Investigating the provision of vitamin B₁₂ in the Micro-Ecological Life Support System Alternative (MELiSSA) loop for long-distance space exploration using algal-bacterial cocultures.
Vitamin B₁₂ (cobalamin) is an essential nutrient for humans as it is required as a cofactor for two enzymes involved in central metabolism, methionine synthase and methylmalonyl CoA mutase. It is a corrinoid molecule, composed of a modified tetrapyrrole ring- with a cobalt ion at the centre and various possible upper and lower axial ligands. Vitamin B₁₂ is synthesised only by certain prokaryotes and the process requires over 20 enzymatic reactions. On Earth, most humans fulfil their vitamin B₁₂ requirement through the consumption of meat, eggs, and dairy products all of which would be difficult to source on long-distance space missions. The Micro-Ecological Life Support System Alternative (MELiSSA) project is a long running initiative from the European Space Agency that aims to develop a biological life support system that could enable long-distance crewed space exploration. The MELiSSA project aims to have five interconnected compartments that perform all the necessary life support functions, thereby reducing the volume of initial resources needed for a long-distance mission. The food production compartment (Compartment IV) is split into two sub-compartments, one inhabited by higher plants and the other by the photoautotrophic cyanobacterium *Limnospira indica*. Higher plants do not make, require, or store vitamin B₁₂, and it has been shown that cyanobacteria synthesise a form of vitamin B₁₂, pseudocobalamin, which is less bioavailable for humans than cobalamin. Therefore, the astronauts may be at risk of developing vitamin B₁₂ deficiency during long space missions. This thesis comprises three main sections. Firstly, a novel microbiological assay for the quantification of vitamin B₁₂ is developed, which is able to distinguish between certain lower axial ligands of the vitamin. The novel microbiological assay relies on the ability of the green alga *Chlamydomonas reinhardtii* to remodel pseudocobalamin to cobalamin through switching of the lower axial ligand from adenine to dimethylbenzimidazole (DMB), when exogenous DMB is provided. Wildtype *C. reinhardtii* is B₁₂-independent as it possesses two isoforms of methionine synthase, one B₁₂-independent (METE) and one B₁₂-dependent (METH). However, there are B₁₂-dependent strains of *C. reinhardtii* (metE7 and metE4), and for these, like humans, cobalamin is more bioavailable. So, by exploiting the remodelling phenotype it is possible to quantify the proportion of cobalamin and pseudocobalamin in a sample using the mutant strains as the bioassay organism. Levels of vitamin B₁₂ down to ~10 pM could be reliably measured. A B₁₂-dependent mutant of the diatom *Phaeodactylum tricornutum* behaved similarly. The novel assay was then used to show that Cobalamin Acquisition Protein 1 (CBA1) is necessary for the uptake of pseudocobalamin by *C. reinhardtii*. In addition, two species of edible microalgae, *Chlorella vulgaris* and *Haematococcus pluvialis* can accumulate both cobalamin and pseudocobalamin. Using *Lobomonas rostrata*, a B₁₂-dependent green alga incapable of remodelling, it was demonstrated that *C. vulgaris* cannot remodel pseudocobalamin to cobalamin, as suggested by the lack of potential remodelling enzymes in the available genome sequence information. Lastly, a CRISPR/Cas9 edited knockout of a putative COBT gene, which encodes for a protein necessary for the activation of DMB, in *C. reinhardtii* was shown to be no longer able to remodel pseudocobalamin. This phenotype strongly indicates a role for CrCOBT in remodelling, likely to be needed for catalysing the activation of DMB. In Chapter 4, the novel microbiological assay was used to quantify the vitamin B₁₂ content of some commercially available algal diet supplements. Spirulina, often derived from *L. indica*, overwhelmingly contained pseudocobalamin, whereas the Chlorella products contained varying mixtures of cobalamin and pseudocobalamin. The MELiSSA loop Compartment V contains humans, who generate B₁₂ containing waste. The microbiological assay was used to show that Compartment I, the anaerobic waste digester, also contains a mixture of B₁₂ variants that changes over time. The assay was utilised to confirm that axenically grown *L. indica* synthesises pseudocobalamin. Compartment II of the MELiSSA loop has historically been proposed to be occupied by the purple non-sulphur bacterium *Rhodospirillum rubrum*, again the assay was used to show that it produces cobalamin. *R. rubrum* is a metabolically flexible bacterium and so its ability to synthesise cobalamin in a range of conditions was investigated, especially those conditions that would allow it to grow in coculture with microalgae. This characteristic was exploited in Chapter 5, where it was shown that the growth of B₁₂-dependent *C. reinhardtii* could be supported by *R. rubrum* in coculture and this mutualism was used to investigate the mechanisms underpinning algal-bacterial interactions. An RNA sequencing experiment was conducted to investigate the differentially expressed genes in *R. rubrum* when grown with the microalga compared to axenic cultures. Among the differentially expressed genes were those involved in the transport of amino acids, and differences in the expression pattern of genes in the two main branches of the porphyrin metabolism were observed. It was also possible to generate cocultures of *C. vulgaris* and *R. rubrum*, with *C. vulgaris* and *R. rubrum* designated as edible by various governmental authorities and the MELiSSA project, respectively. These cocultures were exposed to radiation to simulate time on the International Space Station, showing that at low doses of radiation the cocultures were stable.
Chlamydomonas reinhardtii, Cocultures, Microalgae, Space exploration, Symbiosis
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