Minimize ISS: PBR

ISS Utilization: PBR@LSR (Photobioreactor@Life Support Rack)

Airbus is bringing another experimental system to the ISS (International Space Station) in the form of the photobioreactor (PBR). The PBR, developed at the University of Stuttgart and built at Airbus on behalf of the German Aerospace Center (Deutsches Zentrum für Luft- und Raumfahrt; DLR), is designed to convert part of the CO2 extracted by the LSR(Life Support Rack) on board the ISS into oxygen and biomass, which could help to save valuable resources during future long-term missions into space. 1)

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Figure 1: Photo of the PBR (Photobioreactor) instrument (bottom left): oxygen and a source of nutrition for astronauts (image credit: Airbus)

Future human research missions are expected to take astronauts to the Moon and Mars. A deciding factor for the success of these missions will be keeping the resources carried to a minimum. As it is both difficult and expensive to send new supplies from Earth, the greatest possible closure of the respective resource cycles for water, oxygen and food is of vital importance. Most waste water is already reprocessed into fresh water on the ISS.

The LSR (Life Support Rack) of ESA has been on the ISS since October 2018. The spaceflight experiments PBR@LSR (Photobioreactor at the Life Support Rack) shall demonstrate the technology and performance of a hybrid life support system under real space conditions during an operation of half a year. PBR@LSR combines a micro algae photobioreactor (PBR) and the carbon dioxide (CO2) concentrator of ESA's LSR (Life Support Rack), also known as the European ACLS (Advanced Closed Loop System). 2)

LSR/ACLS was launched on the HTV-7 (H-II Transfer Vehicle-7) "Kounotori-7" flight of JAXA on 22 September 2018. The new system recycles half of the carbon dioxide thereby saving about 400 l of water that needs to be launched to the International Space Station each year. The facility is a Space Station-standard 2 m tall rack. Although the system is made to demonstrate the new technology, it will be part of the Space Station's life support system and produce oxygen for three astronauts, and operated for at least 1 year over 2 years to demonstrate its performance and reliability. 3)

The system traps carbon dioxide from the air as it passes through small beads made from a unique amine developed by ESA for human spaceflight. Steam is then used to extract the carbon dioxide and process it in a Sabatier reactor to create methane and water. Electrolysis then splits the water back into oxygen while the methane is vented into space.

The system is a huge step for human spaceflight as space agencies prepare for exploring further from Earth. Sustainable life-support systems are needed for longer missions such as to the lunar Gateway that is the next structure to be built by the partners of the International Space Station. Foreseen as a staging post for missions to the Moon and even Mars the Gateway will be further away from Earth so harder and more expensive to ferry supplies.

The Advanced Closed Loop System hardware is part of ESA's goal to create a closed life-support system, including water recovery and food production, eventually to keep astronauts in space indefinitely without costly supplies from Earth.

Process

ACLS can produce water from carbon dioxide exhaled by astronauts inside spacecraft. This water then is used to produce oxygen for the crew. It has three major functions:

• The Carbon dioxide Concentration Assembly (CCA) concentrates carbon dioxide from cabin air and keeps carbon dioxide within acceptable levels.

• The Oxygen Generation Assembly (OGA) is an electrolyzer that separates water into oxygen and hydrogen.

• The recycling step takes place in the Carbon dioxide Reprocessing Assembly (CRA) or ‘Sabatier reactor'. Hydrogen, coming from the Oxygen Generation Assembly, and carbon dioxide react over a catalyst to form water and methane. The water is condensed and separated from the product gas stream and fed back to the electrolyzer. Methane is vented into space together with excess carbon dioxide, which explains why only 50% of the recovered CO2 is actually recovered.

Facts

• LRS/ACLS is installed in the US Destiny module

• Can generate about 50% of the water needed for oxygen production on the Space Station

• Sent to space on Japan's HTV-7 vehicle

• Built by Airbus

• Built as International Standard Payload Rack, 1.57 m3 - about 2 m high, 1 m wide, and 85.9 cm deep.

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Figure 2: In the fall of 2018, the LSR was installed by Alexander Gerst in the US Destiny module (image credit: ESA)

Some background: Microalgae cultivation in space enables an essential step to close the carbon loop in future advanced LSS (Life Support Systems), which is important for future and far-distant exploration missions. Utilization of photosynthesis and the combination with existent physicochemical technologies offer a wide potential of benefit for LSS. Chlorella vulgaris (C.vulgaris) as a promising microalgae species allows for cultivation in pumped loops to produce oxygen and edible biomass from carbon dioxide and water. Further nutrients such as ammonium and phosphate are needed. Microalgae offer various advantages compared to higher plants for first integrating steps of biological components into the LSS. Microalgae have a higher harvest index (> 90%), higher light exploitation (9% of microalgae, higher plants 4-6%, 19% upper biological limit), more rapid growth (five to ten times), lower water demand, mostly higher photosynthetic quotient (PQ), and a well controllable metabolism . Long-term cultivation and stability of the algae culture are one of the most critical development steps. 4)

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Figure 3: Photobioreactor infographic (image credit: Airbus)

 

Launch: The SpaceX CRS-17 (Commercial Resupply Service-17) with a Dragon spacecraft on a Falcon 9 Block 5 rocket was launched on 04 May 2019 (02:48 EST, or 06:48 UTC) from the Space Launch Complex 40 at Cape Canaveral Air Force Station in Florida. Major payloads on this flight were: 5) 6)

OCO-3 (Orbiting Carbon Observatory-3) of NASA

STP-H6-XCOM (Space Test Program-Houston 6-X-ray Communication)

PBR (Photobioreactor)

Hermes Facility

Organs on Chips

Orbit: Near circular orbit, altitude of ~ 400 km, inclination = 51.6º.

