Abstract
The filamentous Cyanobacterium Arthrospira is commercially produced and is a functional, high-value, health food. We identified 5 low temperature and low light intensity tolerant strains of Arthrospira sp. (GMPA1, GMPA7, GMPB1, GMPC1, and GMPC3) using ethyl methanesulfonate mutagenesis and low temperature screening. The 5 Arthrospira strains grew rapidly below 14 °C, 43.75 μmol photons m−2 s−1 and performed breed conservation at 2.5 °C, 8.75 μmol photons m−2 s−1. We used morphological identification and molecular genetic analysis to identify GMPA1, GMPA7, GMPB1 and GMPC1 as Arthrospira platensis, while GMPC3 was identified as Arthrospira maxima. Growth at different culture temperatures was determined at regular intervals using dry biomass. At 16 °C and 43.75 μmol photons m−2 s−1, the maximum dry biomass production and the mean dry biomass productivity of GMPA1, GMPB1, and GMPC1 were 2057 ± 80 mg l−1, 68.7 ± 2.5 mg l−1 day−1, 1839 ± 44 mg l−1, 60.6 ± 1.8 mg l−1 day−1, and 2113 ± 64 mg l−1, 77.7 ± 2.5 mg l−1 day−1 respectively. GMPB1 was chosen for additional low temperature tolerance studies and growth temperature preference. In winter, GMPB1 grew well at mean temperatures <10 °C, achieving 3258 mg dry biomass from a starting 68 mg. In summer, GMPB1 grew rapidly at mean temperatures more than 28 °C, achieving 1140 mg l−1 dry biomass from a starting 240 mg. Phytonutrient analysis of GMPB1 showed high levels of C-phycocyanin and carotenoids. Arthrospira metabolism relates to terpenoids, and the methyl-d-erythritol 4-phosphate pathway is the only terpenoid biosynthetic pathway in Cyanobacteria. The 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) gene from GMPB1 was cloned and phylogenetic analysis showed that GMPB1 is closest to the Cyanobacterium Oscillatoria nigro-viridis PCC711. Low temperature tolerant Arthrospira strains could broaden the areas suitable for cultivation, extend the seasonal cultivation time, and lower production costs.
Similar content being viewed by others
References
Aikawa S, Joseph A, Yamada R et al (2013) Direct conversion of Spirulina to ethanol without pretreatment or enzymatic hydrolysis processes. Energy Environ Sci 6:1844–1849. doi:10.1039/c3ee40305j
Andrade MR, Grande RS (2008) Outdoor and indoor cultivation of spirulina platensis in the extreme south of Brazil. Z Naturforsch C 63:85–90
Andreas B, Panwan KD, Lothar K (2004) Phylogenetic realationship of Arthrospira, Phormidium and Spirulina strains from Kenyan and Indian waterbodies. Algol Stud 113:37–56. doi:10.1127/1864-1318/2004/0113-0037
Avila-Leon M, Matsudo C, Sato S et al (2012) Arthrospira platensis biomass with high protein content cultivated in continuous process using urea as nitrogen source. J Appl Microbiol 112:1086–1094
Baurain D, Renquin L, Grubisic S et al (2002) Remarkable conservation of internally transcribed spacer sequences of Arthrospira (Spirulina) (Cyanophyceae, Cyanobacteria) strains from four continents and of recent and 30-year-old dried samples from African. J Phycol 38:384–393. doi:10.1046/j.1529-8817.2002.01010.x
Belay A (1997) Mass culture of Spirulina outdoors the earthrise farms experience. In: Vonshak A (ed) Spirulina platensis (Arthrospira): physiology, cell-biology and biotechnology. Taylor and Francis, London, pp 131–158
Belay A (2008) Spirulina (Arthrospira): production and quality assurance. In: Gershwin ME, Belay A (eds) Spirulina in human nutrition and health. CRC Press, Boca Raton, pp 1–26
Castro GF, Rizzo RF, Passos TS et al (2015) Biomass production by Arthrospira platensis under different culture conditions. Food Sci Technol (Campinas) 35:18–24
Cavalier-Smith T (1999) Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree. J Eukaryot Microbiol 46:347–366
Chaturvedi R, Fujita Y (2006) Isolation of enhanced eicosapentaenoic acid producing mutants of Nannochloropsis oculata ST-6 using ethyl methane sulfonate induced mutagenesis techniques and their characterization at mRNA transcript level. Phycol Res 54: 208–219
Chen CY, Kao PC, Tsai CJ et al (2013) Engineering strategies for simultaneous enhancement of C-phycocyanin production and CO2 fixation with Spirulina platensis. Bioresour Technol 145:307–312
