Methods and kits for investigating the microgravity effects on animal / plant cells under extraterrestrial culture conditions, and culture methods thereof for supporting manned space missions.

Abstract

The present invention describes a technique and method for simulating extraterrestrial conditions to investigate the effects of microgravity and a CO2-rich atmosphere on the cultivation of animal and plant cells using extraterrestrial sources to produce edible biomass for the sustainment of human space missions, as well as material kits and equipment for carrying out the same.

Description

[Technical Field]

[0001] This invention relates to the cultivation of plant cells and their growth media under microgravity conditions for obtaining protein-rich edible biomass to sustain long-term manned space missions, and more particularly to an apparatus or method for obtaining materials from extraterrestrial sources and a simulation thereof on Earth. This invention also relates to the cultivation of animal cells under microgravity conditions using the apparatus described above.

[0002] It is well known that several companies and organizations are interested in conducting manned spaceflight, lunar missions, and space travel over the next 40 years. Specifically, within the framework of current space exploration programs, this is often identified by the acronym ISRU (In Situ Resource Utilization). This acronym relates to the use of extraterrestrial resources already available on the Moon, in space, and / or Mars, which will enable longer manned mission durations and cost reductions.

[0003] Most ISRU technologies consist of physicochemical methods for generating oxygen and propellant from the Martian surface and atmosphere, but these themselves cannot contribute to the production of food necessary to supply the crew.

[0004] Within this framework, a novel technology called ECLSS—Environmental Control and Life Support System—is being developed for the production of food and water through the recycling of liquid and solid waste generated by astronauts involved in research activities conducted aboard the International Space Station (ISS).

[0005] Since 1988, with the aim of implementing the ECLSS paradigm on a real scale, the ESA (European Space Agency) has been working on a project called MELISSA (Micro Ecological Life Support System Alternative), which involves using microorganisms such as algae, bacteria, and fungi to develop a closed-loop process (i.e., producing all the materials the crew needs solely through waste and energy recycling) that creates suitable conditions within the crew cabin to enable its members to survive and work during long-term permanent missions on the Moon and other planets.

[0006] The ultimate goal of the MELISSA project is to achieve a self-sustaining system, but modeling simulations show that even the minimum goal of obtaining 20% ​​of the crew's food needs through waste recycling is unattainable with current technology.

[0007] Similar results have been obtained with other ECLSS systems, demonstrating that, with the latest technology, these systems are not entirely self-sufficient and require the integration of external inputs of oxygen, food, and water to meet the needs of astronauts. Considering deep space crew missions, such integrated resources cannot be continuously supplied from Earth due to the associated high mission costs and therefore must be produced by utilizing resources available in-situ. However, food production cannot avoid the use of microbial and biotechnology techniques.

[0008] In this context, a recent field of research known by the acronym bio-ISRU is being developed to study the possibility of producing food in-situ through bioengineering techniques, including the use of Martian topsoil and atmosphere. Bio-ISRU techniques can be divided into two main categories: those that rely on methylotropic bacteria and those that use autotrophic microorganisms.

[0009] The first technical group is based on the use of methanol, which can be produced in situ via physicochemical ISRUs, to obtain protein-rich edible biomass from microorganisms such as Pichia pastoris and Methylophilus methyloprophus or engineered Escherichia coli and Bacillus subtilis.

[0010] The second category of bio-ISRU processes is based on rock-weathering microalgae or cyanobacteria that can photosynthetically convert N2 and CO2 available in the Martian atmosphere into suitable edible biomass, along with S, P, Fe, Zn, Na and other micronutrients in the topsoil, by relying on water and light available on-site. The use of cyanobacteria and microalgae has a further positive effect in producing photosynthetic oxygen, which is important for the crew, and can integrate the amount produced through physicochemical ISRU processes.

[0011] International Publication No. 2013014606 describes the bio-ISRU process, including algae and cyanobacteria. This process includes the following two main sections: -CO2, H2O, N2, and Ar are extracted from the extraterrestrial atmosphere and topsoil by WAVAR, TSA, and MPO units and then used to produce O2, H2, CO, HNO3, NH3, and NH4NO3 via physicochemical ISRU processes in the "Physicochemical Section"; and, - Edible microalgae and / or cyanobacteria from Earth are cultured in a photobioreactor located inside a dome, the dome being heated to at least 10°C and having an internal pressure of at least 0.8 bar of CO2 produced in the physicochemical section, the photobioreactor being exposed to artificial or natural light and supplied with culture broth, HNO3 and bubbling CO2, the culture broth being an aqueous liquid phase of a slurry consisting of dehydrated extraterrestrial topsoil and H2O acidified with HNO3, the dehydrated extraterrestrial topsoil, H2O, CO2 and HNO3 being produced in the "physicochemical section" in the "biological section".

[0012] One object of the present invention is to provide an improved and more efficient bio-SRU process compared to that described in International Publication No. 2013014606.

[0013] Another object of the present invention is to provide a materials kit and method for simulating the growth of cells on Earth under extraterrestrial conditions in order to study the feasibility of proposed bio-ISRU processes during long-duration manned space missions.

[0014] Definitions and Abbreviations bio-ISRU: Bioengineering technology for food production using on-site resources ECLSS: Environmental Control and Life Support Systems ESA: European Space Agency ISS: International Space Station ISRU: On-site resource utilization LSB: Laboratory-scale bioreactor LSP: Laboratory-scale photobioreactor MELISSA: Microecological Life Support System Alternative MPO: Microwave oven for pizza RPM: Random Positioning Machine TSA: Temperature Swing Adsorbent WAVAR: Water vapor adsorption reactor

[0015] The subject of the present invention is an apparatus for simulating the growth of cells on Earth under extraterrestrial conditions at a given extraterrestrial location, said apparatus comprising - a thermally insulated jar attached to a 3D clinostat or random positioning machine (RPM), which can accommodate at least one laboratory scale bioreactor (LSB) and is equipped with a pressure gauge, a gas inlet and a gas outlet; - a cylinder for storing a gas simulating the extraterrestrial atmosphere, said cylinder having an outlet that can be fluid-connected to the inlet of said jar and comprising.

[0016] A further subject of the present invention is a method for simulating the growth of cells under extraterrestrial conditions on Earth, said method comprising using the above-described simulation apparatus. In particular, the method of the present invention for the cultivation of edible microorganisms - comprises preparing a culture medium by mixing a liquid topsoil leachate obtained by leaching with acidic water and a simulants of extraterrestrial topsoil with a simulant of diluted astronaut urine and micronutrients, the micronutrients being micronutrients that are not available by ISRU at an extraterrestrial location and are known to be essential for the growth of the strain to be cultivated.

[0017] The simulation apparatus and method of the present invention have been successfully used for simulating and investigating the growth of cells of various plant and animal cell lines under simulated extraterrestrial conditions.

[0018] The simulation experiments carried out using the apparatus of the present invention according to the method of the present invention provided evidence that the biomass productivity is very high compared to conventional operating conditions on Earth when using the operating conditions of the method of the present invention.

[0019] A further subject of the present invention is a bio-ISRU method for producing photosynthetic edible biomass and oxygen for maintaining a long-term manned extraterrestrial mission, said method comprising - The step of preparing an extraterrestrial growth medium by mixing topsoil leachate with diluted astronaut urine from ECLSS and other micronutrients brought from Earth that are essential for the growth of edible biomass but are not available on-site, - A step of introducing extraterrestrial growth medium and inoculant material of edible biomass brought from Earth into a photo-bioreactor. Includes.

[0020] Simulation experiments of Arthrospira platensis cultivation conducted using the apparatus of the present invention according to the method of the present invention provided evidence that, using the operating conditions of the method of the present invention, biomass productivity is significantly higher compared to the operating conditions described in International Publication No. 2013014606. This evidence clearly demonstrates the relevant improvements provided by the present invention to the latest technology. The simulation experiments provided evidence that the method of the present invention is advantageously feasible and enables the production of food and oxygen on Mars.

[0021] A further subject of the present invention is astronaut food containing edible biomass obtained by the method of the present invention.

[0022] A further subject of the present invention is a material kit particularly adapted for carrying out the method of the present invention during a long-duration manned space mission, the material kit is -A system for transporting diluted astronaut urine from ECLSS to containers for preparing extraterrestrial growth media; - Micronutrients essential for the growth of edible biomass and unavailable in extraterrestrial locations. It is equipped with.

