We present the complete genome sequence of Debaryomyces hansenii DSM 3428, assembled using long- and short-read sequencing data. This genome provides a valuable reference for investigating the genetic basis of stress tolerance and biotechnologically relevant traits in this yeast.

Patient-derived organoids offer greater human translational relevance than mouse models or immortalized cell lines, owing to their multicellular composition and patient-specific responses. Here, we present a protocol for establishing a human in vitro rectal tumor model for radiotherapy studies using patient-derived tumor organoids (PDTOs) and monolayer derivatives integrated into a microphysiological system (MPS). We describe steps for cultivating PDTOs, their transition into monolayers, and transfer into an MPS. This protocol enables radiotherapy modelling, toxicity testing, and adjuvant therapies screening.

Biohybrid electrodes combine photosynthetic microorganisms with conductive substrates to facilitate light-driven photocurrent generation and fuel-forming reactions. While effective charge transfer at the biological-synthetic interface remains a critical challenge, the use of polydopamine (PDA) at cyanobacteria-diamond interfaces has remained unexplored. In this work, we demonstrate PDA as a multifunctional interfacial layer on semiconducting boron-doped diamond (BDD) to immobilize Limnospira indica cyanobacteria and enhance extracellular electron transfer. PDA modification enabled robust cell immobilization and significantly increased photocurrent densities compared to bare BDD. Furthermore, we observed strain-dependent photoresponses: the straight-trichome strain (P2) achieved a peak photocurrent density of 1020 nA/cm(2) at higher PDA deposition cycles, whereas the helical strain (P6) peaked at 560 nA/cm(2) with fewer cycles. Mechanistic investigations, including control assays and membrane-restricted interfaces, confirmed that the enhanced photocurrent originates primarily from the photosynthetic activity of L. indica, with PDA facilitating a similar to 50% contribution from direct electron transfer pathways. These findings establish PDA as a versatile material for optimizing cyanobacteria-diamond biohybrid electrodes, providing fundamental mechanistic insights into extracellular electron transfer that will guide the future design of bioelectrochemical energy conversion systems.

Colorectal cancer (CRC) remains a major health burden, underscoring the need for continued research to improve screening methods and treatment outcomes. The colitis-associated azoxymethane (AOM) and dextran sodium sulphate (DSS) mouse model has proven valuable for CRC research, as it closely mimics human CRC development and captures the complex interactions between the gut microbiome and immune system. However, variations in AOM/DSS dosing reported in literature can influence baseline conditions, thereby affecting the outcome and interpretation of experimental and therapeutic interventions. This study evaluated the impact of varying AOM and DSS doses on CRC development and the gut microbiome, metabolome and lipidome in mouse faeces (n = 29). Inflammation-driven CRC progression was more pronounced in mice treated with high-dose AOM (12.5 mg/kg) combined with high-dose DSS (2

Understanding how photosynthetic microorganisms exchange electrons with electrodes is central to advancing biophotoelectrochemical systems. Here, we investigate bias-dependent photocurrent generation in biohybrid electrodes containing Limnospira indica immobilized in a PEDOT:PSS composite on fluorine-doped tin oxide (FTO). By systematically varying irradiance, applied potential, oxygen availability, and mediator composition, we show that photocurrent arises from multiple electron-transfer pathways whose engagement depends sensitively on electrical polarization. Under anodic polarization (+0.5 to +0.9 V), photocurrent shows a trend of increase with light intensity while photon-to-current efficiency declines systematically, consistent with regulatory constraints within the photosynthetic chain. In contrast, cathodic polarization (-0.5 to -0.9 V) induces distinct multiphasic transient behavior: at -0.5 V, reproducible spike-peak-trough sequences and delayed post-illumination currents reflect competition between photosynthetic electron-transfer pathways and oxygen-dependent metabolic sinks, and oxygen removal simplifies but reduces the cathodic response. At -0.9 V, cathodic currents become largely oxygen-independent yet remain strictly PSII-dependent, as DCMU abolishes both photocurrent development and dark recovery. These results demonstrate that L. indica engages multiple potential-selective electron-transfer regimes and that transient photocurrent features carry mechanistic information inaccessible from steady-state measurements, informing the rational design of improved biohybrid photoelectrodes.

