Abstract
The green alga Pseudokirchneriella subcapitata is widely used in ecotoxicity assays and has great biotechnological potential as feedstock. This work aims to characterize the physiology of this alga associated with the aging resulting from the incubation of cells for 21 days, in the OECD medium, with continuous agitation and light exposure, in a batch mode. After inoculation, cells grow exponentially during 3 days, and the culture presents a typical green color. In this phase, “young” algal cells present, predominantly, a lunate morphology with the chloroplast occupying a large part of the cell, maximum photosynthetic activity and pigments concentration, and produce starch as a reserve material. Between the 5th and the 12th days of incubation, cells are in the stationary phase. The culture becomes less green, and the cells stop dividing (≥ 99% have one nucleus) and start to age. “Old” algal cells present chloroplast shrinkage, an abrupt decline of chlorophylls content, and photosynthetic capacity (Fv/Fm and ɸPSII), accompanied by a degradation of starch and an increase of neutral lipids content. The onset of the death phase occurs after the 12th day and is characterized by the loss of cell membrane integrity of some algae (cell death). The culture stays, progressively, yellow, and the majority of the population (~93%) is composed of live cells, chronologically “old,” with a significant drop in photosynthetic activity (decay > 75% of Fv/Fm and ɸPSII) and starch content. The information here achieved can be helpful when exploring the potential of this alga in toxicity studies or in biotechnological applications.
Key points
• Physiological changes of P. subcapitata with chronological aging are shown
• “Young” algae exhibit a semilunar shape, high photosynthetic activity, and accumulated starch
• “Old”-live algae show reduced photosynthetic capacity and accumulated lipids
Graphical Abstract
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this published article.
References
Andersson A, Keskitalo J, Sjödin A, Bhalerao R, Sterky F, Wissel K, Tandre K, Aspeborg H, Moyle R, Ohmiya Y, Bhalerao R, Brunner A, Gustafsson P, Karlsson J, Lundeberg J, Nilsson O, Sandberg G, Strauss S, Sundberg B, Uhlen M, Jansson S, Nilsson P (2004) A transcriptional timetable of autumn senescence. Genome Biol 5:R24. https://doi.org/10.1186/gb-2004-5-4-r24
Armstrong GA, Hearst JE (1996) Genetics and molecular biology of carotenoid pigment biosynthesis. FASEB J 10:228–237. https://doi.org/10.1096/fasebj.10.2.8641556
Aswathi Mohan A, Robert Antony A, Greeshma K, Yun J-H, Ramanan R, Kim H-S (2022) Algal biopolymers as sustainable resources for a net-zero carbon bioeconomy. Bioresour Technol 344:126397. https://doi.org/10.1016/j.biortech.2021.126397
Atikij T, Syaputri Y, Iwahashi H, Praneenararat T, Sirisattha S, Kageyama H, Waditee-Sirisattha R (2019) Enhanced lipid production and molecular dynamics under salinity stress in green microalga Chlamydomonas reinhardtii (137C). Mar Drugs 17:484. https://doi.org/10.3390/md17080484
Benasla A, Hausler R (2020) Growth and production of lipids in Raphidocelis subcapitata immobilized in sodium alginate beads. Energies 13:506. https://doi.org/10.3390/en13020506
Bondioli P, Della Bella L, Rivolta G, Chini Zittelli G, Bassi N, Rodolfi L, Casini D, Prussi M, Chiaramonti D, Tredici MR (2012) Oil production by the marine microalgae Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33. Bioresour Technol 114:567–572. https://doi.org/10.1016/j.biortech.2012.02.123
Borowitzka MA (2013) High-value products from microalgae—their development and commercialisation. J Appl Phycol 25:743–756. https://doi.org/10.1007/s10811-013-9983-9
