渗透与非渗透性抗冻剂联用技术对铜绿微囊藻的超低温保藏研究
DEVELOPMENT ON A CRYOPRESERVATION PROTOCOL OF MICROCYSTIS AERUGINOSA USING PENETRATING AND NON-PENETRATING CRYOPROTECTANTS
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摘要: 铜绿微囊藻(Microcystis aeruginosa Kütz)是一种在全世界分部很广的淡水水华蓝藻。在实验室长期的研究工作中, 为了使铜绿微囊藻能维持稳定的生理学特征, 通常使用超低温保藏技术长期冻存藻细胞。研究发现, 同时使用渗透性和非渗透性的抗冻剂比只使用传统的渗透性保护剂能显著提高Microcystis aeruginosa超低温保藏的存活率。以三株铜绿微囊藻为材料进行二步法超低温保藏, 对4 种抗冻剂(甲醇、二甲亚砜、丙三醇、聚乙烯吡咯烷酮), 两种降温速率(-1 /℃min、-0.5℃/min), 第一步温度设置(-30℃、-40℃、-80℃)进行筛选; 用流式细胞仪和细胞计数检测存活率, 并监测冻后相关生理参数、PSII、细胞色素、生长曲线等以确保该方法可保持藻株的活性和生理状态。结果表明, 5%的二甲亚砜和30%的聚乙烯吡咯烷酮(PVP)同时使用时能达到最好效果, 并能保持生理活性与冻前一致。Abstract: Microcystis aeruginosa Kütz is a freshwater bloom-forming cyanobacterium that is found all over the world. Owing to their adverse effects on water quality and human health, these blooms in water of reservoirs and recreational water systems are a cause of great concern, M. aeruginosa became to a popular research object. Previous studies have revealed significant differences between the physiological parameters and stress responses of individual cells in lab and wild type colonial of Microcystis in fields. Therefore, to avoid the loss of natural characteristics, an efficient technology or method would have to be used for preservation of M. aeruginosa. Cryopreservation is a useful method to preserve cells and organs for a long time in lab. It was thus used on M. aeruginosa in order to keep the physiological characteristics stable at long-term studies. In the present study, cryopreservation of three strains M. aeruginosa (PCC7806, FACHB-905 and FACHB-942) was accomplished successfully. Combination of three penetrating cryoprotective additives (CPA) (methanol, dimethylsulphoxide (Me2SO), glycerol) and non-penetrating polyvinylpyrrolidone (PVP) could improve viability almost 20% than only using the same penetrating CPA. The optimal protocol of cryopreservation was screened from CPAs above and four concentrations of each, two cooling rates (-1 /min ℃ n and -0.5℃/min), and the first-step temperature degreed (-30℃, -40℃ and -80℃) of traditional two step methods. In addition, cell viability was determined by performing flow cytometry with fluorescein diacetate (FDA). The results showed that an optimal cooling protocol was at cooling rate of 0.5℃/min to -40℃ and combination of penetrating CPA (Me2SO, 5%) and non-penetrating CPA (PVP, 30%). The ratios of viable cells in the FACHB-942, PCC-7806 and FACHB-905 were approximately (62.72 ± 0.15)%, (66.3 ± 0.65)%, and (71.69 ± 0.39)%, respectively. However, the ratios of viable cells in these strains were all only about 45%. At this protocol of cryopreservation, physiological parameters of three strains M. aeruginosa such as PSII, the cells pigments and specific growth rates were analyzed before preservation and after thaw, results about the comparison showed that M. aeruginosa could keep its physiological characteristics stable.
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Keywords:
- Cryopreservation /
- Microcystis aeruginosa /
- Cryoprotective additives
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[1] [A]. In: Method in enzymology [C]. Kluwer Academic Publishers,Dordrecht. 1988, 167
[1] Chen W, Peng L, Wan N, et al. Mechanism study on thefrequent variations of cell-bound microcystins in cyanobacterialblooms in Lake Taihu: Implications for water qualitymonitoring and assessments [J]. Chemosphere, 2009, 77:1585-1593
[2] Yoshimasa Y, Hiroyuki N. The formation and degradation ofcyanobacterium Aphanizomenon flos-aquae blooms: the importanceof pH, water temperature, and day length [J].Limnology, 2005, 1: 1-6
[3] Shen H, Song L R. Comparative studies on physiologicalresponses to phosphorus in two phenotypes of bloomformingMicrocystis [J]. Hydrobiologia, 2007, 592: 475-486
[4] Wu Z X, Song L R. Physiological comparison between colonialand unicellular forms of Microcystis aeruginosa Kütz.(Cyanobacteria) [J]. Phycologia, 2008, 47: 98-104
[5] Crutchfield A M, Diller K R, Brand J J. Cryopreservation ofChlamydomonas reinhardtii (Chlorophyta) [J]. EuropeanJournal of Phycology, 1999, 34: 43-52
[6] Peng L Y, Xiao Y M, Liu Y. Effect of cryopreservation andshort-term storage of Chinese giant salamander sperm [J].Acta Hydrobiologica Sinica, 2011, 35(2): 326-331 [彭亮跃,肖亚梅, 刘筠. 低温和超低温保存对中国大鲵成熟精子的影响. 水生生物学报, 2011, 35(2): 326-331]
[7] Wei P, Zhu J Q, Yan J Q, et al. Sperm cryopreservation andthe DNA damage detection in Pagrosomus major [J]. ActaHydrobiologica Sinica, 2010, 34(5): 1049-1053 [魏平, 竺俊全, 闫家强, 等. 真鲷精子的超低温冻存及 DNA 损伤的检测. 水生生物学报, 2010, 34(5): 1049-1053]
