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Morphology and Growth of Arthrospira Platensis During Cultivation in a Flat-Type Bioreactor

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Morphology and Growth of Arthrospira Platensis During Cultivation in a Flat-Type Bioreactor life Communication Morphology and Growth of Arthrospira platensis during Cultivation in a Flat-Type Bioreactor Conrad H. G. Jung 1, Steffen Braune 2, Peter Waldeck 3, Jan-Heiner Küpper 1,2, Ingolf Petrick 3 and Friedrich Jung 2,* 1 Carbon Biotech Social Enterprise AG, 01968 Senftenberg, Germany; [email protected] (C.H.G.J.); [email protected] (J.-H.K.) 2 Institute of Biotechnology, Molecular Cell Biology, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, Germany; [email protected] 3 Institute of Materials Chemistry, Thermodynamics, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, Germany; [email protected] (P.W.); [email protected] (I.P.) * Correspondence: [email protected] Abstract: Arthrospira platensis (AP) is a cyanobacterium with a high economic value and is nowadays one of the most important industrially cultivated microalgae. Knowledge of its growth is essential for the understanding of its physiology and yield. The growth of AP biomass occurs through two mechanisms: (1) propagation by fragmentation of trichomes, and (2) the trichomes are extended by binary fission until they reach their mature status. These phases are visualized by live cell light and laser scanning microscopy, demonstrating the different phases of AP growth. Keywords: Arthrospira platensis; Spirulina; life cycle; morphology; fragmentation; fission Citation: Jung, C.H.G.; Braune, S.; Waldeck, P.; Küpper, J.-H.; Petrick, I.; 1. Introduction Jung, F. Morphology and Growth of Arthrospira platensis during Arthrospira platensis (AP) is a species of cyanobacterial phylum. Cyanobacteria typi- Cultivation in a Flat-Type Bioreactor. cally carry out oxygenic photosynthesis with water as an electron donor and use carbon Life 2021, 11, 536. https://doi.org/ dioxide as a carbon source. This cyanobacterium (often called blue-green alga) grows as 10.3390/life11060536 filamentous, helicoidal trichomes, performs oxygenic photosynthesis and reproduces by binary fission [1]. AP has a long history of use as food and gained considerable popularity Academic Editor: Fabia U. Battistuzzi in the human health food industry due to its therapeutic properties, including antioxidant, anti-inflammatory, immune-modulatory and anticancer activities [2–4]. In many countries Received: 16 April 2021 of Asia, it is used as a protein supplement, as human health food and as feed for poultry Accepted: 1 June 2021 and aquaculture. Nowadays, the successful commercial exploitation of AP due to its high Published: 9 June 2021 nutritional value, chemical composition and the safety of the biomass has made it one of the most important industrially cultivated microalgae [5]. Publisher’s Note: MDPI stays neutral Knowledge of its physiology is essential for understanding its growth status. The with regard to jurisdictional claims in main factors for the growth of AP are light and CO2 or HCO3¯, respectively [6–8]. The published maps and institutional affil- life cycle of AP (see Figure1) was first described by Ciferri [ 9]. Here, we investigated the iations. morphology of AP (strain: SAG21.99, Göttingen, Germany) during growth in a bioreactor from the 1st day up to the 7th day via different live cell imaging techniques. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Life 2021, 11, 536. https://doi.org/10.3390/life11060536 https://www.mdpi.com/journal/life LifeLife2021 2021,,11 11,, 536 x FOR PEER REVIEW 22 of 88 FigureFigure 1.1. LifeLife cyclecycle ofof ArthrospiraArthrospira platensisplatensis (modified(modified accordingaccording toto [[9]).9]). TheThe asteriskasterisk (*)(*) indicatesindicates necridia.necridia. 2.2. Materials and Methods APAP used for cultivation cultivation was was obtained obtained from from the the “The “The Culture Culture Collection Collection of ofAlgae Algae at atGoettingen Goettingen University” University” (strain: (strain: SAG21.99). SAG21.99). AP wereAP culturedwere cultured in a flat-type in a flat-type (2 cm) vertical (2 cm) verticaltransparent transparent bioreactor bioreactor consisting consisting of a flexibl of ae flexiblepolyethylene polyethylene (PE, food (PE, safe food grade) safe grade)sleeve sleevewith a with1.0 L aworking 1.0 L working volume. volume. The PE Thesleeve PE was sleeve pressed was pressedbetween between two adaptable two adaptable polyme- polymethyl-methacrylatethyl-methacrylate plates (see plates the (seegreen the reactor green in reactor Figure in 2). Figure For the2). experiments, For the experiments, Zarrouk ◦ Zarroukmedium mediumwas used was [10]. used The [growth10]. The