Brányiková I, Maršálková B, Doucha J, Brányik T, Bišová K, Zachleder V, Vítová M (2011) Microalgae-novel highly efficient starch producers. Biotechnol Bioeng 108:766–776. https://doi.org/10.1002/bit.23016
Breuer G, Martens DE, Draaisma RB, Wijffels RH, Lamers PP (2015) Photosynthetic efficiency and carbon partitioning in nitrogen-starved Scenedesmus obliquus. Algal Res 9:254–262. https://doi.org/10.1016/j.algal.2015.03.012
Cheng D, Li D, Yuan Y, Zhou L, Li X, Wu T, Wang L, Zhao Q, Wei W, Sun Y (2017) Improving carbohydrate and starch accumulation in Chlorella sp. AE10 by a novel two-stage process with cell dilution. Biotechnol Biofuels 10:75. https://doi.org/10.1186/s13068-017-0753-9
Cid A, Prado R, Rioboo C, Suarez-Bregua P, Herrero C (2013) Use of microalgae as biological indicators of pollution: looking for new relevant cytotoxicity endpoints. In: Johnsen MN (ed) Microalgae: Biotechnology, microbiology and energy. Nova Science Publishers Inc, New York, pp 311–324
Consalvey M, Perkins RG, Paterson DM, Underwood GJC (2005) PAM fluorescence: a beginners guide for benthic diatomists. Diatom Res 20:1–22. https://doi.org/10.1080/0269249X.2005.9705619
Damoo DY, Durnford DG (2021) Long-term survival of Chlamydomonas reinhardtii during conditional senescence. Arch Microbiol 203:5333–5344. https://doi.org/10.1007/s00203-021-02508-y
Del Río E, García-Gómez E, Moreno JG, Guerrero M, García-González M (2017) Microalgae for oil. Assessment of fatty acid productivity in continuous culture by two high-yield strains, Chlorococcum oleofaciens and Pseudokirchneriella subcapitata. Algal Res 23:37–42. https://doi.org/10.1016/j.algal.2017.01.003
Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta - Bioenerg 1020:1–24. https://doi.org/10.1016/0005-2728(90)90088-L
Drábková M, Matthijs HCP, Admiraal W, Maršálek B (2007) Selective effects of H2O2 on cyanobacterial photosynthesis. Photosynthetica 45:363–369. https://doi.org/10.1007/s11099-007-0062-9
Fai PB, Grant A, Reid B (2007) Chlorophyll a fluorescence as a biomarker for rapid toxicity assessment. Env Toxicol Chem 26:1520–1531. https://doi.org/10.1897/06-394R1.1
Fernandes B, Teixeira J, Dragone G, Vicente AA, Kawano S, Bišová K, Přibyl P, Zachleder V, Vítová M (2013) Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri. Bioresour Technol 144:268–274. https://doi.org/10.1016/j.biortech.2013.06.096
Fernández C, Asselborn V, Parodi ER (2021) Toxic effects of chlorpyrifos, cypermethrin and glyphosate on the non-target organism Selenastrum capricornutum (Chlorophyta). An Acad Bras Cienc 93:e20200233. https://doi.org/10.1590/0001-3765202120200233
Florea M (2017) Aging and immortality in unicellular species. Mech Ageing Dev 167:5–15. https://doi.org/10.1016/j.mad.2017.08.006
Fröhlich KU, Madeo F (2000) Apoptosis in yeast-a monocellular organism exhibits altruistic behaviour. FEBS Lett 473:6–9. https://doi.org/10.1016/s0014-5793(00)01474-5
Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L, Amelio I, Andrews DW, Annicchiarico-Petruzzelli M, Antonov AV, Arama E, Baehrecke EH, Barlev NA, Bazan NG, Bernassola F, Bertrand MJM, Bianchi K, Blagosklonny MV, Blomgren K, Borner C, Boya P, Brenner C, Campanella M, Candi E, Carmona-Gutierrez D, Cecconi F, Chan FK-M, Chandel NS, Cheng EH, Chipuk JE, Cidlowski JA, Ciechanover A, Cohen GM, Conrad M, Cubillos-Ruiz JR, Czabotar PE, D’Angiolella V, Dawson TM, Dawson VL, De Laurenzi V, De Maria R, Debatin K-M, DeBerardinis RJ, Deshmukh M, Di Daniele N, Di Virgilio F, Dixit VM, Dixon SJ, Duckett CS, Dynlacht BD, El-Deiry WS, Elrod JW, Fimia GM, Fulda S, García-Sáez AJ, Garg AD, Garrido C, Gavathiotis E, Golstein P, Gottlieb E, Green DR, Greene LA, Gronemeyer H, Gross A, Hajnoczky G, Hardwick JM, Harris IS, Hengartner MO, Hetz C, Ichijo H, Jäättelä M, Joseph B, Jost PJ, Juin PP, Kaiser