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]).

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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 measurements of the trichomes were carried out with ImageJ (National Institute of Health, GeometryGeometry measurementsmeasurements ofof thethe trichomestrichomes werewere cacarriedrried outout withwith ImageJImageJ (National(National InstituteInstitute ofof Bethesda, MD, USA) [12]. Figure3 shows the growth curve of AP over seven days. Health,Health, Bethesda,Bethesda, MD,MD, USA)USA) [12].[12]. FigureFigure 33 showshowss thethe growthgrowth curvecurve ofof APAP overover sevenseven days.days.

3.53.5 3.03.0 2.52.5 2.02.0 1.51.5 1.01.0 0.50.5 Optical density [760 nm] [760 density Optical Optical density [760 nm] [760 density Optical 0.00.0 00 24487296120144 24487296120144 TimeTime [h][h]

Figure 3. Growth curve for Arthrospira platensis in the described bioreactor over seven days. For each Figure 3. Growth curve for Arthrospira platensis in the described bioreactorbioreactor overover sevenseven days.days. ForFor eacheach timetime point,point, threethree individualindividual measmeasurementsurements ofof thethe opticaloptical densitydensity (a(att 760760 nm)nm) ofof thethe cellcell solutionsolution areare time point, three individual measurements of the optical density (at 760 nm) of the cell solution are givengiven asas staggeredstaggered blackblack pointpoint sysymbols.mbols. TheThe dasheddashed lineslines indicateindicate aa linearlinear trendtrend betweenbetween thethe meanmean given as staggered black point symbols. The dashed lines indicate a linear trend between the mean valuesvalues atat eacheach timetime point.point. TheThe graygray verticalvertical barsbars indicateindicate thethe memeasurementasurement timetime points.points. values at each time point. The gray vertical bars indicate the measurement time points.

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3.3. Results Results and and Discussion Discussion AA flat flat panel-type bioreactor withwith aa depth depth of of 2 2 cm cm (minimizing (minimizing the the cell-induced cell-induced shading) shading)was was used used to avoid to avoid self-shading self-shading and toand achieve to achieve many many hormogonia. hormogonia. To avoid To avoid phototoxicity, phototoxicity,sufficient sufficient nutrient nutrient concentrations concentrations are needed. are needed. This was This attained was attained by full Zarrouk by full Zarrouk medium mediumand an additionaland an additional 2% CO 22%in CO the2 aeratingin the aerating gas flow. gas flow. The high The aerationhigh aeration rate ofrate 200 of L/h200 L/hguaranteed guaranteed a homogeneous a homogeneous culture culture and and light light availability. availability. ThoseThose light-limited light-limited AP AP were were mature mature (see (see 1. 1. in in Figure Figure 11)) and started to divide asas soonsoon asas the the light light intensity intensity increased. increased. Basically, Basically, the the growth growth of of AP AP biomass biomass occurs occurs through through two two mechanisms:mechanisms: (1) (1) propagation propagation occurs occurs by by fragmentation fragmentation of of trichomes, trichomes, and and (2) (2) the the trichomes trichomes areare extended extended by by binary binary fiss fissionion [13,14] [13,14] until until they they re reachach their their mature mature status. FigureFigure 4 shows the development of AP over thethe cultivationcultivation period.period. At the firstfirst dayday afterafter the the formation formation of of necridia, necridia, only only very very few few short short trichome trichome fragments fragments of of AP AP cells cells (see (see thethe arrow arrow in in the the figure figure for for day day 1) 1) were found. Necridia Necridia are are specialized specialized cells cells within within the the filamentfilament formed formed during during the the propagation propagation of ofAP, AP, which which is accompanied is accompanied by cell by celllysis lysis and andthe formationthe formation of debris of debris (see (seeFigures Figures 5A–H5A–H andand 6A–E).6A–E). Due Due to the to the fragmentation fragmentation of the of the tri- chometrichome at the at the necridia, necridia, short short chains chains of ofcells cells (of (of up up to to 10 10 cells), cells), the the hormogonia, hormogonia, originate originate (see(see red red arrows arrows in in Figures Figures 4 and5 5I–K).I–K). TheseThese cellcell aggregatesaggregates movemove awayaway fromfrom thethe parentalparental filamentfilament and and give give rise rise to to a a new new trichome. trichome. The The cells cells in in the the hormogonium hormogonium lose lose the the attached attached portionsportions of of the the necridia necridia with with a a substantial substantial cell cell debris debris formation formation (see (see Figure Figure 55A–H).A–H). Laser scanningscanning microscopy microscopy revealed revealed (Figure (Figure 66E)E) that the former cytoplasmatic content retained itsits fluorescence fluorescence properties properties in in the the extracellu extracellularlar space. space. However, However, visualization visualization was was possible possible onlyonly when when the the sample sample exhibited exhibited a a substantiall substantiallyy stronger stronger laser laser excitation, excitation, compared compared to to the the otherother images images (compare (compare Figure Figure 66B–E).B–E).

