{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T05:07:22Z","timestamp":1778044042023,"version":"3.51.4"},"reference-count":26,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2019,9,26]],"date-time":"2019-09-26T00:00:00Z","timestamp":1569456000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2019,9,26]],"date-time":"2019-09-26T00:00:00Z","timestamp":1569456000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Industrial scale-up of microalgal cultures is often a protracted step prone to culture collapse and the occurrence of unwanted contaminants. To solve this problem, a two-stage scale-up process was developed \u2013 heterotrophically <jats:italic>Chlorella vulgaris<\/jats:italic> cells grown in fermenters (1<jats:sup>st<\/jats:sup> stage) were used to directly inoculate an outdoor industrial autotrophic microalgal production unit (2<jats:sup>nd<\/jats:sup> stage). A preliminary pilot-scale trial revealed that <jats:italic>C. vulgaris<\/jats:italic> cells grown heterotrophically adapted readily to outdoor autotrophic growth conditions (1-m<jats:sup>3<\/jats:sup> photobioreactors) without any measurable difference as compared to conventional autotrophic inocula. Biomass concentration of 174.5\u2009g\u2009L<jats:sup>\u22121<\/jats:sup>, the highest value ever reported for this microalga, was achieved in a 5-L fermenter during scale-up using the heterotrophic route. Inocula grown in 0.2- and 5-m<jats:sup>3<\/jats:sup> industrial fermenters with mean productivity of 27.54\u2009\u00b1\u20095.07 and 31.86\u2009\u00b1\u20092.87\u2009g\u2009L<jats:sup>\u22121<\/jats:sup> d<jats:sup>\u22121<\/jats:sup>, respectively, were later used to seed several outdoor 100-m<jats:sup>3<\/jats:sup> tubular photobioreactors. Overall, all photobioreactor cultures seeded from the heterotrophic route reached standard protein and chlorophyll contents of 52.18\u2009\u00b1\u20091.30% of DW and 23.98\u2009\u00b1\u20091.57\u2009mg\u2009g<jats:sup>\u22121<\/jats:sup> DW, respectively. In addition to providing reproducible, high-quality inocula, this two-stage approach led to a 5-fold and 12-fold decrease in scale-up time and occupancy area used for industrial scale-up, respectively.<\/jats:p>","DOI":"10.1038\/s41598-019-50206-z","type":"journal-article","created":{"date-parts":[[2019,9,26]],"date-time":"2019-09-26T20:12:13Z","timestamp":1569528733000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":132,"title":["Heterotrophy as a tool to overcome the long and costly autotrophic scale-up process for large scale production of microalgae"],"prefix":"10.1038","volume":"9","author":[{"given":"A.","family":"Barros","sequence":"first","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1369-2099","authenticated-orcid":false,"given":"H.","family":"Pereira","sequence":"additional","affiliation":[]},{"given":"J.","family":"Campos","sequence":"additional","affiliation":[]},{"given":"A.","family":"Marques","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3101-693X","authenticated-orcid":false,"given":"J.","family":"Varela","sequence":"additional","affiliation":[]},{"given":"J.","family":"Silva","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2019,9,26]]},"reference":[{"key":"50206_CR1","doi-asserted-by":"publisher","first-page":"1406","DOI":"10.1016\/j.biortech.2009.09.038","volume":"101","author":"O Jorquera","year":"2010","unstructured":"Jorquera, O., Kiperstok, A., Sales, E. A., Embirucu, M. & Ghirardi, M. L. Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors. Bioresour. Technol. 101, 1406\u20131413 (2010).","journal-title":"Bioresour. Technol."},{"key":"50206_CR2","doi-asserted-by":"publisher","first-page":"1194","DOI":"10.1002\/cssc.201100473","volume":"4","author":"EA Quadrelli","year":"2011","unstructured":"Quadrelli, E. A., Centi, G., Duplan, J.-L. & Perathoner, S. Carbon dioxide recycling: emerging large-scale technologies with industrial potential. ChemSusChem. 4, 1194\u20131215 (2011).","journal-title":"ChemSusChem."},{"key":"50206_CR3","doi-asserted-by":"publisher","first-page":"294","DOI":"10.1016\/j.biotechadv.2007.02.001","volume":"25","author":"Y Chisti","year":"2007","unstructured":"Chisti, Y. Biodiesel from microalgae. Biotechnol. Adv. 25, 294\u2013306 (2007).","journal-title":"Biotechnol. Adv."},{"key":"50206_CR4","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1263\/jbb.101.87","volume":"101","author":"P Spolaore","year":"2006","unstructured":"Spolaore, P., Joannis-Cassan, C., Duran, E. & Isambert, A. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87\u201396 (2006).","journal-title":"J. Biosci. Bioeng."