Jamonline / 2(4); 2012 / 301-311 Research Article

Madhuri Sharon et al

Journal of Atoms and Molecules

An International Online Journal
ISSN 2277 1247

EXTRACELLULAR BIO-SYNTHESIS OF GOLD NANOPARTICLES USING

Escherichia coli AND DECIPHERING THE ROLE OF LACTATE-DEHYDROGENASE

USING LDH KNOCK OUT E. coli

Goldie Oza, Sunil Pandey, Madhuri Sharon* N. S. N. Research Centre for Nanotechnology and Bionanotechnology, Jambhul Phata, Ambernath (W) 421 505, Maharashtra, India Received on: 15-08-2012 Abstract: This paper is devoted to apprehend the exploitation of biological machinery and optimum biosynthesis parameters using Escherichia coli exudates for gold nanoparticle (GNP) synthesis and its modus operandi. The most influential parameters for the synthesis of GNPs were pH 7.35, 300C and 100 ppm aurochlorate salt. The results were verified using UV-Vis spectroscopy, XRD and High Resolution Transmission Electron Microscopy. HRTEM micrographs as well as the SPR peaks of UV-Vis spectra showed that the size of the GNPs ranged from 25-30 nm. The HRTEM demonstrated that at inherent pH of reactant spherical GNPs were formed whereas when pH was adjusted to 4, formation of anisotropic nanostructures occurred. The mechanism of GNP synthesis was decoded using Lactate dehydrogenase (LDH) knock out E.coli. It was found that LDH knock out bacteria was incapable of synthesizing GNP. The role of LDH was further demonstrated when its activity got reduced from 73.5umol mg -1 min-1 to 24umol mg-1 min-1 in normal E.coli.
Keywords: Biosynthesis, E.coli, E.coli LDH knock out, Gold nanoparticles, XRD, Lactate

Revised on: 25-08-2012

Accepted on: 29-08-2012

dehydrogenase. * Corresponding author Madhuri Sharon, Email: sharonmadhuri@gmail.com Introduction: Biosystems are considered to be paradigm for the fabrication of gold

nanoparticles (GNP) which is an area of burgeoning interest to material scientists. The All rights reserved 2011 www.jamonline.in 301

Jamonline / 2(4); 2012 / 301-311 most captivating criteria of GNPs are its chemical uniform composition, dispersity. size, Such shape and

Madhuri Sharon et al thiosulfate complex. The precipitation of gold was due to the formation and release of Hydrogen sulfide as an end product of metabolism and occurred via three possible mechanisms i.e. 1) adsorption and reduction of gold on iron sulfide surface 2) Bacterial electron transport also reduces gold and 3) after the death of the bacteria its metabolic enzymes can also reduce gold ions. They were successful in the formation of spherical aggregates containing octahedral gold. In another communication, Du et.al used E. coli DH5 for the synthesis of GNPs, which can be applied on direct electrochemistry of hemoglobin [4]. Konishi et.al used mesophilic anaerobic bacterium Shewanella algae with H2 as an electron donor for intracellular precipitation of gold at 250C and pH 7. The reductive precipitation was a fast process, producing insoluble nanoparticles of 10-20 nm size within 30 min [5]. GNP bio-synthesis have also performed with M. charantia [6], A. racemosus [7] and their mechanisms were studied exhibiting nitrate reductase as the key enzyme in plants involved in GNP synthesis. A detailed account of living system used for synthesis of plethora of metal nanoparticles can be understood by referring authors exhaustive review [8]. In the present work, GNPs are synthesized using Escherichia coli, a gram-negative bacterium. Different parameters such as pH, temperature and concentration were studied in detail. The modus operandi of synthesis was www.jamonline.in 302

nanoparticles

possess optical, electronic, optoelectronic, mechanical, electrical properties which have received researchers. increased The attention amongst and

various

physical

chemical methods have led to the generation of toxic molecules and hence there is a need of green methodologies for the development of nanoparticles. Biosynthesis of

nanoparticles is the emerging example of intersection biotechnology environmentally material of nanotechnology thus benign For and

developing technologies instance, in

research.

