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Melanin mediated synthesis of gold nanoparticles by Yarrowia lipolytica
Materials Letters 95 (2013) 149–152
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Melanin mediated synthesis of gold nanoparticles by Yarrowia lipolytica Mugdha Apte, Gauri Girme, Resmi Nair, Ashok Bankar, Ameeta Ravi Kumar, Smita Zinjarde n Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India
a r t i c l e i n f o
abstract
Article history: Received 21 July 2012 Accepted 26 December 2012 Available online 2 January 2013
Yarrowia lipolytica is known to synthesize cell-associated gold nanoparticles. Melanin, a pigment associated with the yeast, was identified as a factor responsible for nanoparticle synthesis. Cellextracted melanin as well as that induced by incubating cells with 3,4-dihydroxy- L-phenylalanine (L-DOPA) mediated the synthesis of gold nanoparticles (AuNPs). These nanoparticles were characterized by a variety of standard techniques. This report identifies one of the molecular mechanisms involved in nanoparticle synthesis by Y. lipolytica. The nanoparticles thus synthesized were effective antibiofilm agents. & 2013 Elsevier B.V. All rights reserved.
Keywords: Melanin Gold nanoparticles Electron microscopy XRD
1. Introduction A variety of microorganisms synthesize nanoparticles [1–3]. Fungi and yeasts are important in this regard [4,5]. There are two reports on the synthesis of AuNPs by the yeast Yarrowia lipolytica in a cellassociated manner [6,7]. These reports, however, have not focused on the molecular mechanisms involved in the synthetic process. Metal– microbe interactions in Y. lipolytica have been reviewed recently [8]. This yeast has the inherent ability to tolerate metals by a variety of mechanisms including the constitutive production of a pigment, melanin [9]. Melanins are high molecular weight dark colored pigments produced by oxidative polymerization of phenolic compounds [10]. They are known to confer survival and competitive advantages on the organisms producing them. The pigment has antioxidant properties, confers heat protection, radiation resistance, binds toxic substances and is considered as a virulence factor in pathogenic fungi [10–12]. We hypothesized that this pigment may be involved in nanoparticle synthesis. In this paper, we demonstrate the synthesis of AuNPs by Y. lipolytica-associated melanin obtained by two methods: (i) extracted from cells and (ii) induced after incubation with L-DOPA. The nanostructures have been characterized and applied as antibiofilm agents. This is the first report on the mechanistic aspect of nanoparticle synthesis in Y. lipolytica.
2. Experiment Synthesis of nanoparticles by melanin extracted from cells: Melanin from the tropical marine yeast Y. lipolytica NCIM 3589 n
Corresponding author. Tel.: þ91 20 25601385; fax: þ91 20 25690087. E-mail address: [email protected] (S. Zinjarde).
0167-577X/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2012.12.087
was isolated by following a standard protocol [9]. The melanin thus obtained was used in experiments on nanoparticle synthesis. Typical reaction mixtures (10 ml) containing 150 mg of melanin and 2.5 mM of gold salt were incubated at 100 1C for 10 min and a color change was monitored. The effect of HAuCl4 concentrations (1.25, 2.5 and 5 mM) and melanin content (37.5, 75, 150 and 300 mg/ml) was also studied. Synthesis of AuNPs by L-DOPA induced melanin: Y. lipolytica cells grown in YNB–glucose medium [6] were centrifuged and re-suspended in double volume of distilled water. Increasing numbers of cells (4, 8, 12, or 16 109) were added to 10 ml reaction mixtures containing 80 mg of L-DOPA in 0.1 M NaOH. Incubation at 30 1C for 18 h mediated the transformation of LDOPA into melanin. After centrifugation at 6000g, the melanin in the supernatant was acid precipitated (with 1 N HCl), washed with distilled water, re-dissolved in 0.1 M NaOH and quantified by measuring absorbance at 400 nm [9,13]. For the synthesis of AuNPs, reaction mixtures containing different melanin concentrations were incubated with 2 mM HAuCl4 boiled for 10 min and a change in color was monitored. All experiments were carried out in triplicates with two biological replicates and representative data is depicted here. Characterization of nanoparticles: Standard procedures and the following equipment were used to characterize the nanoparticles: (i) UV–Visible spectra-Jasco V-530 spectrophotometer, (ii) X-ray diffraction (XRD) analysis-D8 Advanced Brucker instrument, and (iii) scanning electron microscope–energy dispersive spectrometer, SEM–EDS-JOEL JSM-6360A [14]. Antibiofilm activity of gold nanoparticles: Antimicrobial activities of the AuNPs derived from the cell-associated melanin preparations against Citrobacter kosarii and Candidia albicans were determined by the plate assay method [15,16]. The antibiofilm activities in 96 well microtitre plates and on glass slides were
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evaluated by using standard protocols [17]. Disruption was monitored by an Axio Scope-A1 fluorescence microscope with ProgRess Capture Pro 2.7 attachment.
