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\def\RunningAuthor{Suguna Selvakumaran et al.}
\firstPage{4084}
\articleType{Original Article}
\receivedDate{04 Dec 2019}
\acceptedDate{10 Jan 2020}
\revisedDate{06 Jan 2020}
\journalVolume{2020, 11}
\journalIssue{3}
\journalDoi{ijrps.v11i3.2610}
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\title{\textbf{Production of Silver Nanoparticles by \textit{Escherichia coli}: Green Approach} \textbf{\space }}
\author{Sivanjale~Ravindrakumar,  
            June~Yeoh~Lee~Xin,  
            Hanan~Rahman~SK,  
            Sabrina~Harudsamy,  
            Shamala~Marimuthu, 
            Suguna~Selvakumaran\textsuperscript{*}~\\[5pt]{Department of Biotechnology, Manipal International University, Nilai, Negeri Sembilan, Malaysia}}

\begin{abstract}
Physical and chemical approaches to synthesize nanoparticles are not suitable for safer medical usage. Thus, green method could be a suitable approach to produce safe and non-toxic nanoparticles. In this study, silver nanoparticles were synthesized by \textit{Escherichia coli }and its biological activities were determined. The produced bacterial biomass (supernatant) was mixed with silver nitrate to produce silver nanoparticles. \textit{Escherichia coli }acting\textit{} as\textit{\space }reducing agents to produce nanoparticles. The colour changes from colourless to brown was observed to indicate the formation of silver nanoparticles. It was further reconfirmed using UV-Vis Spectroscopy, where the peak at 300 nm confirmed the formation of AgNPs. The antimicrobial and cytotoxicity activity of synthesized nanoparticles was tested. At 1 mg/ml concentration, the nanoparticles were effective against all the micro-organisms tested, \textit{Bacillus subtilis } (12.7\ensuremath{\pm}2.5 mm) \textit{Staphylococcus aureus } (13.7\ensuremath{\pm}2.3 mm), \textit{Pseudomonas aeruginosa} (14.3\ensuremath{\pm}2.3 mm) and \textit{Aspergillus niger } (12.7\ensuremath{\pm}2.1 mm)\textit{. }With decreasing concentration, the antimicrobial activity also decreased. Similar results were obtained when the nanoparticles were tested for cytotoxicity ability on HeLa cells. With increasing concentration, more of the tumor cells were inhibited. Based on the result obtained, it can be concluded that \textit{Escherichia coli} can be used in the production of silver nanoparticles. It was found that synthesized nanoparticles possess effective antimicrobial and cytotoxicity activities. Thus, these properties could make them an advancing field in the medical world.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}silver nanoparticles,\newline green synthesis,\newline antimicrobial,\newline cytotoxicity
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Suguna~Selvakumaran~\\ Phone:\ +60149114289~\\  Email:\ suguna.selvakumaran@miu.edu.my
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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        \makeatother
        
    
\section{Introduction}
Research and development in the field of nanoscience and nanotechnology has caught large attention in the past decades \unskip~\citep{827362:19797482}. Nano sized particles are usually 1-100nm in size and these nanoparticles have various properties \unskip~\citep{827362:19797466}, such as very high surface area and volume ratio \unskip~\citep{827362:19797462}. This property helps to increase their biological effectiveness in various applications, such as antimicrobial \unskip~\citep{827362:19797476}, anti-tumour, anti-viral, anti-fungal \unskip~\citep{827362:19797465} disinfectant \unskip~\citep{827362:19797471} (Poonam and Sanjiv Kumar, 2019), biosensor materials \unskip~\citep{827362:19797476} and cosmetic products \unskip~\citep{827362:19797480}. Among the various metals such as zinc, copper, gold and platinum, silver is a one of the most commonly used NPs \unskip~\citep{827362:19797468}.

Physical and chemical methods of synthesis involve high energy radiation, laser, expensive equipment \unskip~\citep{827362:19797467} and use of highly toxic and hazardous chemicals which can pose potential environment and biological risk \unskip~\citep{827362:19797468}. Biosynthesis is an alternative route for nanoparticle synthesis. This uses plant or plant extracts \unskip~\citep{827362:19797478}, micro-organisms such as bacteria \unskip~\citep{827362:19797466} and fungi \unskip~\citep{827362:19797479} and enzyme \unskip~\citep{827362:19797470}. These substances are abundant in nature and can be easily obtained. This method is suitable for large scale synthesis \unskip~\citep{827362:19797472}, faster synthesis rate \unskip~\citep{827362:19797473}, pure particles \unskip~\citep{827362:19797474}, sustainable to the environment and much less toxic. These particles can then be extracted and purified to be used.