The spacecraft will take two days to reach the space station before installation on May 6. When it arrives, astronaut David Saint-Jacques of the Canadian Space Agency will grapple Dragon, with NASA astronaut Nick Hague serving as backup. NASA astronaut Christina Koch will assist by monitoring telemetry during Dragon's approach. After Dragon capture, mission control in Houston will send commands to the station's arm to rotate and install the spacecraft on the bottom of the station's Harmony module.

The Dragon spacecraft will spend about four weeks attached to the space station, returning to Earth with more than 1900 kg of research, hardware and crew supplies.

 


 

PBR Demonstration Experiment

The ISS experiment Photobioreactor at the Life Support Rack (PBR@LSR, Figure 3) shall demonstrate the synergetic combination of the biotechnological component microgravity adapted photobioreactor (µgPBR) and the physicochemical LSR (Life Support), formerly known as ACLS (Advanced Closed Loop System). Inside the PBR the microalgae Chlorella vulgaris (C.vulgaris) is cultivated in a non-axenic manner. Figure 4 shows the process schematic of PBR@LSR. 7)

The PBR@LSR experiment and its development was initiated in 2014 by the German Aerospace Center (DLR) and the Institute of Space Systems (IRS) of the University of Stuttgart with Airbus Defence and Space as prime for the flight hardware. The objective is to demonstrate the functionality and feasibility of a hybrid LSS (Life Support System) in a real space environment during six months operation. The experiment shall also show that the long-term cultivation of C.vulgaris with a high biomass density and turn over is possible in space. Surplus CO2 from LSR (or as a backup solution from a buffer) shall be delivered to the PBR without affecting LSR performance. Inside the PBR, the CO2 is converted into O2. The O2 (together with residual air) is then delivered to cabin air. Regular liquid exchanges, performed with a special liquid exchange device, allow harvesting of algal biomass and provide fresh nutrients. Several algae samples will be taken at different time intervals and returned to ground for further analyses (the determination of µg (<10-3 g) and radiation influence on C.vulgaris physiology). Several sensors allow evaluation of photosynthetic and the facility performance of the experiment.

The main research focuses of PBR@LSR are: the verification of the hybrid system approach, the stability of the gas conversion (CO2 from LSR into O2 to cabin air), the production of algal biomass and operational handling in a non-axenic cultivation system. For the first time, these flight data will reveal the long-term performance, the system stability and reliability as well as the biological stability of a high density and high turn over algae culture (embedded in a synergistically integrated biotechnological LSS component) under space conditions. Besides the introduction of the general µgPBR system design and the used microalgae strain C.vulgaris SAG 211-12, in the following sections a first long term non-axenic cultivation approach within a µg-capable PBR system is presented. Entirely conceivable problems due to successive biofilm layering, based on experimental data and observations, are discussed.

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Figure 4: FM drawing of µg-adapted membrane PBR in MDL-1 (image credit: PBR@LSR Team)

General µgPBR (microgravity adapted Photobioreactor) system design

The µg-adapted membrane PBR FM is shown in Figure 3. The PBR and periphery parts are customized to the standard MDL (Mid-Deck Locker) size for an Express Rack on the ISS. Visible components are the front panel for crew interaction, the experiment compartment (EC, green, in the middle part), peristaltic pump and super absorber unit. A second MDL (MDL-2, not shown) contains a backup CO2 bottle, for experiment periods with limited excess to the LSR. The schematic setup of PBR@LSR is shown in Figure 5.

The majority of the components are located within the gas and water tight EC (Equipment Compartment). The EC contains a total volume of ~10 liter and provides access to the algae suspension loop (ASL) and the defined gas atmosphere. The functional group ASL is composed of two µ g-adapted photobioreactor flow chambers (µgPBR; Figure. 5) which are individually covered with a fluorethylenepropylene (FEP) gas exchange membrane, tubing and connectors, sensors to monitor pH and biomass density (via absorbance measurement at λ = 660 nm) and access ports for feeding and harvesting via a separate LiED (Liquid Exchange Device). Vtotal of the ASL is ~650 ml filled with algae suspension. Lighting is realized by a dichromatic red/blue LED panel placed outside the ASL. Algae are lighted through the gas exchange membrane (transmission >98%). Gases were pulsed into the EC by a pulse chamber and homogeneously distributed within the EC by air circulation fans. Gas evolution through the experiment is monitored by several gas sensors: pCO2 (COZIR Wide Range 20%, Cozir®, Gas Sensing Solutions, UK, sensing principle: nondispersive infrared detector), pO2 (FIGARO SK25F, Figaro®, Osaka, Japan, principle: sensing galvanic cell), pressure (p), relative humidity (rh) and temperature (T). O2 and rh within the EC are actively regulated by absorbers, T is controlled by a cold plate connected to the ISS cooling water loop (16-23 °C). The connection between the µgPBR and LSR (former name ACLS) will be realized by a specific interface (I/F), which allows the transfer of excess CO2 to external experiments. Nominally, the µgPBR will be supplied with CO2 from LSR. LSR is was launched on JAXA HTV-7 flight on 22 September 2018 and was accommodated in the US lab "Destiny" in November 2018 (Figure 2).

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Figure 5: FM (Flight Module) setup of PBR@LSR (image credit: PBR@LSR Team)

Chlorella vulgaris (C.vulgaris) – A small cell with great potential

Microalgae are uni- or multi-cellular, aquatic, eukaryotic microorganisms. For photoautotrophic growth, they conduct photosynthesis given by the top-level formula (1) of:

6 CO2 + 12 H2O + ΔH → C6H12O6 + 6 H2O + 6 O2→(Equation 1), — where ΔH = 2870 kJ mol-1 glucose

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Figure 6: FM drawing of meandric µg-adapted PBR chamber (image credit: PBR@LSR Team)

Equation 1 is a key ability for production of O2 and edible biomass (glucose, C6H12O6) from CO2 and water (H2O) in an ECLSS (Environmental Control and Life Support System) of a space station by using light energy. The auspices of long-term space missions make it important to investigate the influence of the space environment including microgravity (µg) and cosmic radiation on microalgal metabolism as well as the efficient cultivation of microalgae in the space environment in a µg-adapted photobioreactor (µgPBR) system. Compared with higher plants, microalgae have a higher harvest index (Hi >95 %) and a five times higher biomass productivity, a higher light utilization (> 10%) and lower water demand. Photoautotrophic cultivation of microalgae is a promising key factor and reasonable technological step from a state of-the-art physio-chemically based LSS to a hybrid LSS due to mass and energy savings and the in-situ biosynthesis of complex and high molecular biomolecules.