[8] Watanabe M M, Sawaguchi T. Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f.aeruginosa [J]. Phycological Research, 1995, 43: 111-116
[9] Mori F, Erata M, Watanabe M M. Cryopreservation ofcyanobacteria and green algae in the NIES-collection [J].Microbiology and Culture Collections, 2002, 18(1): 45-55
[10] Hwang S W, Hudock G A. Stability of Chlamydomonasreinhardi in liquid nitrogen storage [J]. Journal of Phycology,1971, 7: 300-303
[11] Jochem F J. Dark survival strategies in marine phytoplanktonassessed by cytometric measurement of metabolic activitywith fluorescein diacetate [J]. Marine Biology, 1999, 135:721-728
[12] Wu Z X, Song L R, Li R H. Different tolerances and responsesto low temperature and darkness between waterbloomforming cyanobacterium Microcystis and a green algaScenedesmus [J]. Hydrobiologia, 2008, 56(1): 47-55
[13] Maxwell K, Johnson G N. Chlorophyll fluorescence - a practicalguide [J]. Journal of Experimental Botany, 2000, 345:659-668
[14] Arnon D I. Copper enzymes in isolated chloroplasts. Polyphenoloxidasein Beta vulgaris [J]. Plant Physiology, 1949,24: 1-15
[15] Abelson J N, Simon M I. Phycobiliproteins in cyanobacteria
[16] Richards F A, Thompson T G. The estimation and characterizationof plankton populations by pigment analyses. II. Aspectrophotometric method for the estimation of planktonpigments [J]. Journal of Marine Research, 1952, 11: 156-172
[17] Wintermans J F G M, De Mots A. Spectrophotometric characteristicsof chlorophyll and their pheophytins in ethanol [J].Biochimica et Biophysica Acta, 1965, 109: 448-453
[18] Saks N M. The preservation of salt marsh algae by controlledliquid nitrogen storage [J]. Cryobiology, 1978, 15: 563-568
[19] Ben-Amotz A, Gilboa A. Cryopreservation of marine unicellularalgae. Part I. A survey of algae with regard to size, cultureage, photosynthetic activity and chlorophyll-to-cell ratio[J]. Marine Ecology Progress Series, 1980, 2: 157-161
[20] Mazur P. Freezing of living cells: mechanisms and implications[J]. American Journal of Physiology-Cell Physiology,1984, 247: 125-142
[21] Fleck R A. The assessment of cell damage and recovery incryopreserved freshwater protists [D]. PhD Thesis, UniversityAbertay Dundee. 1998
[22] Morris G J. Cryopreservation of 250 strains of chlorococcalesby the method of two-step cooling [J]. British PhycologicalJournal, 1978, 13: 15-24
[23] Morris G J. The cryopreservation of Chlorella. 1. Interactionsof rate of cooling, protective additive and warning rate[J]. Archives of Microbiology, 1976, 107: 57-62
[24] Day J G, Harding K. Chapter 6 Cryopreservation of Algae[A]. In: Reed B M (Eds.), Plant Cryopreservation: A PracticalGuide [C], Springer, 2008, 95-116
[25] Benhra A, Ferard J F, Vasseur P. Factorial design to optimizethe viability of the alga Scenedesmus subspicatus after cryopreservation[J]. Cryo-Letters, 1994, 15: 269-278
[26] Meryman H T, Williams R J, Douglas M S. Freezing injuryfrom solution effects and its prevention by natural orartificial cryoprotection [J]. Cryobiology, 1977, 14: 287-302
[27] Zdenek H. Protectants used in the cryopreservation of microorganisms[J]. Cryobiology, 2003, 46: 205-229
[28] Sadakane H, Ishibashi K, Yoshimoto M, et al. Effects of lowtemperature, light, and O2 on chilling-sensitive and -resistantstrains in Chlorella ellipsoidea [J]. Plant and Cell Physiology,1981, 22: 657-666
[29] Sathyendranathe S, Lazzara L, Prieur L, Variations in thespectral values of specific absorption of phytoplankton [J].Limnology and Oceanography, 1987, 32: 403-415
[30] Gahan P B. Plant Histochemistry and Cytochemistry [M].London: Academic Press. 1984, 42-47
[31] Li J, Song L R. Applicability of the MTT assay for measuringviability of cyanobacteria and algae, specifically for Microcystisaeruginosa (Chroococcales, Cyanobacteria) [J].Phycologia, 2007, 46: 593-599
[32] Day J G, Harding K C, Nadarajan J, et al. Cryopreservation:Conservation of Bioresources at Ultra Low Temperatures [A].In: Walker J M, Rapley R (Eds.), Molecular BiomethodsHandbook [C]. Humana Press. 2008, 917-947
[33] Jochem F J. Dark survival strategies in marine phytoplanktonassessed by cytometric measurement of metabolic activitywith fluorescein diacetate [J]. Marine Biology, 1999, 135:721-728
[34] Latour D H, Giraudet H, Berthon J L. Frequency of dividingcells and viability of Microcystis aeruginosa in sediment of aeutrophic reservoir [J]. Aquatic Microbial Ecology, 2004, 36:117-122
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