Ciferri O (1983) Spirulina, the edible microorganism. Microbiol Rev 47(4):551–578
Cohen Z (1997) Morphology, ultrastructure and taxonomy of Arthrospira (Spirulina) maxima and Arthrospira (Spirulina) platensis. In: Vonshak A (ed) Spirulina (Arthrospira) platensis: physiology, cell-biology and biotechnology. Taylor and Francis, London, pp 175–204
Colla LM, Bertolin TE, Costa JAV (2004) Fatty acids profile of Spirulina platensis grown under different temperatures and nitrogen concentrations. Z Naturforsch C 59:55–59
Colla LM, Reinehr CO, Reichert C, Costa JAV (2007) Production of biomass and nutraceutical compounds by Spirulina platensis under different temperature and nitrogen regimes. Bioresour Technol 98:1489–1493
Depraetere O, Deschoenmaeker F, Badri H et al (2015a) Trade-off between growth and carbohydrate accumulation in nutrient-limited Arthrospira sp. PCC 8005 studied by integrating transcriptomic and proteomic approaches. PLoS ONE 10(7):e0132461. doi:10.1371/journal.pone.0132461
Depraetere O, Pierre G, Noppe W et al (2015b) Influence of culture medium recycling on the performance of Arthrospira platensis cultures. Algal Res 10:48–54
Ding ML, Yu ZQ (1997) Spirulina industry in China: present status and future prospects. J Appl Phycol 9:25–28
Doan TTY, Obbard JP (2012) Enhanced intracellular lipid in Nannochloropsis sp. via random mutagenesis and flow cytometric cell sorting. Algal Res 1:17–21
Eriksen NT (2008) Production of phycocyanin-a pigment with applications in biology, biotechnology, foods and medicine. Appl Microbiol Biotechnol 80:1–14
Gao KS, Li P, Watanabe T, Walter Helbling E (2008) Combined effects of ultraviolet radiation and temperature on morphology, photosynthesis, and DNA of Arthrospira (Spirulina) platensis (cyanophyta). J Phycol 44:777–786
Guedes AC, Amaro HM, Malcata FX (2011) Microalgae as sources of carotenoids. Mar Drugs 9:625–644
Gutiérrez-Salmeán G, Fabila-Castillo L, Chamorro-Cevallos G (2015) Nutritional and toxicological aspects of Spirulina (Arthrospira). Nutr Hosp 32(1):34–40
Halfmann C, Gu L, Zhou R (2014) Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO2 and H2O. Green Chem 16:3175–3185. doi:10.1039/c3gc42591f
Hu ZM, Zeng XQ, Wang AH et al (2004) An efficient method for DNA isolation from red algae. J Appl Phycol 16:161–166
Koru E (2012) Earth food Spirulina (Arthrospira): production and quality standarts. In: El-Samragy Y (ed) Food additive. InTech, Rijeka, pp 191–203
Kuddus M, Singh P, Thomas G, Al-Hazimi A (2013) Recent developments in production and biotechnological applications of C-phycocyanin. Biomed Res Int. doi:10.1155/2013/742859
Kudoh K, Kawano Y, Hotta S et al (2014) Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis. J Biosci Bioeng 118:20–28
Langlois A, Lebel O (2010) To cyclopropanate or not to cyclopropanate? A look at the effect of cyclopropanation on the performance of biofuels. Energy Fuels 24:5257–5263
Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: measurement and characterization by UV–VIS spectroscopy. In: Wrolstad RE (ed) Current protocols in food analytical chemistry. Wiley, New York, pp F4.3.1–F4.3.8
Madhulika S, Rizwana T, Rajendra S et al (2016) Influence of light intensity, temperature and CO2 concentration on growth and lipids in green algae and cyanobacteria. Indian J Exp Biol 54:482–487
Markou G, Georgakakis D (2011) Cultivation of filamentous cyanobacteria (blue-green algae) in agro-industrial wastes and wastewaters: a review. Appl Energy 88:3389–3401
Markou G, Iconomou D, Muylaert K (2016) Applying raw poultry litter leachate for the cultivation of Arithrospira platensis and Chlorella vulgaris. Algal Res 13:79–84
Mühling M, Somerfield P, Harris N et al (2006) Phenotypic analysis of Arthrospira (Spirulina) strains (Cyanophyceae). Phycologia 45:148–157
Ogato T, Kifle D (2014) Morphological variability of Arthrospira (Spirulina) fusiformis (Cyanophyta) in relation to environmental variables in the tropical soda lake Chitu, Ethiopia. Hydrobiologia 738:21–33
Pattanaik B, Lindberg P (2015) Terpenoids and Their Biosynthesis in Cyanobacteria. Life (Basel) 5:269–293. doi:10.3390/life5010269
