JBNB  Vol.3 No.3 , July 2012
Biomineralization of Zinc-Phosphate-Based Nano Needles by Living Microalgae
Abstract: Up to now, chemical synthesis routes only provide restricted opportunities for the formation of structured nano particles. In contrast, living microorganisms generate nano materials of well defined shapes by the precise control of biomineralization. Here we reveal new principles for the generation of functional nano materials through the process of biomineralization. We used the detoxification mechanism of the unicellular alga Scenedesmus obliquus to generate a techno logically interesting zinc-phosphate-based nano material. The algae were incubated in media with a sublethal zinc concentration (6.53 mg Zn dm-3) for 4 weeks. Using BF-and ADF-STEM imaging combined with analytical XEDS we could show that nano needles containing phosphorus and zinc were formed inside the living cells. Further more, the cells incubated with zinc show a strong fluorescence. Our findings indicate that the algae used polyphosphate bodies for detoxification of the zinc ions, leading to the generation of intracellular zinc-phosphate-based nano needles. Beside the technological application of this material, the fluorescent cells can be used for labeling of e.g. biological probes. This new experimental protocol for the production of an inorganic functional material can be applied also for other substances.
Cite this paper: G. Santomauro, V. Srot, B. Bussmann, P. A. van Aken, F. Brümmer, H. Strunk and J. Bill, "Biomineralization of Zinc-Phosphate-Based Nano Needles by Living Microalgae," Journal of Biomaterials and Nanobiotechnology, Vol. 3 No. 3, 2012, pp. 362-370. doi: 10.4236/jbnb.2012.33034.

[1]   T. Akamatsu and T. Kasuga, “Proton Conductivities of Zinc Phosphate Glass-Derived Hydrogels Controlled by Water Content,” Journal of the Electrochemical Society, Vol. 154, No. 2, 2007, pp. 258-262.

[2]   B. Yan and X. Xiao, “Hydrothermal Synthesis, Controlled Microstructure, and Photoluminescence of Hydrated Zn3(PO4)2: Eu3+ Nanorods and Nanoparticles,” Journal of Nanoparticle Research, Vol. 11, No. 8, 2009, pp.2125-2135. doi:10.1007/s11051-008-9578-6

[3]   M. Roming, C. Feldmann, Y. S. Avadhut and J. Schmedt auf der Günne, “Characterization of Noncrystalline Nano-materials: NMR of Zinc Phosphate as a Case Study,” Materials Chemistry, Vol. 20, No. 18, 2008, pp. 5787- 5795. doi:10.1021/cm800805f

[4]   J. D. Wang, D. Li, J. K. Liu, X. H. Yang, J. L. He and Y. Lu, “One-Step Preparation and Characterization of Zinc Phosphate Nanocrystals with Modified Surface,” Soft Nanoscience Letters, Vol. 1, 2011, pp. 81-85. doi:10.4236/snl.2011.13015

[5]   A. Q. Yuan, S. Liao, Z. F. Tong, J. Wu and Z. Y. Huang, “Synthesis of Nanopar-ticle Zinc Phosphate Dihydrate by Solid State Reaction at Room Temperature and Its Thermochemical Study,” Ma-terials Letters, Vol. 60, No. 17-18, 2006, pp. 2110-2114. doi:10.1016/j.matlet.2005.12.082

[6]   P. Parhi, V. Ma-nivannan, S. Kohli and P. McCurdy, “Room Temperature Metathetic Synthesis and Characterization of α-Hopeit, Zn3(PO4)2?4H2O,” Materials Research Bulletin, Vol. 43, No. 7, 2008, pp. 1836-1841. doi:10.1016/j.materresbull.2007.07.005

[7]   S. Yan, W. He, C. Sun, X. Zhang, H. Zhao, Z. Li, W. Zhou, X. Tian, X. Sun and X. Han, “The Biomimetic Synthesis of Zinc Phosphate Nanoparticles,” Dyes and Pigments, Vol. 80, No. 2, 2009, pp. 254-258. doi:10.1016/j.dyepig.2008.06.010