[0023] Detailed description of the invention According to the present invention, the term "extraterrestrial conditions" means conditions found on Mars, the Moon, or a planet, implicitly meaning microgravity and a CO2-rich atmosphere.

[0024] According to the apparatus of the present invention, the jar is preferably a transparent jar, and at least one LSB is preferably a transparent laboratory-scale photobioreactor (LSP), which consists of a flask made of a transparent, non-cytotoxic, biologically inert, and non-degradable material that can ensure the transmission of light required by algae for photosynthesis. Preferably, untreated polystyrene should be used for this purpose. The LSP should be provided with a vent cap, preferably made of high-density polyethylene. A 0.2 μm non-wetting membrane should be sealed to the cap, which has the function of providing consistent sterile gas exchange while minimizing the risk of contamination. Thus, these caps are useful to ensure the backdiffusion and removal of CO2 required by algae in the broth culture, as well as photosynthetic oxygen, which, if accumulated in high concentrations in the liquid, can inhibit the growth of microalgae and promote photooxidation. The size of the LSP should be compatible with the size of the extraterrestrial dome simulant and the size of the clinostat or RPM. The shape of the unit may be cylindrical or prismatic, insofar as it allows for easier injection and access to the flask for pipetting.

[0025] According to the apparatus of the present invention, at least one clinostat or RPM must impart to the jar (and the sample within it) a motion characterized by the resulting acceleration vector, the module having a time-dependent average value close to zero. Clinostats and RPMs are based on different principles. A clinostat relies on keeping the cells uniformly suspended by continuously rotating the system to reduce sedimentation. This is achieved by rotating a sample on a plane along a circular orbit. Conversely, a random positioning machine simulates microgravity by randomly rotating the sample around two rotation axes. Thus, the sample is constantly reoriented, and the resulting time-averaged gravitational acceleration is close to zero or can be programmed to simulate known gravitational conditions of extraterrestrial locations as closely as possible (for example, the gravity of Mars is known to be about 1 / 3 of Earth's gravity). For algal culture, a random positioning machine should be preferred over a classical clinostat.

[0026] According to the apparatus of the present invention, the jar that functions as a simulant for an extraterrestrial dome preferably consists of a cylindrical jar that is large enough to accommodate at least one input LSP and can be attached to a clinostat or RPM. It is preferably made of a transparent material to allow light to be transmitted to the culture broth and to allow photosynthesis to occur. The material is also preferably non-cytotoxic, biologically inert, and non-degradable. The jar should be provided with at least one inlet connected via a suitable pipe to a cylinder containing a simulant for an extraterrestrial atmosphere (preferably CO2). The jar should be provided with an opening system to allow for easy insertion and removal of the LSP at the end of the experiment. Furthermore, the dome is preferably provided with a pressure gauge that can measure the pressure inside the jar.

[0027] The method of the present invention using the apparatus of the present invention is preferably, The steps involve preparing a culture broth that is as close as possible to the optimal medium for the cell line to be grown, and, if possible, simulating extraterrestrial ISRUs; The present invention comprises the steps of adding culture broth to the LSP of the apparatus, followed by adding inoculant material for the microalgae or cyanobacteria strain to be cultured; The steps include: placing the LSP inside a jar of the device that simulates an extraterrestrial dome; The present invention includes the steps of attaching a jar to a clinostat or RPM; The present invention involves connecting the outlet of the cylinder to the gas inlet of the jar; The steps include: allowing a simulant of extraterrestrial atmosphere to circulate through the jar with the gas outlet open for a sufficient amount of time to cleanse the atmosphere inside the jar; The steps include closing the jar outlet while continuing to ventilate a simulant of an extraterrestrial atmosphere until an internal pressure of at least 0.8 bar is achieved inside the jar; Steps to turn on a clinostat or RPM to simulate microgravity and Includes.

[0028] A method of the present invention using the apparatus of the present invention for cultivating edible biomass preferably further comprises the step of preparing a culture medium by mixing a liquid topsoil leachate obtained by leaching with acidic water and a simulant of extraterrestrial topsoil with a simulant of diluted astronaut urine and micronutrients that are unavailable by ISRU and essential for the growth of the cultured strain.

[0029] A method of the present invention using the apparatus of the present invention for cultivating edible biomass preferably includes the following steps: a) A step of preparing a simulant of extraterrestrial topsoil; b) A step of obtaining a topsoil slurry by contacting a simulant of extraterrestrial topsoil with an leachate, wherein the leachate is water acidified with HNO3; c) A step of filtering the topsoil slurry to obtain solid discharged topsoil and liquid topsoil leachate; d) Steps to prepare a simulant of astronaut urine; e) A step of diluting the urine simulant with water to simulate the dilution determined by the wash water in most ECLSS. In this way, a simulant of ECLSS wastewater is obtained. e') Finally, if the salinity of the wastewater simulant is too high and unsuitable for the growth of microalgae, dilute it further; f) A step of preparing a culture medium containing micronutrients optimal for the growth of the strain to be cultured; g) A step of mixing the ECLSS wastewater simulant with topsoil leachate and culture medium to obtain a culture broth; h) Adding culture broth to the LSP of the apparatus of the present invention, followed by adding inoculant material for the microalgae or cyanobacteria strain to be cultured; i) The step of placing the LSP in a jar of the apparatus for simulating an extraterrestrial dome; j) The step of attaching a jar to the clinostat or RPM of the apparatus of the present invention; k) Connecting the outlet of the cylinder of the apparatus of the present invention to the gas inlet of the jar; l) Allow a simulant of extraterrestrial atmosphere to circulate through the jar with the gas outlet open for a sufficient amount of time to purify the air inside the jar; m) The step of closing the jar outlet while continuing to ventilate the simulant of an extraterrestrial atmosphere until an internal pressure of at least 0.8 bar is achieved inside the jar; n) Turn on a clinostat or RPM to simulate microgravity, and at the same time illuminate the dome simulator with natural or artificial light to promote photosynthesis.

[0030] Extraterrestrial surface soil simulants can be prepared according to the Colorado School of Mines' Planetary Simulant Database (https: / / simulantdb.com / ). In the case of Martian surface soil simulants, it is preferable that the chemical composition is that of the JSC Martian surface soil or Mojave Martian simulant described by the supplier or in the scientific literature (Peters, et al. Icarus. 197 (2008) 470-479. https: / / doi.org / 10.1016 / j.icarus.2008.05.004). Typically, the main elements in these simulants are silicon, aluminum, iron, magnesium, and calcium, but the main crystalline phase can be magnetite, anorthite, hematite, fostelite, enstanitite, wollastonite, etc. (Corrias, et al.; Acta Astronaut. (2012). https: / / doi.org / 10.1016 / j.actaastro.2011.07.022).

[0031] The leachate should preferably be water acidified by adding HNO3 to achieve a maximum solution pH of 8.0. The topsoil simulant and the resulting liquid should then be placed together with the leachate in a laboratory-scale reactor equipped with a stirring system. The solid-to-liquid weight ratio should preferably be at least 1 / 10 g / g. The stirring level should preferably be 100 rpm or higher. A contact time of at least 24 hours should be allowed before proceeding to the next step. During this step (b), several micronutrients necessary for algal growth, such as Fe, Zn, Ca, Na, and Mg, are transferred from the solid to the liquid phase. The next step (c) consists of separating the liquid supernatant from the discharged topsoil simulant. It should be noted that the supernatant should be filtered until a low turbidity level corresponding to a maximum optical density of about 0.05 is achieved.

[0032] Step d) involves preparing a simulant of astronaut urine following the procedure by Sarigul et al. (Sci.Rep.9(2019)1-11.https: / / doi.org / 10.1038 / s41598-019-56693-4). This step is important because urine produced by astronauts during manned missions to Mars or other extraterrestrial locations is one of the main sources of nitrogen and phosphorus macronutrients essential for the growth of microalgae. In fact, according to Sarigul et al. (Sci.Rep.9(2019)1-11.https: / / doi.org / 10.1038 / s41598-019-56693-4), human urine is characterized by the composition shown in the following section of this document. Therefore, it would be important to take a certain amount of urine from the ECLSS and use it to produce a "culture broth". For hygienic purposes, urine is usually discharged from cabin crew along with an appropriate amount of wash water. In this regard, step (e) consists of diluting the urine simulant with an amount of water that simulates the wash water used in the ECLSS. Different volumes of wash water can be used depending on the specific ECLSS that may be considered. To avoid osmotic shock to microalgae caused by the high salinity of the urine, if the water dilution in step e) is too low, a further dilution step in step e') is foreseen at an optional rate. A preferred dilution ratio is 1 part urine simulant to 10 parts water.