The controlled cultivation in a PBR requires a complex infrastructure consisting of illumination, nutrients supply, gas exchange, thermal control, media/solution control, harvesting and stowage/processing. In addition to the technical realization of a sufficient cultivation environment, the choice of the microalgae species and the development of an individualized cultivation process are crucial to provide sufficient growth rates at high biomass concentrations through long cultivation periods. Besides optimized and reproducible growth dynamics, the following factors have to be taken into account for a sustainable process assessment: Cell morphology, physiology and impacts on biomass composition, cell-cell interaction, photosynthetic yield (CO2, O2, evolution or ΦPSII), regeneration potential and genetic stability under space conditions.

Unicellular green algae meet the requirements for application in a LSS in space19,20. Since 2010, several algae species have been investigated at the IRS Stuttgart for usability in space applications, Chlorella vulgaris particularly showed very good results due to its versatility. The eukaryotic green algae Chlorella vulgaris (Chlorophyta) is an immotile single cell organism of spherical shape with a diameter of 2-15 µm (see also Figure 7). Depending on culture growth status and culture condition C. vulgaris can form small cell aggregate structures24. C.vulgaris shows a wide temperature and pH tolerance and grows within a wide range of CO2 concentrations. By choosing a selective lighting strategy or by variation of medium composition, the growth behavior and proliferation of C. vulgaris can be actively controlled. Due to high resistance of the algae to bacterial cross contamination the cultivation process can be performed in a non-axenic manner. This is an important factor for a robust cultivation process serviced by the space crew. Biomass from C. vulgaris is also a nutritive food source containing 10-18 % carbohydrates, 40-58 % proteins and 14-25 % fats with addition of several vitamins, minerals and mono/polyunsaturated fatty acids, i.a. ω3 and ω6 fatty acids. Due to theoretical and experimental studies and space flight experience, C.vulgaris (SAG 211-12) emerges to be a promising candidate for a long-term cultivation experiment (> 180 d) on the ISS.

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Figure 7: Chlorella vulgaris SAG211-12 at Institute of Space Systems, Stuttgart University (image credit: Stuttgart University)

Long term cultivation of C.vulgaris in µg-adapted membrane racetrack PBR

The long-term cultivation in µgPBRs (microgravity adapted photobioreactors) was conducted with the wild type microalgae strain C. vulgaris (SAG 211- 12) obtained from the Department of Experimental Phycology and Culture Collection of Algae (EPSAG), University of Goettingen, Germany. For inoculation only cells at the end of the exponential growth phase of the preculture with a living cell count LCC = 98-100% and mobile single (non-plasmolytic) morphology were taken. No clustering was observed. To make storage conditions representing conditions of the later cultivation scenario on ISS, the cells were dark adapted before inoculation to maximize photosynthetic activity. Light energy was provided by red/blue (R/B) LED panels, which meet the requirements for photoautotrophic growth of C. vulgaris. C. vulgaris absorbs light energy primarily through chlorophyll a/b in the red (660-680 nm) and blue (435-475 nm) spectral range.

The culture was non-axenic but free of major contaminants and from other algae strains, resulting in C.vulgaris being the dominant species within the closed loop system. The cultivation was conducted in fed-batch mode according to the parameters in Table 1. Mobile algal cells were perfused through two serial flat bed, meandering raceway reactors of the closed ASL by a peristaltic pump (Figure 5). Before inoculation, the EC was initially flushed with N2. A defined CO2 atmosphere was established. Background nutrient solution was a diluted seawater nitrogen medium (DSN). In the current study, dry-biomass concentration (X) was determined by measurement of optical density (OD) at 750 nm and 680 nm using a Hach spectrophotometer (DR 2800). OD and X were correlated and calculated according to Equation 2.

Parameter

Setting

Vloop

~650 ml

T

27ºC (±2)

pH

7 (±2)

CO2 in EC (Experiment Compartment)

7-10 vol. %

O2 in EC

10-25 vol. %

rh in EC

50-80 vol. %

PPFD average

100-400 µmol/(m2s)

[NH4+-N]

100-700 mg/l

[PO43-]

50-300 mg/l

Table 1: Cultivation parameters during the long-term cultivation phase of PBR@LSR

X = (0.2312 x OD680) + (0.2886 x OD750)/2 — [g/l] →Equation 2

For determination of nutrient uptake ([NH4 +-N] and [PO43-]) the culture supernatant (SN) was collected. SN samples were prepared and medial ion concentrations were measured according to manufacturer protocols (Hach GmbH, Berlin, Germany). Total cell counts (TCC), living cell counts (LCC) and bacterial stainings were performed according to protocols.

Nominal long term operation in µgPBR

The long term cultivation was conducted for 186 d. In the first phase, day 0 to day 40 (D0-D40), a molar distribution of R/B photons of 50% each was set as baseline. A few hours after inoculation (X= 1,46 g/L) a decrease of biomass within the PBR loop down to Xmobil = 0,03 g/L could be observed ( Figure 8; D0). The fast apparent loss of biomass can be explained by adhesion of the algal cells within the flow channels resulting in biofilm creation through the whole surface of the chamber and gas exchange membrane. A possible explanation for this behavior could be the adaption of the cells to the new cultivation conditions and environment. Sedimentation processes enhanced local accumulation of the algae cells. This could be confirmed by observations in former experiments. Nevertheless a minor fraction of mobile cells could grow within the liquid mobile phase with a mean growth rate of 0.007 g/L/d (D2-40), see also cells at D0 and D34. Kim et al. described the influence of different light wavelengths on the growth behavior of C. vulgaris. Although the related signaling pathways are not fully understood yet, it is shown 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 an enhanced proliferation of cells.