In this project, \textit{Escherichia coli (E. coli)}, was used as a source to bio synthesis silver nanoparticles (AgNPs). \textit{E. coli } is a gram-negative bacillus bacterium that is mostly used for laboratory purposes. The bacterium is expected to reduce silver ions to metal silver to produce silver NPs. The synthesized NPs was checked and analyzed for their antimicrobial activity against various strains and their cytotoxicity effect on cancer cells such as HeLa cells, to evaluate their effectiveness for safe therapy purposes.
    
\section{Materials and Methods}
\textbf{Production of Biomass }

Master plate for \textit{E. coli } was obtained from MIU laboratory, Department of Biotechnology. A single colony was taken from the master plate and streaked using 4-quadrat streaking on a nutrient agar plate and incubated at 37\ensuremath{^\circ}C for 24 hours. For the biomass production a single colony of \textit{E. coli } from the sub-cultured plate was inoculated in LB broth and incubated at 37\ensuremath{^\circ}C for 24 hours. After 24 hours, the broth was centrifuged at 9000 rpm for 10 min and pellet and the supernatant were collected separately. The supernatant was used for the synthesis of AgNPs \unskip~\citep{827362:19797474}.

\textbf{Synthesis of Silver Nanoparticles (AgNO\ensuremath{_{3}}) }

Collected supernatant was added separately to the reaction flask containing 1 mM silver nitrate (AgNO\ensuremath{_{3}}).  The flask was completely covered with aluminum foil to prevent light penetration and left in the incubator at 37\ensuremath{^\circ}C for 48 hours. AgNO\ensuremath{_{3}} was used as control. The change in colour from yellow to brown showed the formation of silver nanoparticles. in the range of 200- 700 nm. After the OD was measured, the solution was centrifuged at 9000 rpm for 15 min. The supernatant was discarded and the pellet containing the silver nanoparticles was allowed to dry at 70\ensuremath{^\circ}C until no moisture was left. The NPs were scrapped off and collected in powder form \unskip~\citep{827362:19797474}. The formation of silver nanoparticles was further confirmed using UV-Vis Spectroscopy. 1 ml of reaction mixture was taken in a quartz cuvette and the corresponding spectra were measured in the range of 200-700 nm. 

\textbf{Antimicrobial Activity}

Synthesized silver nanoparticles by \textit{E. coli was } tested for its antimicrobial properties against \textit{Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa} \textbf{\space }and \textit{Aspergillus niger} \textit{via} disc diffusion method\textbf{. }The pure cultures of organism were sub cultured on nutrient broth and incubated at 37\ensuremath{^\circ}C for 24 hours. After 24 hours the growth of microorganisms was monitored every 4 intervals using UV spectrometer until the optical density reached 0.8- 0.1 at 595nm. This represents 1x10\ensuremath{^{8}} CFU/ml.  After inoculation and cultivation of different target bacteria on top of nutrient agar, discs were placed in selected area on different plates. For the antifungal assay, \textit{Aspergillus niger } from the master plate was swabbed using a sterile cotton swap into a PDA plates and discs  were placed in selected area on different plates. 10 \ensuremath{\mu }l of AgNPs with different concentrations (1mg/ml, 0.1mg/ml, 0.01mg/ml) were loaded on the top of discs. Ampicillin and distills water were used as positive and negative control respectively. All the plates were sealed with parafilm and incubated at 37\ensuremath{^\circ}C for 48 hours. The diameter of the clear zone (mm) was measured and recorded to evaluate antimicrobial activity \unskip~\citep{827362:19797461}.

\textbf{Cytotoxicity Test }

The cytotoxicity test was carried using MTT assay. In a 96 titer well plate of 200\ensuremath{\mu}l capacity, pre-cultured cell suspension was added to each well along with different concentration (1 mg/ml, 0.1 mg/ml, 0.01 mg/ml) of AgNPs. The cells with AgNPs were then incubated at 37\text{\textordmasculine}C in 5\% CO\ensuremath{_{2}} incubator for 24 hours. 1 Mm of silver nitrate was used as positive meanwhile Dimethyl Sulfoxide (DMSO) was used as negative control \unskip~\citep{827362:19797464}. After 24 hours of incubation, 10\ensuremath{\mu}l of MTT solution was added to each well and again incubated at 37\text{\textordmasculine}C in 5\% CO\ensuremath{_{2}} incubator for 4 hours. The plates were then taken out followed by removal of spent medium and replacement with DMSO. The wells were then read at 620 nm (cell line/sample) absorbance using ELISA reader. This reading is the optical density of the wells containing cells that determines the presence of living cells \unskip~\citep{827362:19797481}.
    
