DOI: 10.3303/CET25117041 Paper Received: 18 December 2024; Revised: 24 February 2025; Accepted: 29 April 2025 Please cite this article as: Gamboni E., Atzori F., Casula M., Fais G., Cincotti A., Traversari G., Cao G., Concas A., 2025, Experimental and Modelling Investigation on the Use of In-situ Available Resources to Produce Food and Supplements on Mars Using Microalgae, Chemical Engineering Transactions, 117, 241-246 DOI:10.3303/CET25117041 CHEMICAL ENGINEERING TRANSACTIONS VOL. 117, 2025 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Fabrizio Bezzo, Flavio Manenti, Gabriele Pannocchia, Almerinda di Benedetto Copyright © 2025, AIDIC Servizi S.r.l. ISBN 979-12-81206-17-5; ISSN 2283-9216 Experimental and Modelling Investigation on the Use of In- Situ Available Resources to Produce Food and Supplements on Mars Using Microalgae Emanuela Gamboni a, Federico Atzori b, Mattia Casula a, Giacomo Fais a,b, Alberto Cincotti a,b, Gabriele Traversari b, Giacomo Cao a,b,c, Alessandro Concas a,b,* aInterdepartmental Centre of Environmental Science and Engineering (CINSA), University of Cagliari, Via San Giorgio 12, 09124 Cagliari, Italy b Department of Mechanical, Chemical and Materials Engineering, University of Cagliari, Via Marengo 2, 09123 Cagliari, Italy. c Center for Advanced Studies, Research and Development in Sardinia (CRS4), Loc. Piscina Manna, Building 1, 09050 Pula (CA), Italy. alessandro.concas@unica.it To enhance the prospects of long-term human missions to Mars, it is crucial to investigate the use of locally available resources to cultivate microalgae as a sustainable source of food for astronauts. This research investigates the growth of Chlorella vulgaris in a culture medium potentially obtainable on Mars using only in- situ available resources, i.e. regolith, atmospheric CO2 and metabolic wastes of crew. Experiments were performed in this view. The obtained results were then interpreted by a novel mathematical model. The nutritional characteristics of C. vulgaris biomass cultivated in such a “Martian” medium were also investigated. 1. Introduction As humanity faces the depletion of Earth's resources and an escalating climate crisis, the need for alternative methods to produce essential supplies has become increasingly urgent. A long-term solution involves relocating human populations to other planets, where survival would depend on harnessing locally available resources. To achieve this, extended crewed missions beyond Earth must be conducted. Mars has emerged as a promising candidate for sustaining human life, offering key resources such as CO₂, water, and regolith. Through in-situ processing, these materials could be converted into vital consumables, including oxygen, water, and food (Casula et al., 2024). However, while current in-situ resource utilization (ISRU) technologies on Mars primarily focus on generating oxygen and propellants via physicochemical processes, they remain insufficient for supporting food production—an essential requirement for long-term human missions. Microalgae, particularly *Chlorella vulgaris*, have gained attention due to their ability to produce oxygen, edible biomass, and nutritional supplements for Mars crews. Recent advancements suggest that microalgae could be a viable solution for producing essential materials, including food, with minimal reliance on Earth-derived resources (Fais et al., 2022). This approach could significantly reduce the payload required for Mars missions, thereby enhancing their technical and economic feasibility. To evaluate the practicality of this method, our study focuses on cultivating *C. vulgaris* in a simulated Martian environment using Martian regolith leachate, atmospheric CO₂, and synthetic human urine. Experiments conducted under Mars-like conditions achieved favorable productivity levels, demonstrating the potential of this cultivation approach. Additionally, we developed a mathematical model to predict biomass growth dynamics under Martian conditions, considering factors such as pH, carbon availability, and limiting nutrient concentrations. This