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Figure 8: First long-term cultivation (186 d) of C.vulgaris in µgPBR. The grey area demonstrates the tendency of algal growth behavior after process adaption. Biomass determinations rely on OD680,750 measurements and correlation only on cells within the mobile phase, N = 1. X, biomass concentration (image credit: Stuttgart University)

To increase the proportion of mobile cells within the suspension loop without disturbing single cell growth, the lighting regime was changed to a molar distribution of R/B = (62.5 % / 37.5 %) resulting in a stepwise release of cell clusters from the PBR chambers and proliferation of single cells within the PBR loop, see also cells at D82, D161 and D178. Mean growth rates of 0.009 g/l/d (D41-80), 0.014 g/l/d (D81-120) and 0.044 g/l/d (D121-180) could be proven. Caused by cell clustering, the heterogeneous cell distribution within the mobile phase led to the scattering of biomass data.

Nevertheless, a mean biomass concentration of 4.6 g/l in the mobile phase could be reached at the end of the experiment and gives evidence for fundamental growth in a high biomass concentration range in a µgPBR for the first time. Until D78 LEs (Liquid Exchanges) were performed in periods D4-46 to reduce biomass concentration within the loop and provide the culture with fresh macronutrients. A cultivation period of 14 days between two LEs was chosen for further cultivation runs.

Assessment of biofilms in non-axenic long term cultures

A stable long term functionality of algae driven PBR systems depends in a large part on the equal distribution of free microalgal cells within the ASL, in particular the racetrack 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 requirements for a controlled and stable photoautotrophic process. Hence, uncontrolled immobilization of cells and extracellular biomolecules could affect the capacity of a culture to produce oxygen and to integrate environmental CO2 into algal biomass (represented by photosynthetic quotient, PQ) and the suspension properties for potential biomass harvesting.

With an increasing duration of the total cultivation approach (includes no intermediate purification of the cultivation environment), the probability for biofilm layering due to direct adhesion of cells, biological deposits, e.g. extracellular polysaccharides (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 non-axenic micoralgae cultures, biofilms based on both algal and bacterial EPS and cell debris, are plausible. According to the microbiome composition and the resulting interactions within the "ecosystem PBR", these EPS based biofilms (EPS proportion often >90% of total biofilm38) could strongly vary in complexity and characteristics. The following environmental parameters were observed to affect EPS production and characteristics of algal biofilms:

Light intensity and temperature: The intensity and composition of light influx affect biomass composition, due to its influence on the carbohydrate metabolism. Is has been shown that high light intensities enhanced the proportion of bulk & soluble EPS39. For the current study the light influx to biomass ratio was chosen to be 40-150 µmol/m2 s per g biomass to prevent light inhibition of mobile cells resulting in a minimal tendency for light induced EPS synthesis. The parameter temperature could also synergistically affect EPS synthesis as it affects light inhibition processes.

Availability of carbon, nitrogen and phosphorous: A high CO2 concentration could prevent EPS accumulation due to the carbon concentrating mechanism (CCM) by which microalgae concentrate medial inorganic carbon under low carbon conditions. For example, for C. kessleri is has been demonstrated that a reduction of the medial CO2 level resulted in an enhanced EPS synthesis. In the current study the CO2 level was regulated between 7-9 vol.% to ensure a stable photosynthetic process, but also to avoid C-depletions within the algal cells.

The impact of medial nitrogen and phosphorous levels on the EPS biogenesis are controversially discussed in the literature. Nevertheless, at N-starving conditions it was proven for C.vulgaris to accumulate carbohydrates. The most frequently observed effect of P-starvation is the switch from protein synthesis to carbohydrate and lipid accumulation. This could potentially result in an enhanced excretion of carbohydrate polymers resulting in the forming of a basal EPS.

Stress response - bacteria and mechanical forces: EPS can be formed in response to stress caused by a bunch of biotic (e.g. bacteria) and abiotic factors. Generally, EPS-induced multicellularity of microalgae could appear as a defense mechanism against cross cultivated bacteria or predators (own data, not shown).

The current ASL contains ~650 ml of culture volume and is driven by a peristaltic pump (Watson Marlow® 114 series). Although, comparing other pump types (e.g. gear pump, membrane pump, peristaltic pump) the peristaltic pump generally reduces mechanical forces to a minimum, 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. The impact of the mechanical stress on the algae cells depends on the algal cell size, cell wall composition, algal regeneration capacity, the total number and the frequency of passes through the pump head and the rotating velocity of the pumphead.

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Figure 9: Racetrack PBR-chamber after cultivation of 186 d. A, heterogenic algal biofilm in raceway PBR chamber. The blue and red borders indicate the positions of the blue and red LED spots; B, relative in-vivo absorbance of C.vulgaris SAG 211-12; C, C.vulgaris in racetrack chamber (image credit: Airbus DS)

A. Biofilm in µgPBR chamber

Both µgPBR chambers represent the major growth compartment of the ASL. Inside the meandric flow channels the light influx, main mixture of the nutrients and cells as well as the gas exchange are realized. A stable long term operation requires a homogeneous suspension circulation without major deposits which could disturb the flow behavior. During the running cultivation process only cell material and biopolymeric structures (e.g. EPS) of the mobile liquid phase could be sampled and analyzed. Free single cells and cell clusters composed of algae cells, bacteria and bulk EPS in sizes between 150-9000 µm2 could be identified. The collection and analysis of immobilized algal or bacterial cells, multicellular adhesion layers or clusters were performed after completion of the experiment (186 d) and manual opening of the reactor chambers, (Figure 9). Previously, residual algae suspension has been collected separately.