[8]   W. He, S. Yan, Y. Wang, X. Zhang, W. Zhou, X. Tian, X. Sun and X. Han, “Biomimetic Synthesis of Mesoporous Zinc Phosphate Nanoparticles,” Journal of Alloys and Compounds, Vol. 477, No. 1-2, 2009, pp. 657-660. doi:10.1016/j.jallcom.2008.10.136

[9]   D. Mandal, M. E. Bolander, D. Mukhopadhyay, G. Sarkar and P. Mukherjee, “The Use of Microorganisms for the Formation of Metal Nanoparticles and their Application” Applied Microbiology and Biotechnology, Vol. 69, No. 5, 2006, pp. 485-492. doi:10.1007/s00253-005-0179-3

[10]   F. Natalio, E. Mugnaioli, M. Wiens, X. Wang, H. C. Schr?der, M. N. Tahir, W. Tremel, U. Kolb and W. Müller, “Silicate-inmediated Incorporation of Titanium Into Spicules from the Demosponge Suberites domuncula,” Cell and Tissue Research, Vol. 339, No. 2, 2010, pp. 429-436. doi:10.1007/s00441-009-0903-x

[11]   L. C. Rai, “Heavy Metal Tolerance in Algae,” In: J. P. Gaur, Ed., Algal Adaptation to Environmental Stresses, Springer, Heidel-berg, 2001, pp. 363-388. doi:10.1007/978-3-642-59491-5

[12]   J. D. Keasling, “Regulation of Intracellular Toxic Metals and Other Cations by Hydrolysis of Polyphosphate,” The New York Academy of Sciences, Vol. 829, 1997, pp. 242- 249. doi:10.1111/j.1749-6632.1997.tb48579.x

[13]   Y. P. Ting, F. Lawson and I. G. Prince, “Uptake of Cadmium and Zinc by the Alga Chlorella vulgaris: Part 1. Individual Ion Species,” Biotechnology and Bioengineering, Vol. 34, No. 7, 1989, pp. 990-999. doi:10.1002/bit.260340713

[14]   K. Knauer, R. Behra and L. Sigg, “Adsorption and Uptake of Copper by the Green Alga Scendesmus subspicatus (Chlorophyta),” Journal of Phycology, Vol. 33, No. 4, 1997, pp. 596-601. doi:10.1111/j.0022-3646.1997.00596.x

[15]   D. R. Crist, R. H. Crist, J. R. Martin and J. R. Watson, “Ion Exchange System in Proton-Metal Reactions with Algal Cell Walls,” FEMS Microbiology Reviews, Vol. 14, No. 4, 1994, pp. 309-314. doi:10.1111/j.1574-6976.1994.tb00104.x

[16]   E. Kiefer, L. Sigg and P. Schosseler, “Chemical and Spectroscopic Characterization of Alga Surfaces,” Environmental Science & Technology, Vol. 31, No. 3, 1997, pp. 759-764. doi:10.1021/es960415d

[17]   T. P. Werner, N. Amrhein and F. M. Freimoser, “Inorganic Polyphosphate Occurs in the Cell Wall of Chlamydomonas reinhardtii and Accumulates during Cytokinesis,” BMC Plant Biology, Vol. 7, No. 7, 2007, pp. 51-62. doi:10.1186/1471-2229-7-51

[18]   G. W. Garnham, G. A. Codd and G. M. Gadd, “Kinetics of Uptake and Intracel-lular Location of Cobalt, Manganese and Zinc in the Estuarine Green Alga Chlorella salina,” Applied Microbiology and Biotechnology, Vol. 37, No. 2, 1992, pp. 270-276. doi:10.1007/BF00178183

[19]   T. E. Jensen, J. W. Rachlin, V. Jani and B. Warkentine, “An X-Ray Energy Dispersive Study of Cellular Compartmentalization of Lead and Zinc in Chlorella saccharophila (Chlo-rophyta), Navicula incerta and Nitzschia closterium (Ba-cillariophyta),” Environmental and Experimental Botany, Vol. 22, No. 3, 1982, pp. 319-328. doi:10.1016/0098-8472(82)90024-7

[20]   C. M. Monteiro, S. C. Fonseca, P. M. L. Castro and F. X. Malcata, “Toxicity of Cadmium and Zinc on Two Microalgae, Scendesmus obliquus and Desmodesmus pleiomorphus, from Northern Portugal,” Journal of Applied Phycology, Vol. 23, No. 23, 2011, pp. 97-103. doi:10.1007/s10811-010-9542-6

