwah Th. Alnuaimi*, Noor T. Hamdan**, Esam Abdalraheem*, Zahraa
Zahraw Aljanabi***
*Ministry of Science& Technology/ Directorate of Environment and Water,
Baghdad-Iraq.
**Biology Department, College of Science, Mustansiriyah University,
Baghdad-Iraq
***Environmental Research Centre, University of Technology, Baghdad, Iraq. Drmarwa520@gmail.com, noor.t.hamdan@uomustansiriyah.edu.iq,
11546@uotechnology.edu.iq
Abstract
This study investigated the silver nanoparticles synthesis by biological
method using Bacillus licheniformis extracts was successfully applied for the
determination and quantitative dismissal of Malathion pesticide, which found in diverse
water samples. Structural and morphological analysis of silver bio nanoparticles studied
following Zeta potential, AFM, FTIR and HPLC analysis. The silver bio nanoparticles
were stable in zeta potential which recorded of -32.22mV. The size and diameter of the
nanoparticles were evaluated in average 144 nm as displayed via Atomic Force
Microscope. The Transform Infrared Spectroscopy (FTIR) was conducted to determine
the various functional groups in the silver bio nanoparticles. The biodegradation of
Malathion by using silver bio nanoparticles was determined quantitatively using HPLC
techniques, which began during seven days. We consider that this strategy has
technological potential in the biodegradation of Malathion pesticide contaminated
water through Green biosynthesis.
Keywords: Silver nanoparticles, Green biosynthesis, Bacillus licheniformis,
Malathion, AFM, FTIR, HPLC and Zeta potential.
Introduction:
Currently among of the organophosphorus pesticides calculated more than 36 %
of the total world market (1). Malathion refers to reduce of household and farming
insects. Numerous reports have revealed to the Malathion contaminates soil, water and
aquatic ecosystems (1). In addition to produce different kind of disorders such as
anemia, child leukemia, kidney disaster and human birth defects, as a result it can
remain in human body for a long time (2).Therefore, The degradation process of
pesticides through malathion takes a large space of interest in ecosystem in the world
wide (3).
The biosynthesis of nanoparticles has great attention in the last ten years ago due
to their intrinsic benefits such as ultrafine size, high surface area to volume ratio,
optical, electrical, magnetic as well as catalytic and thermal features (4). Silver
nanoparticles (SNPs) is termed as a silver mineral ranging between 10-100 nm. They
entranced the attention of researchers in different fields everywhere the world due to
the unique chemical and physical features and they are generally introducing in many

application such as in medicine, horticulture, habitat remediation, food technology as
well as management of water (5).
An array of physical and chemical methods are used for the synthesis of
nanoparticles, In spite of that often are highly-priced and involve the use of risky
chemicals, which ultimately limits potential uses of the nanoparticles. Therefore, there
is growing interest to develop clean, nontoxic, environmentally and economically
viable methods for synthesis of nanoparticles produced by mechanisms of biological
origin. Consequently, biological procedure of nanoparticles has been explored. That
meaning it,s employing microorganisms provide advantages over more traditional
chemical and physical methods because the synthesis techniques use nontoxic and
environment-friendly agents, reactions appear at room temperature or lower, and the
approach demands little or no input of energy (6).
However, not many studies have been carried out on silver bio nanparticles
in the degradation of the malathion pesticide in water. Hence, a detailed study has been
carried out to estimate the degradation potential of the malathion via silver bio
nanoparticle and the critical evaluation of the results are presented in this research.
Material and Methods
Microorganisms:
The local bacterial isolate Bacillus licheniformis was obtained from the department of
plant protection , Faculty of agriculture / Baghdad university which identified by the
same department.
Chemicals:
Malathion: Technical grade malathion was obtained from Ministry Of Agriculture
AgNO3: AgNO3 was obtained from" Sigma Aldrich" company (St. Louis, USA) and
media get from "Oxoid" (UK).
Nutrient broth; Nutrient agar were obtained from Hi media, India.
Synthesis of silver bio nanoparticle (Ag NO3)
Preparation AgNO3 solution
The preparation of AgNO3 solution(1mM) consistued of: 16.987g of AgNO3 was added
to 100ml of distilled water and one mole of this solution poured then in to 1000ml of
distilled water to make 1mM solution.The AgNO3 solution stored in dark room due to
have the photosensitive. Therefore, their solution were prepared in an amber bottle for
further analysis. These B.licheniformis isolate were thus applied to further enormous
production by using optimized fermentation media and then grown in their media for
72 h at 36°C. The Extraction and washing of biomass from B.licheniformis culture.
Firatly, their culture was centrifuged at 150rpm for 15min to obtain the biomass which
was used as a starting material for the production of nanoparticles. After that, the
biomass washed twice with NaCl solution(0.5%) and then washed with TE buffer and
distilled water (once each). Finally, it then completed in 3 ml of injection water(7). The
washed biomass(1ml) was added in to 99ml of 1mM silver nitrate solution in the flask
and was neutralized the pH 8.5 by NaOH. After that, the flask then put in a horizontal
shaking water bath at 36°C,150rpm for 72 h in dark room. The occurrence of yellowish
brown colour in flask was be a sign of production of AgNO3. After that. The mixture
solution was transported in to dish for drying. Finally, these dried particles composed