Piorrect M, Klaus-Hinnerk B, Pohl P (1984) Biomass production, total protein, chlorophylls, lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes. Phytochemistry 23:207–216
Price GD, Badger MR (1989) Isolation and characterization of high CO2-requiring-mutants of cyanobacterium Synechococcus the PCC7942. Plant Physiol 91:514–525
Qiao C, Tian XY, Li SY (2013) The classification and morphological anatomical structure of Spirulina (Arithrospira). In: Qiao C, Li SY (eds) Spirulina (Arithrospira) in Alkaline Lakes of the Erdos Plateau. Science Press, Beijing, pp 43–76
Raina P, Anshuman G, Anamika T (2011) Impact of environmental factors on the biomass production of spirulina in different conditions. VEGETOS 24:142–148
Richmond A (1986) Microalgae of economic potential In: Richmond A (ed) Handbook of microalgal mass culture. CRC Press, Boca-Raton, Florida, pp 199–243
Rio-Chanona EAD, Zhang DD, Xie YP et al (2015) Dynamic simulation and optimization for Arthrospira platensis growth and C-phycocyanin production. Ind Eng Chem Res 54:10606–10614
Rohmer M (2003) Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosyntheis. Elucidation and distribution. Pure Appl Chem 75:375–387
Rout NP, Khandual S, Gutierrez-Mora A et al (2015) Divergence in three newly identified Arthrospira species from Mexico. World J Microbiol Biotechnol 31:1157–1165
Silva AFD, Lourenco SO, Chaloub RM (2009) Effects of nitrogen starvation on the photosynthetic physiology of a tropical marine microalga Rhodomonas sp. (Cryptophyceae). Aquat Bot 91:291–297
Spolaore P, Joannis-Cassana C, Duran E et al (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96
Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9(3):297–304
Tomaselli L (1997) Morphology, ultrastructure and taxonomy of Arthrospira (Spirulina) maxima and Arthrospira (Spirulina) platensis. In: Vonshak A (ed) Spirulina (Arthrospira) platensis: physiology, cell-biology and biotechnology. Taylor and Francis, London, pp 79–99
Toyoshima M, Aikawa S, Yamagishi T et al (2015) A pilot-scale floating closed culture system for the multicellular cyanobacterium Arthrospira platensis NIES-39. J Appl Phycol 27:2191–2202
Trabelsi L, Ben Ouada H, Bacha H, Ghoul M (2009) Combined effect of temperature and light intensity on growth and extracellular polymeric substance production by the cyanobacterium Arthrospira platensis. J Appl Phycol 21:405–412
Vonshak A, Tomaselli L (2000) Arthrospira (Spirulina): systematics and ecophysiology. In: Whitton BA, Potts M (ed) The ecology of Cyanobacteria. Kluwer, Dordrecht, pp 505–522
Vonshak A, Laorawat S, Bunnag B, Tanticharoen M (2014) The effect of light availability on the photosynthetic activity and productivity of outdoor cultures of Arthrospira platensis (Spirulina). J Appl Phycol 26:1309–1315
Wilmotte A, Turner S, Van de Peer Y, Pace NR (1992) Taxonomic study of marine oscillatoriacean strains (cyanobacteria) with narrow trichomes II. Nucletide sequence analysis of the 16S ribosomal RNA. J Phycol 28:828–838
Xie YP, Jin YW, Zeng XH et al (2015) Fed-batch strategy for enhancing cell growth and C-Phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation. Bioresour Technol 180:281–287
Yang ZY, Pan WS, Nie Y, Luo H, Wang JW (2014) Breeding of Shiraia sp. strain with high-yield hypocrellin by EMS mutation. Chin J Bioprocess Eng. doi:10.3969/jissn.1672-3678.2014.02.005
Yoshikawa N, Belay A (2008) Single-laboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in Spirulina (Arithrospira) supplements and raw materials by spectrophotometry. J AOAC Int 91(3):524–529
Zittelli GC, Tomasello V, Pinzani E et al (1996) Outdoor cultivation of Arithrospira platensis during autumn and winter in temperate climates. J Appl Phycol 8:293–301
Acknowledgements
We thank the Jiangsu Cibainian Nutrition Food Co., Ltd Dongtai City Top Bio-Engineering Co., Ltd for providing Arthrospira strains.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Guan, J., Shen, S., Wu, H. et al. Biomass and terpenoids produced by mutant strains of Arthrospira under low temperature and light conditions. World J Microbiol Biotechnol 33, 33 (2017). https://doi.org/10.1007/s11274-016-2199-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-016-2199-9