many

microorganisms, plants and algae have been exploited for nanoparticle synthesis. Facile biosynthesis of gold nanoparticles using extracellular enzymes secreted by

Pseudomonas aeruginosa ATCC 90271 was done by Husseiny et al [1]. They have hypothesized the molecular mechanism of nanoparticle dependent synthesis reductase. In using another NADHwork, also

Rhodopseudomonas

capsulata

synthesized gold nanoparticles extracellularly at pH values ranging from 7 to 4. Formation of spherical GNPs in the range of 10-20 nm were observed at pH value of 7 whereas a number of nanoplates were observed at pH 4 [2]. Lengke et al [3] could find a sulfate

reducing bacteria which can synthesize intracellularly elemental Gold using Gold

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Jamonline / 2(4); 2012 / 301-311 depicted using Lactate dehydrogenase knock out Escherichia coli. Lactate dehydrogenases play critical role in gold nanoparticle

Madhuri Sharon et al 500mL of Erlenmeyer flask was taken for GNP synthesis Fabrication of Gold nanoparticle: The supernatant obtained from the above procedure was used for GNP synthesis. The desired pH of the reaction medium was adjusted by adding 1 M NaOH solution or 1 M HCl solution. In order to optimize the

synthesis which could be analysed using normal E.coli and LDH knock out E.coli. Further lactate dehydrogenases were also quantified after nanoparticle synthesis using normal E.coli. Materials and Methods Preparation of Bacterial sample to be used for synthesis of GNP - A loopful of Escherichia coli culture obtained from

GNP formation, the impact of different pH (2, 3, 4 & 6) on synthesis of GNPs was studied at 370 and 100 C. The parameters obtained from the above two experiment were kept constant to comprehend the optical as well as morphological features of GNPs. The most influential concentration of aurochlorate was found to be 100 ppm; hence for all the experiments this concentration was used. Lactate dehydrogenase assay: The assay was performed by recording decrease in absorbance at 340nm which was due to the oxidation of NADH. One unit leads to the oxidation of one micromole of NADH per minute at 250C and pH 7.3, under the specified conditions. 6.6mM NADH was dissolved in 0.2M Tris HCl buffer at pH 7.3. Then 30mM Sodium pyruvate was added which was then converted to Lactic acid that involves oxidation of NADH [9]. Characterization of Nanoparticles: UV-Vis Measurements: was carried out on a dual beam spectroscopy Lambda 25 Perkin Elmer, USA using deionized water as the reference. The colloidal solution was then

NCIM, Pune; was inoculated in 250ml conical flask containing 100ml sterile Nutrient Broth. The inoculated medium was incubated at 370C in a rotary shaker at 120 rpm for 24 hours. After 24 hours, the culture was centrifuged to separate bacterial cells.

Centrifugation was done at 5000 rpm for 10 minutes. Supernatant and pellet were

separated. The supernatant obtained after centrifugation was used for nanoparticles synthesis. A strain of E.coli, which is defective in Lactate dehydrogenase enzyme i.e. (LDH knock out), was procured from American Type Cell Cultures. They were first grown in Nutrient broth and then its supernatant was used for GNP synthesis. Chemicals and Glassware: Chemical used for the synthesis of GNP were chloroauric acid (HAuCl4) (Sigma-Aldrich). 100mL of 1mM aqueous HAuCl4 solution in

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Jamonline / 2(4); 2012 / 301-311 added into a quartz cuvette cell followed by immediate spectral measurements. The SPR peaks were assessed for size and distribution of gold nanoparticles. Transmission Electron Microscopy:

Madhuri Sharon et al XRD Measurements: Crystallographic information about the

samples was obtained from X-ray diffraction (XRD) using (PANalytical, Philips PW 1830, The Netherlands) operating at 40 kV and a current of 30 mA with Cu K radiation ( = 1.5404 ) and the 2 scanning range was of 30-80 at 2 min-1. Prior to recording XRD pattern the colloidal suspension containing metal nanoparticles was dried on a small glass cover slip. Results and Discussions Impact of different pH on the formation of GNPs was studied at 30C using 100 ppm Aurochlorate and exudates of Escherichia coli. 7.35 was found to be the most suitable pH at 300 C (Table 1).