3. Results and discussion Y. lipolytica has an inherent capacity to produce the brown pigment melanin [18–21]. Melanin is known to interact with and sequester heavy metals [8]. The cell-associated melanin obtained by following a standard procedure [9] was used to synthesize AuNPs. Fig. 1a shows that with 1.25 mM of HAuCl4 and 150 mg of melanin, no characteristic color or peak was observed (Fig. 1a: tube and line 1). However, with 2.5 and 5.0 mM HAuCl4, light orange and wine red colors, respectively (indicating the synthesis of AuNPs) and characteristic peaks at 540 nm were obtained (Fig. 1a, tubes and lines 2 and 3). Fig. 1b shows the effect of melanin on the synthesis of AuNPs when 2.5 mM HAuCl4 was used. As seen in Fig. 1b (tube and line 1) with 37.5 mg of melanin, there was no change in color or peak. With 75 and 150 mg of melanin, purple and light orange color, respectively, and peaks at 540 nm were observed (Fig. 1b, tubes and lines 2 and 3). The result obtained with 150 mg is consistent with the observation in Fig. 1a (line and tube 2) wherein, the contents of the reaction mixture were similar. With 300 mg of the pigment, a brown color and no distinct peak were observed (Fig.1b, tube and line 4) implicating the presence of micro-sized gold plates as also reported earlier [7]. Melanin from Y. lipolytica was also obtained by biotransformation of L-DOPA. Candida albicans, a related yeast, is known to synthesize melanin when incubated with L-DOPA [22,23]. We hypothesized that if increasing number of Y. lipolytica cells would be incubated with constant L-DOPA concentration, then, an increasing quantity of melanin would be produced. As seen in Fig. 1c, when cell numbers were increased, the content of melanin increased until a certain level. Tube 1 in Fig. 1c shows a control tube displaying a yellow color. Tube 2 is a representative test
sample (with 16 109 cells) wherein a distinct brown color is visible. When melanin preparations containing increased content of the pigment were incubated with 2.0 mM of the HAuCl4, increasingly darker shades of pink color were observed. Compared to control samples (Fig. 1d, tube 1), the absorbance at 540 nm also appropriately increased (Fig. 1d, tubes 2–5). AuNPs are known to display a variety of colors depending on the size of the nanoparticles. The earlier reports on this yeast system have described a cell-associated production of AuNPs. To release these nanoparticles, additional protocols need to be employed [7]. Extracellular synthesis methods produce large quantities of nanoparticles in a form that is relatively free of other cellular proteins. Since melanins are natural biopolymers with strong anti-oxidant properties, they were exploited in the current investigation. The AuNPs synthesized by both the melanin samples were characterized. Fig. 2a and b shows representative SEM images of gold structures obtained with the melanin extracted from the cells (150 mg melanin and 2.5 mM gold salt). Since SEM samples were air-dried, aggregation due to the coffee-ring phenomenon was observed [14,15]. Representative SEM images of nanoparticles obtained with L-DOPA induced melanin (Fig. 1d, tube 5) are shown in Fig. 2c and d. In this case, distinct spherical nanoparticles were observed. Fig. 2e is the EDS profile showing the elemental composition of a representative field. Peaks at 1.7 and 2.2 keV (corresponding to binding energies of AuMz and AuMr, respectively) were observed. Other signals (silica: from silicon wafer, K and Na) were also seen in control samples that were not treated with HAuCl4. These results are consistent with an earlier report on the synthesis of AuNPs by Stenotrophomonas maltophilia [24]. The X-ray diffraction patterns of the nanostructures showed intense peaks due to (111) and (200) Bragg reflections at 2y ¼38.191 and 44.541, respectively (Fig. 2f). These observations are similar to earlier reports on the XRD patterns obtained with cell-associated gold nanoparticles [6,7]. The melanin mediated AuNPs were evaluated for antimicrobial activity. Zones of inhibition were observed as also described
Fig. 1. Visual observations and UV–Visible spectra of reaction mixtures containing (a) different concentrations of the gold salt (lines and tubes 1, 2, 3 with 1.25, 2.5, 5.0 mM, HAuCl4, respectively) and 150 mg of melanin and (b) different contents of melanin (lines and tubes 1, 2, 3, 4 with 37.5, 75, 150, 300 mg melanin, respectively) and 2.5 mM HAuCl4, (c) synthesis of L-DOPA induced melanin by Y. lipolytica cells. Inset: (1) control lacking cells, and (2) test showing brown colored melanin. (d) Visual observations and UV–Visible spectra of reaction mixtures containing increasing melanin contents obtained from increasing cell numbers after incubation with 2.0 mM HAuCl4.