was used in further characterization (8).
Silver bio nanoparticles (AgNO3) Characterization:
The supernatant of bacterial isolate containing synthesized silver bio nanoparticles
was applied in the characterization as mentioned below:
1. Zeta potential
The synthesized nanoparticles stability was evalutated in terms of zeta potential via
the zeta potential analyzer ranging from -160 mV to +160 mV, and the data was plotted
as graph (9,10).
2. Atomic Forced Microscopy (AFM)
The sample of thin film of nanoparticles was placed on a glass slide by adding 100
μl of the sample on the slide, and then waited to dry for 5 min. The slides were then
checked using the AFM (11).
3. Fourier transform infrared spectrometer ( FT-IR)
The FT-IR analysis was performed with silver nanoparticles and malation solution
were evaluted at the range of 500-4000 cm=1 region and at 8 cm-1 of a resolution using
Fourier transform infrared spectrometer (Shimadzu). The sample containing
synthesized silver nanoparticles (1 mg) was incorporated with KBr (300 mg) to make
a hydraulic pellet press and then analyzed in a FTIR spectroscopy. The occurance of
the peaks noted to the functional groups in particles synthesized (12).
4. HPLC analysis
Twenty gram of sample containing synthesized particles was added to the mixture
consisting of 20µL D.W and 50ml acetone in the flask, after that transported to rotary
shaker at 150rpm for 2h .Then, mixture filter rewashing by acetone three times and
filtrate was collected to the evaporate acetone solution in the flask. The residual
material was carried to the separating funnel composing of equal volume of the
supernatant dichloromethane. The organic layer of the dichloromethane was collected
and allowed to dry via rotary evaporator at 30 °C. Dried residue material was added to
1 ml acetonitrite and was filtrated in 0.45 filter. Then, subjected to HPLC analysis.
Results and Discussion
1. Zeta potential
The results of zeta potential values of the synthesised nanoparticles –was 32.22mV
for AgNPs (fig. 1).
Zeta,potential is a key pointer,of the stability and steadiness, of colloidal nanomaterial. The
size,of the zeta potential indicates the,degree of electrostatic,repulsion between,similarly
charged particles . For molecules and particles that are small,enough,a high zeta potential,will
give stability and steadiness, i.e., the solution,will resist aggregation. When,the potential,is
small, attractive,forces may,exceed Therefore, colloids materials with elevated zeta potential
(positive or negative) are electrically stabilized while colloids materials with low zeta potential
Trend to flocculate or coagulate (13,14). Generally, the zeta potential of the nanoparticles
should be either highest than +30 mV or lower than -30 mV (15,16). So from this

results, showed stability while the rest nanoparticles were very near from normal
stability range.
This finding is agree with (12), he found the zeta potential of green synthesized
AgNPs -31.10 ± 0.42 mV with Bacillus Subtilis. The Zeta potential distributed with
range of -18.9mV indicated the stable in nature of AgNPs synthesized using Bacillus
thuringiensisextract (17).
Figure (1): The zeta potential value of AgNPs.
2. AFM
The results of AFM analysis showed both the two dimensional and three dimensional
view of silver bio nanoparticles they were spherical in shape, single or in aggregates,
AFM analysis were also showed that the average size of particles 133nm .On the
another hand, the different diameters of AgNPs appeared starting from 1 to 300nm and
the high average size range of the AgNPs diameter was 62 nm (fig. 2).
Singh was reported (18) that the silver particle identifying by atomic force
microscope was irregular polygon in shape and calculated of average size range of the
silver particls diameter was to be 69.9 nm.
a. 2-D profile of AgNPs Agglomeration (5x5um).