Examination of the nanoparticle morphology by high-resolution analytical transmission electron microscopy (TEM) was performed on a Carl Zeiss Micro imaging, GmbH, Germany with an electron kinetic energy of 200 kV. For sample preparation, 2-3 drops of the colloidal gold solution were dispensed onto a carbon-coated 200-mesh copper grid and dried under ambient condition before examination.

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Jamonline / 2(4); 2012 / 301-311 pH Time taken for Change in color 2 3 4 5 6 7 Inheren t pH 7.35 8 24 h 24 h 24 h 24 24 h No change in color 24hrs Observations UV-Visible spectrum Broad plateau Broad plateau Broad hump at 585 nm Broad plateau broad hump at 610nm -peak at 540nm XRD

Madhuri Sharon et al

HR-TEM

Crystalline structure Crystalline structure Crystalline structure Crystalline structure Crystalline structure Crystalline structure Crystalline structure 30-50nm anisotropic & isotropic GNP

30nm spherical GNP

No change in No peak color 10 No change in No peak color Table 1: Impact of pH on the Biosynthesis of gold nano particles at 300C using100 ppm Aurochlorate and Escherichia coli exudates

Observations made in table 1 suggested that for GNP biosynthesis pH 7.35 was most suitable. Hence to study the impact of different temperature the pH was kept constant at 7.35 (Table2).

Temperature 20C RT (30C)

Visual Change in color in > 24 h Change in color in < 24 h Change in color within 20 min Change in color in < 5 sec

Observations UV-Vis Peak Broad feeble peak at 590 nm Intense peak at 555 nm Hump at 567 nm Flat absorption spectrum

XRD Crystalline

TEM

Crystalline

Both isotropic & anisotropic gold nanoparticles Both isotropic & anisotropic gold nanoparticles

37 C

Crystalline

100 C

Crystalline

Table 2 : Impact of temperature on synthesis of Gold nanoparticles using Escherichia coli at pH 7.35 using100 ppm aurochlorate All rights reserved 2011 www.jamonline.in 305

Jamonline / 2(4); 2012 / 301-311 It was found that with increase in temperature, the time taken for GNP formation decreased, the minimum being 5 sec at 1000C. However, though at 300 C it took longer (24hrs) for color to change; the UV-Vis spectra showed better formation of GNP at 30C , whereas at 370C that peak disappeared into a broad hump at 567 nm. UV Visible Spectroscopic Studies of Impact of pH on Biosynthesis of GNPs: The exudates of Escherichia coli possess both reducing and capping agents which leads to the efficient biosynthesis of gold

Madhuri Sharon et al ions also possess positive charge which leads to less interaction of gold ions with the positively charged functional groups, thus forming very large sized nanoparticles of anisotropic structures. Further such

nanoparticles are also unstable and start agglomerating due to instability in the double layer formed. The reason behind

agglomeration is Steric destabilization which leads to Ostwald ripening. This is a clear indication that in acidic pH, reducing property of the bacterial exudates is exhibited but the capping mechanism is inactive or very weak. As pH rises to 4, there is more sterically stable nanoparticles, but with little larger size as can be seen from the broad TSPR peak at 585nm (Fig 1b). The larger size can be due to the coalescence of smaller nuclei since there is presence of smaller nanoparticles which can be seen in TEM. As pH is further raised to 7.35, which is also the inherent pH of the bacterial exudates, there is formation of wine coloured nanoparticles which can be depicted by a broad hump existing at 530nm indicating negligible reducing capacity of the reducing agents.(Fig 1 b). At pH 8 and 10, there is no nanoparticle formation (Fig 1 b).