[0033] The culture medium in step (f) is preferably Zarrouk medium if the strain being cultured is Arthrospira platensis.

[0034] In step (g), the ECLSS wastewater simulant, topsoil leachate, and culture medium are preferably mixed in a ratio of 1:1:1v / v.

[0035] The inoculum preferably consists of a small amount of one of the following algal strains: Gloeocapsa strain OU_20, Leptolyngbya strain OU_13, Phormidium strain OU_10, Chroococcidiopsis 029; Arthrospira platensis; Synechococcus elongatus; Anabaena cilindrica; Chlorella vulgaris; Nannochloris Eucaryotum; or a genetically modified strain thereof. However, due to the higher nutritional properties required for astronauts (Soni et al., (2021). Food Supplements Formulated with Spirulina. In Algae (pp. 201-226). Springer, Singapore, doi: 10.1007 / 978-981-15-7518-1_9), the inoculum should preferably consist of either the Arthrospira platensis strain or the Spirulina platensis strain.

[0036] When inoculating, the strain should preferably be in the exponential growth stage, and a pure culture, i.e., the absence of bacteria, rotifers, or different strains, should be ensured.

[0037] Step (h) involves supplying the inoculant to a laboratory-scale optical bioreactor (LSP) that has been pre-supplied with the generated "culture broth" as shown in point (g). The amount of inoculant added to a unit volume of culture broth should be such that the optical density (wavelength 650 nm) of the resulting solution is in the range of 0.05 to 0.15.

[0038] Step (i) involves placing the LSP in a transparent, insulated jar to simulate an extraterrestrial dome hosting biological processes, thereby recreating Earth-like thermal and pressure conditions. To promote effective photosynthesis of microalgae, the refracted light emission is at least 30 μmol m³. -2 s -1 Preferably 100 μmol m -2 s -1 The jar must be transparent as it should have an intensity close to that of [a specific light source]. Microalgae photosynthesis is the phenomenon that underlies the growth and replication of algae used as food, as well as the production of oxygen needed by the crew. However, algae also require a dark period to carry out the Calvin cycle reaction (the so-called dark reaction). For this reason, the light source should be turned off for at least 8 hours per day.

[0039] Preferably, the dome simulant is at least 20 μE m -2 s -1 Equivalent to, but preferably 100 μmol m -2 s -1 The LSP is irradiated with a light bulb capable of providing photosynthetically active radiation of an intensity close to that of the LSP. Preferably, the light bulb is placed near the clinostat or RPM. Preferably, the light bulb is connected to a timer that can turn on the light source with a photoperiod of 12 hours / day.

[0040] Step (j) involves mounting the "extraterrestrial dome simulant," which includes the "injected LSP," onto a clinostat or random positioning machine (RPM). The RPM, which is better described in the kit section, is a tool that can replicate the microgravity inside the LSP through 3D motion characterized by acceleration that is either close to zero (approximately 0.05g) or as close as possible to the gravity of an extraterrestrial location.

[0041] In step (k), the extraterrestrial dome simulant is connected to a gas cylinder via appropriate ports and pipes. Preferably, the gas cylinder contains a gas to simulate an extraterrestrial atmosphere, and when simulating a Martian atmosphere, preferably the gas cylinder contains pure CO2. CO2 is necessary for the growth of microalgae and is a natural resource available on Mars or other extraterrestrial locations. In practice, as reported, for example, in International Publication No. 2013014606, the Martian atmosphere consists mainly of CO2 (about 95% v / v) that can be separated from other gases, pressurized, and supplied to the Martian dome. Preferably, a value of at least 0.8 bar is specified for the pressure of CO2 in the dome. For this reason, in step (l) of the method of the present invention, it is preferably foreseen that the CO2 in the insulated jar is vented until such a pressure level is achieved. A recent paper (Verseux et al. 2021, Front.Microbiol.doi:10.3389 / fmicb.2021.611798.) demonstrates that microalgae can grow even under lower CO2 pressures, and therefore lower CO2 pressures can also be used. After achieving such pressure values, the jar should be sealed (step m).

[0042] Step (n) consists of starting the experiment by turning on a clinostat or random positioning machine, turning on the lights, and then waiting for at least 12 hours to pass under the lights.

[0043] Preferably, the dome simulant should be emptied daily when the lights are turned off and left empty in the dark for 12 hours. Then, when the lights are switched back on, CO2 should be reinjected into the Martian dome simulant and it should be sealed (step m), as described in step (l). This sequence should be repeated throughout the entire experimental period.

[0044] This operation must be performed because, when photosynthesis is not occurring (i.e., during the dark period), CO2 is not absorbed by the algae, and therefore the concentration of CO2 in the gas phase becomes too high, significantly acidifying the culture broth to an unacceptable degree, which in turn inhibits algal growth. Subsequently, after a 12-hour dark period, the jar should be filled with CO2 and sealed as reported in step (l). Preferably, all analyses necessary to monitor algal growth and other parameters should be performed during the dark period when the lights are off and the jar is empty (no CO2). The minimum set of parameters to be monitored includes the optical density of the culture, biomass concentration, and pH concentration.

[0045] The apparatus and method of the present invention can be used to simulate the growth of any cell line under extraterrestrial conditions, similar to the methods described above for edible microbial culture. All cell lines whose growth can be simulated using the apparatus and method of the present invention are from ECACC (https: / / www.phe-culturecollections.org.uk / products / celllines / generalcell / search.jsp?searchtext=human%20cell%20lines&dosearch=true) and ATCC (https: / / www.atcc.org / cell-products / animal-cells).

[0046] Further plant cell lines are cultured using the apparatus and method of the present invention, preferably the cell lines are Chlorella sorokiniana, Chlorella zofigensis, Coccomyxa sp., Synechococcus sp., Pseudochloris wilhelmii, Chlorella protothecoides, Euglena gracilis, Chlamydomonas reinhardtii, Isochrysis galbana, Neochloris oleoabundans, Scenedesmus obliquus, Dunaliella salina, Nannochloropsis oculate, Chlorella pyrenoidosa, Botryococcus braunii, Phaeodactylum tricornutum, Tetraselmis sp., Thalassiosira pseudonana, Haematococcus pluvialis, Nannochloropsis oceanica, Spirulina maxima, Pavlova salina Salina), Porphyridium marinum, Tetraselmis inconspicua, Cyanophora paradoxa, Thalassiosira rotula, Amphora sp., Odontella aurita, Attheya sp., Chromulina ochromonoides, Diacronema vlkianum, Chaetoceros sp., Navicula pelliculosa, Odontella mobiliensis, Porosira pseudodenticulata Selected from the group consisting of (pseudodenticulata).

[0047] Further cell lines that can simulate growth under extraterrestrial conditions using the apparatus and methods of the present invention are: H1, H9, embryonic stem cells, human; HEK-293, adenovirus-transformed embryonic kidney, human; HeLa, epithelial cells, human; HL60, human, promyelocytic leukemia cells, human; MCF-7 breast cancer, human; A549, lung cancer, human; A1~A5-E, amniotic membrane, human; ND-E, esophagus, human; CHO, ovary, Chinese hamster; 3T3, fibroblasts, mouse; BHK21, fibroblasts, Syrian hamster; MDCK, epithelial cells, dog; E14.1, embryonic stem cells (mouse), mouse; COS, kidney, monkey; DT40, lymphoma cells, chicken; S2, macrophage-like cells, Drosophila; GH3, pituitary tumor, rat; L6, myoblasts, rat; Sf9 and Sf21, ovary, Fall armyworm (Spodoptera frugiperda) ; These are human and animal cell lines selected from a group consisting of ZF4 and AB9 cells, embryonic fibroblasts, zebrafish; 1184, dermal fibroblasts, human; E6.1 clone, Jurkat cells, human; THP1 cells, human; SH-SY5Y, neuroblastoma cells, human; iPSCs, stem cells, human; BRISTOL8, B lymphocytes, human.