b. 3-D profile of AgNPs Agglomeration .
c. AgNPs average size range.
Figure (2): (a) AFM image showed two dimintional of AgNPs. (b) AFM image showed three dimintional of AgNPs.
(c) Showed coloumn AFM digram of size range of
AgNPs.
3.FTIR
FTIR measurements were evaluted to identify the potential the functional groups in
the sample.The FTIR spectra of biomolcules of nanoparticles demonstrated eleven
distinct peaks, reporting 547.78, 609.51, 1087.85, 1238.30, 1392.61,1446.61, 1546.91,
1651.07, 2931.80, 3309.85, 3414.00 cm-1 as indicated in figure(3).The peak at 547.78
cm-1 mentions to C-Br stretch while the peak at 609.51 cm-1 correspond to C-H bend
stretch vibration of acetylenic.The vibrations observed at 1087.85 cm-1 may be
indicative of C-O-C stretch. The peak at 1238.30 cm-1,which is characteristic of C-O
stretch. The 1392.61 cm-1 refers to C-H stretch. The crest of the curve at 1446.61 cm-
1 and 1546.91 cm-1 were revealed to C=C stretch. The tip of the curve at 1651.07 cm-1
was assigned to the –C=C vibration of alkenyl. The peaks at 2931.80 and 3309.85
cm-1 were predicted to O  H stretch vibration of carboxylic acid. Finally, the peak at

3414.00 cm-1 observed to N  H stretch vibration of amine.
These peaks mentioned above were diminished in the treated sample of the malation
but some peaks were appeared to 1384.89, 1512.19, 1639.49, 3444.87 and 3502.73 cm-
1 which were assigned to C-H, C=C, C=O, O-H and O-H stretch respectively as
observed in fig 4.
The biochemical interaction between Ag+ and protein molecules was presented
using FTIR to measure to the of silver ions to atom reduction . Thease interactions were
studied by (12).
Figure (3): The FTIR spectra of Bacilluslicheniformis extracts.
Figure (4): The FTIR spectra of Silver bionanoparticles of Bacillus licheniformis
extracts.
4.HPLC
During degradation of malathion by Silver bionanoparticles of
Bacilluslicheniformis extract The remaining malathion was accounted using HPLC for
7 days in liquid culture.
The results revealed that the AgNPs of Bacilluslicheniformis extract is extremely
efficient in degrading malathion which reported of 90% for 7days of the treatment
whrease in control diminished in the degradation of malathion.
Results observed that the consumption of malathion was significantly rising in
treatment sample 1 as compared to 2 and 3, as seen in figures (5, 6,7,8). The rate of
degradation of malathion was increased with time in all treatments rather than standard

especially in sample 1 was significantly higher after 7days of incubation.
AgNPs interaction with malathion in solution leads annihilation of malathion without
formation above harmful products.
The results of (19) indicated the potential of Bacillus licheniformis enzymes used
as biodegrader in the bioremediation of malathion-contaminated soil.
Figure 5 : HPLC chart of standard malathion
Figure 6 : HPLC chart of concentration of sample 1 after 7 days incubation.

Figure 7: HPLC chart of concentration of sample 2 after 7 days incubation.
Figure 8 : HPLC chart of concentration of sample 3 after 7 days incubation.
Conclusion
Based on the results and discussion , it has been reported that Silver bionanoparticles
of Bacilluslicheniformis extracts can be used for the removal of the malathion
contaminated water in the areas where the malathion pesticide contamination is
prevalent.
Reference
1) Yang GJ, Huang JL, Meng WJ, Shen M, Jiao XA (2009). A reusable capacitive
immunosensor for detection of Salmonella spp. based on grafted ethylene
diamine and self-assembled gold nanoparticle monolayers. Analytica chim acta
647: 159-166.
2) Zeljezic, D., & Garaj-Vrhovac, V. (2002). Sister chromatid exchange and
proliferative rate index in the longitudinal risk assessment of occupational
exposure to pesticides. Chemosphere, 46(2), 295-303.
3) Strong P. J. and Burgess J. E., (2008), Treatment methods for wine-related and
distillery wastewaters: a review, Bioremediation journal, 12, pp 70-87.