\section{Results and Discussion}
\textbf{Visual observation and UV- visible spectroscopy of Synthesized AgNPs}

Green synthesis is an alternative and sustainable route for synthesis of NPs since the biological components such as microorganisms and plant extracts are abundant and easy to obtain. This allows to produce large number of pure particles that can be used safely \unskip~\citep{827362:19797468,827362:19797477}. 


\begin{table*}[!htbp]
\caption{\boldmath {Antimicrobialactivity of silver nanoparticle synthesized from \textit{Escherichia coli }} }
\label{tw-ca1cb4495388}
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\begin{tabulary}{\linewidth}{p{\dimexpr.2253\linewidth-2\tabcolsep}p{\dimexpr.156\linewidth-2\tabcolsep}p{\dimexpr.1347\linewidth-2\tabcolsep}p{\dimexpr.144\linewidth-2\tabcolsep}p{\dimexpr.1653\linewidth-2\tabcolsep}p{\dimexpr.1747\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{6}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Mean Diameter of the clear zone/mm}\\
\rowcolor{kwdboxcolor}Organism & \multicolumn{5}{p{\dimexpr(.7747\linewidth-2\tabcolsep)}}{\cAlignHack Concentration of AgNPs (mg/ml)}\\
\rowcolor{kwdboxcolor} & \cAlignHack 1 mg/ml & \cAlignHack 0.1 mg/ml & \cAlignHack 0.01 mg/ml & \cAlignHack Positive control & \cAlignHack Negative control\\
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\textit{Bacillus subtilis } &
  \cAlignHack 12.7 \ensuremath{\pm}2.5 &
  \cAlignHack 0 \ensuremath{\pm}0 &
  \cAlignHack 0\ensuremath{\pm} 0 &
  \cAlignHack 28\ensuremath{\pm}10.6 &
  \cAlignHack 0\ensuremath{\pm}0\\
Staphylococcus aureus  &
  \cAlignHack 13.7\ensuremath{\pm} 2.3 &
  \cAlignHack 0\ensuremath{\pm} 0 &
  \cAlignHack 0\ensuremath{\pm} 0 &
  \cAlignHack 42.3\ensuremath{\pm}4.6 &
  \cAlignHack 0\ensuremath{\pm}0\\
Pseudomonas aeruginosa  &
  \cAlignHack 14.3 \ensuremath{\pm}2.3 &
  \cAlignHack 6\ensuremath{\pm} 5.3 &
  \cAlignHack 0\ensuremath{\pm} 0 &
  \cAlignHack 27.0 \ensuremath{\pm}20.0 &
  \cAlignHack 0\ensuremath{\pm}0\\
Aspergillus niger  &
  \cAlignHack 12.7\ensuremath{\pm} 2.1 &
  \cAlignHack 6 \ensuremath{\pm}1 &
  \cAlignHack 0 \ensuremath{\pm}0 &
  \cAlignHack 0\ensuremath{\pm}0 &
  \cAlignHack 0\ensuremath{\pm}0\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

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\caption{\boldmath {Synthesis of AgNPs by \textit{Escherichia coli} (A) before and (B) after 48 hours}}
\label{f-dc357987c0b4}
\end{figure}
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\caption{\boldmath {Absorption spectra of AgNPs obtained by reactionof \textit{E. coli }and AgNO\ensuremath{_{3}}.}}
\label{f-be75eae993ba}
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\caption{\boldmath {Cytotoxicity effect ofAgNPs on HeLa cells}}
\label{f-e44315367cbd}
\end{figure}
\egroup
In this experiment, the yellow bacterial supernatant solution with AgNO\ensuremath{_{3}} turned slightly brown after 48 hours with brown deposits at the bottom of the flask (Figure~\ref{f-dc357987c0b4} ) due to the reduction of silver metal ions Ag\ensuremath{^{+}} into silver nanoparticles Ag\ensuremath{^{0}}. The colour change in this experiment was explained clearly due to the excitation of electrons. Accordingly, similar results were obtained by \unskip~\cite{827362:19797474} and \unskip~\cite{827362:19797476} where the colour change indicates the formation of AgNPs. The exact mechanism of the reduction process is still never understood yet where \unskip~\cite{827362:19797473} proposed that the exo-chemicals released from the bacteria may be present in the supernatant that acts as reducing agents to reduce the ions. The formation of AgNPs was further confirmed using  UV-Vis Spectroscopy in the range of 200 to 700 nm.