Copper-based materials are actively explored for their potential as antimicrobial agents. However, recent studies show that sublethal concentrations of Cu ions can induce the viable-but-nonculturable (VBNC) cell state in certain bacteria, hampering contamination control, and monitoring. In this study we contribute to the unravelling of this largely enigmatic phenomenon by determining the time-resolved proteome of Cu-treated Cupriavidus metallidurans CH34 during VBNC induction and resuscitation. High-throughput quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed at multiple sample time points, revealing the cellular adaptations that trigger VBNC formation and the characteristic spontaneous recovery of culturability. Entry into the VBNC state correlated with a widespread response to oxidative stress as well as downregulated pyruvate metabolism. The expression of specific metal resistance determinants changed with Cu exposure time and culminated in the strong upregulation of proteins linked to periplasmic Cu ion detoxification during the resuscitation phase. We suggest that this delayed induction of Cu resistance proteins is paralleled by the gradual reconstitution of energy reserves through metabolization of intracellular polyhydroxybutyrate, as supported by flow cytometric fluorescence measurements. Furthermore, Cu-treated cells showed upregulation of several motility and chemotaxis proteins, and increased cell motility was observed phenotypically. Our results reveal a highly dynamic proteomic response, provide fundamental insights into the VBNC state and emphasize the advantages of time-resolved proteomic analysis.

The Micro-Ecological Life Support System Alternative (MELiSSA) project of the European Space Agency develops a biological recycling loop for manned long-term space flight. The air revitalisation and food production are carried out by vascular plants and a photobioreactor containing the cyanobacterium Limnospira indica. In the space flight experiment ARTHROSPIRA-C, cultures of L. indica are run in a one-week batch mode (45 mu mol photons m- 2 s-1) followed by four semi-continuous cycles of two weeks length. Each cycle has a different, predefined light intensity following an increasing regime (45-55-70-80 mu mol photons m- 2 s-1). In this study, two ground science verification tests (SVT and delta-SVT) were conducted in a laboratory setting to test the reliability and functionality of the hardware- and software of ARTHROSPIRA-C. The SVT explored all four cycles and light intensities and delta-SVT was an additional test where only cycle 1 and 2 were performed. delta-SVT was used to investigate anomalies during SVT. These experiments revealed oxygen production rates between 0.10 f 0.03 and 0.45 f 0.01 mmol O2 L- 1 h- 1 and biomass production rates between 0.008 f 0.000 and 0.021 f 0.002 g L- 1 h- 1 while demonstrating sustained photosynthetic activity at all tested light intensities. In addition, proteomics analysis revealed light intensity-induced effects on multiple pathways, whereas the lipidomic analysis reported no alterations. This study delves into the ground tests conducted during ARTHROSPIRA-C, paving the way for a forthcoming successful flight experiment scheduled aboard the International Space Station in autumn 2024.

The joint National Aeronautics and Space Administration and European Space Agency Mars Sample Return (MSR) Campaign is a proposed multi-mission effort to bring selected geological samples from Mars to Earth for the purpose of scientific investigation. Significant parts of these investigations could be affected by Earth-sourced contamination that is either misinterpreted as having a martian origin or that masks a martian signal. The Mars 2020 Perseverance rover implemented strict contamination control requirements to limit contamination of the samples during sample collection. Contamination control and contamination knowledge requirements have not yet been established for the samples after they arrive on Earth. The MSR Sample Receiving Facility (SRF) Contamination Panel (SCP) was tasked with defining the terrestrial biological, organic, and inorganic contamination limits for martian samples during their residence inside the SRF. To reach our recommendations, the SCP studied (i) the previously proposed limits and rationale of the Organic Contamination Panel, (ii) cleanliness levels achieved for sampling hardware by the M2020 mission, (iii) recent improvements in analytical technology and detection limits, (iv) updated information regarding the organic content of martian samples (e.g., from the Sample Analysis at Mars instrument on the Curiosity rover and laboratory analyses of martian meteorites), and (v) information about the composition and geologic context of samples being collected by the Perseverance rover for return to Earth.

Ensuring adequate nutrition is essential for long-duration space missions where Earth resupply is limited or unfeasible. Bioregenerative Life Support Systems (BLSS), such as ESA’s MELiSSA, aim to sustain astronauts by recycling resources and cultivating crops. However, plant-based diets in BLSS may lack key micronutrients. This study identified nutritional gaps in crop-based BLSS diets, revealing deficiencies in several micronutrients, including cobalamin (vit B₁₂), riboflavin (vit B₂), and calciferol (vit D). We screened microorganisms for genomic potential to produce these micronutrients and filtered candidates based on inclusion in the EFSA Qualified Presumption of Safety and Novel Food lists. Organisms were prioritized based on biosynthetic capability and robustness. The result is a ranked list of microbial candidates capable of addressing nutritional deficiencies in BLSS diets. These findings support the integration of resilient, nutrient-producing microbes into space food systems, offering a strategic path toward self-sufficient and health-supportive nutrition for future deep space missions.