WJ, Karin M, Kaufmann T, Kepp O, Kimchi A, Kitsis RN, Klionsky DJ, Knight RA, Kumar S, Lee SW, Lemasters JJ, Levine B, Linkermann A, Lipton SA, Lockshin RA, López-Otín C, Lowe SW, Luedde T, Lugli E, MacFarlane M, Madeo F, Malewicz M, Malorni W, Manic G, Marine J-C, Martin SJ, Martinou J-C, Medema JP, Mehlen P, Meier P, Melino S, Miao EA, Molkentin JD, Moll UM, Muñoz-Pinedo C, Nagata S, Nuñez G, Oberst A, Oren M, Overholtzer M, Pagano M, Panaretakis T, Pasparakis M, Penninger JM, Pereira DM, Pervaiz S, Peter ME, Piacentini M, Pinton P, Prehn JHM, Puthalakath H, Rabinovich GA, Rehm M, Rizzuto R, Rodrigues CMP, Rubinsztein DC, Rudel T, Ryan KM, Sayan E, Scorrano L, Shao F, Shi Y, Silke J, Simon H-U, Sistigu A, Stockwell BR, Strasser A, Szabadkai G, Tait SWG, Tang D, Tavernarakis N, Thorburn A, Tsujimoto Y, Turk B, Vanden Berghe T, Vandenabeele P, Vander Heiden MG, Villunger A, Virgin HW, Vousden KH, Vucic D, Wagner EF, Walczak H, Wallach D, Wang Y, Wells JA, Wood W, Yuan J, Zakeri Z, Zhivotovsky B, Zitvogel L, Melino G, Kroemer G (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 25:486–541. https://doi.org/10.1038/s41418-017-0012-4
Gantt E (1975) Phycobilisomes: light-harvesting pigment complexes. Bioscience 25:781–788. https://doi.org/10.2307/1297221
Gao Y, Yang M, Wang C (2013) Nutrient deprivation enhances lipid content in marine microalgae. Bioresour Technol 147:484–491. https://doi.org/10.1016/j.biortech.2013.08.066
Gershon H, Gershon D (2000) The budding yeast, Saccharomyces cerevisiae, as a model for aging research: a critical review. Mech Ageing Dev 120:1–22. https://doi.org/10.1016/s0047-6374(00)00182-2
Gifuni I, Olivieri G, Pollio A, Franco TT, Marzocchella A (2017) Autotrophic starch production by Chlamydomonas species. J Appl Phycol 29:105–114. https://doi.org/10.1007/s10811-016-0932-2
Guiry MD, Guiry GM (2018) AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; Accessed on 27 July 2022
Hashimoto H, Uragami C, Cogdell RJ (2016) Carotenoids and photosynthesis. In: Stange C (ed) Carotenoids in nature: biosynthesis, regulation and function. Springer International Publishing, Cham, Switzerland, pp 111–139
Horton P, Ruban A (2005) Molecular design of the photosystem II light-harvesting antenna: photosynthesis and photoprotection. J Exp Bot 56:365–373. https://doi.org/10.1093/jxb/eri023
Hossain N, Mahlia TMI, Saidur R (2019) Latest development in microalgae-biofuel production with nano-additives. Biotechnol Biofuels 12:125. https://doi.org/10.1186/s13068-019-1465-0
Humby PL, Snyder ECR, Durnford DG (2013) Conditional senescence in Chlamydomonas reinhardtii (Chlorophyceae). J Phycol 49:389–400. https://doi.org/10.1111/jpy.12049
Kamalanathan M, Pierangelini M, Shearman LA, Gleadow R, Beardall J (2016) Impacts of nitrogen and phosphorus starvation on the physiology of Chlamydomonas reinhardtii. J Appl Phycol 28:1509–1520. https://doi.org/10.1007/s10811-015-0726-y
Khan MI, Shin JH, Kim JD (2018) The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb Cell Fact 17:36. https://doi.org/10.1186/s12934-018-0879-x
Koller M, Muhr A, Braunegg G (2014) Microalgae as versatile cellular factories for valued products. Algal Res 6:52–63. https://doi.org/10.1016/j.algal.2014.09.002
Li T, Gargouri M, Feng J, Park J-J, Gao D, Miao C, Dong T, Gang DR, Chen S (2015) Regulation of starch and lipid accumulation in a microalga Chlorella sorokiniana. Bioresour Technol 180:250–257. https://doi.org/10.1016/j.biortech.2015.01.005
Liang Y, Beardall J, Heraud P (2006) Changes in growth, chlorophyll fluorescence and fatty acid composition with culture age in batch cultures of Phaeodactylum tricornutum and Chaetoceros muelleri (Bacillariophyceae). Bot Mar 49:165–173. https://doi.org/10.1515/BOT.2006.021