M. Apte et al. / Materials Letters 95 (2013) 149–152
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Fig. 2. SEM images of AuNPs synthesized by (a, b) cell-associated melanin, (c, d) L-DOPA induced melanin, (e) representative EDS profile and (f) representative XRD spectrum.
Fig. 3. Disruption of (a)–(d) Citrobacter kosarii and (e)–(h) Candidia albicans biofilms by AuNPs (a and e: control; b, c, d and f, g, h with increasing AuNPs concentrations).
earlier with other bio-inspired AuNPs [25]. The biofilm disruption activities of these nanoparticles in microtitre plates were 95% and 90% for C. kosarii and C. albicans, respectively. Representative images of biofilm disruption on glass slides are shown in Fig. 3. The biofilm form of growth is characteristic of several pathogens [26]. Since cells in this form are resistant towards antimicrobial agents [27,28], there is a need to look for alternative disrupting agents. Compared to control samples (Fig. 3a and e), biofilm formation was hampered with increasing concentrations of the AuNPs (Fig. 3b–d; f–h). These results are consistent with the observations on silver nanoparticles being effective against Pseudomonas aeruginosa and Staphylococcus epidermidis biofilms [29].
4. Conclusions To conclude, we report a simple method to synthesize AuNPs using melanin derived from Y. lipolytica. Although the yeast is reported to synthesize nanoparticles in a cell-associated manner, by the current protocol, AuNPs could be synthesized in an extracellular fashion. These nanoparticles are effective antibiofilm agents.
Acknowledgment The authors thank University of Pune for financial help under the BCUD Research Grant and The University Grants Commission for financial assistance under UPE Phase II. References [1] Bhattacharya D, Gupta RK. Crit Rev Biotechnol 2005;25:199–204. [2] Thakkar KN, Mhatre SS, Parikh RY. Nanomed: Nanotechnol Biol Med 2010;6:257–62. [3] Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P. Appl Microbiol Biotechnol 2006;69:485–92. [4] Gade A, Ingle A, Whiteley C, Rai M. Biotechnol Lett 2010;32:593–600. [5] Dhillon GS, Brar SK, Kaur S, Verma M. Crit Rev Biotechnol 2012;32:49–73. [6] Agnihotri M, Joshi S, Kumar AR, Zinjarde S, Kulkarni S. Mater Lett 2009;63:1231–4. [7] Pimprikar PS, Joshi SS, Kumar AR, Zinjarde SS, Kulkarni SK. Colloids Surf B: Biointerfaces 2009;74:309–16. [8] Bankar AV, Kumar AR, Zinjarde SS. Appl Microbiol Biotechnol 2009;84:847–65. [9] Ito H, Inouche M, Tohoyama H, Joho M. Biometals 2007;20:773–80. [10] Jacobson ES. Clin Microbiol Rev 2000;13:708–17. [11] Revankar SG, Sutton DA. Clin Microbiol Rev 2010;23:884–928. [12] Eisenman HC, Casadevall A. Appl Microbiol Biotechnol 2012;93:931–40. [13] Ganeshkumar C, Mongolla P, Pombala S, Kamle A, Joseph J. Lett Appl Microbiol 2011;53:350–8. [14] Bankar AV, Joshi BS, Kumar AR, Zinjarde SS. Mater Lett 2010;64:1951–3.