[0048] According to the apparatus and method of the present invention, the most preferred cell lines to be cultured are selected from the group consisting of: erythrocyte cell culture, human;C20A4; chondrocyte, human;1301; T-cell leukemia, human;1306, 161BR; dermal fibroblast, human;F-36P myelopathy syndrome, leukemia, human;H9; T cell, human;HeLa; epithelial cell, human;E6.1 clone; Jurkat cell, human;SH-SY5Y; neuroblastoma cell, human;iPSC; stem cell, human;1184; dermal fibroblast, human;hMSC; mesenchymal stem cell, human;mBMSC; bone marrow-derived mesenchymal stem cell, rat;ADSC; adipose-derived stem cell, human;mESC; embryonic stem cell, mouse;MG-63; osteosarcoma cell line, human;HUVEC; and human umbilical vein endothelial cells, human.

[0049] According to the method of the present invention, each cell line is cultured in a culture medium as close as possible to its optimal medium, and, if possible, by simulating an extraterrestrial ISRU.

[0050] To simulate the growth of human and animal cells under extraterrestrial conditions, it is preferable to perform the above method, and steps a to g can be replaced by the step of preparing a culture broth that is as close as possible to the optimal medium for the cell line to be grown, and the step of simulating an extraterrestrial ISRU, if possible, and the irradiation in step n) can be omitted.

[0051] The methods and kits related to the present invention operate in relation to ECLSS sections that are necessarily present in extraterrestrial locations during long-duration manned space missions, and thus represent an ideal completion aimed at achieving an autonomous integrated system. Accordingly, the methods are based on the utilization of extraterrestrial resources such as atmosphere, soil, and solar radiation, the main features of which have been reported in the literature, e.g., Nanagle [Nat. Biotechnol. (2020). https: / / doi.org / 10.1038 / s41587-020-0485-4] and Rapp [https: / / doi.org / 10.1007 / 978-3-319-72694-6]. In particular, relatively large amounts (about 9% wt / wt) of hydrated water have been detected in Martian soil (Rieder, R., et al. Science 306, 1746-1749 (2004)).

[0052] The method of the present invention is preferably, a'. The step of assembling at least one geodetic dome on extraterrestrial soil and placing at least one optical bioreactor inside the dome; b'. Assembling a physicochemical section comprising a photovoltaic panel, at least one WAVAR unit, at least one TSA unit, and at least one MPO unit to extract water from extraterrestrial soil and atmosphere, extract dehydrated and pressurized CO2, N2, and Ar, and produce NH3, O2, H2, HNO3, and NH4NO3; c'. A step of blowing the heated, pressurized, and dehydrated CO2 generated in step (b') into the dome until it reaches a pressure of at least 0.8 bar and a temperature of at least 10°C, preferably 10-15°C; d'. A step of preparing an leachate by mixing HNO3 produced in the physicochemistry section with water; e'. Preferably, the step of leaching the dehydrated topsoil from the physicochemical section with an leachate at a solid / liquid weight ratio of 1:5 for at least one Martian day (sol); f'. A step of filtering the topsoil slurry to obtain topsoil leachate and leachate topsoil; g'. The step of preparing an extraterrestrial growth medium by mixing topsoil leachate with diluted astronaut urine from at least one ECLSS section, HNO3 produced in the physicochemistry section, and other micronutrients brought from Earth that are essential for the growth of edible biomass but are not available in the field; h'. A step of preparing an inoculum of edible microalgae or cyanobacteria brought in from Earth; i'. The step of supplying extraterrestrial growth medium to a photobioreactor, followed by supplying inoculant to obtain a biological slurry; j'. The biological slurry is exposed to CO2 and a light source capable of promoting photosynthesis within the dome, thereby resulting in the formation of new biomass algae and photosynthetic oxygen; k'. The steps include separating algal biomass from the used "culture broth" by centrifugation and extracting photosynthetic oxygen by degassing; l'. The step of introducing oxygen into the ECLSS section to further dehydrate the algal biomass for use as a food or nutritional supplement along with the food produced in the ECLSS section; m'. The step of splitting the used "culture broth" into two streams called α1 and α2; n'. The step of recirculating the spent culture broth stream α1 to at least one photobioreactor; o'.Optionally, the step of transporting Stream α2 together with ammonium nitrate (NH4NO3) produced in the physicochemical section, together with fresh topsoil, together with appropriate amounts of humic and fulvic acid brought in from the earth, and together with human metabolic waste from ECLSS, into the dome where the vegetables are being grown. Includes.

[0053] The extraction process using the physicochemical section preferably includes the following steps: b'-i. Step of assembling an outdoor photovoltaic panel that heats the inside of the dome and generates the energy necessary to power the plant unit; b'-ii. Steps for assembling the outdoor temperature swing adsorption unit (TSA); b'-iii. Steps for assembling the WAVAR unit outdoors for dehydration of the Martian atmosphere; b'-iv. Steps to assemble at least one pizza microwave outdoors; b'-v. Step of blowing the Martian atmosphere into a WAVAR unit operating outdoors to extract water from the atmosphere; b'-vi. A step to transport the Martian atmosphere to a TSA unit, where CO2 separation and pressurization are carried out via adsorption-desorption cycles to zeolite material at various temperatures. In the TSA unit, a secondary gas flow consisting mainly of N2 and Ar is also generated; b'-vii. The step of storing the secondary gas stream of N2 and Ar generated as shown in step (g') into a suitable tank from which it can be used as a buffer gas for analytical instruments used during sampling campaigns conducted for scientific purposes during the mission; b'-viii. Heating CO2 blown into at least one dome via a heating system powered by the photovoltaic panel until a temperature of 10°C or higher is achieved inside the dome; b'-ix. A step of excavating Martian topsoil and transporting it to an MPO system that operates indoors and extracts adsorbed water and hydrated water from the topsoil using microwaves.

[0054] In particular, WAVAR and TSA units operate outdoors. These units are preferably mechanically protected by appropriate structures from potential damage caused by meteorites and / or solids transported during typical sandstorms characteristic of extraterrestrial environments while operating under extraterrestrial thermal and pressure conditions. Such structures can be obtained in the field by specific technologies, such as those described in, for example, International Publication No. 2012 / 014174.

[0055] The MPO unit operates indoors.

[0056] The method of the present invention preferably includes, in the first step (a'), a dome in which an indoor plant unit necessary for carrying out the method is installed and assembled. Inside the dome, thermal and pressure conditions (temperature and pressure) are set up such that the aggregation state of the reactants and products is exactly like that observed on Earth for the same compounds, by a technique better specified below.

[0057] Step (e') preferably involves supplying a topsoil stream along with a water stream into a reactor where a liquid and a solid come into contact to form a continuously stirred slurry, thus enabling effective contact between the liquid and solid phases. The goal of such a step is to transfer all macronutrients (P, S, C) and micronutrients (Fe, Mg, Si, etc.) contained in the topsoil to the liquid phase. In this way, a “topsoil leachate” is produced that, when integrated with other nutrients, can sustain the autotrophic algal growth phenomenon. Preferably, the contact time to ensure effective transfer of nutrients to the liquid phase is about 24 hours.

[0058] Step (f') of this method includes solid-liquid separation, which may be carried out by a suitable filtration system (i.e., a filter plate or filter bag).

[0059] Therefore, operation step (f') produces two separate streams, the first being leachate from the topsoil and the second being a liquid called "topsoil leachate." The second stream is mixed with astronaut urine diluted with wash water from the ECLSS to obtain a solution containing nutrients suitable for biomass. In fact, human urine typically contains important macronutrients such as ammonium, nitrates, phosphates, and orthophosphates, which are generally limiting factors for biomass growth. Thus, the use of this metabolic waste can greatly improve the ability of the resulting medium to sustain microalgae growth. Any other nutrients that are not available in-situ and are necessary to obtain a balanced growth medium can be brought in from Earth.

[0060] Preferably, the inoculum consists of the following algal strains: Gloeocapsa strain OU_20, Leptolyngbya strain OU_13, Phormidium strain OU_10, Chroococcidiopsis 029; Arthrospira platensis; Synechococcus elongatus; Anabaena cilindrica; Chlorella vulgaris; Nannochloris Eucaryotum or genetically modified strains thereof. However, due to its superior trophic properties, the Arthrospira platensis strain should be preferred.