Figure~\ref{f-be75eae993ba} shows the absorption spectra of AgNPs obtained by reaction of \textit{E. coli} and AgNO\ensuremath{_{3}}. The result showed maximum absorption was observed at 300 nm. Considering similar protocols by \unskip~\cite{827362:19797474} and \unskip~\cite{827362:19797476}, the peak was expected to be 380nm - 400 nm. 

\textbf{Antimicrobial Activity}

 The antimicrobial properties of synthesized AgNPs was tested against 3 bacteria and 1 fungus using different concentration (Table~\ref{tw-ca1cb4495388}). The antimicrobial activity of AgNPs clearly increasing with increasing concentrations. AgNPs with 1 mg/ml, showed high antimicrobial activity against all tested strains, where zone of inhibition of \textit{B. subtilis, S. aureus}, \textit{P. aeruginosa }and\textit{ A. niger} was 12.7 \ensuremath{\pm}2.5, 13.7\ensuremath{\pm} 2.3, 14.3 \ensuremath{\pm}2.3 and 12.7\ensuremath{\pm} 2.1 respectively\textbf{. }At 0.1 mg/ml only \textit{P. aeruginosa } and \textit{A. niger } showed positive response. The diameter clearly decreased, and at 0.01 mg/ml concentration, the AgNPs were not effective at all against any microorganisms.

 It was found that gram negative bacteria displayed better zone of inhibition compare to gram positive. Being a gram-negative bacterium\textit{, P. aeruginosa } has a higher tolerance of external pressure due to their outer membrane. Yet this was explained by \unskip~\cite{827362:19797475} the outer membrane layer in gram negative bacteria makes it easy for silver to bind, since they have a higher affinity than the peptidoglycan monomers. Therefore, gives an easy opportunity to destroy the bacteria. This makes the gram-negative bacteria susceptible to be destroyed even at lower concentration than gram positive bacteria \unskip~\citep{827362:19797469}. The fungi have a cell wall made of chitin molecules. Chitin has similar affinity to that of peptidoglycan to bind to the cell wall and enter the fungi. The antimicrobial mechanism of AgNPs has various theories. One mechanism is that silver is toxic and has high affinity to bind to any component in the microbial cell wall. Once bound to the cell wall, silver can enter the cell and cause disruption of DNA during synthesis phase in the cell cycle \unskip~\citep{827362:19797466}.

\textbf{Cytotoxicity Activity}

Cytotoxicity activity of synthesized AgNPs on Hela cells are displayed in Figure~\ref{f-e44315367cbd}. The result showed that the silver nanoparticles were able to inhibit the growth of Hela cells. In a dose dependent manner where, as the concentration increased the inhibition ability of AgNPs also increasing. Hence, AgNPs synthesized by \textit{E. coli} can kill cancer cell. According to \unskip~\cite{827362:19797463}, AgNPs are particles that are very small and can pass through the blood vessels to reach the cancer site. Once AgNPs reach the cancer cells it induces alterations in the cell morphology. Besides that's, AgNPs decrease the metabolic activity and increase oxidative stress leading to DNA damage that eventually leads to apoptosis \unskip~\citep{827362:19797460}. 
    
\section{Conclusion}
Herein, we concluded that on the successful synthesis of silver nanoparticles by using \textit{Escherichia coli } which is free from harmful reducing agents. The result of visual observation and UV- visible spectroscopy has confirmed the formation of AgNPs formation. Synthesized AgNPs showed excellent antimicrobial activity against gram positive, gram negative and fungi as well as excellent cytotoxic effects against Hela cell line. Thus, silver nanoparticles synthesized by using \textit{Escherichia coli } could be a suitable antimicrobial and anticancer drug. 
\section*{Acknowledgement}We would like to thank the Department of Biotechnology, Manipal International University for support of this study

\textbf{Funding Source}

This Manuscript was supported by Manipal International University, Nilai.

\textbf{Conflicts of Interest }

The authors declare that they have no conflicts of interest


    

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