An exploratory study on the possibilities of microalgal biotechnology to obtain the essential 6Li isotope as fusion fuel.

Future energy supply needs to overcome two challenges: environmental impact and dependence on geopolitically unstable countries. A very promising alternative is based on lithium, an element for batteries, and whose isotope 6Li will be essential in nuclear fusion. The objective of this research has been to determine if it is possible to achieve isotopic fractionation of lithium through a process mediated by microalgae. For this purpose, Chlamydomonas reinhardtii was selected and grown in presence of 5 mg/L of lithium. Results revealed that this specie survives at the selected lithium concentration, discriminates isotopes and preferentially capture 6Li (6δ = 10.029 ± 3.307) through a process independent of the cellular growth. Concomitate recovered up 0.206 mg/L of lithium along a process of 21 days. The result of this study lets to affirm that Chlamydomonas reinhardtii might be used to obtain lithium enriched in the lighter isotope.

Microbial biominers: Sequential bioleaching and biouptake of metals from electronic scraps.

Electronic scraps (e-scraps) represent an attractive raw material to mine demanded metals, as well as rare earth elements (REEs). A sequential microbial-mediated process developed in two steps was examined to recover multiple elements. First, we made use of an acidophilic bacteria consortium, mainly composed of Acidiphilium multivorum and Leptospidillum ferriphilum, isolated from acid mine drainages. The consortium was inoculated in a dissolution of e-scraps powder and cultured for 15 days. Forty-five elements were analyzed in the liquid phase over time, including silver, gold, and 15 REEs. The bioleaching efficiencies of the consortium were >99% for Cu, Co, Al, and Zn, 53% for Cd, and around 10% for Cr and Li on Day 7. The second step consisted of a microalgae-mediated uptake from e-scraps leachate. The strains used were two acidophilic extremotolerant microalgae, Euglena sp. (EugVP) and Chlamydomonas sp. (ChlSG) strains, isolated from the same extreme environment. Up to 7.3, 4.1, 1.3, and 0.7 µg by wet biomass (WB) of Zn, Al, Cu, and Mn, respectively, were uptaken by ChlSG biomass in 12 days, presenting higher efficiency than EugVP. Concerning REEs, ChlSG biouptake 14.9, 20.3, 13.7, 8.3 ng of Gd, Pr, Ce, La per WB. Meanwhile, EugVP captured 1.1, 1.5, 1.4, and 7.5, respectively. This paper shows the potential of a microbial sequential process to revalorize e-scraps and recover metals and REEs, harnessing extremotolerant microorganisms.

The Upcoming 6Li Isotope Requirements Might Be Supplied by a Microalgal Enrichment Process.

Lithium isotopes are essential for nuclear energy, but new enrichment methods are required. In this study, we considered biotechnology as a possibility. We assessed the Li fractionation capabilities of three Chlorophyte strains: Chlamydomonas reinhardtii, Tetraselmis mediterranea, and a freshwater Chlorophyte, Desmodesmus sp. These species were cultured in Li containing media and were analysed just after inoculation and after 3, 12, and 27 days. Li mass was determined using a Inductively Coupled Plasma Mass Spectrometer, and the isotope compositions were measured on a Thermo Element XR Inductively Coupled Plasma Mass Spectrometer. The maximum Li capture was observed at day 27 with C. reinhardtii (31.66 µg/g). Desmodesmus sp. reached the greatest Li fractionation, (δ6 = 85.4‰). All strains fractionated preferentially towards 6Li. More studies are required to find fitter species and to establish the optimal conditions for Li capture and fractionation. Nevertheless, this is the first step for a microalgal nuclear biotechnology.

Fluctuation analysis to select for Samarium bio-uptaking microalgae clones the repurposing of a classical evolution experiment.

Rare Earth Elements (REE) increasing demand prompts the research of biotechnological approaches to exploit secondary resources. We made use of the adapted Fluctuation analyses experiment to obtain Chlamydomonas reinhardtii ChlA strains resistant to Samarium (Sm) as the reference REE. The starting hypothesis was that adaptation to metal-containing media leads to an enhanced metal uptake. ChlA was able to adapt to 1.33·10-4 Sm M and pH~3 by pre-existing genetic variability, allowing the evolutionary rescue of 13 of the 99 populations studied. The rescuing resistant genotypes presented a mutation rate of 8.65·10-7 resistant cells per division. The resulting resistant population contradicted the expected fitness cost associated with the adaptation to Sm, selection resulted in larger and faster-growing resistant cells. Among the three isolated strains studied for Sm uptake, only one presented uplifted performance compared to the control population (46.64 µg Sm g-¹ of wet biomass and 3.26·10-7 ng Sm per cell, mainly bioaccumulated within the cells). The selection of microalgae strains with improved tolerance to REEs by this methodology could be a promising solution for REES sequestration. However, increased tolerance can be independent or have negative effects on uptake performance and cellular features studied are not directly correlated with the metal uptake. SUMMARY SENTENCE: Repurposing a classic laboratory evolution experiment to select for microalgae Samarium adapted strains for metals recovery and biotechnology approaches. DATA AVAILABILITY STATEMENT: All data generated or analyzed during this study are included in this published article (and its raw files).