Cultures of Limnospira indica were exposed to low-dose rate g-irradiation for 8 weeks to simulate 2 months of a Mars transit irradiation. Two experiments were conducted: in the first, 5% v/v inoculations were used over 2-week batches; in the second, 25% v/v inoculations over 1-week batches. The cultures were continuously illuminated (45 mmol photons m-2 s-1, LEDs). A transient hormesis effect was observed in experiment 1, with irradiated cultures showing higher dry weight (1.88 +/- 0.05 g L-1) than controls (1.70 +/- 0.06 g L-1) on day 14. Irradiated cultures also had fewer pigments. Experiment 2 showed similar, though less pronounced, results. These findings suggest that Limnospira indica would not be negatively affected by cosmic radiation during Mars transit, though further validation under space flight conditions is needed. The resilience of Limnospira indica to chronic low-dose radiation supports its potential for oxygen and food production in life support systems for manned space missions.

Cyanobacterium Limnospira indica is being explored for oxygen production and carbon dioxide removal from air in future space stations. Before activation in space, it undergoes a transport and storage phase lasting from one to several weeks, during which it must remain dormant. This study examines the effects of dormancy in dark and cold conditions on L. indica's photosynthetic performance and biomass composition after storage. Results showed that storage negatively affects photosynthetic growth and biomass composition, but the impact depends on factors such as initial cell concentration, medium pH, cell pigment content, nutrient and gas availability, and storage duration. Storage was also tested under simulated microgravity conditions, but no adverse effects of reduced gravity were observed when healthy cultures were used.

Human space exploration faces different challenges. Topics like Bioregenerative Life Support Systems, In Situ Resource Utilization, and radiation protection, still require for more suitable solutions to be applied in long-term space exploration. Synthetic biology could be a powerful tool for enabling human exploration of space and planets. This paper explores key topics including resource utilization, life support systems, radiation protection, and human health, providing recommendations for short-, mid-, and long-term advancements in space exploration.

Recently, studies have emerged exploring the potential application of fecal microbiota transplantation (FMT) in pre-clinical settings. Here, we present a protocol for FMT for mice housed in a specific pathogen-free (SPF) facility. We describe steps for sample collection, microaerophilic processing of freshly collected fecal pellets, and administration through oral gavage. We then detail procedures for the engraftment of the bacterial community. This protocol focuses on age- and gender-matched, healthy donor mice using a mobile and cost-effective alternative to an anoxic cabinet.

Metagenome community analyses, driven by the continued development in sequencing technology, is rapidly providing insights in many aspects of microbiology and becoming a cornerstone tool. Illumina, Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) are the leading technologies, each with their own advantages and drawbacks. Illumina provides accurate reads at a low cost, but their length is too short to close bacterial genomes. Long reads overcome this limitation, but these technologies produce reads with lower accuracy (ONT) or with lower throughput (PacBio high-fidelity reads). In a critical first analysis step, reads are assembled to reconstruct genomes or individual genes within the community. However, to date, the performance of existing assemblers has never been challenged with a complex mock metagenome. Here, we evaluate the performance of current assemblers that use short, long or both read types on a complex mock metagenome consisting of 227 bacterial strains with varying degrees of relatedness. We show that many of the current assemblers are not suited to handle such a complex metagenome. In addition, hybrid assemblies do not fulfil their potential. We conclude that ONT reads assembled with CANU and Illumina reads assembled with SPAdes offer the best value for reconstructing genomes and individual genes of complex metagenomes, respectively.

Regenerative life support systems for space crews recycle waste into water, food, and oxygen using different organisms. The European Space Agency’s MELiSSA program uses the cyanobacterium Limnospira indica PCC8005 for air revitalization and food production. Before space use, components’ compatibility with reduced gravity was tested. This study introduced a ground analog for microgravity experiments with oxygenic cyanobacteria under continuous illumination, using a random positioning machine (RPM) setup. L. indica PCC8005 grew slower under low-shear simulated microgravity, with proteome analysis revealing downregulation of ribosomal proteins, glutamine synthase, and nitrate uptake transporters, and upregulation of gas vesicle, photosystem I and II, and carboxysome proteins. Results suggested inhibition due to high oxygen partial pressure, causing carbon limitation when cultivated in low-shear simulated microgravity. A thicker stagnant fluid boundary layer reducing oxygen release in simulated microgravity was observed. These findings validate this RPM setup for testing the effects of non-terrestrial gravity on photosynthetic microorganisms.