Figure 4. Representative overview images of the morphological characteristics of Arthrospira platensis (strain: SAG21.99) Figure 4. Representative overview images of the morphological characteristics of Arthrospira platensis (strain: SAG21.99) during the seven days’ cultivation. Red arrows indicate short trichome fragments of Arthrospira platensis. Bright field microscopy,during the Axio seven Scope, days’ Zeiss cultivation. Microimaging Red arrows GmbH. indicate short trichome fragments of Arthrospira platensis. Bright ﬁeld microscopy, Axio Scope, Zeiss Microimaging GmbH.

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Figure 5. Representative images of light-induced necridia formation (A,B: intermediate cells, C: terminal cell), (D–H) frag- Figure 5. Representative images of light-induced necridia formation (A,B: intermediate cells, C: terminal cell), (D–H) mentation and (I–K) hormogonia/trichomes in light-limited AP (strain: SAG21.99). (G–H) Simultaneous multiple frag- fragmentation and (I–K) hormogonia/trichomes in light-limited AP (strain: SAG21.99). (G–H) Simultaneous multiple mentation of AP at very high illumination (2000 µmol/(m2 * s)) and sufficient HCO3¯. (A–F,I–K) Bright field microscopy, 2 BZ-X810,fragmentation Keyence, of AP Japan, at very (H high) phase illumination contrast (2000microscopy,µmol/(m Axio* s)) Scope, and sufﬁcient Zeiss Microimaging HCO3¯.(A –GmbH,F,I–K) BrightGermany. ﬁeld microscopy, BZ-X810, Keyence, Japan, (H) phase contrast microscopy, Axio Scope, Zeiss Microimaging GmbH, Germany.