model could help determine optimal conditions for maximizing biomass productivity to meet the nutritional needs of a long-duration Mars mission crew. Ultimately, this study aims to demonstrate that *C. vulgaris* can thrive and generate substantial amounts of algae-based food using only in-situ resources during a crewed Mars mission. This breakthrough would 241 significantly improve mission feasibility by drastically reducing payload requirements and increasing self- sufficiency for long-term human habitation on Mars. 2. Materials and Methods 2.1 Microalgal strain Cells of C. vulgaris CCALA 269 were obtained from the Culture collection of autotrophic organisms (CCALA, Czech Republic). The strain was maintained in Bold Basal Medium (BBM) at 20 °C in a 5-L laboratory bottle under a light intensity, measured by a suitable luxmeter, of 40 µmolph m−2 s−1. A photoperiod of 12 h/12 h day/night was adopted. Cultures were agitated at 70 rpm by using a rotary shaker. 2.2 Simulant of culture medium obtainable on Mars A growth medium potentially attainable on Mars (MM) using in-situ available resources was produced by mixing the leachate of a regolith simulant (RL) with diluted synthetic human urine (SU) as reported by Fais et al. (2022). The regolith leachate (RL) was obtained by contacting a suitable amount of JSC Mars-1 regolith simulant with a given volume of distilled water (pH 6.80) and then stirring the solid-liquid mixture for 24 h at 25 °C. SU was produced according to Sarigul et al. (2019) and diluted with ultrapure water at a ratio of 1:1te-0 v/v. Subsequently, RL and diluted SU were mixed (1:1 v/v) to obtain the MM. Different dilutions of MM with bicarbonate-enriched BBM were then evaluated during as growth media for C. vulgaris cultivation. Specifically, the dilutions are reported in the following table. Table 1. Volumetric composition of the investigated media Medium MM0 MM20 MM40 MM60 MM100 BBM (vol %) 100 80 60 40 0 MM (vol %) 0 20 40 60 100 2.3 Growth experiments An inoculum (5 mL) of C. vulgaris CCALA 269 in exponential phase was fed to 15 vented-cap, transparent flasks filled with 25 mL each of one among the five experimental growth media described above, in triplicate. The strain was cultivated at 25 ± 1 °C under continuous stirring at 70 rpm using a white light intensity of 80 µmolph m−2s−1 provided from above. Microalgal growth was monitored through daily OD measurements. Biomass concentrations were obtained from OD via a calibration line providing OD as function of biomass. pH was also assessed daily. To reproduce the use of Mars atmosphere an apparatus was set up consisting of a transparent insulated jar wherein pure CO2 (100% vol) was continuously blown in. The transparent flasks were disposed within the mentioned jar to simulate a Martian dome filled with pressurized Martian atmosphere according to the process proposed by Fais et al. (2022). 2.4 Harvesting and biomass characterization After 17 days of cultivation time, microalgal cultures were harvested and centrifuged. Supernatant was then removed, and pellets were resuspended in distilled water. The procedure was repeated three times. The obtained pellets were subsequently frozen at −80 °C, then lyophilized and finally grinded to powder by mortar and pestle. Different amounts of microalgal powder were then subjected to further different processing depending on the biochemical parameter to be determined. Briefly, carbohydrates were analysed through a slightly modified version of the method by Dubois et al. (1956). The total content of soluble protein was evaluated as reported by Lowry et al. (1951). After cell disruption the total lipid content were extracted and determined according to the procedure by Folch et al., (1957). Finally, total carotenoids content was evaluated through the method by Zavrel et al. (2015). The details on the specific analyses being performed can be found elsewhere (Casula et al., 2024 b). 