High density long-term cultivation of Chlorella vulgaris SAG 211-12 in a novel microgravity-capable membrane raceway photobioreactor for future bioregenerative life support in SPACE

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High density long-term cultivation of Chlorella vulgaris SAG 211-12 in a novel microgravity-capable membrane raceway photobioreactor for future bioregenerative life support in SPACE Harald Helischa, , Jochen Kepplera, Gisela Detrella, Stefan Belza, Reinhold Ewalda, Stefanos Fasoulasa, Arnd G. Heyerb ⁎

a b

Institute of Space Systems, University of Stuttgart, Pfaffenwaldring 29, 70569 Stuttgart, Germany Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57 70569 Stuttgart, Germany

ARTICLE INFO

ABSTRACT

Keywords: Chlorella vulgaris Microalgae Xenic cultivation Membrane photobioreactor Hybrid life support system ISS space flight experiment

Hybrid life support systems are of great interest for future far-distant space exploration missions to planetary surfaces, e.g. Mars, planned until 2050. By synergistically combining physicochemical and biotechnological algae-based subsystems, an essential step towards the closure of the carbon loop in environmental control and life support systems (ECLSS) shall be accomplished, offering a wide beneficial potential for ECLSS through the utilization of oxygenic photosynthesis: O2 and potential human food can be formed in-situ from CO2 and water. The wild type green alga Chlorella vulgaris strain SAG 211-12 was selected as model microorganism due to its photoautotrophic growth, high biomass yield, cultivation flexibility and long-term cultivation robustness. The current study presents for the first time a stable xenic long-term processing of microalgae in a novel microgravity capable membrane raceway photobioreactor for 188 days with the focus on algal growth kinetics and gas evolution. In particular, culture homogeneity and viability were monitored and evaluated during the whole cultivation process due to their putative crucial impact on long-term functionality and efficiency of a closed cultivation system. Based on a specially designed cyclic batch cultivation process for SAG 211-12, a successive biomass growth up to a maximum of 12.2 g l−1 with a max. global volumetric productivity of 1.3 g l−1 d−1 was reached within the closed loop system. The photosynthetic capacity was assessed to a global molar photosynthetic quotient of 0.31. Furthermore, cultivation parameters for a change from batch to continuous processing at high biomass densities and proliferation rates are introduced. The presented µgPBR miniature plant and the developed high throughput cultivation process are planned to be tested under real space conditions within the PBR@LSR project (microgravity and cosmic radiation) aboard the International Space Station with an operation period of up to 180 days to investigate the impact on long-term system stability.

Acronyms/Abbreviations Acronyms/Abbreviations ASL Chl CTR DLR DSN EC ECLSS

⁎

Algae suspension loop Chlorophyll Carbon dioxide transfer rate German Aerospace Center (Deutsches Zentrum für Luft- und Raumfahrt) Diluted seawater nitrogen medium Experiment compartment Environmental control and life support system

EPS EPSAG

Extracellular polymeric substances Department of Experimental Phycology and Culture Collection of Algae ESA European Space Agency FEP Fluorethylenepropylene FPA-PBR Flat panel airlift photobioreactor IRS Institute of Space Systems (Institut für Raumfahrtsysteme) ISS International Space Station ISRU In-situ resource utilization LED Light emitting diode LSR Life Support Rack NASA National Aeronautics and Space Administration

Corresponding author. E-mail address: [email protected] (H. Helisch).

https://doi.org/10.1016/j.lssr.2019.08.001 Received 11 February 2019; Received in revised form 6 August 2019; Accepted 7 August 2019 2214-5524/ © 2019 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

Please cite this article as: Harald Helisch, et al., Life Sciences in Space Research, https://doi.org/10.1016/j.lssr.2019.08.001

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Nomenclature p(i) rh pH T λi φi ΦPSII CO2 CxHxOx OD OTR PAR PBR PBR-A/B p/c PE PFD PMMA PMP PTFE PS PQ ROS SD TRL µg µgPBR

CH4 H2 H2O N2 NH4+ NH4+-N NO3− O2 PO43− PO43−-P

(partial) pressure relative humidity potential of hydrogen temperature wavelength gas proportion photosynthetic yield carbon dioxide organic molecule Optical density Oxygen transfer rate Photosynthetic active radiation Photobioreactor Photobioreactor chambers A/B Physico-chemical Polyethylene Photon flux density Polymethylmethacrylate Polymethlpentene Polytetrafluoroethylene Photosynthesis Photosynthetic quotient Reactive oxygen species Standard deviation Technology readiness level Microgravity Microgravity capable photobioreactor

methane hydrogen water nitrogen ammonium cation nitrogen in ammonium nitrate anion oxygen phosphate anion phosphorous in phosphate

energy production/stowage by regenerative fuel cells (Belz et al., 2014). Thus, photosynthesis-based systems could find application in closed loop space systems (e.g. for transfer flights) or in in-situ resource utilization (ISRU) facilities directly on extraterrestrial planetary surfaces, e.g. Mars (Messerschmid et al., 1999; Meyen et al., 2016; Verseux et al., 2016). Further advantages of biological organisms compared to technical systems are the ability for self-regeneration, replication and adaption to novel environmental conditions up to evolutionary development within the cultivation housing. 1.2. Axenic or xenic microalgae cultivation in space? To involve living organisms as realistic future components of a bioregenerative ECLSS, the following requirements have to be considered (Eckart, 1996; Gitelson et al., 2003; Schmid-Staiger et al., 2009): Cultivability in microgravity, robustness/tolerance towards cosmic radiation, high cultivation system utilization or efficiency (includes high light utilization yields, high volumetric biomass productivities or carrying capacities and constant O2 production yield), reliability of the biological system towards malfunction, long-term stability of metabolic processes (bioprocess stability), nutritional value of biomass, controllability and reproducibility of growth processes, low susceptibility to bacterial contaminations (xenic breeding) and low crew interaction effort or high level of automation. Microalgae meet the requirements best, as they can be cultivated in axenic or xenic (Lakaniemi et al., 2011, 2012) manner in terrestrial open (Bergmann and Trösch, 2016) or µg-capable closed photobioreactors (PBRs) (Helisch et al., 2016, 2018; Keppler et al., 2018) with high volumetric biomass concentrations and efficient conversion rates of extracellular substances into biomass (Zhu et al., 2008; Chisti, 2010). The usage of axenic or defined xenic processing are currently discussed: Because they could lead to a culture breakdown, bacteria in algal cultures are often described as contaminations (Wang et al., 2013; Bacellar Mendes and Vermelho, 2013). Nevertheless, algal-bacterial interactions were also reported to be beneficial, as the bacterial community might act in a symbiotic manner: Ramanan et al. reported about parasitic, mutualistic or even symbiotic interactions between microalgae and bacteria (Ramanan et al., 2016). Heterotrophic bacteria are described to have a primary function of decomposition in xenic culture, and are also reported to be involved in algal growth promotion. Within a mutualistic relationship, heterotrophic bacteria can deliver dissolved inorganic carbon, while microalgae supply fixed organic carbon (Cho et al., 2015; Ramanan et al., 2016). Additionally, it was reported that, in exchange for organic carbon, bacteria are able to deliver vitamin B12 to microalgae, a major limiting vitamin for algal growth (Croft et al., 2005). Furthermore, Azospirillum and Mesorhizobium were demonstrated to provide ammonia to algae, which are not able to assimilate elementary nitrogen (Bashan and Holguin, 1998). Finally, mutualistic interactions are reported for a co-culture of Chlorella vulgaris and Rhizobium sp., where Rhizobium was described to promote algal growth (Kim et al., 2014a). Parasitic interactions between bacteria and microalgae are also described. They are based on the competition for

1. Introduction 1.1. Physicochemical and biotechnological ECLSS Space agencies’ plans for future human space exploration include returning to the Moon and further missions to Near Earth Objects and Mars (Henn, 2013; ESA, 2015; NASA, 2015; ISECG, 2018). These human long-term, deep-space mission scenarios require environmental control and life support systems (ECLSS) with a high level of regeneration and closure in order to minimize resupply demands. Current physicochemical ECLSS technologies (e.g. Sabatier process or electrolysis) are extensively tested and provide a high technology readiness level (TRL) (Abney and Mansell, 2010; Bockstahler et al., 2017). However, these technologies are strictly limited to their clearly defined underlying physicochemical reactions to produce only simple basic substances like oxygen (O2), methane (CH4), hydrogen (H2), or elemental carbon (C) (Swickrath and Anderson, 2013). Living organisms, e.g. cyanobacteria, microalgae or higher plants offer the essential advantage to synthesize a variety of complex and higher-energy organic molecules (CxHxOx) and bioactive secondary metabolites from inorganic minerals (e.g. ammonium (NH4+), nitrate (NO3−), phosphate (PO43−), sulfate (SO42−) salts) by oxygenic photosynthesis (Temple et al., 1998; Heldt and Piechulla, 2015; Larkum, 2016; da Silva Vaz et al., 2016). The resulting potentially edible plant biomass could basically be used as dietary additive for the crew (Ganzer and Messerschmid, 2009; Belz et al., 2013). Consequently, plants or plantlike microorganims offer the convincing potential to produce fresh potentially edible biomass in-situ in closed environmental systems at any place and at any time. In particular, this could be of increasing importance for stowage or radiation-sensitive food components with increasing mission durations up to several years (Hendrickx et al., 2006; Lehto et al., 2006; Zwart et al., 2009; Perchonok et al., 2012). The apparent by-product O2 can be utilized for crew respiration or for 2

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nutrients and light or could include lysis of algal cells by bacteria for the utilization of intracellular compounds (Ramanan et al., 2016). Concluding, it is not fully clarified if bacterial microbiomes could be necessary for the establishment and maintenance of the long-term stability of an algae-based micro-ecosystem. However, there is currently no evidence in literature for a stable axenic cultivation process in a completely closed cultivation system for longer periods, e.g. months. By choosing a single microalgae species as dominant microorganism in xenic culture, the complexity of the ecosystem could remain manageable due to the selected specific processing parameters (Lakaniemi et al., 2012).