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[15] Bankar AV, Joshi BS, Kumar AR, Zinjarde SS. Colloids Surf B: Biointerfaces 2010;80:45–50. [16] Bankar AV, Joshi BS, Kumar AR, Zinjarde SS. Colloids Surf A: Physicochem Eng Aspects 2010;368:58–63. [17] Dusane DH, Rajput JK, Kumar AR, Nancharaiah YV, Venugopalan VP, Zinjarde SS. Lett Appl Microbiol 2008;47:374–9. [18] Carreira A, Paloma L, Loureiro V. Int J Food Microbiol 1998;41:223–30. [19] Gardini F, Tofalo R, Belletti N, Iucci L, Suzzi G, Torriani S, et al. Food Microbiol 2006;23:641–8. [20] Carreira A, Ferreira LM, Loureiro V. J Appl Microbiol 2001;90:372–9. [21] Carreira A, Ferreira LM, Loureiro V. Appl Environ Microbiol 2001;67:3463–8. [22] Morris-Jones R, Go´mez BL, Diez S, Uran M, Morris-Jones SD, Casadevall A, et al. Infect Immun 2005;73:6147–50.
[23] Walker CA, Go´mez BL, Mora-Montes HM, Mackenzie KS, Munro CA, Brown AJ, et al. Eukaryotic Cell 2010;9:1329–42. [24] Nangia Y, Wangoo N, Goyal N, Shekhawat G, Suri CR. Microb Cell Fact 2009;8:39. [25] Suresh AK, Pelletier DA, Wang W, Broich ML, Moon JW, Gu B, et al. Acta Biomater 2011;7:2148–52. [26] Karunakaran E, Mukherjee J, Ramalingam B, Biggs CA. Appl Microbiol Biotechnol 2011;90:1869–81. [27] Stewart P. Lancet 2001;358:135–8. [28] d’Enfert C. Curr Drug Targets 2006;7:465–70. [29] Kalishwaralal K, Barathmanikanth S, Pandian SRK, Deepak V, Gurunathan S. Colloids Surf B Biointerfaces 2010;79:340–4.
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Melanin mediated synthesis of gold nanoparticles by Yarrowia lipolytica Mugdha Apte, Gauri Girme, Resmi Nair, Ashok Bankar, Ameeta Ravi Kumar, Smita Zinjarde n Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India
a r t i c l e i n f o
abstract
Article history: Received 21 July 2012 Accepted 26 December 2012 Available online 2 January 2013
Yarrowia lipolytica is known to synthesize cell-associated gold nanoparticles. Melanin, a pigment associated with the yeast, was identified as a factor responsible for nanoparticle synthesis. Cellextracted melanin as well as that induced by incubating cells with 3,4-dihydroxy- L-phenylalanine (L-DOPA) mediated the synthesis of gold nanoparticles (AuNPs). These nanoparticles were characterized by a variety of standard techniques. This report identifies one of the molecular mechanisms involved in nanoparticle synthesis by Y. lipolytica. The nanoparticles thus synthesized were effective antibiofilm agents. & 2013 Elsevier B.V. All rights reserved.
Keywords: Melanin Gold nanoparticles Electron microscopy XRD
1. Introduction A variety of microorganisms synthesize nanoparticles [1–3]. Fungi and yeasts are important in this regard [4,5]. There are two reports on the synthesis of AuNPs by the yeast Yarrowia lipolytica in a cellassociated manner [6,7]. These reports, however, have not focused on the molecular mechanisms involved in the synthetic process. Metal– microbe interactions in Y. lipolytica have been reviewed recently [8]. This yeast has the inherent ability to tolerate metals by a variety of mechanisms including the constitutive production of a pigment, melanin [9]. Melanins are high molecular weight dark colored pigments produced by oxidative polymerization of phenolic compounds [10]. They are known to confer survival and competitive advantages on the organisms producing them. The pigment has antioxidant properties, confers heat protection, radiation resistance, binds toxic substances and is considered as a virulence factor in pathogenic fungi [10–12]. We hypothesized that this pigment may be involved in nanoparticle synthesis. In this paper, we demonstrate the synthesis of AuNPs by Y. lipolytica-associated melanin obtained by two methods: (i) extracted from cells and (ii) induced after incubation with L-DOPA. The nanostructures have been characterized and applied as antibiofilm agents. This is the first report on the mechanistic aspect of nanoparticle synthesis in Y. lipolytica.