[0061] Step (i') involves supplying inoculum to a photobioreactor, which is simultaneously supplied with the “Mars growth medium”. The mixture obtained in the photobioreactor is hereafter referred to as the “biological slurry”. In accordance with step (j'), the CO2 required by the biomass to carry out photosynthesis is extracted from the atmosphere, which consists of pure CO2 inside the dome, through a suitable opening in the photobioreactor, which is preferably covered by a semipermeable membrane that allows for the diffusion of CO2 toward the biological slurry and the backdiffusion of oxygen produced by photosynthesis. Photosynthesis is carried out by the algae thanks to the luminous flux provided by a light source in accordance with step (j') of the method. The luminous flux can be supplied by directly exposing the culture to solar radiation incident on the Martian surface, or preferably by a suitable system such as a light collector and optical fibers. Thus, the photosynthetic process results in the production of new microalgae, which leads to an increase in the algal biomass concentration in the culture.

[0062] According to a preferred embodiment, the photobioreactor operates in a fed-add mode. Thus, biomass cultivation is carried out within the photobioreactor until the biomass concentration reaches an appropriate value corresponding to the stationary phase of the biomass growth kinetics. Once the stationary phase is reached, an appropriate amount of "biological slurry" is withdrawn and subjected to a dehydration process to separate the biomass from the used "culture broth". The amount of biological slurry withdrawn from the photobioreactor is then replaced with an equal amount of fresh "culture broth" to resupply the nutrients consumed during biomass growth. Aliquots of the medium required to replace the withdrawn amount of biological slurry can also be obtained by recirculating the solution according to step (n'). Once the fresh "culture broth" recovery and reintegration operation step is performed, microalgae growth is restarted in batch mode. The removal and reintegration operation should be repeated periodically, preferably once a day, at the same time to ensure growth for at least 25 hours (a Martian day) in batch mode, for example.

[0063] Step (k') of this method includes transferring the "biological slurry" extracted daily to a solid-liquid separation step performed by a suitable centrifuge system. This solid-liquid separation allows for the separation of algal biomass from the used "culture broth".

[0064] The spent culture broth can be recycled to the head of a photobioreactor to reduce the amount of water required for inlet. According to a preferred embodiment, another aliquot of the spent culture broth, including the residual content of relevant nutrients, can be used for greenhouse irrigation purposes in the process described in International Publication No. 2013014606.

[0065] Solid algal biomass separated by centrifuge can be further dehydrated by microwave and then used as food by astronauts.

[0066] In step (l'), the oxygen produced by the biomass through photosynthesis is separated from the biological slurry via a suitable degassing system and then transferred to an ECLSS unit, where it can be used for crew cabin air regeneration. Simultaneously, the microalgae biomass is further dehydrated and then transferred to the ECLSS, where it can be used as astronaut food. Thus, a further subject of the present invention is astronaut food containing microalgae biomass obtained by the method of the present invention.

[0067] Step (o') involves transporting the different products of the previously described methods within a dome that operates as a greenhouse in which plants and vegetables can be grown.

[0068] The material kit of the present invention is preferably, - At least one geodetic dome for housing different plant units used in the physical chemistry group of the above procedure, - At least one photovoltaic system for generating the energy required to heat the internal atmosphere of at least one dome, and the energy required to power the operation of the plant unit, - An adsorption process, followed by desorption by microwave heating, is carried out for the extraction of water from the Martian atmosphere in at least one WAVAR unit based on the use of zeolite, -At least one TSA unit comprising at least one zeolite adsorbent bed and at least one radiator, wherein at least one radiator ensures heat exchange with the Martian environment and the execution of adsorption-desorption cycles at variable temperatures, thereby enabling the separation and pressurization of CO2 from other gases (mainly N2 and Ar) constituting the Martian atmosphere, and the pressurized pure CO2 produced by the TSA unit can be blown into at least one dome until a suitable pressure is achieved inside the dome; - At least one excavator and at least one conveyor belt for excavating Martian surface soil and transporting it to the next processing unit, - At least one MPO unit containing at least one magnetron for extracting adsorbed water and hydrated water from Martian surface soil by microwave heating, - At least one unit for mixing water extracted from the topsoil with an appropriate amount of nitrate produced in the physicochemical section, - At least one leachate reactor operating in continuous mode for leaching topsoil through a mixture of water and nitrate, - A unit comprising at least one "filter plate" for solid-liquid separation of a slurry stream flowing out of a leachate reactor, which generates a liquid stream called "topsoil leachate" and a solid stream of "leachate topsoil", - At least one unit for obtaining a so-called "culture broth" by mixing "topsoil leachate" with urine diluted with wash water produced by astronauts in the ECLSS, - The following algal strains: at least one tank for storing the gas mainly composed of N2 and Ar generated in the TSA unit as a result of CO2 separation, - At least one of the following: Gloeocapsa strain OU_20, Leptolyngbya strain OU_13, Phormidium strain OU_10, Chroococcidiopsis 029; Arthrospira platensis; Synechococcus elongatus; Anabaena cylindrica; Chlorella vulgaris; Nannochloris Eucaryotum or at least one of their genetically engineered strains, - At least one unit for preparing the inoculum of the algal strain, - At least one photobioreactor for generating algal biomass, - Nutrients from the Earth that are essential for the growth of at least one algal strain but are not available for use by ISRU, - At least one unit for separating the algal biomass from the used "culture broth" and the oxygen generated in the photobioreactor, - At least one unit for dehydrating the algal biomass, - (Optionally) at least one geodesic dome used as a greenhouse for cultivating edible plants and is provided with.

[0069] Preferably, the dome is made of a framework of aluminum beams with a circular cross-section. Preferably, the covering of the geodesic dome is made of a sheet of ETFE (ethylene tetrafluoroethylene) having a surface density of 0.2 kg / m 2 and high mechanical and thermal resistance.

[0070] Preferably, at least one photovoltaic system generates the energy necessary to power all operating steps of the method of the present invention, including the step of heating the atmosphere inside the dome. From an electrical standpoint, the photovoltaic system is preferably divided into separate sections (arrays), each of which is about 40 m 2 It has a surface and a yield of approximately 11% of solar radiation converted into electricity.

[0071] The use of TSAs utilizing variable-temperature adsorption / desorption cycles on zeolites is proposed for the separation, injection, and compression of CO2 within domes, following the principles described above for TSA units. A unit suitable for extracting water from the Martian atmosphere may be the one described by Williams, JD et al. (Journal of British Interplanetary Society, 1995, 48, 347-354).

[0072] At least one excavator and at least one belt conveyor must excavate extraterrestrial topsoil and transport it to the MPO unit. The excavator consists of a vehicle independently powered by a photovoltaic rechargeable battery or by a small photovoltaic system housed in the same vehicle.

[0073] At least one slurry reactor should be stirred and coated with an acid-resistant coating. Preferably, the size of the reactor should be such that it provides a residence time of at least 24 hours. The slurry exiting the reactor is transferred to a step in which solid-liquid separation is performed. For this purpose, the method of the present invention includes the use of at least one filter for separating the solid phase of the slurry from the liquid.

[0074] Different types of photobireactors can be used, but tubular ones should be preferred. The tubes should be made of PET (polyethylene terephthalate) as they must be transparent to photosynthetically active radiation. Preferably, the tubes should have a diameter of less than 0.2 m. The photobireactor should operate primarily in flow-add mode. The light flux necessary to promote photosynthesis can be supplied by directly exposing the photobireactor to solar radiation incident on the extraterrestrial surface, or preferably by a suitable focusing system such as a light concentrator and optical fibers that transmit light to a dome in which the photobireactor is housed.

[0075] If the kit contains the Arthrospira platensis algal strain, the nutrients from earth are preferably boric acid and a complexing agent, more preferably H3BO3 and EDTA.

[0076] Next, the amount of "biological slurry" periodically withdrawn from the photobioreactor should undergo solid-liquid separation. Preferably, the unit for separating algal biomass from the used "culture broth" is carried out by at least one centrifuge. [Brief explanation of the drawing]

[0077] [Figure 1] This figure shows the scheme of the apparatus of the present invention. [Figure 2] This is a graph of gravitational acceleration achieved by a clinostat. [Figure 3] This figure shows the time-varying algal biomass concentration during a specific experimental test, aimed at separating the effects of each operating condition of the method against base case experiment Zm_air_1g. (A) Effect of using Martian culture medium; (B) Effect of using simulated Martian atmosphere; (C) Effect of microgravity. Figure (D) shows the effect of a synergistic combination of two of the three operating conditions of the present invention's method against the base case condition. [Figure 4]This figure shows the effect of simulating the operating conditions for implementing the method of the present invention (Mm40_CO2_μg) on ​​Mars, with respect to (A) changes in biomass concentration over time and (B) final biomass productivity after 22 days of cultivation. [Figure 5] This figure shows a comparison of the results obtained under the operating conditions (Mm40_CO2_μg) used to simulate the method of the present invention, and the results obtained under the operating conditions (RL_CO2_μg) used to simulate the method described in the existing International Publication of the Latest Technology, brochure No. 2013014606, regarding (A) the change in biomass concentration over time and (B) the final biomass productivity after 22 days of cultivation, with the results obtained under the operating conditions (RL_CO2_μg) used to simulate the method described in the existing International Publication of the Latest Technology, brochure No. 2013014606. [Figure 6] This figure shows a flow sheet of a method according to an example of the present invention.