In diverse terrestrial research and more than thirty international space flight experiments since the 1950′s, the resilience of the genus Chlorella to space conditions has been widely demonstrated in small scale cell culture approaches (Myers, 1953; Golueke and Oswald, 1963; Eley and Myers, 1964; Niederwieser et al., 2018). Up to date, no high throughput algae cultivation process with high proliferation rates, high biomass productivity, high carrying capacity and continuous O2 production was demonstrated. Likewise, no closed PBR system has been presented that would support a stable long-term bioprocessing through months. 1.4. The goals of current research

1.3. Chlorella vulgaris - oxygen re-utilization and food production in space

The aim of this study is to assess a novel high throughput xenic photoautotrophic cultivation process of the wildtype green alga Chlorella vulgaris SAG 211-12 in a novel µg-capable membrane raceway photobioreactor miniature plant (µgPBR) for the first time. Particularly, this work focuses on the long-term system stability for 188 d, tested on Earth. To evaluate the long-term processing, growth kinetics, gas evolution, and the particulate distribution of algal cells within the suspension were assessed throughout the whole experiment time. The results of this study shall serve as data basis for the processing of C.vulgaris in a planned long-term cultivation experiment on the International Space Station (ISS). As technology demonstrator, the µgPBR shall give evidence for the long-term performance (max. 180 d), system stability and reliability of a microalgae-based cultivation facility under space conditions. By the synergetic connection to the current physicochemical life support system of the ISS, the µgPBR will create the first known working hybrid life support system (hECLSS) model containing a bioregenerative engine, Chlorella vulgaris.

The green alga genus Chlorella is one of the most investigated and widely characterized algae worldwide with diverse application possibilities such as component for biofuels, animal feed and aquaculture, human nutrition, wastewater treatment or as bio-fertilizer in agriculture (Chisti, 2007a; Guccione et al., 2014; Safi et al., 2014; Otondo et al., 2018). This alga is widely distributed in various habitats such as marine water, fresh or brackish water and soil. Chlorella vulgaris is a type of this genus, usually occurring unicellular and immobile with a mean cell diameter of 2–20 µm (Yamamoto et al., 2004, 2005) and was first described by Beyerinck (1890). The cell body is largely filled by a single chloroplast with a centrally located pyrenoid (Goncalves et al., 2013). Yamada and Sakaguchi and Gerken et al. found a notably thick and rigid cell wall structure as common among several Chlorella species, the composition of which may vary across species or due to the respective cultivation environment (Yamada and Sakaguchi, 1982; Gerken et al., 2013). Because of their small cell size and spherical shape, C.vulgaris cells have the potential to be cultivated in all known photobioreactor types. In return, a suitable post-cultivation treatment (downstream processing) is commonly necessary to make intracellular nutrients available (Komaki et al., 1998; Janczyk et al., 2007). As nutritive food supplement, biomass of C.vulgaris is rich in protein (up to 58%) and contains all essential amino acids (Becker, 2007). It is also rich in unsaturated fatty acids, carotenoids, dietary fibers, vitamins, minerals and other bioactive molecules (Safi et al., 2014). Depending on the provided carbon source (e.g. organic glucose or inorganic CO2) C.vulgaris can grow heterotrophic, photoautotrophic or mixotrophic (Scarsella et al., 2010). Due to its high biomass productivity and strong CO2 fixation ability (de Morais and Costa, 2007; Chiu et al., 2008; Fulke et al., 2010; Borkenstein et al., 2011; Yewalkar et al., 2011), Chlorella is the most commonly used alga for sequestration of CO2. Douskova and Lívanský demonstrated for Chlorella to have high CO2 sequestration and fixation rates of up to 4.4 g l−1 d−1 in photoautotrophic culture (Doucha and Lívanský, 2009), what basically offers a great potential for the quantitative CO2 biomitigation of flue and exhaust gases or air treatment in closed rooms. Hence, besides other well characterized microalgal genera like Athrospira sp. or Chlamydomonas sp., Chlorella sp. are outstandingly suitable to be used as model organisms for space application due to their cultivation flexibility and remarkable robustness. Regarding utilization in Earth orbit or deep space, microalgae require regeneration capacity towards cosmic radiation. In several radiation experiments, Rea et al. described that Chlorella sp. could survive continuous exposure to ionizing irradiation under preservation of more than 90% of their initial photosynthetic capacity. Comparing other species, Chlorella preserved the highest oxygen evolving capacity (Rea et al., 2008). Posner and Sparrow reported Chlorella showing comparatively high survival rates after acute irradiation peaks (Posner and Sparrow, 1964). Regardless of the chosen microalgae, a suitable shielding will be required for the cultivation compartment to minimize potential negative impacts of primary (neutron or γ-radiation) and secondary radiation sources (e.g. reactive oxygen species, ROS).

2. Material and methods 2.1. Microalgae strain and medium 2.1.1. Cultivation medium All cultivations were conducted in a freshwater-like modified inorganic diluted seawater nitrogen medium (DSN) according to Pohl et al. (1987), Atkinson and Bingman (1998), prepared in sterile deionized water. DSN effects rapid algal growth and is therefore predestined for algal mass culture, essential for possible space applications. All chemicals used were of analytical grade. Basal medium contained (in g l−1) KCl, 1.8; NaCl, 0.95; C6H5FeO7 H2O, 0.082; Na2EDTA 2 H2O, 0.022; FeCl3 6 H2O, 0.016 and micronutrients (in mg l−1) MnCl2 4 H2O, 0.8; ZnSO4 7 H2O, 0.2; CoSO4 7 H2O, 0.2; Na2MoO4 2 H2O, 0.2; CuSO4 5 H2O, 0.02. For microalgae scale-up and culture maintenance macronutrients were set to 388 mg l−1 for [NH4+-N] and to 65 mg l−1 for [PO43−-P]. Medium pH was initially adjusted to pH 7 by titration with 0.1 M HCl or 0.1 M NaOH. During µgPBR cultivation, cultures were periodically supplied with macronutrients by the provision with complete medium containing concentrations of 776 to 2329 mg l−1 for [NH4+-N] and 65 to 196 mg l−1 for [PO43−-P]. 2.1.2. Microalgae strain, culture scale-up and maintenance The wild type Chlorella vulgaris strain SAG 211-12 (authentic Beijerinck strain (Beyerinck, 1890)) was obtained from the Department of Experimental Phycology and Culture Collection of Algae (EPSAG), University of Goettingen, Germany. The culture was not axenic but free of major contaminants and from other algae strains (data not shown). For the culture scale-up, the cells were directly inoculated in 50 ml suspension culture flasks (Bio-One, Greiner, Vtotal = 20 ml) using sterile DSN (see Section 2.1.1). The flasks were incubated at 85 rpm (Digital Orbital Shaker, Heathrow Scientific), 23 ( ± 2) °C and a continuous mean photosynthetic photon flux density (PFD) of 57 µmol photons m−2 s−1 using a commercial LED-panel (red, λ = 630 nm; blue, λ = 465 nm; Deckey). An additional CO2 source was not supplemented. 3

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Five cultures were collected and transferred into a 500 ml suspension culture flask after 14 d. Cells were diluted with DSN (Vtotal = 400 ml) and incubated under the same conditions for 14 d. The suspension was then transferred into a 5 l cultivation flask (Schott AG, Germany) and diluted in complete DSN to a final suspension volume of 4 l for culture maintenance. Algae suspensions were maintained at analogous conditions. Additionally, sterile filtered compressed air (filter pore size 0.22 µm, Carl Roth GmbH + Co. KG, Germany) with a flow rate of 150 l h−1 and a CO2 fraction of 0.052% (v/v) was provided by a volume flow controller (max. 10 l min−1, FR2A15, Brooks Instrument GmbH, Germany) to ensure an adequate suspension mixing.