Figure 9 shows algal biofilm within the open PBR chamber. According to the defined position of red and blue LEDs on the LED-panel, areas of photobleached algae could be observed only under blue light spots. This pattern could be explained by the relatively high energy intake of short-wave radiation. Carefully removed top cell layers showed a bleaching impact down to a layer depth of at least ~1 mm. Cells under red spots were apparently not stressed. According to this observation ~17 % of the immobilized algal material was calculated to be influenced or even damaged. Excess light exposure could lead to a degradation or destruction of algal light-harvesting pigments, e.g. Chlorophyll a and b (Chl a/b) of C.vulgaris.

Vitality assays of isolated photobleached cells (Figure 9 A) showed a LCC >80%. The residual ~20% biomass could be assumed as dead cell material due to photoinhibition and photooxidation as a result of immobilization. The viable bleached C.vulgaris cells (Figure 10) are not further able to grow photoautotrophically, but compensate energy demands by changing their metabolism into respiration (via oxygenase activity of RuBisCO). Using organic carbon sources, e.g. free floating or EPS-bound cellular debris, these cells survive as oxygen consumers/CO2 producers. Due to further EPS-driven crosslinking, a propagation of the consumers is a realistic problem for the long term efficiency of the O2 producing and carboxylating system, measurable by a decreasing algal photosynthetic activity quantified by the photosynthetic quotient, PQ.

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Figure 10: Cluster of photobleached but viable C.vulgaris cells, crosslinked by bulk EPS (image credit: University of Stuttgart)

The PQ could be used as an adequate online indicator for influences of environmental process parameters on microalgal physiology, in the current case the increased gradual biofilm layering within the PBR chamber. For the current experiment, light deficiency impacts due to a potential high mobile biomass concentration can be excluded (Figure 8). PQ values were calculated according to Equation 3:

PQ = O2 production/CO2 consumption x M(CO2)/M(O2) [/] →(Equation 3) — (where M = molar mass, M(CO2) = 44,0095 g/mol and M(O2) = 31,9988 g/mol)

Figure 11 shows the photosynthetic activity of the C.vulgaris culture during the long term cultivation experiment. Due to culture adaption a decrease was observed until D 40. After changing the lighting regime, the higher absolute energy influx of red wavelength could increase the mass related PQ to a maximum (PQ ~0.8). An physiological adaption to the new set up was observed, resulting in a PQaverage = 0.351 after D 80. In accordance with the stepwise release of cells into the mobile phase, the lighting distribution has been improved. This results in an increased mean PQ at the late experiment phase (D150-D186).

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Figure 11: Photosynthetic activity of C.vulgaris SAG211-12 during long term cultivation. PQ values are averaged over 24 h. Dashed line, change of lighting regime (image credit: University of Stuttgart)

B. Biofilm on FEP gas exchange membrane

The FEP membrane shall provide a constant exchange of CO2 and O2 between the PBR chambers and the EC (Figure 13 A). As shown for the PBR chambers, a pattern of photobleached and non-bleached cells could be observed on the membrane side after long term operation (Figure 12). The hydrophilic membrane side was oriented to the aqueous medium and thereby provided potential anchor points for a cell binding directly by surface proteins (adhesion) or in-directly by carbohydrate chains of the EPS. This allows a successive accumulation of algal cells or their immobilized proliferation within the EPS matrix directly on the membrane. A plane EPS covering throughout the entire membrane could be verified, independent of the membrane orientation to the upper or lower side of the PBR chamber (Figure 13 B).

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Figure 12: FEP membrane on raceway PBR chamber after 186 d: "Pattern of (non)-bleached cells"(image credit: University of Stuttgart)

This gives evidence for the biofilm exposure to occur mainly due to adhesion events. Sedimentation effects only enhance the layering. Similar covering could also be observed after shorter cultivation periods (data not shown). The careful removing of several algal cell layers also showed that on the basal EPS layer the majority of rod-shaped bacteria are localized. This suggests that the EPS was bacteria-induced. Predominantly gram-negative bacteria were identified (Gram staining, Figure 13 C).

According to this, the accumulation of algal cell multilayers occurred by crosslinking with the EPS. To receive a deeper understanding of the algae-bacterial physiological interactions and their influence on the cultivation process, a detailed characterization of the bacterial microbiome by next generation sequencing (NGS) is currently in progress. Despite an obvious membrane covering, the constant CO2 consumption and O2 production through the entire cultivation gives first evidence for the long-term functionality of the gas transfer in the non-axenic bioprocess. The assessment of the relative gas transfer efficiency will be given after a process optimization towards a homogeneous distributed cell culture is realized. At least, this is necessary for a constant reliable controlling of the photosynthetic process and biomass density by cyclic harvesting (LiEx).

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Figure 13: Layered biofilm on gas transfer membrane. A, O2/CO2 transfer principle through FEP membrane; B, layering of bacterial/algal biofilm; C, Gram staining of bacterial cell layer. The arrowheads indicate the edge of the membrane (image credit: University of Stuttgart)

C. Biofilm in biomass sensor

The absorbance, A is a measure for the opacity of a liquid, and can be correlated with the biomass concentration (in g/l), allowing the in-situ measurement of algal culture growth. Commercial biomass density sensors are available, but they are too heavy or big for this space application. Therefore, an biomass-sensor has been developed at the IRS, specifically for the PBR@LSR experiment.

Operation principle and chamber assembly: The biomass sensor measures the amount of light "lost" in a specific length of algae suspension. Therefore, the sensor is equipped with two LEDs with a specific wavelength (λ = 660 nm) that work independently. Each LED sends an initial flux Io through the suspension. A sensor next to the LEDs measures this flux density. On the other side of the algae solution (with a predefined specific liquid depth, d ~4 mm), another light sensor measures the remaining photon flux density (Figure 14). Following the fundamental principles of the law of Lamber-Beer, the absorbance A can be calculated according to Equation. 4:

A1 = log (I0/I1) for LED-1 and A2 = log (I0/I2) for LED-2 [/] →(Equation 4)

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Figure 14: Working principle optical density sensor (image credit: PBR@LSR Team)

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Figure 15: Measuring chamber of the biomass sensor (flow cell), image credit: PBR@LSR Team

The measured absorbance values were correlated to manually premeasured OD660 (Hach, DR2800). The biomass sensor will be integrated into the FM-ASL as well as the refurbished on-ground reference µg-capable PBR-ASL for the parallel in-situ measurement of cell density during the flight experiment.

