Our results demonstrate that the two Chlorophyta microalgal strains studied fractionate DU. Each strain displayed a different enrichment factor but both reflect a strong fractionation of the light 235U isotope in the cellular pellet. The marine strain TmmRU, despite presenting higher cell volume and organic weight36,37, presented a lower U uptake rate. However, the enrichment factor was significantly higher than in the freshwater ChlGS, despite being exposed to a lower 235U content DU (~0.0022 235U). The similar U isotopic composition found in the cellular pellets of the different replicates of each strain, irrespective of the culture time and the total U incorporated by the cells, raises the issues of preferential U uptake paths. The pathways leading to U uptake by the cells are not well documented18, and the joint complexity derivate of the simultaneously of several processes28,38 ‒ redox reactions, ligand exchange, diffusion, adsorption ‒ make interpretations of U fractionation origin extremely complex. Furthermore, U speciation and redox state may influence the fractionation process. Whatever the route, as previously described by Baselga-Cervera et al.39, U is bound to the outer wall and transported across the cell wall and membrane, becoming distributed inside the cell. These data suggest that U carried by the cellular pellet accomplishes isotopic partitioning, resulting in 235U being concentrated by the cells and the surrounding environment being enriched in 238U.

Consistent with our results, the isotopic ratio n(235U)/n(238U) value found in the U mine water suggest a possible enrichment process. Only microbial life has been detected in this pond39,40, and therefore biomass does not pass to higher trophic levels. Thus, 235U, as one of the lighter isotopes of U, could preferentially enter cells. When cells break, U enriched in 235U might be liberated to the water. The remaining U enriched in 238U bound to the cell wall might sediment in the bed of the pond. Additionally, bacteria that might be present in this pond and can contribute to this result. Reductive bacteria can induce U fractionation; the reaction products (U(IV)) are enriched in 238U, rendering the residual dissolved U enriched in 235U25,41. Combined effects of different microorganisms may have led to this result.

Microbial isotopic behaviour in elements with higher mass numbers, such as U, is typically poorly studied compared to light elements because of the need for more sophisticated and precise analyses. Recent evidence in the field has demonstrated U isotopic fractionation mediated by bacteria and neuron-like human cells. Biotic reduction studies with metal-reducing bacterial isolates show an enrichment in the heavier 238U isotope into the solid U(IV) byproduct26,42,43 that is not dependent on microbial sorption. Conversely, the isotopic behaviour displayed by neuron-like human cells showed a preferential 235U incorporation28. Paredes et al.28 suggested two possible isotopic fractionation processes based on the enrichment direction and U bioaccumulation: a mass-dependent “zero-point energy” mechanism29 and the mass-independent “nuclear field shift”. In the case of our study, as the two processes are not mutually exclusive and operate in the same fractionation direction, we cannot determine the precise contribution of each mechanism proposed. High-precision determination of other U isotopes, such as 234U ‒ commonly concentrated during 235U enrichment ‒ could provide insights into the fractionation mechanism at the cellular level and the proposed biological preference for lighter isotopes, but it is complex due to the abundances limitation [natural 234U abundance is comprehended between 0.000050–0.000059]. Reported isotope fractionation ratios for 235U/238U during U reduction by bacteria have ranged from −0.31 to −0.99‰26,42,43, and 0.38 ± 0.13‰ in neuron-like line cells29. We found that the cellular pellet was enriched in 235U relative to the supernatant with DU by 23.6 ± 12.5‰ and 370.4 ± 103.9‰ for the ChlGS and TmmRU strains, respectively. This fractionation behaviour is consistent in its direction with that observed in neuron-like cells, but the fractionation factors are significantly higher. One potential application of the observed microalgae-induced U isotopic variation is for DU waste re-enrichment along the U fuel cycle for nuclear fission energy. The current DU stockpiles worldwide would render one-third of natural-equivalent U after several cycles of re-enrichment, reducing DU tail and U-mine production. Currently, only centrifuge separation and gaseous diffusion have operated at commercial scale, even though several enrichment processes have been demonstrated historically and in the laboratory44,45,46,47. Both enrichment processes present important drawbacks: water reactivity by-products rendering hazardous compounds highly corrosive, as well as significant amounts of energy consumption, considerable costs and generate DU as a low-level waste product48. Reprocessing U tails with current techniques, despite its potential, is not economically and energetically feasible and does not ease the problem of final tail disposition49. Particularly interesting would be to develop a viable biological process to enrich DU, recovering natural-equivalent U from tailings waiting for final disposal without high energy demand and the associated carbon footprint. Hypothetically, multiple stages of microalgal bioaccumulation of DU, cell harvesting and U resuspension into the next higher step would enrich the DU to the desired amount, up to the 235U natural content or even above. The predicted advantages of this biological process are the reduced cost and low energy requirements, turning DU in a resource that even the U-tail problem decreases only marginally. Further experimentation may resolve the expected value range for U fractionation mediated by microalgal bioaccumulation and the efficiency of the enrichment process. In addition, other microalgal species might display a different isotopic behaviour, considering the significant differences exhibited by the two Chlorophyta species.

Our findings might also have implications for the identification of biotic signatures using isotope tools to study ancient microbial life and understand global uranium flux. Significant uncertainties remain regarding isotopic signatures owing to the ambiguity in the interpretation of the signs. Development of analytical techniques has opened the possibility of studying small U isotope natural variation. Uranium is the heaviest element for which natural variations have been reported50. U isotopic fractionation in nature occurs, with opposite directions, in both anoxic/euxinic and oxic environments and can be associated with chemical transformations such as adsorption, speciation or redox chemistry22. Microorganisms probably have a part in the U cycling and deposit formation in nature51,52,53. The large isotope fractionation that occurs during microalgal U uptake suggests the role of microalgae in the conservative behaviour of U and a tight range of isotope composition in modern oceans. For geological implications, recent studies of biotic redox transformation of U with metal-reducing bacteria induced U isotopic fractionation enriching for 238U in the reaction products25,26, contradicting previous studies27 and consistent with environmental studies and U-reduced depositional samples22,41,43,53,54. U isotopic fractionation mediated by bacterial enzymatic reduction raises as a tangible biosignature in the rock record for specific metabolic groups and onto the timing emergence of specific metabolisms. In oxic sedimentary environments, sample fractionation occurs towards lighter isotope composition, and intriguingly, banded-iron formation samples present the lightest U composition studied22. The banded-iron formations’ lighter values could indicate microbial phototroph co-precipitation by adsorption of iron and U, supporting the previously suggested microbial implication55. Thus, the preferential accumulation of the fissile 235U isotope in sediments might be a proxy for the activity and presence of microalgae. U fractionation biological fingerprints in ancient sedimentary rocks would provide insights into ancient microbial activity and establish temporal constraints. Additionally, our findings might provide insights into the Oklo phenomenon on the assumption of a microbial contribution in the initiation of the natural nuclear reactors40,56,57. Several factors probably contribute to these unique chain reactions. Two thousand million years ago – the same time proposed for Oklo’s event58,59,60,61 – 235U made up approximately 3% of the natural U, a condition that makes possible the starting of a fission reaction. However, a U-rich mineral deposit needs to be formed to obtain a critical mass. Most likely, the presence of increasing oxygen in the Earth’s atmosphere enabled the U flux and subsequent concentration in U ore bodies62. The direction and magnitude of the observed microalgal U isotopic fractionation during bioaccumulation could support the biological hypothesis of the origin of the natural reactors.