3. Mathematical Model The evolution of main macro- and micro-nutrients’ concentration during cultivation was analysed to identify the growth limiting nutrient (data not shown). At the end of the growth phase, when the stationary one took place, the intracellular concentration of iron, evaluated through proper analyses and mass balances (data not shown) was lower than the minimum iron cell quota for C. vulgaris growth according to the literature (Concas et al., 2021). Therefore, the following mathematical model was developed considering iron and inorganic carbon concentration along with pH as factors controlling C. vulgaris growth. 242 3.1 Model Equations The model equations are the mass balances for: (1) total microalgal biomass concentration (b); (2) the limiting substrate (i.e. S = [Fe]) in the liquid bulk; (3) Fe in the intracellular compartment; (4) CO2 in the gas phase; (5) the non-carbonic alkalinity (Alk); (6) [H+] in the liquid bulk and finally (7) [CO2] in the liquid bulk. Eqs (1) to (3) derive from the application of the well-known Droop model to the concerned system (Concas et al., 2021) while Eq. (7) is obtained from the application of the electroneutrality principle to the investigated system. Finally Eqs. (4) to (5) and (7) to (8) are obtained considering mass transfer of CO2 from gas phase to liquid and its subsequent speciation in a liquid with a given pH. Eq (5) also considers that inorganic carbon is consumed by microalgae in the liquid phase. Eqs (6) and (7) are obtained through the novel approach proposed by Atzori et al. (2023) that allows to avoid solving a differential algebraic system of equations.     1      TICmin max TIC TIC CqdX X dt q C K    = −  +  (1)       Fe Fe F x e F ma e C C X d Cdt K = − + (2)     1        min max ma Fe Fe Fe x qdq q qCdt K C     = − −  +   (3) ( )2 2 2 2,     F F L CO l CO CO L dP K P H CO RT dt V a V V  − − − = (4)   ( ) 2 2 2,    TIC F l CO CO L TIC L d C V dX K P H CO Y dt V dt a  = − −  (5)    alk d Alk dX Y dt dt = − (6) ( )  2 2  1 1 2 1 1 2           [ ]    2     C C C TIC C C C d Alk H K H K Kd H H dt dt d Cg H K H K K dt + + + + + +      + +              =          + +     (7) ( )             3 2, 1 2, 1 2, 2, 1 2 2, 1 2   4 2 TIC TIC w L TIC C L C L TIC L C C L C C d C d C H H k dt dt Hd CO d C d Alk K CO H K CO H dt dt dtg H d C d Alk CO K K CO K K dt dt + + + + + +      +                  = + +                       + +       (8) ( ) 2 2 1 1 2          C C C C C Cg H H K H K K H K K K+ + + +       = + + +        (9) The detailed symbol significance is reported in the nomenclature. The system of ordinary differential equations (ODEs) is solved along with the initial conditions of each variable via numerical routines available in MATLAB. 4. Results and discussion Figure 1 shows the comparison between experimental model results obtained when cultivating C. vulgaris in the investigated media (Table 1) under a pure CO2 atmosphere. In all cases, the biomass concentration starts growing without showing a significant lag phase until the growth stops when the internal cell quota of Fe achieved the minimum value capable to sustain growth. From the practical point of view, the relevant aspect deductible from Figure 1 is that the C. vulgaris cultivated in MM100, i.e. a medium obtainable by exploiting only in-situ available resources on Mars, grew till the end of the experiment at a rate very close to the one observed when using BBM medium. Indeed, the biomass productivities (Figure 1) evaluated at the end of the experiments were all very close (around 0.1 g L-1 day-1) without a statistically significant difference. The capability of C. vulgaris to grow in medium obtainable using only in-situ available resources only is very important in view of its application on Mars also because it would reduce dramatically the need of compounds to bring from Earth and, correspondingly, the payload of the mission. 