The sensor consist of a measurement chamber (Figure 15), which is divided in two parts, the frame (3D printed, Accura® ABS Black) and 2 translucid covers at each side (polycarbonate). An O-ring ensures the tightness of the chamber. This chamber will be integrated into the algae-loop. The LEDs and the sensors are mounted at both sides of this chamber, in LED/Sensors housing, specifically designed for this sensor. The LEDs and sensors are controlled using a microcontroller (Arduino©).

Biofilm formation testing: The most critical aspect to be considered in a long-term use of the sensor, is the accumulation of EPS and cells on the transparent surfaces of the measurement chamber, which might have an influence on the sensor signal over time. A biomass-sensor prototype (flow cell version) has been used for over 50 days integrated in the ASL under current operation conditions. This should allow the synthesis of an adequate biofilm layer. After finishing the experiment, basal DSN and algae suspension (OD660 = 10.49) have been pumped through the flow chamber and have been measured, before and after cleaning the sensor.

The A difference for the medium was found to be marginal (ΔA660 = 0.02), whereas the tested suspension showed a ΔA660 = 1. This equals a relative sensor error of 9.87 % after 50 d of cultivation. This could be explained by the higher sensitivity of the biomass-sensor to variations of Chl a/b (in-vivo absorbance maxima in the red spectral range for Chl a = 670-680 nm and Chl b = 650 nm), than to other cellular structures like carbohydrates, e.g. cellulose (A= 260 nm). In both cases the value before cleaning was higher due to biofilm formation. In conclusion, a successive biofilm layering has to be considered, which could result in a sensor signal drift.

Conceivable countermeasures

For long term non-axenic cultivation operations in µgPBRs a successive synthesis of bacterial and algal biofilm within the ASL has to be considered. Especially deposits on interaction surfaces with the environment (EC or LED), like the flow channels of the PBR chambers, the gas exchange membrane and the measurement chamber of the biomass sensor are in the focus of the current work. Therefore, a reduction of biofilms to a minimum is a major goal in the context of bioprocess control and upcoming optimizations for the technical realization, not only of the current experiment, but for the future development of µg capable (membrane) PBR system technologies.

To reduce bacteria or algae induced biofilms within the running cultivation process and to reach a higher level of mobile cells, several strategies or treatments are conceivable. In Table 2 µg-relevant strategies and their advantages/disadvantages are presented. In principle, several strategies could be combined to use synergetic treatment effects. All suggested treatments are based on a non-axenic wild type C.vulgaris culture. Cultures, based on genetically modified microorganisms, were not considered.

Principle

Method

Effect

+

-

Constriction of flow channel width (PBR chamber design)

Increased flow velocity, enhanced Reynolds number, but laminar flow

Enhanced mixing, cluster breaking in PBR chamber

Enhanced mechanical stress due to turbulent flow, no effect on existing biofilm

Flow breaker

Local change of flow dynamics, local turbulences

Enhanced mixing, cluster breaking in PBR chamber, potential for flashing light effect

Enhanced mechanical stress due to turbulent flow, no effect on existing biofilm

Flow principles

Mechanical removing of cell/biofilm

Abrasion of already consisting deposits & biofilm, cluster breaking in PBR loop

Potential cell immobilization on particles, artificial adhesion nuclei, risk of clogging

PBR chamber surface treatment

Smoothing of surfaces

Reduced anchor sides for adhesive molecules

Possibly cost and work intensive

Physical

Pulsed electric field

Selective electroporation

Selective in-situ bacteria inactivation /lysis, non-invasive

Possible uncontrolled fusion of algal cell organelle membranes, changes of photosynthesis apparatus or cellular damage

Low energy electron beam

Partial disinfection

Selective in-situ bacteria inactivation /lysis, non-invasive

Currently limited knowledge of free electrons influencing algal cell metabolism, viability or productivity

Cross flow filtration

Separation and removal of bacteria

Selective in-situ bacteria removal, combinable with continuous cultivation and DSP (harvesting)

Necessity of multi level filtration systems bypass loop

Selective lighting

Enhanced proliferation, reduced clustering

Prevention anoxygenic photosynthesis, in-situ algae cell remobilization, reduction of current bacteria number, non-invasive

Highly specific for individual algae species or even strain

Biological

Enzymatic treatment

Digestion of EPS components

Utilization of indigestible carbohydrates (cellulose) into glucose of EPS proteins in free aminoacids

Invasive, short enzyme half life, expensive, post treatment purification necessary

Antibiotic treatment

Reduction of bacteria number

Cyclic sharp decrease of total bacteria number, potential avoidance of bacterial endotoxins

Invasive, potential of bacteria to generate resistances, unexpected negative impacts on algal metabolism (e.g. chloroplasts); no inhibition of spore germination; no safe usage as food source

Axenic cultivation (selective bioprocessing)

Algae strain as singular microorganism, no microbiome

Higher algal growth rates, higher photosynthetic yield, pure algae biomass

High cultivation effort, not feasible for long term application, potentially only restricted biomass composition (e.g. B12 synthesis)

Chemical

Surface hydrophobing

Reduced adhesion spots on ASL surfaces

Reduced planar biofilm spreading, reduced starting points for release of bulk EPS/cell clusters

Putative biochemical incompatibilities

Selective media design, e.g., N-source, pH

Shift of selective pressure within the µgPBR

Promoted algal growth, reduced bacteria number, reduced bacterial EPS

Highly specific for individual algae species or even strain

Immobilization, e.g. by algimate drops

No free cells within the ASL

Immobilized but mobile algae cells, cyclic cleaning and full recovery of algal cells possible

No quantitative algal biomass harvesting, possible accumulation of bacteria within the ASL possible, risk of clogging

Table 2: Conceivable countermeasures against biofilm synthesis in membrane µgPBRs before and during PBR operation

In the following, the possibility for an improvement of cultivation processes is illustrated and discussed by the example of the hardware component µgPBR chamber. In dependence of the manufacturing process quality, like milling of the flow channels from a massive polycarbonate plate, this results in the formation of individual groove profile. The defined surface roughness of the cultivation chambers could serve as niches for algal cells (2-15 µm, depending on maturation status), but in particular for the smaller bacterial cells (mean dm ~ 1 µm). The niches support cell deposition due to microscopic wake space and could form the basis for the synthesis of cross-linking EPS.