[0078] Experiment Section 1. Materials and Methods 1.1. Microorganism maintenance conditions Monoalgae cultures of Cyanobacterium Spirulina were obtained from the TOLO Green Farm algal culture in Arborea, Sardinia, Italy. The strains were maintained under sterile conditions in the laboratory of the Interdisciplinary Center for Environmental Science and Technology (CINSA) at the University of Cagliari in Sardinia, Italy. The cultures were kept in 250 mL Erlenmeyer flasks containing 150 mL of Zarrouk medium (Table 1), sealed with aluminum foil covered with cotton wool. [Table 1]

[0079] The flask containing the culture medium was autoclaved at 121°C for 15 minutes before inoculation. The culture was maintained under photoautotrophic conditions and incubated at 20±1°C in a thermostat-controlled chamber. The photoperiod was set to 25 μmol / m². 2 The system was illuminated with white light (using a Delta OHM HD 2302.0 light meter) at a fixed 12:12 light and dark period. Stirring was set to 100 rpm.

[0080] 1.2. Preparation and composition of Martian medium (MM) MM was prepared by mixing leachate of Martian surface soil simulant (JSC MARS-1) and synthetic human urine (MP-AU) according to the literature (Sarigul, et al. Sci.Rep. (2019) https: / / doi.org / 10.1038 / s41598-019-56693-4). The main components of JSC MARS-1 used in this study are reported in Table 2 as oxide weight %. The mineral phase of JSC MARS-1 identified by XRD analysis mainly consists of tectosilicate plagioclase feldspar, pyroxene, iron oxides (magnetite and hematite), ilmenite, and olivine (Peters, et al. Icarus. 197 (2008) 470-479. https: / / doi.org / 10.1016 / j.icarus.2008.05.004). [Table 2]

[0081] The leachate was prepared by contacting 15 g of topsoil simulant (diameter < 1 mm) with 150 ml of ultrapure water with a pH of 6.80 in a 250 ml capped Erlenmeyer flask. The solid-liquid mixture was stirred at 200 rpm for 24 hours at 25°C using an orbital shaker (Stuart SSM1, Bio sigma). The resulting solution was filtered by gravity using absorbent paper. The supernatant was analyzed using inductively coupled plasma atomic emission spectroscopy (Varian 710-ES ICP OES) to determine Al, Ca, Fe, K, Mg, Mn, Na, P, Si, and Ti. The results are shown in Table 3. [Table 3]

[0082] The operating conditions were a high-frequency generator power of 1.2 kW and a frequency of 40 MHz, and Ar (99.996% purity) was used for both plasma (15 L / min), nebulizer (200 kPa), and optical feed (1.5 L / min). The spray chamber was a double-pass glass cyclone. The applied power and pressure were 600 W and 100 PSI for 13 minutes. A calibration curve was calculated at 5 points, and R 2 Values ​​≥0.999 were considered acceptable. Synthetic human urine (MP-AU) was prepared according to the literature (Sarigul, et al. Sci.Rep. (2019) https: / / doi.org / 10.1038 / s41598-019-56693-4) and then diluted with ultrapure water at a ratio of 1:10 to meet the nitrogen requirements of microalgae. The chemical composition of the diluted human urine simulants is shown in Table 4.

[0083] Finally, a mixture of 1 part Martian soil leachate and 1 part diluted urine (1:1v:v) was prepared to create the so-called Martian medium (MM). It had a conductivity of 850 μS / cm and a pH of 7.4 at 25°C. MM (20%, 40%, 60%, and 80%) was diluted in Zarrouk medium, which also served as the experimental control medium. MM and its dilutions were sterilized at 121°C for 15 minutes before use. Table 5 shows the composition of the obtained Martian medium in terms of macronutrients, and Table 6 shows its composition in terms of metals. Some metals, such as Zn, Fe, Mg, Si, Mn, and K, can function as micronutrients for algae. [Table 4] [Table 5] [Table 6]

[0084] 1.3. Growth experiments to identify the optimal MM content in the culture medium and to determine the effect of simulated Martian conditions. Preliminary tests were conducted using growth media consisting of mixtures of MM and Zarrouk medium (ZM) with MM volume percentages equal to 0, 20, 40, 60, and 80% v / v, respectively. From these experiments (data not shown), it was identified that the best growth medium, including the use of MM, contained 40% v / v MM and 60% v / v ZM (Mm40). Subsequently, different experiments were conducted to evaluate the effect of operating conditions simulating one of the methods implemented on Mars. Both the isolation and synergistic effects of all operating conditions in the method implemented on Mars were evaluated. Table 7 summarizes the experiments conducted in this case. These experiments allowed us to determine the feasibility of the method of the present invention.

[0085] The following characteristics were common to all these experiments: Batch culture experiments were performed in clear, ventilated cap flasks filled with 40 ml of culture solution. The experiment involved 100 μmol m³ of culture solution on the irradiated surface of the culture flask. -2 s -1 The lighting was set up in a triple configuration. The optical density at the start of the experiment was approximately 0.2 at a wavelength of 650 nm. [Table 7]

[0086] Cell morphology was examined using 40x and 100x (Leica DM750) optical microscopes coupled with a Leica EC3 digital camera (Leica Microsystems, Wetzlar, Germany), and the Leica Application Suite (version 3.4.0, Leica Microsystems). All operations were performed under a microbiological safety cabinet to avoid environmental contamination. The atmosphere consisted of air, and gravity was equal to 1g. During the experiment, cyanobacteria growth was monitored by microplate reader ELISA (TECAN, Sunrise®, Tecan Trading AG, Switzerland) for chlorophyll-optical density (OD) absorbance of cultures at a wavelength of 650nm. Biomass concentration C x (gL -1Calibration curve C was obtained by gravimetric analysis of biomass concentration in a known culture volume that had been previously centrifuged at 4000 rpm for 15 minutes and dried at 105°C for 24 hours. x pH was calculated from OD measurements using the OD ratio. pH was measured daily using a pH meter (Basic 20, Crison). pH was also measured daily using a pH meter (XS Instruments, Calpi, MO, Italy).

[0087] 1.4. Simulation of the atmosphere inside the Martian dome (Whitley jar gasification system) To investigate the potential of using CO2 from the Martian atmosphere, further experiments were conducted to grow microalgae in an atmosphere consisting of pure CO2. For this purpose, a Don Whitley workstation was used, which can provide excellent conditions for sample processing, incubation, and inspection without exposure to atmospheric oxygen. The workstation allows for handling samples in a sustainable environment, where parameters can be changed to create the conditions necessary to grow a culture in a jar in the presence of approximately 100% CO2 in just two minutes. The resulting full-color touchscreen control panel allows the operator to monitor in real time whether the criteria necessary for culture growth have been met. The workstation is connected to both a CO2 cylinder and a polycarbonate jar. The jar has a capacity of 2.5L (height 24cm, diameter 17cm) and is 75cm. 2 It can accommodate eight flasks, each with a base. The jar has a built-in fault detector that generates an alarm if it is leaking.

[0088] 1.5. Simulation under microgravity conditions To investigate whether the growth and metabolism of microalgae can be influenced by the microgravity conditions achieved in space and on Mars, further experiments were conducted under microgravity conditions at the Institute of Biomedical Research, University of Sassari, Sardinia, Italy. A 3D Random Positioning Machine (RPM, Fokker Space, Netherlands) was used to simulate microgravity (μg). The 3D Random Positioning Machine (RPM) was a microgravity simulator based on the principle of "gravity vector averaging," built by Dutch Space (formerly Fokker Space) in Leiden, Netherlands. The 3D RPM consists of two vertical frames that rotate independently. The direction of the gravity vectors is constantly changed so that the average of the gravity vectors simulates a microgravity environment. The 3D RPM is 10 -3 It provides simulated microgravity of less than g. The dimensions of the 3D RPM are limited to 1000 × 800 × 1000 mm (length × width × height). The mechanical stage can accommodate up to 12 flasks simultaneously, and the samples should be positioned within 10 cm of the rotation center before being placed on the 3D RPM.