243 Figure 1. Microalgae biomass concentration and pH time evolution in the cultures with different percentage of Martian Medium. Last figure summarizes the biomass productivities achieved with the different media. The experimental data in Figure 1 were well simulated by the proposed model when using the model parameters in Table 1. It should be noted that most parameters values were taken from the literature since only two of them, 𝝁𝒎𝒂𝒙 and 𝝆𝒎𝒂𝒙 , were suitably tuned by using the function fmincon in MATLAB. It should be noted that the values obtained of the two adjusted parameters are consistent with those reported in the literature for similar systems. It should be noted that fitting was performed using only two series of data, i.e. ph Vs time and X Vs time for MM0, while in the remaining simulations the obtained values of the above parameters were kept constant so that simulations could be considered predictions. The mean square errors (MSE) were in the range 0.02 - 0.12 for the biomass variable and 0.32-0.38 for pH thus demonstrating the good predictive capability of the model. 244 Table 2. Model parameters Parameter Value Units Reference 𝑯𝑪𝑶𝟐 3.4 × 10−4 𝑀 𝑃𝑎−1 Atzori et al., 2023 𝑲𝒍𝑎 2.93 × 10−4 ℎ𝑟−1 Klöckner et al. 2013 𝒀𝑻𝑰𝑪 4.1 × 10−2 𝑚𝑜𝑙 𝑔−1 Concas et al., 2021 𝒀𝑨𝒍𝒌 1.00 × 10−5 𝑚𝑜𝑙 𝑔−1 Concas et al., 2021 𝒌𝒘 1.01 × 10−14 𝑀2 Bates and pinching 1949 𝑲𝑪 10−2.77 𝑀 Atzori et al., 2023 𝑲𝟏𝑪 10−6.1 𝑀 Atzori et al., 2023 𝑲𝟐𝑪 10−9.2 𝑀 Atzori et al., 2023 𝒃𝟎 1.4 − 2.2 × 10−1 𝑔 𝐿−1 Experimental value 𝒔𝟎 1.3 − 1.9 × 10−3 𝑔 𝐿−1 Experimental value 𝒒𝟎 5.5 × 10−3 - Concas et al., 2021 𝑲𝑺 9.5 × 10−6 𝑔 𝐿−1 Concas et al., 2021 𝒒𝒎𝒊𝒏 4 × 10−4 - Concas et al., 2021 𝝁𝒎𝒂𝒙 2.73 × 10−2 ℎ𝑟−1 This work 𝝆𝒎𝒂𝒙 6.49 × 10−7 ℎ𝑟−1 This work In Figure 2 the content of total carbohydrates, proteins, lipids and carotenoids in the biomass cultivated in the different media is shown. Except for slight and not statistically significant variations, the macro-biochemical composition of the obtained biomass was not affected by the percentage of MM. Figure 2. Effect of MM percentage in the growth media on the biochemical composition of the obtained biomass. Since the composition of C.vulgaris biomass obtained using only MM as the growth medium (MM100) was more or less similar to the one obtained using typical growth medium (MM0 that is BBM) and the latter is generally recognized as safe (GRAS) by the US Food and Drugs Administration (Gouda et al., 2022), it can be deduced an edible biomass capable to feed the astronauts can be potentially produced by relying only on in-situ available resources on Mars. 5. Conclusions C.vulgaris was capable to grow in a medium potentially obtainable using only in-situ available resources during a crewed mission (MM100) on Mars without relevant differences respect to the growth in a classical growth medium. Good productivities and nutritional characteristics were obtained at the end of cultivation period thus demonstrating the feasibility of using C. vulgaris as a source of food for the astronauts involved in the mission. A suitable mathematical model was capable to well capture the experimental results thus representing a potentially useful tool to design the implementation of this technology on Mars. 0 5 10 15 20 [g /1 0 0 g ] MM0 MM20 MM40 MM60 MM100 Carbohydrates 0 10 20 30 40 [g /1 0 0 g ] MM0 MM20 MM40 MM60 MM100 Proteins 0 10 20 30 [g /1 0 0 g ] MM0 MM20 MM40 MM60 MM100 Lipids 0 50 100 150 200 [m g /1 0 0 g ] MM0 MM20 MM40 MM60 MM100 Carotenoids 245 Nomenclature a – interfacial area. m2 m–3 Alk – non carbonic Alkalinity, g L-1 CFe – iron bulk concentration, g L-1 CTIC – total inorganic carbon, g L-1 HCO2 – henry constant CO2, M atm-1 K1C – dissociation constant of H2CO3, M K2C – dissociation constant of HCO3 –, M Kl – mass transfer coefficient, m s-1 KTIC – half saturation of C, mol L-1 KFe – half saturation of Fe, g L-1 PCO2 – pressure of CO2, atm q – iron cell quota, g g-1 S – iron bulk concentration, g L-1 T – temperature, K VF – volume of flasks, L VL – volume of liquid, L X – biomass concentration, g L-1 Yalk – Alkalinity yield, mol g-1 Yalk – Alkalinity yield, mol g-1 max – Max specific growth rate, day-1 max – Max specific absorption rate of Fe, day-1- Acknowledgments This work has been developed within the framework of the project eINS- Ecosystem of Innovation for Next Generation Sardinia (cod. 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