[21]   B. N. Tripathi and J. P. Gaur, “Physiological Behavior of Scendesmus Sp. During Exposure to Elevated Levels of Cu and Zn after Withdrawal of Metal Stress,” Protoplasma, Vol. 229, No. 1, 2006, pp. 1-9. doi:10.1007/s00709-006-0196-9

[22]   R.-Q. Yu and W.-X. Wang, “Biokinetics of Cadmium, Selenum, and Zinc in Freshwater Alga Scendesmus obliquus Under Different Phosphorus and Nitrogen Conditions and Metal Transfer to Daphnia magna,” Environmental Pollution, Vol. 129, No. 3, 2004, pp. 443-456. doi:10.1016/j.envpol.2003.11.013

[23]   H. H. Omar, “Bioremoval of Zinc Ions by Scendesmus obliquus and Scendesmus quadricauda and Its Effect on Growth and Metabolism,” International Biodeterioration & Biode-gradation, Vol. 50, No. 2, 2002, pp. 95-100. doi:10.1016/S0964-8305(02)00048-3

[24]   B. T. A. Muyssen and C. R. Janssen, “Zinc Acclimation and Its Effect on the Zinc Tolerance of Raphidocelis subcapitata and Chlorella vulgaris in Laboratory Experiments,” Chemosphere, Vol. 45, No. 4-5, 2001, pp. 507- 514. doi:10.1016/S0045-6535(01)00047-9

[25]   B. N. Tripathi, S. K. Mehta, A. Amar and J. P. Gaur, “Oxidative Stress in Scendesmus Sp. during Short- and Long-Term Exposure to Cu2+ and Zn2+,” Chemosphere, Vol. 62, No. 4, 2006, pp. 538-544. doi:10.1016/j.chemosphere.2005.06.031

[26]   N. Taka-mura, F. Kasai and M. M. Watanabe, “Effects of Cu, Cd and Zn on Photosynthesis of Freshwater Benthic Algae,” Journal of Applied Phycology, Vol. 1, No. 1, 1989, pp. 39-52. doi:10.1007/BF00003534

[27]   V. Cepák, P. P?ibyl, M. Vítová and V. Zachleder, “The Nucleocytosolic and Chloroplast Cycle in the Green Chlorococcal Alga Scendesmus obliquus (Chlorophyceae, Chlorococcales) Grown under Various Temperatures,” Phycologia, Vol. 46, No. 3, 2007, pp. 263-269. doi:10.2216/06-39.1

[28]   D. Schmitt, A. Müller, Z. Cs?g?r, F. H. Frimmel and C. Posten, “The Adsorption Kinetics of Metal Ions onto Different Microalgae and Si-liceous Earth,” Water Research, Vol. 35, No. 3, 2001, pp. 779-785. doi:10.1016/S0043-1354(00)00317-1

[29]   S. L. Faucheur, F. Schildknecht, R. Behra and L. Sigg, “Thiols in Scendesmus vacuolatus upon Exposure to Metals and Metalloids Aquatic,” Aquatic Toxicology, Vol. 80, No. 4, 2006, pp. 355-361. doi:10.1016/j.aquatox.2006.10.002

[30]   A. Kornberg, N. N. Rao and D. Ault-Riché, “Inorganic Polyphosphate: A Molecule of Many Functions,” Annual Review of Bio-chemistry, Vol. 68, 1999, pp. 89-125. doi:10.1146/annurev.biochem.68.1.89

[31]   Y. Komine, L. L. Eggink, H. Park and J. K. Hoober, “Vacuolar Granules in Chlamydomonas reinhardtii: Poly-phosphate and a 70-kDa Polypeptide as Major Components,” Planta, Vol. 210, No. 6, 2000, pp. 897-905. doi:10.1007/s004250050695

[32]   F. A. Ruiz, N. Marchesini, M. Seufferheld, Govindjee and R. Docampo, “The Polyphosphate Bodies of Chlamydomonas reinhardtii Possess a Proton-pumping Pyrophos- phatase and Are Similar to Acidocalcisomes,” The Jour- nal of Biological Chemistry, Vol. 276, 2001, No. 49, pp. 46196-46203. doi:10.1074/jbc.M105268200