peristaltic pump (Watson Marlow® 114 series, Watson Marlow GmbH Germany; used pump tubing, Gore® Sta-Pure® PCS, W. L. Gore & Associates, Inc., USA) and is composed of two double sided photobioreactor core assemblies (see Section 2.2.1) tubing and connectors, sensors to monitor pH (PreSens FTC-SU-HP5-S, PreSens® Precision Sensing, Germany) and biomass density (developed at Institute of Space Systems Stuttgart, IRS). Feeding and harvesting are performed via access ports by using a separate liquid exchange device (LiED). PBR-A and PBR-B are arranged in series. The Vtotal of the ASL, about 650 ml, is filled with algae suspension. Lighting is carried out by a dichromatic red/blue LED panel placed outside the ASL (see Section 2.2.1). Gases are pulsed into the EC by a pulse chamber and homogeneously distributed within the EC by air circulation fans. This provides a tangential gas flow between the FEP membrane and the lighting units. CO2 is provided by a pulse camber connected to an external gas bottle. After implementation in an existing physico-chemical (p/c) ECLSS a µgPBR plant is planned to receive CO2 from a carbon dioxide removal and concentration assembly (CCA) aboard the closed system, e.g. spacecraft (Bockstahler et al., 2017). Gas evolution and process parameters through the experiment are monitored by several gas sensors: pCO2 (COZIR Wide Range 20%, Cozir®, Pewatron AG, Switzerland), pO2 (FIGARO SK25F, Figaro®, Japan), pressure, p (XFGM-6100KPGWSR, Pewatron AG, Switzerland), relative humidity, rh (SHT21, Sensirion, Switzerland) and temperature, T (LM35 BZ, B+B Thermo-Technik GmbH, Germany). Absorbers are used to regulate O2 (AnoxiBug oxygen scavenger, Hanwell Solutions Ltd., UK) and rh (Silicagel E Organe, GIEBEL FilTec GmbH, Germany) actively within the EC. T is controlled by a cold plate connected to the cooling water circuit.

2.1.3. Pre-culture design and inoculum stowage The preparation of pre-cultures was conducted in a polymethyl methacrylate (PMMA) flat panel airlift photobioreactor (FPA, Subitec® GmbH, Germany) with a cultivation volume of 6 l. Subitec® FPA-PBRs were chosen as they are developed to obtain high biomass productivities in a open PBR system under lab scale conditions (Bergmann and Trösch, 2016). C.vulgaris cells were cultivated in complete DSN (see Section 2.1.1) and fed-batch mode with a continuous mean PFD = 250 µmol photons m−2 s−1 using a sodium steam lamp (Plantastar 600 W, Osram, Germany). Supply air was introduced with a flow rate of 200 l h−1 and a CO2 fraction of 8% (v/v). Temperature and pH were monitored using a pH/thermal sensor (InPro R3253i/SG/120, Mettler Toledo, USA). Temperature was kept constant at 26.0 ( ± 0.5) °C by a control unit (M300 analytical transmitter, Mettler Toledo, USA) connected to a cooling water circuit. Algae samples were taken at early and late log phase to provide an inoculum (Vtotal = 200 ml) with a maximal proliferation rate and high biomass concentration under given process parameters. Samples were stored in sealed syringes at 4 °C without lighting before inoculation into the µgPBR.

2.2.3. Mode of operation The µgPBR was operated in repeated batch mode for 188 d. The ASL was filled with complete DSN and warmed up to an operating T = 25.5 ( ± 1.0) °C. The EC was initially flushed with N2, and a constant CO2 level between 7–9% (v/v) was adjusted. The reactor was inoculated with 2 × 100 ml C.vulgaris suspension pre-cultured as described in Section 2.1.3. Suspension flow velocity was set to 200 ml min−1. Cells were continuously lighted with dynamic red/blue light (see Section 2.2.1) in molar photon ratios of R50 : B50 (6 d week−1) and R70 : B30 (1 d week−1) at continuous mean PFDtotal = 200–300 µmol photons m−2 s−1. Optical density (OD) was measured daily (see Section 2.3.1), nitrogen and phosphorus were regularly monitored (see Section 2.3.2). Monthly, viability of the cells within the ASL was determined. Every 14 d 200 ml suspension was harvested and fresh complete DSN supplied.

2.2. Membrane photobioreactor design and operation 2.2.1. Photobioreactor design The µgPBR assembly is composed of two double sided raceway reactor cores (see Fig. 1) which contain the algae suspension. The raceway flow channel design was chosen (transparent Lexan® polycarbonate; channel depth, 3 mm; channel width, 10 mm) to increase the surface to volume ratio resulting in an enhanced photon influx and gas exchange surface. Both reactor cores are sealed by a gas permeable fluorethylenepropylene membrane (DuPont™ Teflon® FEP Fluoroplastic Film 100 C; thickness, 25 µm) allowing sufficient gas transfer of CO2 and O2 between the gaseous phase outside the assembly (see Section 2.2.2, experiment compartment, EC) and the liquid phase inside the reactor. The membrane surfaces are optically transparent allowing a transmission of > 96% within the spectrum of photosynthetically active radiation (PAR) spectrum (Fig. 2). The LED Panel design was oriented towards the in-vivo absorption spectrum of C.vulgaris SAG 211-12 (λmax = 435–475 nm and 660–680 nm; Fig. 3). Red (Oslon SSL LH CPxP GF, λmax = 660 nm, Osram, Germany) and blue (Oslon SSL LB CP7P-GZHX-35, λmax = 470 nm, Osram, Germany) LEDs are evenly distributed on the LED panel and are controlled separately for each color, enabling the possibility for setting different molar light distributions and total PFD. Each side of each reactor chamber is lighted through the gas exchange membrane. 2.2.2. Principles and functionality The setup of the µg-capable PBR miniature plant is shown in Fig. 4. The majority of the components are located within the water and gas tight experiment compartment (EC). The EC contains a total gas volume of ∼10 l and provides access to the algae suspension loop (ASL) and a defined gas atmosphere. The functional group ASL is driven by a

Fig. 1. Raceway reactor core design, total dimensions of single chamber: 210 × 320 mm (Keppler et al., 2017). 4

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2.3.2. Dissolved ammonium and phosphate Concentrations of dissolved NH4+-N and PO43−-P were monitored throughout the FPA-PBR and µgPBR cultivations every two days. Samples were centrifuged at 10,000 x g for 10 min (centrifuge type 5430, rotor FA-45-30-11, Eppendorf AG, Germany) and the SN was collected. SN was filtered (pore size of 0.22 μm; CME-Rotilabo® syringe filter, Carl Roth GmbH + Co. KG, Germany). Sample preparation and measurement of dissolved ions were performed according to the respective manuals (Hach GmbH, Germany). NH4+-N and PO43−-P in [mg l−1] were measured by using quantitative colorimetric cuvette tests (type LCK 303, λmeasurement = 694 nm or LCK 049, λmeasurement = 435 nm, respectively, Hach GmbH, Germany) and a spectrophotometer (type DR 3900, Hach, Germany).

Fig. 2. Cross-section of halved µgPBR assembly. Culture depth, 3 mm, gas circulation between LED panel and FEP-membrane by crossflow fans, FEP,fluorethylenepropylene (Keppler et al., 2017).

2.3.3. Algal viability The viability of algae cells was determined by using a modified selective cell staining assay according to Black and Berenbaum, 1964 and Imase et al., 2013. Samples were diluted to give X = 0.23 g l−1 and analyzed in triplicate. Untreated algae within the log phase were used as a viability control (+ Ctrl), and thermally inactivated algae within the log phase (T = 80 °C for 15 min, cooled to RT, type D1302, digital dry bath, Labnet International, Inc., USA) as a staining control (– Ctrl). Samples were centrifuged at 10,000 x g for 4 min (centrifuge type 5430, rotor FA-45-30-11, Eppendorf AG, Germany) and the SN was discarded. The cell pellet was successively washed two times by repeated resuspension of the cells in deionized H2O, centrifugation and discarding of the SN. Cells were resuspended in 500 µl deionized H2O. For cell staining, 500 µl Eosin Y disodium salt solution (Sigma-Aldrich, USA) were added to reach a final concentration of 10 mg ml−1. Samples were carefully inverted and incubated for 20 min at room temperature and without light. Samples were diluted with 4 ml deionized H2O, briefly inverted and centrifuged at 10,000 x g for 2 min. SN was discarded and cells were resuspended in 5 ml deionized H2O. Cells were successively washed further two times and finally resuspended in 500 µl complete DSN. Living cell counts (LCC, unstained viable green cells) and total cell counts (TCC, green and red colored cells) were determined by using a microscope (DM 750, Leica Microsystems, Germany) and a Thoma hemacytometer according to manufactuer protocols (Brand® GmbH, Germany). Culture viability was calculated according to Eq. (3):