Longo VD, Shadel GS, Kaeberlein M, Kennedy B (2012) Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab 16:18–31. https://doi.org/10.1016/j.cmet.2012.06.002
Machado MD, Soares EV (2012) Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata. Appl Microbiol Biotechnol 95:1035–1042. https://doi.org/10.1007/s00253-012-4185-y
Machado MD, Soares EV (2014) Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress. Aquat Toxicol 147:1–6. https://doi.org/10.1016/j.aquatox.2013.11.017
Machado MD, Soares EV (2020) Reproductive cycle progression arrest and modification of cell morphology (shape and biovolume) in the alga Pseudokirchneriella subcapitata exposed to metolachlor. Aquat Toxicol 222:105449. https://doi.org/10.1016/j.aquatox.2020.105449
Machado MD, Lopes AR, Soares EV (2015) Responses of the alga Pseudokirchneriella subcapitata to long-term exposure to metal stress. J Hazard Mater 296:82–92. https://doi.org/10.1016/j.jhazmat.2015.04.022
Madadi R, Maljaee H, Serafim S (2021) Microalgae as contributors to produce biopolymers. Mar Drugs 19:466. https://doi.org/10.3390/md19080466
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence - a practical guide. J Exp Bot 51:659–668. https://doi.org/10.1093/jexbot/51.345.659
McLean RJ (1968) Ultrastructure of Spongiochloris typica during senescence. J Phycol 4:277–283. https://doi.org/10.1111/j.1529-8817.1968.tb04696.x
McLean RJ (1969) Rejuvenation of senescent cells of Spongiochloris typica. J Phycol 5:32–37. https://doi.org/10.1111/j.1529-8817.1969.tb02572.x
Merchant SS, Kropat J, Liu B, Shaw J, Warakanont J (2012) TAG, You’re it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. Curr Opin Biotechnol 23:352–363. https://doi.org/10.1016/j.copbio.2011.12.001
Monici M (2005) Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Annu Rev 11:227–256. https://doi.org/10.1016/S1387-2656(05)11007-2
Msanne J, Xu D, Konda AR, Casas-Mollano JA, Awada T, Cahoon EB, Cerutti H (2012) Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry 75:50–59. https://doi.org/10.1016/j.phytochem.2011.12.007
Müller P, Li X-P, Niyogi KK (2001) Non-photochemical quenching. A response to excess light energy. Plant Physiol 125:1558–1566. https://doi.org/10.1104/pp.125.4.1558
Nagappan S, Das P, AbdulQuadir M, Thaher M, Khan S, Mahata C, Al-Jabri H, Vatland AK, Kumar G (2021) Potential of microalgae as a sustainable feed ingredient for aquaculture. J Biotechnol 341:1–20. https://doi.org/10.1016/j.jbiotec.2021.09.003
Naselli-Flores L, Padisák J (2022) Ecosystem services provided by marine and freshwater phytoplankton. Hydrobiologia 28:1–16. https://doi.org/10.1007/s10750-022-04795-y
Nyström T (2003) Conditional senescence in bacteria: death of the immortals. Mol Microbiol 48:17–23. https://doi.org/10.1046/j.1365-2958.2003.03385.x
OECD (2011) Test No 201: Freshwater alga and cyanobacteria, growth inhibition test. Organization for Economic Cooperation and Development, Paris, France. https://doi.org/10.1787/9789264069923-en
Patil V, Källqvist T, Olsen E, Vogt G, Gislerød HR (2007) Fatty acid composition of 12 microalgae for possible use in aquaculture feed. Aquac Int 15:1–9. https://doi.org/10.1007/s10499-006-9060-3
Petralia RS, Mattson MP, Yao PJ (2014) Aging and longevity in the simplest animals and the quest for immortality. Ageing Res Rev 16:66–82. https://doi.org/10.1016/j.arr.2014.05.003
Puzanskiy R, Tarakhovskaya E, Shavarda A, Shishova M (2018) Metabolomic and physiological changes of Chlamydomonas reinhardtii (Chlorophyceae, Chlorophyta) during batch culture development. J Appl Phycol 30:803–818. https://doi.org/10.1007/s10811-017-1326-9
Ritchie RJ (2008) Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents. Photosynthetica 46:115–126. https://doi.org/10.1007/s11099-008-0019-7