medium growth was medium initially was sterilized initially at sterilized 121 °C in at a 121HV-50C inautoclave a HV-50 (SYSMEX autoclave VX-95, (SYSMEX Sysmex, VX-95, Norderst Sysmex,edt, Norderstedt, Germany) Germany)for 15 min. for The 15 bioreactor min. The bioreactorwas inoculated was inoculatedwith AP cells with (0.19 AP cellsg/L) from (0.19 g/L)a light-limited—because from a light-limited—because of the shading of the of shadingthe high of cell the density—back-up high cell density—back-up bioreactor bioreactor in which inthe which AP had the already AP had alreadyreached reachedthe sta- thetionary stationary growth growth phase. phase. At this At stage this of stage develo of development,pment, AP cells AP were cells transferred were transferred into a intobio- areactor bioreactor filled filled with withZarrouk Zarrouk medium, medium, aerated aerated with air with supplemented air supplemented with 2% with CO 2%2 and CO il-2 andluminated illuminated with a with blue-red a blue-red LED LEDlamp lamp (AP673L, (AP673L, Valoya, Valoya, Helsinki, Helsinki, Finland) Finland) set to set 250 to 2 250µmol/(mµmol/(m2 * s) at* the s) at bioreactor the bioreactor surface surface for up forto seven up to days. seven Stirring days. Stirring of the culture of the suspen- culture suspensionsion was carried was carried out using out usingsix tubes six tubesso that so sufficient that sufficient mixing mixing of the of culture the culture medium medium was µ wasachieved. achieved. Air was Air waspumped pumped through through a membrane a membrane filter filter (Millipore; (Millipore; 0.45 0.45 µm porem pore size, size, 10 10cm cm diameter) diameter) and and moistened moistened by by passaging passaging it itth throughrough distilled distilled water, water, with with a flowflow raterate ofof 200200 L/h,L/h, respectively. respectively. Aeration was adjusted using area flowflow meters.meters. TheThe appropriateappropriate airair volumevolume flowflow waswas measuredmeasured inin pretestspretests soso thatthat thethe pHpH valuevalue ofof thethe growthgrowth mediummedium waswas maintained between pH 9 and pH 10.0 for the duration of the experiment. The filling level maintained between pH 9 and pH 10.0 for the duration of the experiment. The filling level was kept constant to compensate for evaporation losses. The temperature in the bioreactor was kept constant to compensate for evaporation losses. The temperature in the bioreactor was maintained at 25 ◦C. Light intensity was measured using a LI-250 light meter with was maintained at 25 °C. Light intensity was measured using a LI-250 light meter with a a LI-190SA pyranometer sensor (LI-COR, Inc., Lincoln, NE, USA). The optical density LI-190SA pyranometer sensor (LI-COR, Inc., Lincoln, NE, USA). The optical density (Ther- (Thermofisher, Genesys 100 Bio, Waltham, MA, USA), temperature (PT1000, Wernberg, mofisher, Genesys 100 Bio, Waltham, MA, USA), temperature (PT1000, Wernberg, Ger- Germany), pH values (EGA 133, Sensortechnik Meinsberg, Meinsberg, Germany) and many), pH values (EGA 133, Sensortechnik Meinsberg, Meinsberg, Germany) and oxygen oxygen concentration (FDA120, Hamilton, Bonaduz, Switzerland) of the culture medium concentration (FDA120, Hamilton, Bonaduz, Switzerland) of the culture medium were were monitored during the cultivation time continuously. A sketch of the bioreactor is monitored during the cultivation time continuously. A sketch of the bioreactor is shown shown in Figure2 (for details, see [11]). in Figure 2 (for details, see [11]). Life 2021, 11, 536 3 of 8 LifeLife 20212021,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 88 FigureFigure 2. 2. SketchSketch of of the the bioreactor bioreactor for for the the production production of of ArthrospiraArthrospira platensisplatensis (adapted(adapted fromfrom [[11]).[11]).11]). OnOn each each experimentalexperimentalexperimental day, day,day, samples samplessamples were werewere taken takentaken from fromfrom the thethe bioreactor bioreactorbioreactor and andand were werewere examined exam-exam- inedviained bright viavia brightbright field andfieldfield phase andand phase contrastphase contrastcontrast microscopy microscopymicroscopy (Axio Scope, (Axio(Axio ZeissScope,Scope, Microimaging ZeissZeiss MicroimagingMicroimaging GmbH, GmbH,Jena,GmbH, Germany; Jena,Jena, Germany;Germany; BZ-X810, BZ-X810,BZ-X810, Keyence, Keyence,Keyence, Japan). Samples JapaJapan).n). Samples wereSamples further werewere studied furtherfurther by studiedstudied laser scanning byby laserlaser scanningmicroscopyscanning microscopymicroscopy (Axio Observer.Z1/7, (Axio(Axio Observer.Z1/7,Observer.Z1/7, Zeiss Microimaging ZeissZeiss MicroimagingMicroimaging GmbH, Jena GmbH,GmbH, Germany). JenaJena Germany).Germany). Geometry
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