Fig. 3. In-vivo absorption spectrum of SAG 211-12 in µgPBR (cultivation day 83). Absorption maxima were identified at λ = 397.9 nm, λ = 431.3 nm and 676.3 nm. The arrowheads indicate the emission maxima and the red/blue areas the total spectral range of the chosen LEDs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Viablity [%] = LCC · TCC

2.3. Analytics

with R2 = 0.99; N = 14

(1)

X [g l 1] = 0.29·OD750

with R2 = 0.99; N = 15

(2)

· 100

with N = 3

(3)

2.3.4. Pigment analysis Pigments were analyzed using a dimethylformamide (DMF) extraction assay modified for C.vulgaris SAG 211-12. The assay was conducted in triplicates. As required samples were diluted to OD680 = 1 for a standardized concentration. Samples of 1 ml suspension were repeatedly centrifuged at 10,000 x g for 10 min and SN was fully discarded. The pellet was directly frozen at T = −20 °C and stored o/n. For pigment extraction the pellet was quickly thawed to RT, shortly vortexed and directly resuspended in 1 ml DMF. The homogenate was incubated in an ultrasonic bath (JPS-10 T, Jietai GmbH, China) with an intensity of 60 W at T = 50 – 55 °C for 30 min followed by a centrifugation an 10,000 x g for 2 min. The green SN was transferred into a DMF stable cuvette (semi micro UV-cuvette, Brand® GmbH + Co.KG, Germany) and absorbance was instantly measured as triplicate at λ = 480 nm, λ = 647 nm and λ = 664.5 nm in a spectrophotometer (type DR 3900, Hach, Germany). Pure DMF was used as blank. Chlorophyll concentrations were calculated according to Eqs. (4)–(6), total carotenoid concentrations according to Eq. (7) (Inskeep and Bloom, 1985; Wellburn, 1994).

2.3.1. Optical density and dry weight Optical density was determined at 750 nm and 680 nm in a spectrophotometer (type DR 3900, Hach, Germany). Deionized water was used as blank. Measurements were conducted in triplicates. Algae samples were diluted in deionized water to reach OD750 ranges between 0.3 and 0.6 or for OD680 between 0.4 and 0.7 during measurements. For determination of dry weight, OD750 and OD680 were measured in triplicates, values were averaged. Samples of 10 ml were centrifuged at 10,000 x g for 10 min (centrifuge type 5430, rotor FA-45–30–11, Eppendorf AG, Germany). Supernatant (SN) was discarded and the cell pellet was successively washed twice in deionized H2O. Cells were resuspended in 1.5 ml deionized H2O and transferred to a pre-weighed aluminum bowl. Cells were dried at 105 °C for 24 h and transferred to a desiccator to cool down for 20 min before dry mass was repeatedly weighted (Moisture analyzer, MB 50, PCE Instruments GmbH, Germany). OD and dry weight were correlated using a linear fitting to determine biomass concentration per volume X [g l−1], see Eqs. (1) and (2):

X [g l 1] = 0.23·OD680

1

c (Chla)[µ gml 1] = 12.70*A664.5

2.79*A647

(4)

c (Chlb)[µ gml 1] = 20.70*A647

4.62*A664.5

(5)

c (Chla + b)[µ gml 5

1]

= 17.90*A647 + 8.08*A664.5

(6)

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Fig. 4. Setup of membrane µgPBR miniature plant, modified according to Helisch et al. (2018). The presented cultivation system fits into a Middeck Locker Equivalent of the ISS. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

c (Chlx + c )[µ gml 1] = [(1000*A 480 ) /245

(2.14*Chla)

2.3.6. Photosynthetic photon flux density PFD was determined within the PAR range using a mobile quantum sensor (type PAR lite, Kipp & Zonen BV, Netherlands) connected to a digital multimeter (type UT71C, UNI-Trend®, China). PFD was measured at evenly distributed points on the respective PBR surface (63 points for FPA-PBR and 360 points for the different red/blue light patterns of the µgPBR). The measured current values were averaged and converted into PFD considering the sensor sensitivity (S), Eq. (8). For the µgPBR, several molar distributions of red and blue wavelengths were set to reach an absolute mean PFD = 200–300 µmol photons m−2 s−1.

(70.16*Chlb)] (7)

2.3.5. Particle analysis and culture condition The condition of living specimen was periodically investigated using in-situ light microscopy (DM 750, Leica Microsystems, Germany) with the focus on cellular morphology, proliferation and purity, aggregation behavior and the excretion of extracellular polymeric substances (EPS). The program ImageJ (software version 1.51d, Fiji, National Institutes of Health, USA) was used for graphical particle analyses. Average sizes of single cells, cell clusters and the percentage distribution were investigated. Nine independent images per sample with a total average number between 1.4 • 105 and 5.6 • 106 cells per sample were analyzed. Microscopic images at 100 x magnification were uploaded to the toolbox. The background was subtracted and the RGB color image was converted to 8-bit. The threshold was set to reduce the image into black and white. The “fill holes” function was used in order to transform the cells into perfectly filled black dots. Cells, which were closely together, were separated visually using the “watershed” function. Finally particles were quantified using the “particle analysis” function. Minimum size was set to 1 µm2, circularity was set to 20%, in order to exclude soiling from analysis.

PFD [µ molphotonsm 2s 1] = mV ·S

1

·1000

= 5.4 µ V·µ mol

with S

1·s·m2

(8)

2.3.7. Photosynthetic capacity The photosynthetic capacity was determined by calculating the gravimetric assimilation rates of CO2 and release rates of O2 due to oxygenic photosynthesis within the µgPBR. Therefore, concentrations of gaseous CO2 and O2 (φ CO2/O2 in% (v/v)) within the gastight EC were measured during the whole cultivation time (see Section 2.2.2). CO2 and O2 concentrations within the EC were converted into gas volumes according to Eq. (9) and converted into gas masses (g) or ammount (mol) according to Eq. (10) and 11. Gravimetric gas conversion rates 6

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were calculated in [g d−1]. The photosynthetic quotient (PQ) as a measure for the photosynthetic capacity was calculated according to Eq. (12). Considering the molar masses (M) of CO2 and O2, the maximum possible molar PQ is 1.

VCO2/O2 [l] =

CO2/ O2

· VEC · 100

1

with

CO2/ O2

= CO2 or O2 in N2 [%(v/v)], VEC = 10.5 l mCO2/ O2 [g] = VCO 2/ O2 · O2

= 1.3kgm

with

CO2/ O2

After inoculation, an adaption phase of Chlorella to the new cultivation environment occurs, which could partly be due to pigment regeneration after light induction. In the experiment, initial mobile biomass (free algae cells) decreased within the ASL from Xstart = 0.75 g l−1 down to X = 0.19 g l−1 during the first 4 days of cultivation. In this phase, only a slight net O2 production qO2 < 0.1 g d−1 was measured. Already one hour after inoculation a commencing light induced regeneration of the photosynthetic apparatus was observed in-vivo (Fig. 5, ASL 1 h). For re-establishing algal cultures, storage of cells needs to be considered.

CO2

(9)

= 1.8kgm 3,

3

(at T = 26 C andp = 0.96bar) nCO2/ O2 [mol] = mCO2/ O2 · MCO2/ O2 MO2 = 31.99gmol

1

3.2. Xenic long-term cultivation in raceway µgPBR

with MCO2 = 44.01gmol 1,

3.2.1. Growth kinetics and biomass productivity Long-term cultivation of C.vulgaris in the µgPBR was conducted in two experiments. The first run (187 d) served to verify principle longterm feasibility of the cultivation system as well as the establishment and final optimization of the bioprocess parameters (Helisch et al., 2018). For the second run, the identical hardware and their configuration were used as at the end of the first experiment. In 188 d of run 2, the optimized bioprocess was characterized by the assessment of relevant growth parameters of the consecutively repeated batch process (Fig. 6). All single 14 batch cycles and the global growth were considered (Table 1). As mentioned in Section 3.1, the “starter cells” needed four days after inoculation to adapt to the new environmental conditions. Biomass recovery followed to a biomass of X = 0.34 g l−1 (∼ 45 % of Xtotal) until the end of batch cycle 1. Although mobile biomass could not reach the initial value within the first batch, C.vulgaris cells in xenic culture remained quantitative mobile within the ASL. In theory, single cells should be homogeneously distributed within the loop without aggregation or adhesion to ensure a high system control (e.g. for feeding and harvesting) and long-term process stability. Interactions between algal cells or with the bacterial microbiome are not considered. The decrease in mobile biomass resulted from bacterial or algal induced adhesion processes as well as cell sedimentation within the cultivation chambers (PBR-A and B) resulting in an apparent biomass loss. C.vulgaris appeared unicellular or in cell clusters with a mean number of 29 cells per cluster, cross-linked by cellular debris of proliferating or dying cells, extracellular polymeric substances (EPS) or other secreted polymers. These are potential starting points for the creation of a planar biofilm through the reactor surface or gas transfer membrane and were previously described for the given system in Helisch et al. (2018). The apparent biomass loss was assumed to result from the establishment