2. Experiment Synthesis of nanoparticles by melanin extracted from cells: Melanin from the tropical marine yeast Y. lipolytica NCIM 3589 n
Corresponding author. Tel.: þ91 20 25601385; fax: þ91 20 25690087. E-mail address: [email protected] (S. Zinjarde).
0167-577X/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2012.12.087
was isolated by following a standard protocol [9]. The melanin thus obtained was used in experiments on nanoparticle synthesis. Typical reaction mixtures (10 ml) containing 150 mg of melanin and 2.5 mM of gold salt were incubated at 100 1C for 10 min and a color change was monitored. The effect of HAuCl4 concentrations (1.25, 2.5 and 5 mM) and melanin content (37.5, 75, 150 and 300 mg/ml) was also studied. Synthesis of AuNPs by L-DOPA induced melanin: Y. lipolytica cells grown in YNB–glucose medium [6] were centrifuged and re-suspended in double volume of distilled water. Increasing numbers of cells (4, 8, 12, or 16 109) were added to 10 ml reaction mixtures containing 80 mg of L-DOPA in 0.1 M NaOH. Incubation at 30 1C for 18 h mediated the transformation of LDOPA into melanin. After centrifugation at 6000g, the melanin in the supernatant was acid precipitated (with 1 N HCl), washed with distilled water, re-dissolved in 0.1 M NaOH and quantified by measuring absorbance at 400 nm [9,13]. For the synthesis of AuNPs, reaction mixtures containing different melanin concentrations were incubated with 2 mM HAuCl4 boiled for 10 min and a change in color was monitored. All experiments were carried out in triplicates with two biological replicates and representative data is depicted here. Characterization of nanoparticles: Standard procedures and the following equipment were used to characterize the nanoparticles: (i) UV–Visible spectra-Jasco V-530 spectrophotometer, (ii) X-ray diffraction (XRD) analysis-D8 Advanced Brucker instrument, and (iii) scanning electron microscope–energy dispersive spectrometer, SEM–EDS-JOEL JSM-6360A [14]. Antibiofilm activity of gold nanoparticles: Antimicrobial activities of the AuNPs derived from the cell-associated melanin preparations against Citrobacter kosarii and Candidia albicans were determined by the plate assay method [15,16]. The antibiofilm activities in 96 well microtitre plates and on glass slides were
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evaluated by using standard protocols [17]. Disruption was monitored by an Axio Scope-A1 fluorescence microscope with ProgRess Capture Pro 2.7 attachment.
3. Results and discussion Y. lipolytica has an inherent capacity to produce the brown pigment melanin [18–21]. Melanin is known to interact with and sequester heavy metals [8]. The cell-associated melanin obtained by following a standard procedure [9] was used to synthesize AuNPs. Fig. 1a shows that with 1.25 mM of HAuCl4 and 150 mg of melanin, no characteristic color or peak was observed (Fig. 1a: tube and line 1). However, with 2.5 and 5.0 mM HAuCl4, light orange and wine red colors, respectively (indicating the synthesis of AuNPs) and characteristic peaks at 540 nm were obtained (Fig. 1a, tubes and lines 2 and 3). Fig. 1b shows the effect of melanin on the synthesis of AuNPs when 2.5 mM HAuCl4 was used. As seen in Fig. 1b (tube and line 1) with 37.5 mg of melanin, there was no change in color or peak. With 75 and 150 mg of melanin, purple and light orange color, respectively, and peaks at 540 nm were observed (Fig. 1b, tubes and lines 2 and 3). The result obtained with 150 mg is consistent with the observation in Fig. 1a (line and tube 2) wherein, the contents of the reaction mixture were similar. With 300 mg of the pigment, a brown color and no distinct peak were observed (Fig.1b, tube and line 4) implicating the presence of micro-sized gold plates as also reported earlier [7]. Melanin from Y. lipolytica was also obtained by biotransformation of L-DOPA. Candida albicans, a related yeast, is known to synthesize melanin when incubated with L-DOPA [22,23]. We hypothesized that if increasing number of Y. lipolytica cells would be incubated with constant L-DOPA concentration, then, an increasing quantity of melanin would be produced. As seen in Fig. 1c, when cell numbers were increased, the content of melanin increased until a certain level. Tube 1 in Fig. 1c shows a control tube displaying a yellow color. Tube 2 is a representative test
