Carbone et al. AMB Expr (2020) 10:170 https://doi.org/10.1186/s13568-020-01110-7 ORIGINAL ARTICLE Open Access Comparison of Galdieria growth and photosynthetic activity in diferent culture systems Dora Allegra Carbone1*, Giuseppe Olivieri2,3, Antonino Pollio4 and Michael Melkonian5,6 Abstract In the last years, the acidothermophilic red microalga Galdieria sulphuraria has been increasingly studied for industrial applications such as wastewater treatment, recovery of rare earth elements, production of phycobilins. However, even now it is not possible an industrial cultivation of this organism because biotechnological research on G. sulphuraria and allied species is relatively recent and fragmented. Having in mind a possible scale-up for commercial applications, we have compared the growth and photosynthetic performance of G. sulphuraria in four suspended systems (Inclined bubble column, Decanter Laboratory Flask, Tubular Bioreactor, Ultra-fat plate bioreactor) and one immobilized system (Twin Layer Sytem). The results showed that G. sulphuraria had the highest growth, productivity and photosynthetic performance, when grown on the immobilized system, which also ofers some economics advantages. Keywords: Galdieria sulphuraria, photobioreactors, biomass, comparison Keypoints 2007; Toplin et al.2008; Castenholz and Mcdermott 2010; Ciniglia et al. 2014; Ciniglia et al. 2017) Comparison of diferent microalgal cultivation sys- Tese extreme environmental conditions strongly limit tems (suspended and immobilized) contaminations that are prevalent in open microalgal Analysis of growth and photosynthetic performance mass cultivation systems. In consequence, these organ- Immobilized cultivation on the Twin layer system isms are of considerable interest for commercial applica- showed the best performance with respect to growth tions (Carfagna et al. 2018; Carbone et al. 2019). and photosynthesis Cyanidiophyceae are divided into three genera, Cya- nidium, Galdieria, and Cyanidioschyzon (Gross 2000; Heilmann and Gross 2001; Ciniglia et al. 2004; Del Introduction Mondo et al.2019) but only Galdieria is known to grow Cyanidiophyceae are a class of red microalgae living in heterotrophically, also achieving a higher biomass density extreme environments (Albertano et al. 2000; Pinto et al. (Gross et al. 1998; Gross and Schnarrenberger 1995; Gra- 2003; Yoon et al. 2004). Tey prevalently thrive in geo- ziani et al. 2013, Vítová et al. 2016); therefore it is con- thermal volcanic areas at temperatures around 40 °C and sidered a promising candidate for industrial applications. at high sulfuric acid concentrations, with ambient pH Indeed, Galdieria has been the subject of diferent values between 1 and 3 (Albertano et al. 2000; Pinto et al. studies in algal biotechnology. It was used for wastewa- ter treatment (Ju et al. 2016; Henkanette-Gedera et al. 2016; da Silva et al. 2016; Carbone et al. 2018; Galasso et al. 2019; Alalwan et al. 2019; Sosa-Hernández et al. *Correspondence: [email protected] 1 Laboratory of Biological Oceanography, Stazione Zoologica ‘‘A. Dohrn’’ 2019) and for recovery of rare earth elements (Minoda of Napoli, Villa Comunale, Napoli I80121, Italy et al. 2015). Moreover, this organism produces high levels Full list of author information is available at the end of the article of phycobiliproteins that are used in diverse medical and © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Carbone et al. AMB Expr (2020) 10:170 Page 2 of 14 w(t) − w(0) cosmetic products (Schmidt et al. 2005; Graverholt and SWI(t) = Eriksen 2007; Sørensen et al. 2013; Eriksen 2018) and in w(0) diferent compounds with antioxidant properties (Carf- agna et al. 2016). where w(t) is the dry weight at day t (more exactly, t is the However, biotechnological research on Galdiera is rel- number of the day when the sampling is taken and meas- atively recent. Te data around the growth of this micro- ured) and w(0) the dry weight at day 0 (g). alga are still fragmentary and even now it is not possible an industrial cultivation of this organism. Terefore, having in mind a possible scale-up and com- Specifc light yield (SLY) mercial applications of G. sulphuraria, in this paper, To consider the light energy necessary for the growth, we the growth and the photosynthetic performance of this used the standard light yield and normalized it. Te for- 1 microalga were systematically compared in fve diferent mula for the specifc light yield (SLY) (photons mol − ) is