(11)

1

PQmolar [/] = nO 2 · nCO2

(10)

(12)

1

2.4. Statistical treatment The process-optimized long-term cultivation of SAG 211-12 in the µgPBR was conducted as a single experiment due to its high preparation effort and exceptional cultivation duration. Various preliminary experiments with individual durations between 7 d and 187 d with a total cultivation time of more than 1000 d were performed to confirm system and basic bioprocess functionality (Helisch et al., 2016; Keppler et al., 2017, 2018). All samples were taken at least as triplicates (biological replica) and measured three times each (technical replica) to consider inhomogeneous cell distributions and individual failures of manual measurements. Values of biological replica were averaged and data scattering was indicated by standard deviations (SD). Microalgae growth was evaluated using the logistic or sigmoid function as it represents all growth phases including cell dying in the culture (Ernst et al., 2005). Data points were fitted using Python (Spyder version 3.7, Anaconda Cloud) according to Eq. (13). The sigmoidal shape is characterized by carrying capacity Xmax [g l−1], the specific growth rate constant, µ [d−1], the turning point, tc [d] and the starting concentration X0 [g l−1].

y (t ) = (Xmax

X0 )/1 + e

µ ·(t tc )

+ X0

(13)

3. Results and discussion 3.1. Influence of stowage conditions on algae inoculum and culture adaption The inoculation represents the most critical step for a stable longterm cultivation process due to the adaption of “starter cells” to the novel PBR environment. For the current study, inocula were prepared as described in Section 2.1.3 and stored for 4 weeks at 4 °C and without lighting to simulate a realistic period from sampling to arrival at the target reactor, e.g. on ISS. Previously, surviving storage periods of more than 6 weeks were proven for sealed xenic SAG 211-12 samples under maintenance of post storage algal biomass productivity (Helisch et al., 2016). A decrease in the absorption capacity of chlorophyll a/b in viable Chlorella cells has been observed when using cooled and dark stowage (Fig. 5, inoculum). Pigments with absorption maxima in the red spectral range were primarily affected. This may be related to a stepwise degradation of chlorophyll a/b down to basal chlorophyllides catalyzed by intracellular chlorophyllase activity in darkness and at low temperature, resulting in a changed in-vivo absorption profile for SAG 21112. This observation was confirmed by Böger, who reported a high temperature insensitivity of chlorophyllase (EC.3.1.1.14) in C.vulgaris SAG 211-11 h (Böger, 1965).

Fig. 5. In-vivo absorption spectrum of SAG 211-12 inoculum (stored at 4 °C without light) and mobile cells within the ASL after 1 h and 188 d of cultivation in µgPBR. For comparison values were normalized to biomass and the absorption minimum at λ = 750 nm. ASL, algae suspension loop. 7

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Fig. 6. Growth profile of C.vulgaris in mobile phase of µgPBR miniature plant. The sigmoidal function relies on the respective carrying capacity of the individual batch cycles (R2 = 0.99). Prediction of tendency for globally decreasing biomass started at cultivation day 152. X, biomass concentration (dry wt). Gray area, global log phase. Each data value represents the average ± SD of n = 3 data points that were measured within one reactor experiment.

and self-stabilization of the algae-bacterial micro-ecosystem within the ASL to allow a stable mutual relationship between the dominant algae and the bacterial microbiome. In all following batches the expected cyclic sigmoidal growth was observed (Fig. 6). Biomass was successively increased with a maximal volumetric productivity of Pmax = 1.02 g l−1 d−1 up to Xmax = 12.2 g l−l. A maximal absolute daily biomass yield of myield,max = 0.66 g d−1 was obtained. As a prediction for global growth behavior of C.vulgaris over 188 d, a sigmoidal function based on the carrying capacity of the respective batch cycles was calculated. An initial adaption related biomass decrease and globally decreasing biomass after day 152 are considered. Regarding global biomass evolution, an adaption and acceleration phase of 42 d, an exponential phase of 30 d, a deceleration phase of 48 d, a stationary phase of 32 d and a decreasing phase of 36 d were determined. Nevertheless, in all individual batches

culture activity was given until the end of the long-term cultivation. The overall biomass decrease after day 152 can be explained by reaching the maximal biomass carrying capacity of the reactor at PFD = 200 µmol photons m−2 s−1 and successive biofilm creation. Growth limitation due to nutrient starvation did not occur (see Section 3.2.2). Based on the calculated characteristic values of the global growth, the optimal biomass concentration for the change from a successive biomass increase to a continuous processing was determined to fall within the late global log phase (end of batch cycle 6) with Xopt = 9.1 g l−1 ( ± 1.6), with µopt = 0.41 d−1 ( ± 0.03), Pmax,opt = 0.92 g l−1 d−1 ( ± 0.10) and myield,opt = 0.6 g d−1 ( ± 0.1) for the selected bioprocess parameters. The resulting irradiance (PFD = 22–33 µmol photons m−2 s−1 per g l−1 biomass) can support high continuous growth rates for strain SAG 211-12 (Degen et al.,

Table 1 Cyclic and global growth of C.vulgaris in µgPBR ASL mobile phase.

Xmax, carrying capacity; µ specific growth rate; Pmax, maximal volumetric biomass productivity; myield, daily biomass yield. Gray area, optimum time for process adaption. Calculation of Pmax and Myield are based on values of Xmax and µ. 8

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2001). Hence, a change to continuous operation mode by adjusting a constant dilution rate D = 0.41 l d−1 corresponding to µopt = 0.41 d−1 at the ceasing global log phase (between batch cycles 6 and 8), would allow a constant high biomass production of up to 10 g l−1 with a volumetric productivity of up to 1 g l−1 d−1 for a xenic C.vulgaris culture in membrane-based µgPBRs. For the given system, Xopt would be clearly below the calculated maximal carrying capacity Xmax = 12.2 g l−1, which was reached in batch cycle 10. Consequently, a limitation within presented parameters is not expected. For comparison, in a gravitydependent high throughput FPA-PBR, the same strain was cultivated at similar conditions up to a Xmax = 12.7 g l−1, but with a Pmax of up to 2 g l−1 d−1. At high light conditions (PFD = 750–800 µmol photons m−2 s−1) a Pmax of even 4 g l−1 d−1 could be reached in a FPA-PBR.

frequently supplied as macronutrients with a N:P ratio of ∼10:1 as NH4+-N appeared to be the limiting growth factor during the single batches. As N:P = 8:1 was reported being an optimal ratio for a maximal N-uptake rate in Chlorella vulgaris (Kapdan and Aslan, 2008), the nutrient concentrations and ratios in the current process were chosen to slightly reduce N-uptake and biotransformation to prevent the system from medial N-depletion during the selected batch periods of 12–14 d. As N-depletion was found to potentially enhance EPS biogenesis and cell cluster formation [78], the process was adapted to ensure a minimal [NH4+-N] = 0.96 mg l−1 (0.069 mmol l−1) within the ASL (equals a cyclic N-removal efficiency of 99.6%). A significant temporary release of cellular N into the medium was not observed, what could be an indicator for protein-N degradation by autolysis and re-utilization. With a minimal [PO43−-P] = 0.58 mg l−1 (0.041 mmol l−1)(equals a cyclic P-removal efficiency of 97.4%), a P-depletion did also not occur. Mean uptake rates for NH4+-N = 36.2 mg l−1 d−1 ( ± 6.4) (2.3 mmol l−1 d−1) and PO43−-P = 2.5 mg l−1 d−1 ( ± 0.7) (0.178 mmol l−1 d−1) were determined for the given parameter set (Table 2). The optimal substrate usage (Yx/s) was found between batch cycles 7 and 8 (Fig. 11) with nitrogen being the limiting substrate. This correlates with the described growth data (Table 1) and supports the concept of a possible adaption to a continuous process at this stage. The cultivation was conducted at constant environmental parameters Taverage = 25.5 °C and pHaverage = 7.3 (Table 3), as these could have a crucial impact on photosynthetic sub-processes (e.g. CalvinBenson cycle, etc.) (Tcherkez et al., 2006). A pH below 8.0 was chosen to minimize NH3, thus excluding poisoning of the culture during the entire cultivation time of 188 d (Tam and Wong, 1996). The decrease of pHaverage in batches 5 to 7 may indicate an increased biotransformation of inorganic NH4+ into cellular glutamate under enhanced release of H+ into the medium (Liu et al., 2015). These findings correlate with the maximal values for Yx/s (Table 2 and Fig. 11) or Pmax (Table 1). To avoid possible limitations of cellular N, cellular pigmentation was analyzed through the whole process [Fig. 12]. According to Bernard (2011) the amount of total chlorophyll can be assumed to be proportional to cellular N. A mean Chltotal ∼ 60 mg pigment g−1 dry mass was observed through the whole cultivation. A pigment increase of up to ∼ 130 mg pigment g−1 dry mass occurred in batch cycles 1013. The enhanced pigment biosynthesis can be explained as culture response to the lower available light quantities per cell. Consequently, a significant decrease in pigmentation was not observed, a resulting Nlimitation was basically excluded.