sample (with 16 109 cells) wherein a distinct brown color is visible. When melanin preparations containing increased content of the pigment were incubated with 2.0 mM of the HAuCl4, increasingly darker shades of pink color were observed. Compared to control samples (Fig. 1d, tube 1), the absorbance at 540 nm also appropriately increased (Fig. 1d, tubes 2–5). AuNPs are known to display a variety of colors depending on the size of the nanoparticles. The earlier reports on this yeast system have described a cell-associated production of AuNPs. To release these nanoparticles, additional protocols need to be employed [7]. Extracellular synthesis methods produce large quantities of nanoparticles in a form that is relatively free of other cellular proteins. Since melanins are natural biopolymers with strong anti-oxidant properties, they were exploited in the current investigation. The AuNPs synthesized by both the melanin samples were characterized. Fig. 2a and b shows representative SEM images of gold structures obtained with the melanin extracted from the cells (150 mg melanin and 2.5 mM gold salt). Since SEM samples were air-dried, aggregation due to the coffee-ring phenomenon was observed [14,15]. Representative SEM images of nanoparticles obtained with L-DOPA induced melanin (Fig. 1d, tube 5) are shown in Fig. 2c and d. In this case, distinct spherical nanoparticles were observed. Fig. 2e is the EDS profile showing the elemental composition of a representative field. Peaks at 1.7 and 2.2 keV (corresponding to binding energies of AuMz and AuMr, respectively) were observed. Other signals (silica: from silicon wafer, K and Na) were also seen in control samples that were not treated with HAuCl4. These results are consistent with an earlier report on the synthesis of AuNPs by Stenotrophomonas maltophilia [24]. The X-ray diffraction patterns of the nanostructures showed intense peaks due to (111) and (200) Bragg reflections at 2y ¼38.191 and 44.541, respectively (Fig. 2f). These observations are similar to earlier reports on the XRD patterns obtained with cell-associated gold nanoparticles [6,7]. The melanin mediated AuNPs were evaluated for antimicrobial activity. Zones of inhibition were observed as also described
Fig. 1. Visual observations and UV–Visible spectra of reaction mixtures containing (a) different concentrations of the gold salt (lines and tubes 1, 2, 3 with 1.25, 2.5, 5.0 mM, HAuCl4, respectively) and 150 mg of melanin and (b) different contents of melanin (lines and tubes 1, 2, 3, 4 with 37.5, 75, 150, 300 mg melanin, respectively) and 2.5 mM HAuCl4, (c) synthesis of L-DOPA induced melanin by Y. lipolytica cells. Inset: (1) control lacking cells, and (2) test showing brown colored melanin. (d) Visual observations and UV–Visible spectra of reaction mixtures containing increasing melanin contents obtained from increasing cell numbers after incubation with 2.0 mM HAuCl4.
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Fig. 2. SEM images of AuNPs synthesized by (a, b) cell-associated melanin, (c, d) L-DOPA induced melanin, (e) representative EDS profile and (f) representative XRD spectrum.
Fig. 3. Disruption of (a)–(d) Citrobacter kosarii and (e)–(h) Candidia albicans biofilms by AuNPs (a and e: control; b, c, d and f, g, h with increasing AuNPs concentrations).
earlier with other bio-inspired AuNPs [25]. The biofilm disruption activities of these nanoparticles in microtitre plates were 95% and 90% for C. kosarii and C. albicans, respectively. Representative images of biofilm disruption on glass slides are shown in Fig. 3. The biofilm form of growth is characteristic of several pathogens [26]. Since cells in this form are resistant towards antimicrobial agents [27,28], there is a need to look for alternative disrupting agents. Compared to control samples (Fig. 3a and e), biofilm formation was hampered with increasing concentrations of the AuNPs (Fig. 3b–d; f–h). These results are consistent with the observations on silver nanoparticles being effective against Pseudomonas aeruginosa and Staphylococcus epidermidis biofilms [29].