types of cultivation systems (one immobilized and four the following: suspended) at the same conditions of temperature and SWI(t) SLY (t) = irradiance. A ∗ t ∗ s ∗ pm Materials and methods where SWI(x) is the specifc weight increase, A the area Algal strain and stock cultures of surface of the bioreactor exposed to the light (m2), t Galdieria sulphuraria strain 064 from ACUF collection is the number of days, s is the number of seconds of illu- (D’elia et al. 2018 http://www.acuf.net) was chosen. Te mination per day (s) (in our case, this number, 50,400, is stock culture was cultivated in Galdieria medium (Gross obtained multiplying the number of illuminations hours, and Schnarrenberger 1995) acidifed by sulfuric acid 14, by the number of seconds in a hour, 3600), pm is the at pH 1.5. Stock cultures were grown in 1 l Erlenmeyer number of the given moles of photosynthetically active fasks and were exposed to an adaptive light intensity photons per second and per square meter (photons mol 1 2 of 30 μmol photons m−2 s−1 with a light/dark cycle of s− m− ) (in our case, pm is the number of the given PAR, 14/10 h. Te temperature was 35 °C. 100, multiplied by 10−6). Analysis of growth Growth rate (GR) We consider several parameters to analyse growth. Tese parameters are depending variables of time, the Te growth rate in the time period is calculated thanks to −1 only independent variable. Some depending variables, the growth rate GR (day ) with this formula: denoted with the term “specifc”, are normalized by divid- w(t+h) Ln w(t) ing by the initial values, to take the diferent inocula into GR(t) = account (conversely, the non-normalized depending vari- h ables can be obtained multiplying the normalized ones by the initial values). We explicitly observe that normali- where Ln is the natural logarithm, w(t+h) is the dry zation is necessary because Twin-Layer S needs inocula weight at day t+ h, w(t) is the dry weight at day t, h is the concentrations very diferent from those used for sus- number of days between two consecutive measures (in pended systems. our case, h is equal to 3). Te considered variables are: coefcient of determina- tion, specifc weight increase, specifc light yield, growth rate. Determination of biomass In liquid cultivation systems, 2 ml of the culture was har- Coefcient of determination vested every three days in triplicate with a sterile syringe Te coefcient of determination (r2) is a measure of how for dry mass determinations and then fltered on a poly- close the data are to the regression line. It was used to carbonate disc using a vacuum pump. In Twin Layer compare the diferent bioreactor systems. System, the polycarbonate discs were taken of from the bioreactor and biomass in the inoculated area was con- Specifc weight increase (SWI) sidered, while the rest was scraped of. All samples were lyophilized in a freeze dryer for two Te specifc weight increase (SWI) was used to analyse hours and weighed with an analytical balance (Sartorius the trend of growth in the diferent bioreactors. Bovenden, Germany). Tis is the formula defning SWI: Carbone et al. AMB Expr (2020) 10:170 Page 3 of 14 Analysis of the photosynthetic state of microalgae two consecutive measures (in our case, h is equal to 3), A Pigment concentration: microalgae were harvested and the area of surface of the bioreactor exposed to the light lyophilised then they were mixed with quartz sand to (m2), s is the number of seconds of illumination per day obtain homogeneous powder. (s) (in our case, this number, 50.400, is obtained multiply- Photosynthetic pigments were extracted overnight with ing the number of illuminations hours, 14, by the num- acetone (Costache et al. 2012). Chlorophyll a and carot- ber of seconds in a hour, 3.600), pm is the number of the enoids were analysed by spectrophotometry (Shimadzu given moles of photosynthetically active photons per sec- UV-2450) (Tomitani et al. 1999). ond and per square meter (photons mol s −1 m−2) (in our case, pm is the number of the given PAR, 100, multiplied 6 Pigment concentration by 10− ), N is the Avogadro number, e is the approximate energy of a photon of 400 nm 173 wave length (kJ) (this Diferent formulae were considered to compare the pho- value is around 4 * 10−22). tosynthetic state of each culture. In this formula, we normalized by w(0) to highlight Tese equations were used: the relevant diferences between the TL-S system and Chl a = 11.75 ∗ (A662) − 2.350 ∗ (A645) suspended systems.