3.2.2. Apparent and hidden growth The absolute biomass produced was assessed after termination of the experiment at day 188 and opening the reactor. Mobile and immobilized biomass of the ASL were collected. For calculations, removed samples and harvested fractions with Xtotal,dry = 12.9 g after 188 d were also considered. Hidden growth due to biomass immobilization was calculated to myield = 0.03 g d−1. Thus, an overall cell mobility of 55.6% (comparing 24.5% before optimization in first long-term cultivation experiment with 186 d) were reached (Fig. 7). The absolute dry mass before and after optimization indicated that the µgPBR plant was working at its upper loading capacity. For µg-capable photobioreactors, especially membrane-based systems, these data give a first impression of possible biomass deposits in the cultivation chambers due to adhesion and biofilm formation (Fig. 8). Nevertheless, microgravity could induce changes in the bacterial and algal metabolism, resulting in an increased biofilm formation (Kim et al., 2013a, 2013b). A long-term operation under µg could give a comprehensive insight into the evolution of biofilms in such cultivation systems. To reduce cell damage and maintain algal cell distribution and unicellularity, continuous stress loads (e.g. mechanical stress) shall be reduced to a minimum. As the ASL is a pumped system, mobile algae cells pass the pump head frequently. Other stress factors were minimized during optimization process (Helisch et al., 2018). Although comparing other pump types (e.g. membrane pump or gear pump), the peristaltic pump was proven to generally reduce mechanical forces for SAG 211-12 to a minimum (Bretschneider et al., 2016), the occurring periodic shear stress due to pressure swings or vibrations could induce certain cellular responses resulting in the enhanced creation of algal or bacterial EPS as protective stress response. Long-term impacts on culture viability in a regenerative cultivation system are not investigated yet. The impact of the mechanical stress on the cells depends on speciesspecific cell size, cell wall composition, cellular regeneration capacity, the total number, and frequency of passes through the pump head and the rotating velocity of the pump head (Vandanjon et al., 1999). Culture viability was periodically measured. Cells from the mobile phase of the ASL were taken during the running experiment, as they are constantly exposed to mechanical stress caused by the pumphead (every 3 min per cell in the current ASL), and processed according to Section 2.3.3. Viabilities between 93.5% and 99% were measured until the end of the long-term cultivation (Fig. 9). Culture viabilities of immobilized cell mass within the chambers PBR-A/B maintained > 97%. Comparing other cultivation systems (e.g. FPA-PBR, viability > 99%) similar viabilities could be reached/maintained for C.vulgaris in the given µgPBR facility over 188 d Furthermore, cell wall debris was only sporadically found within the mobile phase during the whole cultivation time. These results imply a high regenerative capacity of SAG 21112 against mechanical stress, essential for long-term application in pumped systems.

3.2.4. Gas evolution and photosynthetic capacity During cultivation a total CO2 mass of 126.8 g (2.88 mol) was

Fig. 7. Immobilization and absolute C.vulgaris dry mass after long-term cultivation (188 d) in µgPBR miniature plant before and after process optimization. The calculations consider total biomass of the mobile and immobile phase of the ASL after harvesting, samples and harvested fractions within two reactor experiments.

3.2.3. Macronutrient uptake and pigmentation After culture adaption in cycle 1, a characteristic pattern of nutrient uptake was observed (Fig. 10A, B). Ammonium and phosphate were 9

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assimilated and a total O2 mass of 28.9 g (0.90 mol) was released. A mean CO2 assimilation rate of qCO2 = 0.67 g d−1 ( ± 0.06) (0.17 mol d−1) and O2 release rate of qO2 = 0.15 g d−1 ( ± 0.04) (0.005 mol d−1) were calculated over 188 d (Table 3). For short-term operation of 12 days, a PQ = 0.46 was proven. A general decrease of the photosynthetic efficiency occurred due to progressive algal-bacterial biofilm development on the gas exchange membrane (Helisch et al., 2018), which may influence the transfer rates for CO2 (CTR) and O2 (OTR) at constant specific exchange surface and suspension volume. The impact of heterotrophic bacteria on gas balancing within the EC was not considered because of their negligible mass proportion of total mobile and immobilized biomass (< 0.8%, see also Section 3.2.4). Hence, for the tested period of 188 d, a continuous and stable CO2 biofixation and O2 synthesis was proven for the given system (Fig. 13A, B) and an extension of the maximal cultivation period seems feasible. Within the batch cycles, the PQ followed a “dome-like pattern” where the PS-capacity increased instantly after harvesting/feeding due to a reduced biomass density and related high PFD availability per cell (Fig. 13C). This confirms the declining PQ not only to depend on biofilm creation but also on the successive biomass increase within the ASL. After a possible process change to continuous operation mode for a running µgPBR (e.g. after batch cycle 6–8) the PQ decrease would depend symptomatically only on progressive biofilm synthesis. Assuming a constant linear (conservative) reduction of photosynthesis capacity, a drop down to 12.5% would be reached after a tmax = ∼ 340 d. An exponential decrease of photosynthesis capacity, caused by a progressive evolving but partially self-regulating biofilm in a stable microecosystem, is a more plausible scenario (unpublished data), resulting in a tmax = ∼ 500 d without a need for purification or exchange of the cultivation chambers. Thus, xenic algal growth within a closed cultivation system is a highly dynamic and complex process at any time, where the photosynthetic capacity depends on the interaction of several process factors. These include for example membrane material and physical properties (kL,O2/CO2-values), current biomass density in the PBR chambers (cyclic and global), algae/bacteria dynamics, availability of light and nutrients, biofilm synthesis (global) or gas composition within the EC.

Fig. 8. Biofilm after long-term cultivation (188 d) in µgPBR miniature plant. A, µgPBR chamber, B, FEP gas exchange membrane.

Fig. 9. Impact of mechanical (pump) stress on culture viability of C.vulgaris (mobile cells) during long-term cultivation in µgPBR miniature facility. µgPBR samples were taken from the mobile liquid phase of the ASL. FPA represents cells from the pre-culture (airlift) and PBR-A/B from both µgPBR chambers after reactor opening and mixing. Samples normalized to FPA-PBR cells. Each bar represents the average ± SD of n = 3 data points that were measured within one reactor experiment.

3.2.5. Cell distribution and clustering behavior A stable long-term functionality of the µgPBR facility depends to a large part on the equal distribution of C.vulgaris within the ASL, in particular the raceway flow channels of the PBR chambers. Especially a homogeneous availability of inorganic macronutrients and the “dispersion” of light energy influx within the algal suspension are basic

Fig. 10. Averaged metabolized ammonium and phosphorous within single batch cycles. A, NH4+-N (R2 = 0.98), B, PO43−-P (R2 = 0.99). Each data value in A and B represents the average ± SD of n = 9 data points that were measured within one reactor experiment, cycles 1–3,7 and 8 are not considered. 10

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Table 2 Nutrient uptake of C.vulgaris in µgPBR ASL mobile phase.

YX/S, yield coefficient biomass/substrate; NH4+-N, ammonium-nitrogen; PO43−-P, phosphate-phosphorous. Gray area, optimum time for process change to continuous operation. YX/S estimations are based on deltas for X and N of single batch cycles of single reactor experiment.

Radical growing cells with maximal cell diameters of up to dmcell ∼ 19 µm were identified at the same time (unicellular and bound in clusters). This can be explained by a temporary location of the cells near to the blue LED lighting spots, where immobilized cells were lighted only monochromatically. After detaching randomly, the cells returned into the mobile ASL phase. This agrees with observations by Kim et al. who reported for C.vulgaris the influence of different light wavelengths on the growth behavior. Although the related signaling pathways are not fully understood yet, it is proven in experiments with monochromatic lighting that blue photons enhance pure cell growth up to a critical size, necessary for efficient cell proliferation. On the other hand, red photons induce enhanced cell proliferation (Kim et al., 2014b). In the following batches, a regeneration towards unicellular mobile cells occurred up to an optimal distribution of > 99% (cycle 5) within the global log phase at constant lighting conditions. Here, the smallest cells had a dmcell ∼ 4.3 µm according to maximal proliferation, described by µmax (Table 1). The relative particle number showed a similar tendency, as in cycle 5 only 0.03% of all particles are present as clusters (Table 4). For the global log phase a mean particle proportion of < 0.5% was observed. The optimal distribution of unicellular C.vulgaris cells and minimal deposits within chambers PBR-A/B confirmed the previously described optimum time for process adaption to a continuous operation. Until end of the experiment a mean clustering of 7.9% ( ± 4.2) was maintained. Biomass samples were analyzed after the opening of µgPBR chambers PBR-A and B, following 188 d of cultivation. A clustering of 16.2 – 23.5% of total biomass was observed. The bacterial load remained at a mass level of ∼ 1%. This data demonstrates for the first time, that C.vulgaris can grow in xenic culture in a high throughput µg-suitable cultivation system as dominant species with mass proportions of 99.6% ( ± 0.2). The stability of the algae-bacterial based ecosystem was maintained during 188 d