Rapid Colonization of Uranium Mining-Impacted Waters, the Biodiversity of Successful Lineages of Phytoplankton Extremophiles.

Anthropogenic extreme environments are emphasized as interesting sites for the study of evolutionary pathways, biodiversity, and extremophile bioprospection. Organisms that grow under these conditions are usually regarded as extremophiles; however, the extreme novelty of these environments may have favor adaptive radiations of facultative extremophiles. At the Iberian Peninsula, uranium mining operations have rendered highly polluted extreme environments in multiple locations. In this study, we examined the phytoplankton diversity, community structure, and possible determining factors in separate uranium mining-impacted waters. Some of these human-induced extreme environments may be able to sustain indigenous facultative extremophile phytoplankton species, as well as alleged obligate extremophiles. Therefore, we investigated the adaptation capacity of three laboratory strains, two Chlamydomonas reinhardtii and a Dictyosphaerium chlorelloides, to uranium-polluted waters. The biodiversity among the sampled waters was very low, and despite presenting unique taxonomic records, ecological patterns can be identified. The microalgae adaptation experiments indicated a gradient of ecological novelty and different phenomena of adaptation, from acclimation in some waters to non-adaptation in the harshest anthropogenic environment. Certainly, phytoplankton extremophiles might have been often overlooked, and the ability to flourish in extreme environments might be a functional feature in some neutrophilic species. Evolutionary biology and microbial biodiversity can benefit the study of recently evolved systems such as uranium-polluted waters. Moreover, anthropogenic extremophiles can be harnessed for industrial applications.

Evidence of microalgal isotopic fractionation through enrichment of depleted uranium.

Resulting from the nuclear fuel cycle, large amounts of depleted uranium (DU) tails are piling up, waiting for possible use or final disposal. To date, the recovery of the residual 235U isotope contained in DU has been conducted only marginally by physical processes. Relative isotope abundances are often mediated by biological processes, and the biologically driven U isotopic fractionation has been previously identified in reducing bacteria. Our results indicate that the cells of two microalgal strains (freshwater Chlamydomonas sp. (ChlGS) and marine Tetraselmis mediterranea (TmmRU)) took up DU from the exposure solutions, inducing U isotopic fractionation with a preference for the fissile 235U isotope over 238U. The n(235U)/n(238U) isotopic fractionation magnitudes (δ235) were 23.6 ± 12.5‰ and 370.4 ± 103.9‰, respectively. These results open up new perspectives on the re-enrichment of DU tailings, offering a potential biological alternative to obtain reprocessed natural-equivalent uranium. Additionally, the findings present implications for identifying biological signatures in the geologic records.

Improvement of the Uranium Sequestration Ability of a Chlamydomonas sp. (ChlSP Strain) Isolated From Extreme Uranium Mine Tailings Through Selection for Potential Bioremediation Application.

The extraction and processing of uranium (U) have polluted large areas worldwide, rendering anthropogenic extreme environments inhospitable to most species. Noticeably, these sites are of great interest for taxonomical and applied bioprospection of extremotolerant species successfully adapted to U tailings contamination. As an example, in this work we have studied a microalgae species that inhabits extreme U tailings ponds at the Saelices mining site (Salamanca, Spain), characterized as acidic (pH between 3 and 4), radioactive (around 4 µSv h-1) and contaminated with metals, mainly U (from 25 to 48 mg L-1) and zinc (from 17 to 87 mg L-1). After isolation of the extremotolerant ChlSP strain, morphological characterization and internal transcribed spacer (ITS)-5.8S gene sequences placed it in the Chlamydomonadaceae, but BLAST analyses identity values, against the nucleotide datasets at the NCBI database, were very low (<92%). We subjected the ChlSP strain to an artificial selection protocol to increase the U uptake and investigated its response to selection. The ancestral strain ChlSP showed a U-uptake capacity of ≈4.30 mg U g-1 of dry biomass (DB). However, the artificially selected strain ChlSG was able to take up a total of ≈6.34 mg U g-1 DB, close to the theoretical maximum response (≈7.9 mg U g-1 DB). The selected ChlSG strain showed two possible U-uptake mechanisms: the greatest proportion by biosorption onto cell walls (ca. 90%), and only a very small quantity, ~0.46 mg g-1 DB, irreversibly bound by bioaccumulation. Additionally, the kinetics of the U-uptake process were characterized during a microalgae growth curve; ChlSG cells removed close to 4 mg L-1 of U in 24 days. These findings open up promising prospects for sustainable management of U tailings waters based on newly evolved extremotolerants and outline the potential of artificial selection in the improvement of desired features in microalgae by experimental adaptation and selection.

Adaptation prevents the extinction of Chlamydomonas reinhardtii under toxic beryllium.