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Figure 6. Representative images of the fragmentation and release of the cytoplasm content into the extra cellular space. Figure 6. Representative images of the fragmentation and release of the cytoplasm content into the extra cellular space. (A) bright field microscopy at a 40-fold primary magnification (BZ-X810, Keyence, Japan). (B–E) Label-free laser scanning (A) bright field microscopy at a 40-fold primary magnification (BZ-X810, Keyence, Japan). (B–E) Label-free laser scanning microscopy of unfixed AP cells. Samples were exited at a 555 nm wavelength. Emissions were detected between 650 nm andmicroscopy 700 nm. of(B) unfixed represents AP a cells. transmitted Samples mode were image. exited (C at,D a) 555 show nm images wavelength. from different Emissions z-levels were of detected the same between x-y position. 650 nm(E) showsand 700 an nm. image (B) with represents enhanced a transmitted fluorescence mode intensities image. to (showC,D) th showe fluorescence images from properties different of z-levelsthe former of thecytoplasmatic same x-y position.content. (ScaleE) shows bar represents an image 10 with µm. enhanced Images were fluorescence taken with intensities an Axio Observer.Z1/7, to show the fluorescence Zeiss Microimaging properties GmbH, of the Germany. former cytoplasmatic content. Scale bar represents 10 µm. Images were taken with an Axio Observer.Z1/7, Zeiss Microimaging GmbH, Germany. The number of hormogonia increased significantly up to day 3 (see red arrows in FigureThe 4). numberAt the same of hormogonia time, the divided increased cells started significantly to grow up immediately to day 3 (see due red to arrowsthe abun- in Figure4). At the same time, the divided cells started to grow immediately due to the dant light and HCO3¯ availability, the main factors inducing AP to grow [15]. During this process,abundant the light trichomes and HCO increased3¯ availability, in length the and main reached factors the inducing typical AP helicoidal to grow [shape.15]. During This trichomethis process, elongation the trichomes occurs through increased multiple in length intercalary and reached cell thedivision typical by helicoidalbinary fission shape. at rightThis trichomeangles toelongation the long axis occurs of the through trichome multiple [16]. intercalary cell division by binary fission at rightSimultaneous angles to the multiple long axis fragmentations of the trichome of [ 16trichomes]. were observed only when AP Simultaneous multiple fragmentations of trichomes were observed only when AP from the stationary growth phase were illuminated with a high photon flux density and from the stationary growth phase were illuminated with a high photon flux density and sufficient HCO3¯. Such an event is shown in Figure 5G,H. Under such conditions, necridia sufficient HCO ¯. Such an event is shown in Figure5G,H. Under such conditions, necridia are formed and3 the highest growth rates occur. Ma et al. have confirmed earlier studies are formed and the highest growth rates occur. Ma et al. have confirmed earlier studies showing that upon exposure of AP to photosynthetic active radiation (e.g., solar, ultravi- showing that upon exposure of AP to photosynthetic active radiation (e.g., solar, ultra- olet), reactive oxygen species are generated [17]. These can oxidize lipids of the cell mem- violet), reactive oxygen species are generated [17]. These can oxidize lipids of the cell brane or the sheath, which can lead to the breakage of the spiral structure. It seems con- membrane or the sheath, which can lead to the breakage of the spiral structure. It seems ceivable that the conditions applied in this study might induce similar processes, leading conceivable that the conditions applied in this study might induce similar processes, lead- to the formation of necridia and the release of the intracellular content. However, shear ing to the formation of necridia and the release of the intracellular content. However, shear stresses or cell-cell contact processes have also been discussed as reasons for AP breakage stresses or cell-cell contact processes have also been discussed as reasons for AP breakage inin bioreactors bioreactors and should be considered as well. After seven days, the trichomes had had lengths lengths between between 100 100 µmµm and 500 µmµm (only very fewfew short short spin-offs spin-offs were were still still visible visible at at that that time) time) with with an an outer outer helix helix diameter diameter between between 20 µm20 µ andm and 60 µm. 60 µ m.This This corresponds corresponds well well with with former former data data by byCiferri Ciferri for for AP AP [9]. [9 However,]. However, it isit isworth worth noting noting that that the the helix helix geometry geometry ofof APAP strainsstrains can can be be influenced influenced by by certain certain environ- envi- ronmentalmental variables variables such such as light,as light, temperature, temperatur pH,e, pH, salinity salinity and and nutrient nutrient availability availability [18 –[18–20], 20],so that so that different different morphologies morphologies can alsocan also result. result. The AP diameters decreased fromfrom 7.57.5 ±± 2.92.9 µmµm on day 1, to 6.56.5 ±± 1.71.7 µmµm on day 2, to 5.75 ± 1.461.46 µmµm on on day day 3, 3, to to 5.25 ± 1.261.26 µmµm on on day day 6, 6, and and to 6.75 ± 1.71.7 µmµm on on day day 7. 7. Thus, Thus, after seven days, the cells almost reached the diameters of those from the initial back-up bioreactor culture.

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4. Conclusions Live cell microscopical observations over the cultivation time demonstrated the growth process of AP in a bioreactor setup. The division of mature spirals is a physi- ological reaction—not a pathological sign—as soon as they are exposed to a high photon flux density and sufficient nutrients. Beyond the classically applied bright field and phase contrast light microscopy, high-resolution laser scanning microscopy can also be utilized for studying nonfixed AP samples. The image-based techniques can be used for funda- mental scientific studies. But also—in addition to recording the optical density—to find the adequate time for harvesting large mature AP spirals with size-adapted meshes and to lead back the filtrate—small cell aggregates or single cells—to the next growth cycle.

Author Contributions: C.H.G.J., S.B., P.W.: conceptualization, methodology, data curation, writing— original draft. J.-H.K., I.P., F.J.: resources, writing—review & editing. All authors have read and agreed to the published version of the manuscript. Funding: The work was financially supported by the Wirtschaftsregion Lausitz of Brandenburg through the grant of the joint project “Multiparametrischer Bioreaktor als Pilotanlage zur Simulation der großtechnischen Produktion der Mikroalge Spirulina platensis” (project number: 19.1.16.2). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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