Fig. 11. Optimal substrate usage in µgPBR ASL. Limiting substrate NH4+-N; cycles 13 and 14 were not considered; YX/S, yield coefficient biomass/substrate.

requirements for a controlled and stable photoautotrophic process. Hence, uncontrolled immobilization of cells and extracellular biomolecules could affect the photosynthetic capacity of the culture and the suspension properties for potential biomass harvesting. With an increasing process duration the probability for biofilm layering due to direct adhesion of cells, biological deposits like extracellular polymeric substances (e.g. polysaccharides or glycoproteins in EPS) or cellular debris increases dramatically. Furthermore, a resulting increase of cell clustering due to interconnection with mobile bulk EPS or the excretion of soluble EPS could have a vast impact on viscosity and flow dynamics of the liquid phase within the ASL. In xenic culture, biofilms based on both algal and bacterial EPS and cell debris are plausible (Chen et al., 2015). According to the microbiome composition and the resulting interactions within the “ecosystem µgPBR”, these EPS-based biofilms (EPS portion often > 90% of total biofilm (Borowitzka et al., 2015)) could strongly vary in complexity and characteristics. During long-term cultivation, algal cell size, cell distribution and clustering behavior in xenic culture were monitored in both, mobile and immobile phase of the µgPBR ASL (Fig. 14). Through all batches, neither potential predators or fungi, nor other algae were identified by microscopy. C.vulgaris cells showed no symptoms of plasmolysis. The stored pre-culture cells (FPA) showed a nearly perfect cell distribution with dmcell ∼ 3.5 µm. Single bacterial cells and algal clusters were hard to find. Transfer into the µgPBR imposed stress to the algae, resulting in the enhanced excretion of EPS and the rapid creation of algal clusters (batch cycles 1 and 2, Fig. 15). This correlated with the apparent loss of biomass during culture adaption (Fig. 6), according to which the cells quantitatively adhered to the lighted µgPBR-chambers and FEP membranes.

3.3. Assessment of the µgPBR miniature facility and comparison to current µg-capable PBR systems The utilization of microalgae for in-situ fixation of CO2 and simultaneous release of O2 for the application in space requires compatible cultivation systems. Different PBR types, as well as microalgae processing approaches, are conceivable (Table 5). All relevant current µg-capable reactor prototypes rely on a hydrodynamic system, driven by liquid pumps or stirrers, to provide a sustainable mixing of cells, nutrients and photons through a liquid suspension loop. In separate gas 11

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Table 3 Gas evolution in µgPBR.

qCO2, CO2 assimilation rate; qO2, O2 release rate. † Measurement data collected in gastight experiment compartment. †† Measurement data collected in liquid phase of ASL. Each data value represents the average of ≥ 2 × 106 data points (data collection every 5 s.) ± SD that were measured within one reactor experiment.

exchange compartments, artificial gas convection currents ensure an equal gas and temperature distribution (Keppler et al., 2018) or distinct CO2 and O2 flows (Posten and Chen, 2016). Besides the homogeneous distribution of cells within the suspension, the efficient and stable gas transfer between liquid and gaseous PBR phase (CO2-influx/O2-efflux) remains the greatest challenge for a stable long-term cultivation in microgravity. In the current study, the gas exchange occurs through gas permeable FEP-membranes. Although the principle was demonstrated to work for other cultivation systems with different membranes (e.g. PTFE, PE, PMP) and other algal species (e.g. Chlamydomonas reinhardtii CC-1690, Arthrospira sp. PCC-8005)

(Cogne et al., 2005; Podhajsky et al., 2014; Posten and Chen, 2016) for short time periods (5 h −17 d (Cogne et al., 2005; Ai et al., 2008; Podhajsky et al., 2014), this study gives the first proof of a reliable longterm usability (> 180 d) of a membrane-based cultivation system. Mass transfer coefficients (kL) of CO2 and O2 through the FEP membrane were previously determined for the given setup in triplicate with kL,CO2 = 8.71 × 10−7 m s−1 ( ± 0.39) and kL,O2 = 4.83 • 10−6 m s−1 ( ± 0.14). The experimental data presented has shown, that an average of 0.667 g d−1 (0.015 mol) of CO2 has been consumed. To achieve this flux over the membrane, a pressure difference of about 34 mbar CO2 was needed to achieve the desired flux. This pressure

Fig. 12. Pigmentation during long-term in µgPBR miniature plant. FPA represents cells from the pre-culture (airlift) and PBR-A/B from both µgPBR chambers after reactor opening and mixing. Each bar represents the average ± SD of n ≥ 5 individual biological samples measured within one reactor experiment. 12

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Fig. 13. Gas evolution in µgPBR miniature plant. A, O2 and CO2 gas concentrations in gastight EC (section within log phase). B, Calculation of absolute mols of consumed CO2 and produced O2 rely on measurements within the gastight EC. O2 transfer through the membrane is much lower than for CO2, indicating a specific limitation for O2. C, Photosynthetic capacity of C.vulgaris. PQ, daily molar photosynthetic quotient. Gray area, optimum time for process change to continuous operation. Each data value in C represents the average of 1.7 × 105 data points (data collection every 5 s.) measured within one reactor experiment.

difference can be easily realized by pulsing pure CO2 into the EC, as it is done during the experiment. If an equimolar flux of O2 over the same membrane is desired, a pressure difference of about 161 mbar O2 would be needed between the liquid and the gaseous phase. If we consider the operational O2 concentration inside the EC being between 12-25 vol.-%, we would need oxygen pressures between 282-414 mbar inside the liquid phase. Such high oxygen partial pressures within algae suspensions are just not feasible. As can be seen by the experimental data, only a mean PQmolar = 0.31 could be reached. This is equivalent to an average O2 partial pressure difference of about 36 mbar. Hence, kL,O2 values for membrane PBR must be significantly higher (kL,O2 ∼ 4.5 × 10−5 m s−1) to allow higher mass fluxes of O2 in order to achieve

higher PQ. Further research on gas transfer materials is required to enable a sufficient O2 transfer between liquid and gaseous phase and ensure that membrane-based systems can remain a reliable alternative for µg applications in comparison to other gas separation systems (e.g. vortex separation). For both, axenic (Podhajsky et al., 2014) or xenic (current study) cultivation, biofilm creation and related partial immobilization of algal biomass in the PBR chambers was reported. However, a significant impact on gas transfer was not identified for the given xenic process during long-term operation. Axenic long-term processing for comparable time periods in µg-capable PBRs are not found to exist in literature. Comparing growth dynamics, the given µgPBR miniature plant

Fig. 14. Mass distribution profile of C.vulgaris in mobile and immobile phase of ASL. FPA represents the particle composition from the pre-culture and PBR-A/B from both µgPBR chambers after reactor opening and mixing. In total, between 2.5 × 105 and 5.6 × 106 particles per cycle were analyzed. Each bar represents 14 microscopy pictures per cycle that were taken within one reactor experiment (n = 14). Minor particles (≤ 2 µm) include small nutrient crystals, cell debris and bacteria. 13

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Fig. 15. Average cell size of C.vulgaris during long-term cultivation in µgPBR miniature plant. In total, between 1.38 × 105 and 3.1 × 106 cells per cycle were analyzed. Each boxplot represents 12 microscopy pictures per cycle that were taken within one reactor experiment (n = 12). Gray area, optimum time for process change to continuous operation. Notches indicate the 95% confidence interval of the median.