[33]   K. Nishikawa, Y. Yamakoshi, I. Uemura and N. Tominaga, “Ultrastructural Changes in Chlamydomonas acidophila (Chlorophyta) Included by Heavy Metals and Polyphosphate Metabolism,” FEMS Microbiology Eco- logy, Vol. 44, No. 2, 2003, pp. 253-259. doi:10.1016/S0168-6496(03)00049-7

[34]   N. Perdrial, N. Liewig, J.-E. Delphin and F. Elsass, “TEM Evidence for Intracellular Accumulation of Lead by Bacteria in Sub-surface Environments,” Chemical Geology, Vol. 253, 2008, pp. 196-204.

[35]   L. Sicko-Goad and D. Lazinsky, “Quantitive Ultrastructural Changes Associated with Lead-Coupled Luxury Phosphate Uptake and Polyphos-phate Utilization,” Archives of Environmental Contami-nation and Toxicology, Vol. 15, No. 6, 1986, pp. 617-627. doi:10.1007/BF01054908

[36]   C. Ballan-Dufran?ais, C. Marcaillou and C. Amiard-Triquet, “Response of the Phytoplantonic Alga Tetraselmis suecica to Copper and Silver Exposure: Vesicular Metal Bioaccumulation and Lack of Starch Bodies,” Biology of the Cell, Vol. 72, 1991, pp. 103-112. doi:10.1016/0248-4900(91)90084-Z

[37]   S. L. Wong, L. Nakamoto and J. F. Wainwright, “Identification of Toxic Metals in Affected Algal Cells in Assays of Wastewaters,” Journal of Applied Phycology, Vol. 6, No. 4, 1994, pp. 405-414. doi:10.1007/BF02182157

[38]   W. Zhang and V. Majidi, “Monitoring the Cellular Response of Stichococcus bacillaris to Exposure of Several Different Metals Using in Vivo 31P NMR and Other Spectroscopic Techniques,” Environmental Science & Technology, Vol. 28, No. 9, 1994, pp. 1577-1581. doi:10.1021/es00058a007

[39]   R. Y. Stanier, R. Kunisawa, M. Mandel and G. Cohen- Bazire, “Purification and Properties of Unicellular Blue- Green Algae (Order Chroococcales),” Bacteriological Re- views, Vol. 35, 1971, pp. 171-205.

[40]   R. Rippka and M. Herdman, “Catalogue of Strains. Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture,” Institut Pasteur, Paris, 1992.

[41]   Z. Tukaj, A. Ba?cik-Remisiewicz, T. Skowroński and C. Tukaj, “Cadmium Effect on the Growth, Photosynthesis, Ultrastructure and Phytochelatin Content of Green Microalga Scendesmus armatus: A Study at Low and Elevated CO2 Concentration,” Envi-ronmental and Experimental Botany, Vol. 60, No. 3, 2007, pp. 291-299. doi:10.1016/j.envexpbot.2006.12.002

[42]   A. R. Spurr, “A Low-Viscosity Resin Embedding Medium for Electron Microscopy,” Journal of Ultrastructure Research, Vol. 26, No. 1-2, 1969, pp. 31-43. doi:10.1016/S0022-5320(69)90033-1

[43]   N. Reid, “Ul-tramicrotomy,” In: A. M. Glauert, Ed., Practical methods in electron microscopy, North-Holland Publishing Company, Amsterdam, 1975, pp. 215-350.

[44]   G. H. Michler and W. Lebek, “Ultramikrotomie, ” In: G. H. Michler, Ed., Ultramikrotomie in der Materialfor- schung, Hanser Verlag, Germany, 2004.

[45]   H. K. Hagler, “Ultrami-crotomy for Biological Electron Microscopy,” In: J. Kuo, Ed., Electron Microscopy, Humana Press, New Jersey, 2007.