nanoparticles. The bio-fabrication of GNPs was done at varying (30C) pH, and incubation optimum

temperature

concentration of 100ppm. At lower pH (2 and 3), there is very mild activity of reducing agents leading to minor change in colour causing a broad hump in UV-Vis spectra. Fig 1 b depicts that at pH 2 and 3, there is a very broad plateau which clearly indicates that the size of the GNPs are too large since at this pH due to large proton concentration, all the functional charges possess positive charge which have got less reduction potential. Gold

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Jamonline / 2(4); 2012 / 301-311

Madhuri Sharon et al

Figure 1: Impact of pH on bio-fabrication of gold nanoparticles using Escherichia coli, as shown by UV-Vis spectra Impact of Temperature on Biosynthesis of GNPs: GNPs were fabricated using exudates of E. coli at various temperatures viz 20,30, 37& 100C. Nanoparticles fabrication was dielectric constant. Further, high ionic

strength coupled with high temperature also might be instrumental in creating a barrier for synthesis of nanoparticles.

speculated at room temperature (30 C) with inherent pH (pH 7.35) wherein the solution gives sharp peak at 555nm.(Fig 2 ) . At lower temperature (20C) with a SPR band centred at 590 nm polydispersed nanoparticles were speculated; as the area under UV-Vis spectrum is broad as seen in Fig 2. This indicates that at lower temperatures, the electron transfer rate of the reducing or nucleating agent, which is a rate determining step, is negligible. On fabrication of GNPs at 37C there was formation of larger GNPs, which is in agreement with the SPR band in UV-Vis spectrum at 567 nm.(Fig 2) As the reaction temperature was raised to 100C, there was no nanoparticle synthesis; which symbolizes a decrement in the refractive index of the solvent which also affects its All rights reserved 2011 www.jamonline.in 307 High Resolution Transmission Electron Figure 2: Impact of temperature on Gold nanoparticle synthesis using Escherichia coli using uv-spectroscopy.

Microscopic Analysis of the Morphology of GNPs exhibited that GNPs that were

synthesized using E. coli exudates were polydispersed shapes) (having several different structures

small

anisotropic

Jamonline / 2(4); 2012 / 301-311 (triangular and icosahedrons) at different pH and different temperatures. Spherical

Madhuri Sharon et al having 30-50nm size. While at 7.35 (the inherent pH of exudates) there was formation of spherical GNPs having size 20nm

structures were formed at pH 2 and 4; triangular, hexagonal nanostructures along with spherical ones were formed at pH of 4,

diameter.

Figure 3: High transmission electron micrographs showing GNPs of (a) 20nm size synthesized at inherent pH of exudates i.e.7.35 and (b) 30-50 nm size at pH 4

Impact

of

temperature

on

size

and

at 30C (Fig 4b). Anisotropic GNPs of size 100nm were formed at room temperature and 37C (Fig 4c). Whereas, at 20C nanoparticles were not synthesized this was confirmed by TEM micrograph (fig 4a.)

morphology of the nanoparticles: HR-TEM of GNPs synthesized at different temperatures shows formation of mixed spherical as well as anisotropic nanostructures

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Jamonline / 2(4); 2012 / 301-311

Madhuri Sharon et al

Figure 4: Impact of temperature on biosynthesis of GNP using E. coli exudates at (a) 20C (b) 30 C and (c) at 37C