[0089] 1.6. Simulation of the simultaneous effects of all operating conditions for the method to be implemented on Mars. To simulate the effects of all operating conditions on the method of the present invention on Mars, the following procedure was employed. To avoid fluid shear, medium a containing 40% v / v MM (Mm40) was carefully packed into a flask without introducing air bubbles (approximately 80 ml). Subsequently, the flask was fixed inside a jar, filled with CO2, and then attached to a 3D RPM. The 3D RPM was operated for at least 23 days in a dedicated room at 25°C. The same culture was grown in parallel at 1 g and placed in a stationary bar containing a control culture to receive the same vibrations of the sample under μg conditions. The 3D RPM was connected to a computer, and the rotation mode and rotation speed were selected via specific software. A random walk mode of 60 degrees / second (rpm) was selected. The culture was grown at 150 μmol m -2 s -1The microalgae were illuminated with white light for 12 hours, and CO2 was administered to them during the light period. A simplified scheme of the experimental steps is shown in Figure 1, while Figure 2 shows a graph of the vector components of acceleration achieved by RPM, and a table reporting the vector sum (G-res) of such components (whose values ​​are always close to 0).

[0090] 2. Experimental Results 2.1. The effects of using Martian culture medium alone This experiment aimed to investigate whether replacing a certain volume of Zarrouk medium with the same volume of Mars medium could affect the growth of Spirulina. For this purpose, preliminary tests were conducted using growth media consisting of mixtures with volume percentages of Mm equal to 0, 20, 40, 60, 80% v / v, and 100%, respectively. These experiments were performed under atmospheric and Earth gravity. From these experiments (data not shown), the best growth medium, including the use of Mm, was identified as containing 40% v / v Mm and 60% v / v Zm (Mm40). Higher percentages resulted in a decrease in the growth rate of the culture. Figure 3A shows a comparison of the changes in biomass concentration obtained when using Zm and Mm40 alone.

[0091] Both cultures are 0.45 gL -1 When concentrations close to the specified value were achieved, no significant effect was observed from the rearrangement of 40% v / v Zm with a corresponding volume of Mm for up to 14 days. Rather, a slight improvement in growth could be observed up to 13 days of culture. After 15 days of culture, both cultures began to decline, likely due to inhibition determined by carbon starvation or the high pH (close to 11) achieved by the system (data not shown). Therefore, to be productive when operating in batch mode, these cultures should be stopped after 15 days of culture.

[0092] 2.2. The standalone effect of using the simulated Martian atmosphere. The objective of this experiment was to isolate the effect of using an atmosphere to simulate the atmosphere that would be realized in a dome hosting the method of the present invention on Mars. According to the method of the present invention, this atmosphere consists of nearly pure CO2 obtained from the Martian atmosphere and pressurized to a pressure equal to at least 0.8 bar. For this reason, these experiments were carried out by inserting a laboratory-scale optical bioreactor (flask) containing a microalgae culture into a jar, and then fluxing pure CO2 until the partial pressure inside the jar was equal to 1 bar. The results obtained are shown in Figure 3B. In this case, the culture medium was always Zarrouk medium.

[0093] The associated effects resulting from replacing the air with CO2 to simulate the Martian atmosphere within the Martian dome simulant could not be observed until day 14 of cultivation. However, from that point onward, the culture using the simulated Martian atmosphere, i.e., CO2, continued to grow, but the biomass concentration of the culture grown in air began to decrease. This demonstrates that from day 14 onward, carbon is the main limiting factor for Spirulina growth. Therefore, the use of an atmosphere to simulate what is predicted by the proposed method is not only feasible but even advantageous due to the resulting reduction in the payload required to transport CO2 cylinders to Mars.

[0094] 2.3. Simulated effects of microgravity alone. This experiment aimed to evaluate how simulated microgravity might affect the growth of Spirulina platensis. This was done by mounting flasks containing Spirulina cultures in Zarrouk medium on a random positioning machine and periodically monitoring their growth. The corresponding results are shown in Figure 3C. A slight improvement in growth rate was observed when the samples were cultured under microgravity conditions. This may be due to a reduction in the effects of gravity-determined sedimentation and aggregation. The latter phenomenon can actually hinder the diffusion of nutrients to the algae. Ultimately, although the gravitational force on Mars is slightly higher than that employed in this experiment, the latter demonstrates that the reduced gravity conditions on Mars not only do not affect the growth of Spirulina, but can even slightly improve it.

[0095] 2.4. The synergistic effect of a combination of two of the three operating conditions of the present invention as implemented on Mars. The objective of these experiments was to explore the effect of applying two of three operating conditions simultaneously, simulating those used in methods realized on Mars, such as Martian atmosphere + microgravity, Martian medium + microgravity, or Martian medium + Martian atmosphere. Figure 3D shows that all possible combinations of operating conditions produced a synergistic effect, resulting in better growth of microalgae compared to the basic case experiment (Zm_air_1g). In particular, the culture using Martian medium under microgravity (Mm40_air_μg) performed better until day 15 of growth, but began to decline from day 16, likely due to carbon deficiency. In contrast, the two cultures using CO2, i.e., the simulated Martian atmosphere used in the method, continued to grow throughout the entire experimental period, thus demonstrating the ability of this strain to benefit from the high carbon concentration in the liquid. The best performance was then obtained using CO2 and microgravity (Zm_CO2_μg). In fact, in the latter experiment, approximately 1.2 gL was obtained after 22 days of cultivation. -1The biomass concentration was achieved.

[0096] 2.5. Simulation of all operating conditions performed on Mars according to the method of the present invention. In this experiment, all operating conditions of the method of the present invention were used, namely, microgravity, CO2. 2大気 And Mm40 is applied simultaneously to verify its feasibility. The results obtained are shown in Figure 4A.

[0097] It can be observed that the simultaneous use of all operating conditions of the present invention not only did not affect the cultivation of microalgae, but also resulted in a corresponding improvement in their growth, exhibiting a synergistic effect. This is likely due to the fact that as the biomass concentration increases (at the end of the experiment), the culture requires more CO2 for photosynthesis, while microgravity conditions prevent aggregation and sedimentation. Furthermore, the Spirulina strain tolerates high CO2 concentrations well, as its photosynthesis significantly increases the culture pH, counteracting the acidifying effect of CO2 that could potentially inhibit growth. The reasons underlying such improvements should be better investigated in further studies, but the experimental evidence is unambiguous and further confirmed by the comparison of productivity achieved by the two curves after 22 days (see Figure 4B).

[0098] Ultimately, based on these results, it can be reasonably stated that although gravity on Mars is slightly higher than that employed in this experiment, the method of the present invention is advantageously feasible and enables the production of food and oxygen. The possibility of using in-situ available resources such as topsoil and atmosphere also results in a corresponding reduction in the payload of the kit to be transported from Earth to Mars to implement the method in-situ.

[0099] 2.6. Comparison of the performance of the method of the present invention with that described in International Publication No. 2013014606 To evaluate improvements to the present invention's method with respect to cutting-edge technologies related to microalgae cultivation in extraterrestrial settings using the bio-ISRU paradigm, further experiments were conducted under the operating conditions described in International Publication No. 2013014606. In the latter, the microalgae growth medium consisted of topsoil leachate concentrated with HNO3 (RL) to provide the nitrates necessary for microalgae growth. Therefore, in this experiment, the growth medium was obtained by adding HNO3 to the leachate to obtain a final nitrate concentration equivalent to that of medium Z. For the remainder, the operating conditions were kept equivalent to those of the present invention, i.e., an atmosphere consisting of pure CO2 and microgravity. A comparison of the results obtained with the configuration of the present invention (Mm40_CO2_μg) and those of International Publication No. 2013014606, called RL_CO2_μg, is shown in Figure 5A for the change in biomass concentration over time, and in Figure 5B for the biomass productivity achieved after 13 days of cultivation. When using the operating conditions reported in International Publication No. 2013014606 (Experiment RL_CO2_μg), it was found that no increase in biomass concentration could be detected until day 14 of cultivation. Conversely, a slight decrease could be observed. Therefore, although the algae could survive under the conditions reported in International Publication No. 2013014606 (the decrease was not related), they could not grow and replicate significantly. Thus, the corresponding productivity after 14 days was slightly negative, but close to zero (see Figure 6B). For this reason, this experiment was terminated after 14 days. Therefore, a comparison with the results obtained using the method of the present invention (Mm40_CO2_μg) is reported for up to 14 days.