4. Conclusions To conclude, we report a simple method to synthesize AuNPs using melanin derived from Y. lipolytica. Although the yeast is reported to synthesize nanoparticles in a cell-associated manner, by the current protocol, AuNPs could be synthesized in an extracellular fashion. These nanoparticles are effective antibiofilm agents.
Acknowledgment The authors thank University of Pune for financial help under the BCUD Research Grant and The University Grants Commission for financial assistance under UPE Phase II. References [1] Bhattacharya D, Gupta RK. Crit Rev Biotechnol 2005;25:199–204. [2] Thakkar KN, Mhatre SS, Parikh RY. Nanomed: Nanotechnol Biol Med 2010;6:257–62. [3] Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P. Appl Microbiol Biotechnol 2006;69:485–92. [4] Gade A, Ingle A, Whiteley C, Rai M. Biotechnol Lett 2010;32:593–600. [5] Dhillon GS, Brar SK, Kaur S, Verma M. Crit Rev Biotechnol 2012;32:49–73. [6] Agnihotri M, Joshi S, Kumar AR, Zinjarde S, Kulkarni S. Mater Lett 2009;63:1231–4. [7] Pimprikar PS, Joshi SS, Kumar AR, Zinjarde SS, Kulkarni SK. Colloids Surf B: Biointerfaces 2009;74:309–16. [8] Bankar AV, Kumar AR, Zinjarde SS. Appl Microbiol Biotechnol 2009;84:847–65. [9] Ito H, Inouche M, Tohoyama H, Joho M. Biometals 2007;20:773–80. [10] Jacobson ES. Clin Microbiol Rev 2000;13:708–17. [11] Revankar SG, Sutton DA. Clin Microbiol Rev 2010;23:884–928. [12] Eisenman HC, Casadevall A. Appl Microbiol Biotechnol 2012;93:931–40. [13] Ganeshkumar C, Mongolla P, Pombala S, Kamle A, Joseph J. Lett Appl Microbiol 2011;53:350–8. [14] Bankar AV, Joshi BS, Kumar AR, Zinjarde SS. Mater Lett 2010;64:1951–3.
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[15] Bankar AV, Joshi BS, Kumar AR, Zinjarde SS. Colloids Surf B: Biointerfaces 2010;80:45–50. [16] Bankar AV, Joshi BS, Kumar AR, Zinjarde SS. Colloids Surf A: Physicochem Eng Aspects 2010;368:58–63. [17] Dusane DH, Rajput JK, Kumar AR, Nancharaiah YV, Venugopalan VP, Zinjarde SS. Lett Appl Microbiol 2008;47:374–9. [18] Carreira A, Paloma L, Loureiro V. Int J Food Microbiol 1998;41:223–30. [19] Gardini F, Tofalo R, Belletti N, Iucci L, Suzzi G, Torriani S, et al. Food Microbiol 2006;23:641–8. [20] Carreira A, Ferreira LM, Loureiro V. J Appl Microbiol 2001;90:372–9. [21] Carreira A, Ferreira LM, Loureiro V. Appl Environ Microbiol 2001;67:3463–8. [22] Morris-Jones R, Go´mez BL, Diez S, Uran M, Morris-Jones SD, Casadevall A, et al. Infect Immun 2005;73:6147–50.
[23] Walker CA, Go´mez BL, Mora-Montes HM, Mackenzie KS, Munro CA, Brown AJ, et al. Eukaryotic Cell 2010;9:1329–42. [24] Nangia Y, Wangoo N, Goyal N, Shekhawat G, Suri CR. Microb Cell Fact 2009;8:39. [25] Suresh AK, Pelletier DA, Wang W, Broich ML, Moon JW, Gu B, et al. Acta Biomater 2011;7:2148–52. [26] Karunakaran E, Mukherjee J, Ramalingam B, Biggs CA. Appl Microbiol Biotechnol 2011;90:1869–81. [27] Stewart P. Lancet 2001;358:135–8. [28] d’Enfert C. Curr Drug Targets 2006;7:465–70. [29] Kalishwaralal K, Barathmanikanth S, Pandian SRK, Deepak V, Gurunathan S. Colloids Surf B Biointerfaces 2010;79:340–4.