Assessing Storage of Stability and Mercury Reduction of Freeze-Dried Pseudomonas putida within Different Types of Lyoprotectant
Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage in 4oC without vacuum. Polyethylene glycol (PEG) pre-treated freeze dry cells and broth pre-treated freeze dry cells after freeze-dry recovered more than 64% (5.0 E+09CFU/ml) and >0.1% (5.60E+07CFU/ml). Freeze-dried P. putida cells in PEG and broth cannot survive after 4 weeks storage. Freeze dry also does not really change the pattern of growth P. putida but extension of lag time was found 1 hour after 3 weeks of storage. Additional time was required for freeze-dried P. putida cells to recover before introduce freeze-dried cells to more complicated condition such as mercury solution. The maximum mercury reduction of PEG pre-treated freeze-dried cells after freeze dry and after storage 3 weeks was 56.78% and 17.91%. The maximum of mercury reduction of tween 80/sucrose pre-treated freeze-dried cells after freeze dry and after storage 3 weeks were 26.35% and 25.03%. Freeze dried P. putida was found to have lower mercury reduction compare to the fresh P. putida that has been growth in agar. Result from this study may be beneficial and useful as initial reference before commercialize freeze-dried P. putida.
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 Morgan, C. A., Herman, N., White, P. A., & Vesey, G. (2006). Preservation of Micro-organismby Drying; A review. Journal of Microbiological Methods, 183-193.
 Castein, H. V., Li, Y., Timmis, K. N., Deckwer, K. N., & Dobler, I. W. (1999). Removal of Mercury from Chlorakali Electrolysis Wastewater by a Mercury Resistance Pseudomonas putida Strain. Applied and Environmental Microbiology, 5279-5284.
 Mortazavi, S., Razaee, A., Khavanin, A., Varmazyar, S., & Jafarzadeh, M. (2005). Removal of Mercuric by Mercury Resistance Pseudomonas puida Strain. Journal of Biological Sciences, 269-273.
 Azoddein, A. B. (2013). Mercury Removal From Petroleum Based Industries Wastewater by Pseuomonas putida ATCC 49128 in Membran Bioreactor. Pahang: University Malaysia Pahang.
 Vijayaraghavan, K., & Yun, Y.-S. (2008). Bacterial Biosorbents and Biosorption. Biotechnology Advances, 266-291.
 Shafeeq, A., Muhammad, A., Sarfraz, W., Toqeer, A., Rashid, S., & Rafiq, M. K. (2012). Mercury Removal Techniques for Industrial Waste Water. World Academy of Science, Engineering and Technology, 12-16.
 Fonseca, P., Moreno, R., & Fernando, R. (2011). Grwoth of Pseudomonas putida at Low Temperature : Global Transcriptomic and Proteomic Analysis. Environmental Microbiology Reports.
 Wenfeng , S., Gooneratne, R., Glithero, N., Weld, R. J., & Pasco, N. (2013). Appraising Freeze-Drying for Storage of Bacteria and Their Reafy Access in a Rapid Toxicity Assessment Assay. Environmental Biotechnology , 10189-10198.
 Poddar, D., Das, S., Jones, G., Palmer, J., Jameson, G. B., Haverkamp, R. G., et al. (2014). Stability of probiotic Lactobacillus paracasei during storage as affected by the drying method. International Dairy Journal, 1-7.
 Miyamoto-Shinohara, Y., Imaizumi, T., Sukenobe, J., Murakami, Y., Kawamura, S., & Komatsu, Y. (2000). Survival Rate of Microbes After Freeze-Drying and Long-Term Storage. Cryobiology, 251-155.
 Morgan, C., & Vesey, G. (2009). Freeze-Drying of Microorganisms. Encyclopedia of Microbiology (Third Edition), 162-173.
 Schoug, A. (2009). A Dry Phase of Life. Uppsala: Swedish University of Agricultural Sciences.
 Cote, R. J. (1998). Current Protocols in Cell Biology . Maryland: John Wiley Sons.
 Palmfedt, J., Radstrom, P., & Hahn-Hagerdal, B. (2003). Optimisation of Initial Cell Concentration Enhances Freeze-Drying Tolerance of Pseudomonas Chlororaaphis. Crybiology, 21-29.
 Chess, B. (2009). Laboratory Application Microbiology : A Case Study Approach. New York: McGraw-Hill.
 Hubalek, Z. (2003). Protectants Used in The Cryopreservation of Microorganisms. Cryobiology, 205-229.
 Song, W., Ma, D., & Wei, X. (2014). Assessing Cell Viability And Biochemical Stability of Freeze-Dried Sulphate -dried Sulphate - reducing bacteria within different types of cryoprotectants and storage conditions. Int.J.Curr.Microbiol.App.Sci, 659-670.
 Santivarangkna, C., Higl, B., & Foerst, P. (2008). Protection mechanisms of sugars during different stages of preparation process of dried lactic acid starter cultures. Food Microbiology, 429-441.
 Kuleshova, L. L., MacFarlane, D. R., Trounson, A. O., & Shaw, J. M. (1999). Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology, 119-130.
 Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membrans and proteins intact bacteria during drying. Environmental Biology, 3592-3597.
 Berner, D., & Viernstein, H. (2006). Effect of protective agents on the viability of Lactococcus lastics subjected to freeze--thawing and freeze-drying. Scientia Pharmaceutica, 137-149.
 Adams, G. (2007). The Principles of Freeze Drying. In J. G. Day, & G. N. Stacey, Cryopreservation and Freeze-Drying Protocols (pp. 15-38). Totowa: Humana Press Inc.
 Wong, P. K., & Chu, L. M. (2003). Treatment of Soild Hazardous Wastes. In L. Y. Kun, Microbial Biotechnology Principles and Applications (pp. 625-652). Singapore: World Scientific Publishing.
 Wesche, A. M., Gurtler, J. B., Marks, B. P., & Ryser, E. T. (2009). Stress, sublethal injury, resuscitation and virulence of bacteria foodborne pathogens. Journal of Food Protection, 1121--1138.
 A. A. M. Azoddein, R. M. Yunus, N. M. Sulaiman, A. B. Bustary, F. A. Azli. (2015). Mercury Removal From Petroleum Based Industries Wastewater By P. Putida, 4(6).