X-ray diffraction pattern that corresponds to the GNPs exhibits Bragg reflections, which could be well manifested on the basis of the face centred cubic (fcc) gold nanostructures. A number of Bragg reflections corresponding to the {1 1 1}, {2 0 0}, {2 2 0} {3 1 1} sets of lattice planes are observed, which had been indexed on the basis of the FCC structures of Au (JCPDS file no.01-1174).The very strong diffraction peak at 38 degrees is considered to be of {1 1 1} facet of the face centred cubic structure, while the diffraction peaks of other gold peaks are found to be much weaker compared to standard gold nanoparticles. It is imperative to note that the ratio of intensity between {2 0 0} and {1 1 1} peaks, {2 2 0} and {1 1 1} peaks as well as {3 1 1} and {1 1 1} peaks are much smaller compared to the intensity ratios of standard GNPs. Deciphering the role of Lactate dehydrogenase using Escherichia coli LDH knock out Lactate dehydrogenase in Escherichia coli is considered to be catalysing the formation of D-lactate from pyruvate. This enzyme is purely NADPH-dependent having very high Michaelis constants for NADPH and pyruvate thus exhibiting sigmoidal kinetics with Figure 5: XRD of the gold nanoparticles on silicon wafer, the characteristic Bragg reflections {1 1 1}, {2 0 0}, {2 2 0} {3 1 1} sets of lattice planes are observed.

respect to pyruvate. A study was performed to apprehend the efficacy of LDH for gold All rights reserved 2011 www.jamonline.in 309

Jamonline / 2(4); 2012 / 301-311 nanoparticle synthesis. This enzyme is a membrane bound as well as secretory enzyme [9]. Hence to decipher its role LDH knock out E.coli was exploited to check its efficacy for gold nanoparticle synthesis. It was found that there is no formation of gold nanoparticle synthesis. This can be confirmed by UVvisible spectra which resulted in

Madhuri Sharon et al controllable tuning of the synthesis of thermodynamically stable gold nanoparticle. Dehydrogenases are expected to be acting as reducing agent for the biosynthesis of GNPs; as proved by the use of LDH Knock out E. Coli. Acknowledgements Authors wish to acknowledge the financial support provided by the authorities of SICES, Ambernath and especially to Mr. K.M.S. Nair (President of nsnRc) and Mr. K.M.K Nair

disappearance of the peak. However, one needs to find other reasons for the synthesis of gold nanoparticle synthesis apart from LDH.

Gold nanoparticle synthesis using E.coli LDH knock out

4 2 0
0 500 1000 ABS

(Member of Governing council, nsnRc) to carry out this project. We also feel gratitude towards Dr. Lalla , UGC-DAE consortium for HR-TEM analysis. References 1) Husseiny MI, AbdEl-Aziz M, Badr Y, Mahmoud MA, Spectrochimica Acta

Fig. 6 UV-Visible spectra of LDH Knock out E.coli exhibiting no Surface Plasmon Resonance peak which is an indicator of gold nanoparticles Conclusion Escherichia coli can be used for rapid synthesis of GNPs. The most influential parameters were found to be inherent pH and room temperatures. The TEM micrographs of GNPs show the presence of both spherical and icosahedral nanostructures. The

Part A, 2007, 67: 1003 2) He S, Guo Z, Zhang Y, Zhang S, Wang J, Gu N, Materials Letters, 2007, 61: 3984 3) Lengke M, Southam G, Geochimica et Cosmochimica Acta, 2006, 70: 3646 4) Du L, Jiang H, Liu X, Wang E, Electrochemistry 2007, 9: 1165 5) Konishi Y, tsukiyama T, Ohno K, Saitoh N, Nomura T, Nagamine S, Communications,

substantial decrease in the LDH activity confirms that dehydrogenases are involved in the reduction of gold ions to GNPs. The methodology presented can be used for a

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Jamonline / 2(4); 2012 / 301-311 Hydrometallurgy, 2006 81: 24 6) Sunil Pandey, Goldie Oza, Ashmi Mewada, Madhuri Sharon, Archives of Applied Science Research,2012,4(2): 1135-1141 7) Sunil Pandey, Goldie Oza, Arvind Gupta, Ritu Shah & Madhuri Sharon, European Journal of Experimental Biology, 2012, 2 (3):475-483 8) Madhuri Sharon, Goldie Oza, Sunil Pandey & Maheshwar Sharon, J. Phytol, 2012, 4 (5) in press 9) Tarmy EM, Kaplan NO, The Jour. Biol. Chem, 1968, 243(10): 2587

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