4) Chowdhury S, Yusof F, Faruck MO, Sulaiman N (2016). Process optimization
of silver nanoparticles synthesis using response surface methodology. Procedia
Eng. 148:992-999.
5) Singh R, Utkarsha US, Sweety AW, Balu AC (2015). Bacteriagenic silver
nanoparticles: Synthesis Mechanism and Applications. Appl. Microbiol.
Biotechnol. 99: 4579- 4593.
6) [6]Fariq, A.; Khan, T. and Yasmin, A. (2017).Microbial synthesis of
nanoparticles and theirpotential applications in biomedicine. Journal ofApplied
Biomedicine. 15: 241-248.
7) Naz, S.; Nawaz, H.; Arshad, U.; Ansari, F.; Shahzadi, R.; Anjum, F. and Bashir.
(2017). Biogenic Synthesis of Silver Nanoparticles and Valuation of their
Antimicrobial Activity against Dengue Larvae. Journal of Plant Pathology and
Microbiology. 8:8.
8) Dash, L. (2013). Biological Synthesis and Characterization of Silver
Nanoparticles using Bacillus thuringiensis. M. thesis, The Institute of Life
Science, National Institute of Technology Rourkela-769008, Orissa, India.
9) Surega, R. (2015). Green synthesis of bioactive silver nanoparticles using plant
extracts and their antinemic properties. PhD. Thesis,College of Agricalture,
Tamil Nadu Agricultural University, Coimbatore.
10) Aljabali , A. A. A.; Akkam, Y. I; Al-Zoubi , M. S.; Al-Batayneh, K. M.; Al-
Trad, B.; Abo Alrob, O.; Alkilany, A. M.; Benamara, M. and Evans, D. J.
(2018). Synthesis of Gold Nanoparticles Using Leaf Extract of Ziziphus
zizyphus and their Antimicrobial Activity. Nanomaterials J., 8(3): 174-188.
11) Hemath Naveen, K.S.; Kumar, G.; Karthik, L. and Bhaskara Rao, K.V. (2010).
Extracellular biosynthesis of silver nanoparticles using the filamentous fungus
Penicillium sp. Arch. Appl. Sci. Res, 2(6): 161-167.
12) Maduabuchi ,E. K. ; Noundou2, X. S.; Ejike U. S. and Atuzie W. (2018).
Biosynthesis, Characterization and Antimicrobial Activity of Silver
Nanoparticles Using Cell Free Lysate of Bacillus Subtilis: A Biotechnology
Approach. American Journal of Nanoscience & Nanotechnology Research,
6:18-27.
13) Sujitha, M.V. and Kannan, S. (2013). Green synthesis of gold nanoparticles
using Citrus fruits (Citrus limon, Citrus reticulata and Citrus sinensis) aqueous
extract and its characterization. Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy, 102: 15-23.
14) [14] González-Ballesteros, N.; Prado-López, S.; Rodríguez-González, J.B.;
Lastra, M. and Rodríguez-Argüelles, M.C. (2017). Green synthesis of gold
nanoparticles using brown algae Cystoseira baccata: Its activity in colon cancer
cells. Colloids and Surfaces B: Biointerfaces, 153: 190-198.
15) Shaban, S.M.; Aiad, I.; El-Sukkary, M.M.; Soliman, E.A.; El-Awady, M.Y.
(2014). One step green synthesis of hexagonal silver nanoparticles and their
biologicalactivity. Journal of Industrial and Engineering Chemistry, 20: 4473-
4481.
16) Samy, M.S. (2016). Studying the effect of newly synthesized cationic
surfactanton silver nanoparticles formation and their biological activity. J Mole
Liq, 216: 137-145.
17) Kumar, M.A. ( 2014). Fabrication and extraction of silver nanoparticle using
Bacillus thuringiensis. MS thesis. National Institute Of Technology Rourkela-
769008, Odisha, India.
18) Singh, N.; Saha, P.; Rajkumar, K. and Abraham J. (2014). Biosynthesis of silver

and selenium nanoparticles by Bacillus sp. JAPSK2 and evaluation of
antimicrobial activity. Der Pharmacia Lettre, 6 (1):175-181.
19) Khan, S.; Habiba, Z.; Usman, I.; Raza, A.; Abdul Rehman, K.;Mohammad, M.;
Muhammad, B.; Mazhar, I. and Tatheer N. (2016). Biodegradation of Malathion
by Bacillus licheniformis strain ML-1. Arch. Biol. Sci., Belgrade, 68(1), 51-59.