In the case of the standard manufactured PBR chambers, the mean groove depth was ~ 9 µm (Figure 16 A). A typical algae cell in the current µgPBR process (size ~ 6 µm) could easily inhere and start crosslinking by EPS or direct cell-cell adhesion (Figure 16 C). After optimization of the manufacturing process, realized by a special fine milling, the mean groove depth could be reduced to ~ 2.8 µm (Figure 16 B). This went hand in hand with a smoothing of the groove profile (Figure 16 D). Thus, this counteracts the fundamental adhesion of algae cells, resulting in a reduction of the chance for the creation of a planar layering by algae.

ISS-PBR_Auto3

Figure 16: Interferogram of milled surface (µgPBR chamber section) in dependence of manufacturing process. Surface section and maximal groove depth before (A), after (B) and surface profile of µgPBR chambers before (C) and after (D) manufacturing optimization. Green circle, schematic algal cell (~ 6 µm); small beige square, schematic bacterial cell (~ 1 µm). Data was collected in cooperation with the Institute for Machine Components (IMA), image credit: University of Stuttgart

The sole optimization of the surface roughness resulted in a slight increase of cell number within the mobile liquid phase of the ASL (data not shown). An accompanying adaption of the bioprocess realized by optimized cultivation conditions currently represents the most promising option for the (re-)mobilization of algae cells of a non-axenic culture in a meandric membrane PBR system. According to significant changes of metabolic responses of different microalgae strains to process parameters like lighting strategy, harvest/feed design, temperature scenario, an individual characterization of the mutual influence of the microalgae physiology and the cultivation environment is crucial for an optimized, sustainable and reliable operation of the µgPBR system. That is especially the case for long term cultivation processes.

Summary and Outlook

To be launched to the International Space Station (ISS) in 2019, PBR@LSR follows the hybrid LSS approach by combining a microalgae PBR and the CO2 concentrator of ESA's LSR. The PBR@LSR experiment and its development was initiated in 2014 by the German Aerospace Center (DLR) Space Administration (grant) and the Institute of Space Systems (IRS) of the University of Stuttgart with Airbus Defence and Space as prime contractor for the flight hardware. The two µg-adapted PBRs, the LiED (Liquid Exchange Device) and the syringes are built by IRS.

The final configuration of PBR@LSR is presented with a focus on process important components of the ASL (Algae Suspension Loop), like the cultivation chamber, the gas exchange membrane and the biomass sensor. The interaction of this components with the non-axenic culture could result in a successive creation of biofilm layers, which could strongly influence the long-term functionality, photosynthetic efficiency and finally the operational handling of a µgPBR. Depending on the individual cultural microbiome, biofilms could vary in structure and characteristics. Regarding this, the biofilm deposits of the presented long-term approach were investigated. Appropriate countermeasures for reduction of biofilm deposits within the given PBR set up were presented and discussed. With the current long term experiment of 186 days basically successful on-ground long-term cultivation in a protoflight breadboard setup of PBR@LSR could be proven. This was verified by the overall biomass growth within the ASL and the net O2 production.

Future work at the IRS (Institute of Space Systems), University of Stuttgart, will include the optimization of the cultivation process within the given PBR set up, pursuing the goal to increase mobility of the total algal cell biomass as well as the photosynthetic capacity. Therefore, a comprehensive characterization of the used C.vulgaris strain SAG 211-12 will be performed in the context of long-term cultivation and individual processing in µg-capable meandric membrane PBR systems.

The flight data of PBR@LSR will, for the first time, reveal the long-term performance, the system stability and reliability as well as the biological stability of a synergetically integrated biotechnological LSS component in a real space environment. Microalgae samples taken during the experiment will be returned to earth and analyzed. The sequencing of isolated genetic material could be highly beneficial for the evaluation of putative alterations of photosynthesis associated C.vulgaris genes, which could have a significant impact on algal photosynthetic performance for permanent application as a biotechnological component in hybrid LSS.

 


 

Some background on PBR@LSR

Astronauts on future long-duration spaceflight missions to the Moon and Mars could rely on microalgae to supply essentials including food, water and oxygen. A new investigation aboard the International Space Station tests using the microalgae Chlorella vulgaris as a biological component of a hybrid LSS (Life Support System). 8)

As humans travel farther from Earth and for longer periods of time, bringing along sufficient supplies of food, water and oxygen becomes a challenge. Packing food that is nutritious and perhaps even tasty may prove harder still.

Current life support systems, such as the LSR (Life Support Rack), use physicochemical processes and chemical reactions to generate oxygen and water and remove carbon dioxide from the space station.

The PBR (Photobioreactor) investigation demonstrates creating a hybrid LSS by adding the biological processes of a microalgae, which has a photosynthetic efficiency up to ten times greater than more complex plants. These tiny plants could take concentrated carbon dioxide removed from the cabin atmosphere and use photosynthesis to produce oxygen and possibly even food for astronauts, according to Norbert Henn, a co-investigator and consultant at the ISR (Institut für Raumfahrtsysteme — Institute of Space Systems) at the University of Stuttgart.