Cyanobacteria designated as Spirulina, Arthrospira and Limnospira are worldwide well-known food and food supplement sources. Despite their long history of use for human consumption, many indigenous strains remain unstudied. Here we performed a genomic and physiological characterization of AlgLouSa11, an Algerian strain isolated from the Taguemart region in Tamanrasset. Its genome was sequenced and compared (via ANI and PaSit4) to 21 genomes of the Arthrospira and Limnospira genera, indicating that AlgLouSa11 belonged to the Limnospira genus, with Limnospira fusiformis KN01 being the closest relative (99.3 %). Growth was scored in three different culture media (Hiri, Zarrouk, and BG-11), with the highest cell density, 4.76 x 105 cells/ml after 11 days, obtained for Hiri medium. The optimal growth temperature was 34 degrees C, with automatic agitation producing the highest yields (5.90 +/- 0.25 g/l). The optimal growth pH was nine, with growth decreasing with increasing alkalinity. Finally, exploring the genome of Limnospira sp. AlgLouSa11 showed the presence of coding sequences involved in the biosynthesis of secondary metabolites such as cyanobactin, terpenes and lanthipeptides.

Approaches to DNA extraction play a crucial role in determining the variability of results obtained through 16S rRNA amplicon sequencing. Particularly, clay-rich samples can impede the efficiency of various standard cultivation-independent techniques. We conducted an inter-laboratory comparison study to thoroughly assess the efficacy of two published DNA extraction methods (kit-based and phenol-chloroform-based) specifically designed for bentonite samples. To this end, we spiked Wyoming MX 80 bentonite with two different mock communities and compared the obtained DNA yield and purity, the presence of contaminants and the community profile. Our findings suggest that both methods are equally viable, with the best choice depending on the specific requirements of the downstream analysis. However, it is crucial to maintain consistency in the chosen method, as comparing results becomes challenging, particularly in the presence of bentonite. In summary, our study emphasizes the significance of standardized DNA extraction methods and underscores the importance of validating these methods using appropriate controls when studying microbial communities with 16S rRNA amplicon sequencing, particularly in environments characterized by low biomass and clay-rich compositions. Additionally, slight modifications to one of the extraction methods can substantially enhance its efficiency.

As humans advance their presence in space and seek to improve the quality of life on Earth, a variety of science questions in support of these two objectives can be answered using the Moon. In this paper, we present a concept for an integrated mission focused on answering fundamental and applied biological questions on the Moon: BioMoon. The mission was designed to investigate the effects of the lunar radiation, gravity, and regolith on biological systems ranging from biomolecules to systems with complex trophic interactions, spanning a range of model organisms. Using common analytical systems and data processing, BioMoon represents a systems-level integrated life sciences mission. It would provide fundamental insights into biological responses to the lunar environment, as well as applied knowledge for In-Situ Resource Utilisation (ISRU), closed-loop life support system development, planetary protection and human health care. The mission was conceived to test biotechnology and sensor technology for lunar and terrestrial application and provide education and outreach opportunities. Although BioMoon was considered in the context of the European Space Agency’s Argonaut (European Large Logistics Lander) concept, the mission design provides a template for any integrated life sciences experimental suite on the Moon and other celestial bodies, implemented either robotically or by human explorers.

Safe geological disposal of radioactive waste requires a thorough understanding of geochemical conditions in the host formation. Boom Clay is a potential candidate in Belgium, where active methanogenesis has been detected in its deep subsurface, influencing the local geochemistry. However, the pathways driving this process and the characteristics of the methanogenic archaea involved remain unclear. We isolated a distinct archaeal strain from Boom Clay pore water and characterized it geno- and phenotypically. Isolate TD41E1-1 belongs to a novel species of the Methanosarcina genus, for which the name Methanosarcina hadiensis sp. nov. is proposed. TD41E1-1 cells are coccus-shaped, irregularly sized cells enveloped by extracellular polymer substances. Growth and substrate utilization experiments and genomic analysis demonstrated that the strain prefers methylated compounds or hydrogen as substrates for methane production. Although it possesses a complete acetoclastic pathway, no growth was observed in the presence of acetate in the tested conditions. Based on its phylogenetic relation to other known Methanosarcina species and on the presence of c-type cytochromes, it can be concluded that the strain likely occupies an intermediate position between type I and type II Methanosarcina species. These findings provide valuable insights for assessing Boom Clay's suitability for geological disposal of radioactive waste.