Sato N, Toyoshima M (2021) Dynamism of metabolic carbon flow of starch and lipids in Chlamydomonas debaryana. Front Plant Sci 12:646498. https://doi.org/10.3389/fpls.2021.646498
Schenk PM, Thomas-Hall SR, Stephens E, Marx UC, Mussgnug JH, Posten C, Kruse O, Hankamer B (2008) Second generation biofuels: high-efficiency microalgae for biodiesel production. BioEnergy Res 1:20–43. https://doi.org/10.1007/s12155-008-9008-8
Schiphorst C, Bassi R (2020) Chlorophyll-xanthophyll antenna complexes: in between light harvesting and energy dissipation. In: Grossman AR, Raven JA (eds) Larkum AWD. Springer International Publishing, Switzerland, pp 27–55. https://doi.org/10.1007/978-3-030-33397-3_3
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. https://doi.org/10.1038/nmeth.2089
Soto P, Gaete H, Eliana Hidalgo M (2011) Assessment of catalase activity, lipid peroxidation, chlorophyll-a, and growth rate in the freshwater green algae Pseudokirchneriella subcapitata exposed to copper and zinc. Lat Am J Aquat Res 39:280–285. https://doi.org/10.3856/vol39-issue2-fulltext-9
Strickland J, Parsons TR (1972) A practical handbook of seawater analysis, 2nd edn. Fisheries Research Board of Canada, Ottawa
Teh KY, Loh SH, Aziz A, Takahashi K, Effendy AWM, Cha TS (2021) Lipid accumulation patterns and role of different fatty acid types towards mitigating salinity fluctuations in Chlorella vulgaris. Sci Rep 11:438. https://doi.org/10.1038/s41598-020-79950-3
US-EPA (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. EPA-821-R-02-012. 5th edn. United States Environmental Protection Agency. Washington, DC, USA
US-EPA (2012) Algal toxicity (OCSPP 850.4500). Ecological effects test guidelines. Office of Chemical Safety and Pollution Prevention. United States Environmental Protection Agency. EPA 712-C-006. Washington, DC, USA
Van den Hoek C, Mann DG, Jahns HM (1998) Algae: an introduction to phycology. Cambridge University Press, Cambridge UK
Van der Grinten E, Pikkemaat MG, van den Brandhof E-J, Stroomberg GJ, Kraak MHS (2010) Comparing the sensitivity of algal, cyanobacterial and bacterial bioassays to different groups of antibiotics. Chemosphere 80:1–6. https://doi.org/10.1016/j.chemosphere.2010.04.011
Walz H (2000) WinControl window software for PAM fluorometers. In: Walz H (ed) Users manual. Heinz Walz GmbH, f Effeltrich Germany
Yang L, Chen J, Qin S, Zeng M, Jiang Y, Hu L, Xiao P, Hao W, Hu Z, Lei A, Wang J (2018) Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii. Biotechnol Biofuels 11:40. https://doi.org/10.1186/s13068-018-1041-z
Zhu S, Huang W, Xu J, Wang Z, Xu J, Yuan Z (2014) Metabolic changes of starch and lipid triggered by nitrogen starvation in the microalga Chlorella zofingiensis. Bioresour Technol 152:292–298. https://doi.org/10.1016/j.biortech.2013.10.092
Funding
This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. Manuela D. Machado thanks FCT for funding through program DL 57/2016-Norma Transitória.
Author information
Authors and Affiliations
Contributions
MM: validation, formal analysis, investigation, writing—original draft preparation, and visualization. ES: conceptualization, validation, writing—original draft preparation, review and editing, visualization, and supervision.
Corresponding authors
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Consent for publication
All authors have read and approved the final version of the manuscript for publication.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Machado, M.D., Soares, E.V. Life and death of Pseudokirchneriella subcapitata: physiological changes during chronological aging. Appl Microbiol Biotechnol 106, 8245–8258 (2022). https://doi.org/10.1007/s00253-022-12267-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-022-12267-5