Our previous studies investigated the synergetic integration of PBR systems into advanced ECLSS architectures for long-duration spaceflight missions, like extended surface missions on the Moon or a journey to Mars (Ganzer and Messerschmid, 2009; Belz et al., 2013, 2014). We conclude, that depending on the exact mission scenario (e.g. eclipse phases and availability of light), the integration of photobioreactors can be highly beneficial in terms of resupply mass savings. Considering a lunar base scenario, the benefit of a synergistically integrated PBRsystem into a p/c ECLSS is present after less than two years of mission duration (Belz et al., 2013), assuming algal biomass could generally be used as potential food supplement with potential health benefits and for CO2 fixation into biomass. Under this assumption and based on the given data a PBR-volume (in continuous mode) of 481.5 l would be required for a microgravity capable cultivation system to ensure an effective daily CO2 reprocessing for one crew member. A PBR-volume of 51.5 l ( ± 14.4) would be suitable to provide biomass as a potential minor food supplement (e.g. 7.8% of human diet, equals 30 g d−1 biomass per crew member). The consideration of an exclusive food production by the presented PBRsystem would not make sense at this time. As unprocessed algae may cause digestive problems and deleterious effects on several body functions depending on species and amount (Gietelson et al., 2003), algal biomass (as potential future human food supplement) has to be processed in the downstream (cell separation from the medium, cell cracking, or the isolation and enrichment of distinct fractions, e.g. proteins, pigments or vitamins). In any case, detailed human or at least mammalian feeding studies with processed algae as a supplemental part of a human diet have to be realized to finally evaluate the actual nutritional value of microalgal biomass. Nevertheless, the potential for an additional flexible in-situ production of algal biomass of the presented photoautotrophic process would basically benefit the primary O2 release. Since resupply-missions are extremely expensive, or in case of far-distant destinations from Earth just not reasonable, the resupply mass saving potential of photosynthetic-driven ECLSS components on long-duration spaceflight missions might be considered in future ECLSS architectures, enabling human presence in far-distant space. Xenic cultivation offers the advantage of reducing the sterility challenge while increasing overall culture robustness, which may have great impact on future operational handling in space. Currently, it is not fully clarified if a basal self-regulating microbiome is essential for the long-term stability of a microalgae culture. However, a negative impact on growth and photosynthetic capacity on Chlorella within six months could be excluded. Inoculation and adaption could be critical steps towards a stable working microalgae culture. Thus, these phases should be reduced to a minimum with regard to the total mission duration. To ensure a stable reactor operation, the establishment of a stable ecosystem within the reactor chambers could be seen as prerequisite, which would be

Table 4 Evolution of algal clusters in µgPBR.

Gray area, optimum time for process adaption. For estimation of clustering all image data of n = 14 microscopy pictures per cycle of one reactor experiment were collected. In total between 2.5 × 105 and 3.1 × 106 particles per cycle were analyzed. † data rely on absolute collected particle number. †† precultue in Subitec© FPA-PBR. ††† c ollected immobilized algae of µgPBR chambers (PBR-A and PBR-B) after long-term cultivation.

(xenic) and the “space microalgae photo-bioreactor” (axenic) (Ai et al., 2008) share mean volumetric productivities, within the same magnitude per µmol photon-influx. Furthermore, comparable photosynthetic yields (ФPS II) of both systems were found (Chen et al., 2015). 4. Conclusion and future perspectives The presented µgPBR miniature plant represents the first facility for the long-term cultivation of green algae in microgravity. The algae strain Chlorella vulgaris SAG 211-12 was repeatedly cultivated dominantly in a defined xenic culture for more than 180 days. A realistic calculation of potential benefits of a photosynthesisdriven ECLSS component requires the coupled consideration of both, O2 production capacity and CO2 fixation in nutritive and potentially edible biomass. The presented reactor system and bioprocess serve as demonstrator for the scale-up of efficient µg-capable cultivation systems and bioprocesses that shall further increase biomass and O2 productivity with minimal increase of system mass, volume or power. 14

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Table 5 Comparison of “state-of-the-art” PBR systems for space application.

Subitec© FPA PBR is listed as standard high throughput cultivation system for terrestrial application; n.d., no data available. PTFE, Polytetrafluoroethylene, FEP, fluorethylenepropylene, PE, polyethylene, PMP, polymethylpentene. † Photosynthetic yield of photosystem II (ФPSII) measured with extracted cells at the end of experiment. †† values estimated according to published data in Cogne et al. (2005) or Podhajsky et al. (2014).

preferred for a potential transfer flight (e.g. to Mars) due to its lowest error rate. This could take place on-ground before launch or in Earth orbit before starting the deep space flight. Although, it was shown that Chlorella vulgaris could be stored cooled and dark for longer periods, (intra)cellular degradation processes could still occur, resulting in an extended culture adaption phase or even in an increased inoculation error. Furthermore, repair mechanisms (e.g. after radiation damage) might be compromised. For a deeper understanding, future research will investigate physiological alterations due to microgravity, to cosmic radiation and related selection pressure within the given high throughput cultivation facility, which could have significant impact on the functionality of the PBR (Kim et al., 2013a, 2013b). Furthermore, impacts on algae-bacterial interactions shall be evaluated. Therefore, a xenic long-term

cultivation (up to 180 d) of SAG 211-12 in an equivalent µgPBR-flightfacility on the International Space Station, ISS (in NASA lab “Destiny”), shall be realized in 2019. Although the ISS is not a perfect analog for deep space, it is much closer than the environment on the Earth's surface and will provide invaluable operational experience (West et al., 2017). An identically treated bioprocess in an identical µgPBR miniature facility will be used as terrestrial reference. By the connection to the existing p/c ECLSS component LSR (ESA's Life Support Rack©, constructed by Airbus Defence and Space, Germany) the first hybrid ECLSS configuration in space will be demonstrated (Fig. 16). CRediT authorship contribution statement Harald Helisch: Conceptualization, Investigation, Methodology.

Fig. 16. Hybrid Environmental Control and Life Support System, hECLSS. The p/c subcomponent include the CO2 removal/concentration unit, the Sabatier reactor and electrolyzer. The waste product CH4 could be reduced by the connection to a PBR. CO2 is integrated in algal biomass during oxygenic photosynthesis, additionally O2 will be generated, modified according to Helisch et al. (2018). 15

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Jochen Keppler: Methodology. Gisela Detrell: Project administration. Stefan Belz: Funding acquisition, Project administration. Reinhold Ewald: Supervision. Stefanos Fasoulas: Writing - review & editing. Arnd G. Heyer: Writing - review & editing.

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Acknowledgements This work is part of the PBR@LSR (“PhotoBioReactor@ LifeSupportRack”) project and the DLR project 50JR1104 funded by Federal Ministry for Economic Affairs and Energy (BMWi) of Germany, within the frame of the national space program, realized and guided by the DLR (Deutsches Zentrum für Luft- und Raumfahrt), German Aerospace Center, Department of Human Spaceflight ISS and Exploration. The experiment and development of the μg-capable PBR (PBR@LSR) facility was initiated in 2014 by the DLR and the Institute of Space Systems (IRS) of the University of Stuttgart with Airbus Defence and Space GmbH as prime for the flight hardware. The authors would also like to thank Subitec© GmbH for the very fruitful support and discussions and ESA for close cooperation and invaluable support. A special thank is given to Norbert Henn and Oliver Angerer (DLR) for enabling the project. Contribution H.H., and S.B designed the study, H.H. and J.K. collected and analyzed the data, H.H. provided C.vulgaris biomass and cultivation concepts, H.H. and J.K. technical prepared the µgPBR facility. G.D. provided gas sensors. H.H., R.E., S.F and A.G.H. developed the study layout, all authors contributed to analysis and writing of the manuscript. Competing Interests Statement The authors declare no financial or commercial competing conflict of interest. Statement of informed consent No conflicts, informed consent, human or animal rights applicable. Authors agreement to authorship and submission All authors agreed to authorship and submission of the manuscript for peer review. References Abney, M.B., Mansell, J.M., 2010. The Bosch Process - Performance of a Developmental Reactor and Experimental Evaluation of Alternative Catalysts 9 NTRS.NASA.gov. Ai, W., Guo, S., Qin, L., Tang, Y., 2008. Development of a ground-based space micro-algae photo-bioreactor. Adv. Space Res. 41, 742–747. Atkinson, M.J., Bingman, C., 1998. Elemental composition of commercial seasalts. J. Aquaric. Aquat. Sci. 8 (2), 39–43. Bacellar Mendes, L., Vermelho, A., 2013. Allelopathy as a potential strategy to improve microalgae cultivation. Biotechnol. Biofuels 6, 152. Bashan, Y., Holguin, G., 1998. Proposal for the division of plant growth-promoting rhizobacteria into two classifications: Biocontrol-PGPB (plant growth-promoting bacteria) and PGPB. Soil Biol. Biochem. 30, 1225–1228. Becker, E.W., 2007. Micro-algae as a source of protein. Biotechnol. Adv. 25, 207–210. Bernard, O., 2011. Hurdles and challenges for modelling and control of microalgae for CO2 mitigation and biofuel production. J. Process Control 21, 1378–1389. Belz, B., Ganzer, B., Messerschmid, E., Friedrich, K.A., Schmid-Staiger, U., 2013. Hybrid life support systems with integrated fuel cells and photobioreactors for a lunar base. Aerosp. Sci. Technol. 24, 169–176. Belz, S., Buchert, M., Bretschneider, J., Nathanson, E., Fasoulas, S., 2014. Physicochemical and biological technologies for future exploration missions. Acta Astronaut. 101, 170–179. Bergmann, P., Trösch, W., 2016. Repeated fed-batch cultivation of thermosynechococcus elongatus BP-1 in flat-panel airlift photobioreactors with static mixers for improved light utilization: Influence of nitrate, carbon supply and photobioreactor design. Algal Res. 17, 79–86.

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