[0100] As is clear, biomass productivity can be significantly increased by using the operating conditions of the present invention. This evidence clearly demonstrates that the present invention represents an improvement over the latest technology.

Claims

1. The following apparatus is used to simulate the growth of cell lines on Earth under microgravity and CO2-rich extraterrestrial conditions: Insulated jars mounted on a 3D clinostat or random positioning machine (RPM), However, the insulated jar can house at least one laboratory-scale bioreactor (LSB) containing cell lines cultured with a culture medium as close to the optimal as possible, can simulate in-situ resource utilization (ISRU), and the jar is equipped with a pressure gauge, a gas inlet and a gas outlet capable of measuring pressure; A cylinder for storing a gas that simulates an extraterrestrial atmosphere rich in CO2, having the inlet of the jar and an outlet to which a fluid can be connected.

2. The apparatus according to claim 1, wherein a 3D clinostat or RPM is programmed to give the jar motion characterized by the resulting acceleration vector, the module of which, as an average value over time, is close to 0 or as close as possible to the gravitational conditions of the extraterrestrial location being simulated.

3. The apparatus according to claim 1 or 2, wherein the jar is transparent and the LSB is transparent, and the apparatus is a laboratory-scale photobioreactor (LSP).

4. The cell line is Gloeocapsa, Leptolingbya, Phormidium, Chroococcidiopsis; Arthrospira platensis; Synechococcus elongatus; Anabaena cilindrica; Chlorella vulgaris; Nannochloris eucariotum The apparatus according to claim 3, wherein the apparatus is at least one algal strain selected from the group consisting of Eucaryotum or genetically modified strains thereof.

5. The cell line is Chlorella sorokiniana, Chlorella zofigensis, Coccomyxa sp., Synechococcus sp., Pseudochloris wilhelmii, Chlorella protothecoides, Euglena gracilis, Chlamydomonas reinhardtii, Isochrysis galbana galbana), Neochloris oleoabundans, Scenedesmus obliquus, Dunaliella salina, Nannochloropsis occulate, Chlorella pyrenoidosa, Botryococcus braunii, Pheodactylum tricornutum, Tetraselmis Thalassiosira pseudona, Haematococcus pluvialis, Nannochloropsis oceanica, Spirulina maxima, Pavlova salina, Porphyridium marinum, Tetraselmis inconspicua, Cyanophora paradoxa Paradoxa, Thalassia rotula, Amphora sp., Odontella aurita, Attheya* * sp. ), * Chromulina ochromonoides , * Diacronema vlkianum , * Chaetoceros sp. , * Navicula pelliculosa , * Odontella mobiliensis , * Porosira pseudodenticulata Cell lines selected from the group consisting of (pseudocenticulata); or H1, H9, human embryonic stem cells; HEK-293, human embryonic kidney transformed with adenovirus; HeLa, human epithelial cells; HL60, human promyelocytic leukemia cells; MCF-7, human breast cancer; A549, human lung cancer; A1-A5-E, human amniotic membrane; ND-E, human esophagus; CHO, Chinese hamster eggs Nest; 3T3, Mouse fibroblasts; BHK21, Syrian hamster fibroblasts; MDCK, Canine epithelial cells; E14.1, Mouse embryonic stem cells; COS, Monkey kidney; DT40, Chicken lymphoma cells; S2, Drosophila macrophage-like cells; GH3, Rat pituitary tumor; L6, Rat myoblasts; Sf9 and Sf21, Fall armyworm (Spodoptera fulgiperda) frugiperda)) ovary; ZF4 and AB9 cells, zebrafish embryonic fibroblasts; 1184, human dermal fibroblasts; E6.1 clone, human Jurkat cells; human THP1 cells; SH-SY5Y, human neuroblastoma cells; iPSC, human stem cells; human erythrocyte culture; C20A4, human chondrocytes; 1301, human T-cell leukemia; 1306, 161BR, human dermal fibroblasts; F-36P, human myelopathy syndrome, leukemia; H9, human T cells; HeLa The apparatus according to any one of claims 1 to 3, wherein the cell line is selected from the group consisting of human epithelial cells; E6.1 clone, human Jurkat cells; SH-SY5Y, human neuroblastoma cells; iPSC, human stem cells; 1184, human dermal fibroblasts; hMSC, human mesenchymal stem cells; mBMSC, rat bone marrow-derived mesenchymal stem cells; ADSC, rat adipose-derived stem cells; mESC, mouse embryonic stem cells; MG-63, human osteosarcoma cell line; HUVEC, and human umbilical vein endothelial cells.

6. A method for simulating cell growth on Earth under microgravity and CO2-rich extraterrestrial conditions at a predetermined extraterrestrial location, comprising using a simulation apparatus according to any one of claims 1 to 5.

7. The method according to claim 6, including the following: A step of preparing a culture broth that is as close as possible to the optimal medium for the cell line to be grown, by simulating an extraterrestrial ISRU; A step of adding the culture broth to the LSB of the apparatus according to any one of claims 1 to 5, followed by adding the inoculant material for the cell line of microalgae or cyanobacteria to be cultured; Steps include placing the LSB inside the jar; A step of attaching the jar to the clinostat or RPM of the apparatus according to any one of claims 1 to 5; The step of connecting the outlet of the cylinder of the apparatus according to any one of claims 1 to 5 to the gas inlet of the jar; The step of passing a CO2-rich extraterrestrial atmosphere simulant into the jar with the gas outlet of the jar open for a sufficient time to clean the atmosphere inside the jar; The step of closing the jar outlet while continuing to allow the extraterrestrial atmosphere simulant to pass through until an internal pressure of at least 0.8 bar is achieved inside the jar; The step of turning on the clinostat or RPM to simulate microgravity.

8. A method according to claim 6 or 7 for cultivating a strain of edible microalgae or cyanobacteria, further comprising the step of preparing a culture medium by mixing a liquid topsoil leachate obtained by leaching with acidic water and a simulant of extraterrestrial topsoil with a simulant of diluted astronaut urine and micronutrients, wherein the micronutrients are known to be unavailable to ISRU in extraterrestrial locations and essential for the growth of the strain to be cultured.

9. The method according to claim 8, including the following: a) Step of preparing a simulant of extraterrestrial topsoil; b) A step of obtaining a topsoil slurry by bringing the simulant of extraterrestrial topsoil into contact with an leachate, wherein the leachate is HNO ３ The step is to use acidified water; c) A step of filtering the topsoil slurry to obtain solid discharged topsoil and liquid topsoil leachate; d) Step of preparing a simulant of astronaut urine; e) Diluting the urine simulant with water in order to simulate the dilution determined by the wash water in the Environmental Control and Life Support System (ECLSS), thereby obtaining a simulant of ECLSS wastewater; e') Finally, if the salinity of the simulant in the wastewater is too high and unsuitable for the growth of the strain, the step of further diluting it; f) A step of preparing a culture medium containing micronutrients optimal for the growth of the strain to be cultured; g) A step of mixing the ECLSS wastewater simulant with the topsoil leachate and the culture medium to obtain a culture broth; h) The step of adding the culture broth to the LSB of the apparatus according to any one of claims 1 to 5, followed by adding the inoculant of the microalgae or cyanobacteria strain to be cultured; i) The step of placing the LSB in a jar of the apparatus for simulating an extraterrestrial dome; j) The step of attaching the jar to the clinostat or RPM of the apparatus according to any one of claims 1 to 5; k) Connecting the outlet of the cylinder of the apparatus according to any one of claims 1 to 5 to the gas inlet of the jar; l) A step of venting a simulant of extraterrestrial atmosphere into the jar with the gas outlet of the jar open for a sufficient time to purify the atmosphere inside the jar; m) Closing the jar outlet while continuing to allow the extraterrestrial atmosphere simulant to pass through until an internal pressure of at least 0.8 bar is achieved inside the jar; n) Turning on the clinostat or RPM to simulate microgravity, and simultaneously irradiating the dome simulant with natural or artificial light to promote photosynthesis.

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