The Institute of Space Systems began research on microalgae for space applications back in 2008 and started work on Photobioreactor in 2014, together with the German Aerospace Center (DLR) and Airbus.

"The use of biological systems in general gains importance for missions as the duration and the distance from Earth increase. To further reduce the dependency on resupply from Earth, as many resources as possible should be recycled on board," said co-investigator Gisela Detrell.

Astronauts activate the system hardware aboard the space station and let the microalgae grow for 180 days. That span of time allows researchers to evaluate the stability and long-term performance of the Photobioreactor in space, as well as the growth behavior of the microalgae and its ability to recycle carbon dioxide and release oxygen, according to co-investigator Jochen Keppler. Investigators plan to analyze samples back on Earth to determine the effects of microgravity and space radiation on the microalgae cells.

"This is the first data from a flight-proven, long-term operation of a biological LSS component," said Keppler. The algae's resilience to space conditions has been widely demonstrated in small-scale cell culture, but this will be the first investigation to cultivate it in a PBR in space.

Chlorella, one of the most studied and widely characterized algae worldwide, is used in biofuels, animal feed, aquaculture, human nutrition, wastewater treatment and bio-fertilizer in agriculture.

"Chlorella biomass is a common food supplement and can contribute to a balanced diet thanks to its high content of protein, unsaturated fatty acids, and various vitamins, including B12," said co-investigator and biotechnologist Harald Helisch at the Institute of Space Systems. As for the taste, he adds, "if you like sushi, you will love it."

The long-term goal is to facilitate longer space missions by reducing total system mass and resupply dependency, said co-investigator Johannes Martin. "To achieve this, future areas of focus include downstream processing of the algae into edible food and scaling up the system to supply one astronaut with oxygen. We'll also be working on interconnections with other subsystems of the LSS, such as the waste water treatment system, and transfer and adaption of the technology to a gravity-based system such as a lunar base."

ISS-PBR_Auto2

Figure 17: The Photobioreactor science team from the Institute of Space Research. Top, left to right: Prof. Reinhold Ewald, Johannes Martin, Prof. Stefanos Fasoulas. Bottom, left to right: Jochen Keppler, Dr. Gisela Detrell, Harald Helisch (image credit: Institute of Space Systems – University of Stuttgart, Germany)

ISS-PBR_Auto1

Figure 18: Chlorella vulgaris cells under the microscope. These microalgae have a variety of uses on Earth and may be part of life support systems on future space voyages (image credit: Institute of Space Systems, University of Stuttgart, Germany)

ISS-PBR_Auto0

Figure 19: The Photobioreactor chamber is used to cultivate microalgae aboard the International Space Station in a demonstration of creating hybrid life support systems that use both biological and physicochemical processes (image credit: Institute of Space Systems, University of Stuttgart, Germany)

 


1) "Airbus brings new experimental technology to the International Space Station," Airbus, 25 April 2019, URL: https://www.airbus.com/newsroom/press-releases/en
/2019/04/photobioreactor-oxygen-and
-a-source-of-nutrition-for-astronauts.html

2) Jochen Keppler, Stefan Belz, Gisela Detrell, Harald Helisch, Johannes Martin, Norbert Henn, Stefanos Fasoulas and Reinhold Ewald, "The final configuration of the algae-based ISS experiment PBR@LSR," 48th International Conference on Environmental Systems, ICES-2018-141, 8-12 July 2018, Albuquerque, New Mexico, URL: https://ttu-ir.tdl.org/bitstream/handle/2346/74122/ICES_2018
_141.pdf?sequence=1&isAllowed=y

3) "Advanced Closed Loop System," ESA, 2 November 2018, URL: https://www.esa.int/Our_Activities/Human_and_Robotic
_Exploration/Research/Advanced_Closed_Loop_System

4) S. Belz, H. Helisch, J. Keppler, G. Detrell, S. Fasoulas, R. Ewald, N. Henn, "Photobioreactor Technology for Microalgae Cultivation To Support Humans in Space with Oxygen and Edible Biomass," 51st ESLAB Symposium – Extreme Habitable Worlds, 2017, URL: https://www.cosmos.esa.int/documents/1566003/1642650/eslab2017-
abstract+36.pdf/18bd2bcf-dc77-c63e-a09c-fa457862951f
, and URL: https://www.cosmos.esa.int/documents/1566003/1580285/06dec1000_
Detrell_ESLAB2017.pdf/a1481be0-d67e-ef15-7558-57bba90d6917

5) Joshua Finch, Courtney Beasley, Sean Potter, "SpaceX Dragon Heads to Space Station with NASA Science, Cargo," NASA Release 19-035, 04 May 2019, URL: https://www.nasa.gov/press-release/spacex-dragon-heads-to
-space-station-with-nasa-science-cargo

6) Stephen Clark, "Launch Schedule," Spaceflight Now, 29 April 2019, URL: https://spaceflightnow.com/launch-schedule/

7) Harald Helisch, Stefan Belz, Jochen Keppler, Gisela Detrell, Norbert Henn, Stefanos Fasoulas, Reinhold Ewald, Oliver Angerer, "Non-axenic microalgae cultivation in space – Challenges for the membrane µgPBR of the ISS experiment PBR@LSR," 48th ICES (International Conference on Environmental Systems), 8-12 July 2018, Albuquerque, New Mexico, USA, URL: https://ttu-ir.tdl.org/bitstream/handle/2346/74157/ICES_2018_186.pdf?sequence=1&isAllowed=y

8) Melissa Gaskill, Michael Johnson, "Building Better Life Support Systems for Future Space Travel," NASA, 26 April 2019, URL: https://www.nasa.gov/mission_pages/station/research/
news/photobioreactor-better-life-support

 


The information compiled and edited in this article was provided by Herbert J. Kramer from his documentation of: "Observation of the Earth and Its Environment: Survey of Missions and Sensors" (Springer Verlag) as well as many other sources after the publication of the 4th edition in 2002. - Comments and corrections to this article are always welcome for further updates (herb.kramer@gmx.net).