[46]   K. Knauer, R. Behra and L. Sigg, “Effects of Free Cu2+ and Zn2+ Ions on Growth and Metal Accumulation in Freshwater Algae,” Environmental Toxicology and Chemistry, Vol. 16, No. 2, 1997, pp. 220-229. doi:10.1002/etc.5620160218

[47]   R. Kumar and D. Goyal, “Waste Water Treatment and Metal (Pb2+, Zn2+) Removal by Microalgal Based Stabilization Pond System,” Indian Journal of Microbiology, Vol. 50, Supp. 1, 2010, pp. 34-40. doi:10.1007/s12088-010-0063-4

[48]   T. Bisalputra and T. E. Weier, “The Cell Wall of Scen- desmus quadricauda,” American Journal of Botany, Vol. 50, No. 10, 1963, pp. 1011-1019. doi:10.2307/2439909

[49]   D. H. Miller, “Cell Wall Chemistry and Ultrastructure of Chlorococcum oleofaciens (Chlorophyceae),” Journal of Phycology, Vol. 14, No. 2, 1978, pp. 189-194. doi:10.1111/j.1529-8817.1978.tb02447.x

[50]   H. Takeda, “Cell Wall Sugars of Some Scendesmus Species,” Phyto-chemistry, Vol. 42, No., 3, 1996, pp. 673-675. doi:10.1016/0031-9422(95)00952-3

[51]   J. Sianoudis, A. C. Küsel, A. Mayer, L. H. Grimme and D. Leibfritz, “Distribution of Polyphosphates in Cell-Compartments of Chlorella fusca as Measured by 31P-NMR- spectros-copy,” Archives of Microbiology, Vol. 144, No., 1, 1986, pp. 48-54. doi:10.1007/BF00454955

[52]   L. Sicko-Goad, “A Morphometric Analysis of Algal Response to Low Dose, Short-Term Heavy Metal Exposure,” Protoplasma, Vol. 110, No., 2, 1982, pp. 75-86. doi:10.1007/BF01281533

[53]   M. A. Smith, “The Effect of Heavy Metals on the Cytoplasmic Fine Structure of Skeletonema costatum (Bacillariophyta),” Protoplasma, Vol. 116, No., 1, 1983, pp. 14- 23. doi:10.1007/BF01294226

[54]   J. W. Rachlin, T. E. Jensen and B. Warkentine, “The Toxicological Response of the Alga Anabaena flos-aquae (Cyanophyceae) to Cad-mium,” Archives of Environmental Contamination and Toxicology, Vol. 13, No., 1, 1984, pp. 143-151. doi:10.1007/BF01055871

[55]   T. Atici, H. Katircio?lu and B. Akm, “Sensitivity of Freshwater Microalgal Strains (Chlorella vulgaris Beijerinck and Scendesmus obliquus (Turpin) Kützing) to Heavy Metals,” Fresenius Environmental Bulletin, Vol. 17, No. 3, 2008, pp. 268-274.

[56]   H. S. Levinson, I. Mahler, P. Blackwelder and T. Hood, “Lead Resistance and Sensitivity in Sta-phylococcus aureus,” FEMS Microbiology Letters, Vol. 145, No. 3, 1996, pp. 421-425. doi:10.1111/j.1574-6968.1996.tb08610.x

[57]   L. E. Ma-caskie, A. C. R. Dean, A. K. Cheetham, R. J. B. Jakeman and A. J. Skarnulis, “Cadmium Accumulation by a Ci-trobacter Sp.: The Chemical Nature of the Accumulated Metal Precipitate and Its Location on the Bacterial Cells,” Journal of General Microbiology, Vol. 133, 1987, pp. 539-544.

[58]   P. E. de Almeida, J. R. M. van Rappard and J. C. Wu, “In Vivo Bioluminescense for Tracking Cell Fate and Function,” American Journal of Physiolo-gy—Heart and Circulatory Physiology, Vol. 301, No. 3, 2011, pp. 663-671. doi:10.1152/ajpheart.00337.2011

[59]   I. Belharouak, H. Aouad, M. Mesnaoui, M. Maazaz, C. Parent, B. Tanguy, P. Gravereau and G. Le Flem, “Crystal Structure and Lu-minescence Properties of Silver in AgM(PO3)3 (M = Mg, Zn, Ba) Polyphosphates,” Journal of Solid State Chemistry, Vol. 145, No. 1, 1999, pp. 97- 103. doi:10.1006/jssc.1999.8227