In 2025, the Artemis II marks the first crewed mission orbiting the Moon, with plans for subsequent missions landing astronauts near the lunar South Pole and NASA aims to reach Mars by the 2030s. The growing interest in space underscores the increasing importance of long-term human presence in space missions. Challenges such as human health and sustainable food preservation persist in establishing settlements on other planetary bodies. Space agencies are developing regenerative life support systems utilizing hydroponic cultivation of plants and microalgae, fueled by crew waste as fertilizers. While biological systems could sustain astronauts, the predominantly vegan diets lack essential micronutrients. To address this, integrating microbial-based food supplements into current bioregenerative systems is crucial for ensuring a balanced diet and maintaining the health of space explorers. The aim of this project is to develop an alternative food system by growing microorganisms in space-related conditions and using their biomass, or products thereof, as food supplements for space travelers on long-duration space missions, e.g. to Mars. We select and study the impact of space conditions on microorganisms that can provide useful micronutrients for future space travelers, which cannot be fully provided by vegan diets. This will be done by selecting a range of candidate beneficial microorganisms. Various options are available, including Bacillus subtilis spp., which can produce riboflavin (vitamin B2) and whose spores have already been tested on Mars analog surfaces (Cortesão et al., 2019). Limosilactobacillus reuteri could be used as supplement of riboflavin (Spacova et al., 2022) and has previously been shown to increase its production of reuterin under simulated microgravity conditions (Senatore et al., 2020). In addition, the yeast Yarrowia lipolytica is a well-known producer of essential amino acids, PUFA, MUFA, and vitamin B complexes (Jach & Malm, 2022). Final strain selection will be based on (i) their ability and efficiency to produce micronutrients, (ii) their safety and health promoting (incl. Radiation protective) properties, (iii) their ability to survive and maintain production efficiency under extreme environments, including ionizing radiation and microgravity, and (iv) their compatibility with bio-based in situ resource utilization techniques (e.g., gas or mineral sources from Martian atmosphere or regolith through biomining) to increase loop-closure. The selected strains will be stored, revived and grown in simulated Martian conditions, to test their long-term stability and preservation as food supplement source. Through international collaborations, we will test these conditions using reduced-gravity simulators, space radiation analogs, and substrates based on lysed cells of bacteria previously grown on regolith simulants, such as Chroococcidopsis sp. (Billi et al., 2021), and Anabaena sp., which has already been used to grow Bacillus subtilis from its inactivated biomass (Verseux, 2018). At the end of this 4-year PhD research project, the expected outcome is to improve the nutritional well-being of future space travelers settling on other planets, and also to generate innovative insights applicable to Earth-based fields such as biotechnology, radioprotection, and environmental science. References Billi, D., Gallego Fernandez, B., Fagliarone, C., Chiavarini, S., & Rothschild, L. J. (2021). Exploiting a perchlorate-tolerant desert cyanobacterium to support bacterial growth for in situ resource utilization on Mars. International Journal of Astrobiology, 20(1), 29–35. https://doi.org/10.1017/S1473550420000300 Cortesão, M., Fuchs, F. M., Commichau, F. M., Eichenberger, P., Schuerger, A. C., Nicholson, W. L., Setlow, P., & Moeller, R. (2019). Bacillus subtilis spore resistance to simulated mars surface conditions. Frontiers in Microbiology, 10(FEB). https://doi.org/10.3389/fmicb.2019.00333 Jach, M. E., & Malm, A. (2022). Yarrowia lipolytica as an Alternative and Valuable Source of Nutritional and Bioactive Compounds for Humans. In Molecules (Vol. 27, Issue 7). MDPI. https://doi.org/10.3390/molecules27072300 Senatore, G., Mastroleo, F., Leys, N., & Mauriello, G. (2020). Growth of Lactobacillus reuteri DSM17938 under Two Simulated Microgravity Systems: Changes in Reuterin Production, Gastrointestinal Passage Resistance, and Stress Genes Expression Response. Astrobiology, 20(1), 1–14. https://doi.org/10.1089/ast.2019.2082 Spacova, I., Ahannach, S., Breynaert, A., Erreygers, I., Wittouck, S., Bron, P. A., Van Beeck, W., Eilers, T., Alloul, A., Blansaer, N., Vlaeminck, S. E., Hermans, N., & Lebeer, S. (2022). Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods. Frontiers in Nutrition, 9. https://doi.org/10.3389/fnut.2022.916607 Verseux, C. (2018). Resistance of cyanobacteria to space and Mars environments, in the frame of the EXPOSE-R2 space mission and beyond. https://doi.org/10.13140/RG.2.2.28437.88808