},{"key":"50206_CR5","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/j.watres.2010.08.037","volume":"45","author":"OP Garcia","year":"2011","unstructured":"Garcia, O. P., Escalante, F. M. E., de-Bashan, L. E. & Bashan, Y. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45, 11\u201336 (2011).","journal-title":"Water Res."},{"key":"50206_CR6","doi-asserted-by":"publisher","first-page":"2379","DOI":"10.1007\/s10811-014-0271-0","volume":"26","author":"C Safi","year":"2014","unstructured":"Safi, C. et al. A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption. J. Appl. Phycol. 26, 2379\u20132387 (2014).","journal-title":"J. Appl. Phycol."},{"key":"50206_CR7","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1007\/s00253-007-1285-1","volume":"78","author":"W Xiong","year":"2008","unstructured":"Xiong, W., Li, X. F., Xiang, J. Y. & Wu, Q. Y. High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Appl. Microbiol. Biotechnol. 78, 29\u201336 (2008).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"50206_CR8","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1007\/s00449-010-0474-y","volume":"34","author":"J O\u2019Grady","year":"2011","unstructured":"O\u2019Grady, J. & Morgan, J. Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol. Bioprocess Biosys. Eng. 34, 121\u2013125 (2011).","journal-title":"Bioprocess Biosys. Eng."},{"key":"50206_CR9","doi-asserted-by":"publisher","first-page":"359","DOI":"10.1023\/A:1007981930676","volume":"9","author":"JC Ogbonna","year":"1997","unstructured":"Ogbonna, J. C., Masui, H. & Tanaka, H. Sequential heterotrophic\/autotrophic cultivation \u2013 an efficient method of producing Chlorella biomass for health food and animal feed. J. Appl. Phycol. 9, 359\u2013366 (1997).","journal-title":"J. Appl. Phycol."},{"key":"50206_CR10","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1016\/S0168-1656(99)00075-9","volume":"35","author":"JC Ogbonna","year":"1999","unstructured":"Ogbonna, J. C., Tomiyama, S. & Tanaka, H. Production of \u03b1-tocopherol by sequential heterotrophic-photoautotrophic cultivation of Euglena gracilis. J. Biotechnol. 35, 213\u2013221 (1999).","journal-title":"J. Biotechnol."},{"key":"50206_CR11","doi-asserted-by":"publisher","first-page":"395","DOI":"10.1023\/A:1011921329568","volume":"13","author":"N Hata","year":"2001","unstructured":"Hata, N., Ogbonna, J. C., Hasegawa, Y., Taroda, H. & Tanaka, H. Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic\u2013photoautotrophic culture. J. Appl. Phycol. 13, 395\u2013402 (2001).","journal-title":"J. Appl. Phycol."},{"key":"50206_CR12","doi-asserted-by":"publisher","first-page":"8","DOI":"10.1016\/j.biortech.2013.06.068","volume":"144","author":"S Kim","year":"2013","unstructured":"Kim, S., Park, J.-E., Cho, Y.-B. & Hwang, S.-J. Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions. Bioresour. Technol. 144, 8\u201313 (2013).","journal-title":"Bioresour. Technol."},{"key":"50206_CR13","first-page":"62","volume":"51","author":"WB Kong","year":"2013","unstructured":"Kong, W. B. et al. Effect of glycerol and glucose on the enhancement of biomass, lipid and soluble carbohydrate production by Chlorella vulgaris in mixotrophic culture. Food Technol. Biotechnol. 51, 62\u201369 (2013).","journal-title":"Food Technol. Biotechnol."},{"key":"50206_CR14","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1007\/s00253-011-3311-6","volume":"91","author":"F Bumbak","year":"2011","unstructured":"Bumbak, F., Cook, S., Zachleder, V., Hauser, S. & Kovar, K. Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. Appl. Microbiol. Biotechnol. 91, 31\u201346 (2011).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"50206_CR15","doi-asserted-by":"publisher","first-page":"356","DOI":"10.1016\/j.biortech.2017.06.110","volume":"243","author":"M Sakarika","year":"2017","unstructured":"Sakarika, M. & Kornaros, M. Kinetics of growth and lipids accumulation in Chlorella vulgaris during batch heterotrophic cultivation: Effect of different nutrient limitation Strategies. Bioresour. Technol. 243, 356\u2013365 (2017).","journal-title":"Bioresour. Technol."},{"key":"50206_CR16","doi-asserted-by":"publisher","first-page":"253","DOI":"10.1089\/ind.2017.0007","volume":"13","author":"T Ghidossi","year":"2017","unstructured":"Ghidossi, T., Marison, I., Devery, R., Gafney, D. & Forde, C. Characterization and optimization of a fermentation process for the production of high cell densities and lipids using heterotrophic cultivation of Chlorella protothecoides. Ind. Biotechnol. 13, 253\u2013259 (2017).","journal-title":"Ind. Biotechnol."},{"key":"50206_CR17","doi-asserted-by":"publisher","DOI":"10.1186\/1754-6834-7-84","volume":"7","author":"A Guccione","year":"2014","unstructured":"Guccione, A. et al. Chlorella for protein and biofuels: from strain selection to outdoor cultivation in a green wall panel photobioreactor. Biotechnol. Biofuels 7, 84 (2014).","journal-title":"Biotechnol. Biofuels"},{"key":"50206_CR18","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1007\/s10811-010-9643-2","volume":"24","author":"J Doucha","year":"2012","unstructured":"Doucha, J. & Livansky, K. Production of high-density Chlorella culture grown in fermenters. J. Appl. Phycol. 24, 35\u201343 (2012).","journal-title":"J. Appl. Phycol."},{"key":"50206_CR19","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1111\/j.1472-765X.2006.02038.x","volume":"44","author":"Z Wu","year":"2007","unstructured":"Wu, Z. & Shi, X. Optimization for high-density cultivation of heterotrophic Chlorella based on a hybrid neural network model. Lett. Appl. Microbiol. 44, 13\u201318 (2007).","journal-title":"Lett. Appl. Microbiol."},{"key":"50206_CR20","doi-asserted-by":"publisher","first-page":"723","DOI":"10.1021\/bp0101987","volume":"18","author":"X-M Shi","year":"2002","unstructured":"Shi, X.-M. & Chen, F. High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture. Biotechnol. Prog. 18, 723\u2013727 (2002).","journal-title":"Biotechnol. Prog."},{"key":"50206_CR21","doi-asserted-by":"publisher","first-page":"437","DOI":"10.1016\/S1389-1723(05)00309-9","volume":"98","author":"H Sansawa","year":"2004","unstructured":"Sansawa, H. & Endo, H. Production of intracellular phytochemicals in Chlorella under heterotrophic conditions. J. Biosci. Bioeng. 98, 437\u2013444 (2004).","journal-title":"J. Biosci. Bioeng."},{"key":"50206_CR22","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1080\/00021369.1974.10861121","volume":"38","author":"H Endo","year":"1974","unstructured":"Endo, H., Nakajima, K., Chino, R. & Shiroa, M. Growth characteristics and cellular components of Chlorella regularis, heterotrophic fast growing strain. Agri. Biol. Chem. 38, 9\u201318 (1974).","journal-title":"Agri. Biol. Chem."},{"key":"50206_CR23","doi-asserted-by":"publisher","first-page":"1711","DOI":"10.1007\/s10811-013-0212-3","volume":"26","author":"C Safi","year":"2014","unstructured":"Safi, C. et al. Extraction of lipids and pigments of Chlorella vulgaris by supercritical carbon dioxide: influence of bead milling on extraction performance. J. Appl. Phycol. 26, 1711\u20131718 (2014).","journal-title":"J. Appl. Phycol."},{"key":"50206_CR24","doi-asserted-by":"publisher","first-page":"144","DOI":"10.3390\/md14080144","volume":"14","author":"H Safafar","year":"2016","unstructured":"Safafar, H., Hass, M. Z., M\u00f8ller, P., Holdt, S. L. & Jacobsen, C. High-EPA biomass from Nannochloropsis salina cultivated in a flat-panel photo-bioreactor on a process water-enriched growth medium. Mar. Drugs 14, 144 (2016).","journal-title":"Mar. Drugs"},{"key":"50206_CR25","doi-asserted-by":"publisher","first-page":"230","DOI":"10.1104\/pp.47.2.230","volume":"47","author":"SI Beale","year":"1971","unstructured":"Beale, S. I. & Appleman, D. Chlorophyll synthesis in Chlorella: regulation by degree of light limitation of growth. Plant Physiol. 47, 230\u2013235 (1971).","journal-title":"Plant Physiol."},{"key":"50206_CR26","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1016\/j.algal.2016.10.014","volume":"21","author":"A Barros","year":"2017","unstructured":"Barros, A. et al. Mass balance analysis of carbon and nitrogen in industrial scale mixotrophic microalgae cultures. Algal Res. 21, 35\u201341 (2017).","journal-title":"Algal Res."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-50206-z.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-50206-z","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-50206-z.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,17]],"date-time":"2022-12-17T13:55:59Z","timestamp":1671285359000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-019-50206-z"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,9,26]]},"references-count":26,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["50206"],"URL":"https:\/\/doi.org\/10.1038\/s41598-019-50206-z","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,9,26]]},"assertion":[{"value":"18 February 2019","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"2 September 2019","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"26 September 2019","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"13935"}}