The current biodiversity crisis represents a historic challenge for natural communities: the environmental rate of change exceeds the population's adaptation capability. Integrating both ecological and evolutionary responses is necessary to make reliable predictions regarding the loss of biodiversity. The race against extinction from an eco-evolutionary perspective is gaining importance in ecological risk assessment. Here, we performed a classical study of population dynamics-a fluctuation analysis-and evaluated the results from an adaption perspective. Fluctuation analysis, widely used with microorganisms, is an effective empirical procedure to study adaptation under strong selective pressure because it incorporates the factors that influence demographic, genetic and environmental changes. The adaptation of phytoplankton to beryllium (Be) is of interest because human activities are increasing the concentration of Be in freshwater reserves; therefore, predicting the effects of human-induced pollutants is necessary for proper risk assessment. The fluctuation analysis was performed with phytoplankton, specifically, the freshwater microalgae Chlamydomonas reinhardtii, under acute Be exposure. High doses of Be led to massive microalgae death; however, by conducting a fluctuation analysis experiment, we found that C. reinhardtii was able to adapt to 33 mg/l of Be due to pre-existing genetic variability. The rescuing adapting genotype presented a mutation rate of 9.61 × 10(-6) and a frequency of 10.42 resistant cells per million wild-type cells. The genetic adaptation pathway that was experimentally obtained agreed with the theoretical models of evolutionary rescue (ER). Furthermore, the rescuing genotype presented phenotypic and physiologic differences from the wild-type genotype, was 25% smaller than the Be-resistant genotype and presented a lower fitness and quantum yield performance. The abrupt distinctions between the wild-type and the Be-resistant genotype suggest a pleiotropic effect mediated by an advantageous mutation; however, no sequencing confirmation was performed.

Rapid adaptation of some phytoplankton species to osmium as a result of spontaneous mutations.

To understand the vulnerability of individual species to anthropogenic contamination, it is important to evaluate the different abilities of phytoplankton to respond to environmental changes induced by pollution. The ability of a species to adapt, rather than its initial tolerance, is the basis for survival under rapidly increasing levels of anthropogenic contamination. High doses of osmium (Os) cause massive destruction of diverse phytoplankton groups. In this study, we found that the coastal chlorophyte Tetraselmis suecica and the continental chlorophyte Dictyosphaerium chlorelloides were able to adapt to a lethal dose of Os. In these species, Os-resistant cells arose as a result of rare spontaneous mutations (at rates of approximately 10(-6) mutants per cell division) that occurred before exposure to Os. The mutants remained in the microalgal populations by means of mutation-selection balance. The huge size of phytoplankton populations ensures that there are always enough Os-resistant mutants to guarantee the survival of the population under Os pollution. In contrast, we observed that neither a haptophyte species from open ocean regions nor a cyanobacterium from continental freshwater were able to adapt to the lethal Os dose. Adaptation of phytoplankton to Os contamination is relevant because industrial activities are leading to a rapid increase in Os pollution worldwide.

Microalgas: ¿los primeros ingenieros nucleares? / Microalgae: the first nuclear engineers?

Self-sustaining nuclear chain reactions ran spontaneously 1.7 billion years ago at Oklo (Gabon, Africa) are a mystery. It was hypothesized that the microalgae concentrated enough rich-uranium in Oklo as for a natural reactor to start operating. The key to understanding as microalgae could do this is in an extremely U-contaminated pond of Saelices uranium-mine (Spain). Some microalgae colonized this extreme pond due to spontaneous mutations of single-genes. These U-resistant microalgae concentrate 115 mg U/g dried-biomass by bio-adsorption and bioaccumulation and are able to enrich uranium producing isotopic fractionation 235U/238U. Consequently, microalgae could be able to build a nuclear reactor in appropriate circumstances (AU)

Las reacciones nucleares en cadena auto-sostenibles que ocurrieron espontáneamente hace 1.700 millones años en Oklo (Gabón, África) son un misterio. Hipotéticamente las microalgas concentraron suficiente uranio enriquecido para que un reactor nuclear natural comenzara a operar. La clave está en un estanque contaminado por uranio en la mina de Saelices (España). Algunas microalgas colonizaron este estanque extremo debido a mutaciones espontáneas de genes individuales. Estas microalgas Uranio-resistentes concentran 115 mg U / g de biomasa seca mediante bio-absorción y bioacumulación, siendo capaces de enriquecer uranio produciendo fraccionamiento isotópico 235U/238U. Estas microalgas podrían construir un reactor nuclear en circunstancias apropiadas (AU)

Effects of adaptation, chance, and history on the evolution of the toxic dinoflagellate Alexandrium minutum under selection of increased temperature and acidification.

The roles of adaptation, chance, and history on evolution of the toxic dinoflagellate Alexandrium minutum Halim, under selective conditions simulating global change, have been addressed. Two toxic strains (AL1V and AL2V), previously acclimated for two years at pH 8.0 and 20°C, were transferred to selective conditions: pH 7.5 to simulate acidification and 25°C. Cultures under selective conditions were propagated until growth rate and toxin cell quota achieved an invariant mean value at 720 days (ca. 250 and ca. 180 generations for strains AL1V and AL2V, respectively). Historical contingencies strongly constrained the evolution of growth rate and toxin cell quota, but the forces involved in the evolution were not the same for both traits. Growth rate was 1.5-1.6 times higher than the one measured in ancestral conditions. Genetic adaptation explained two-thirds of total adaptation while one-third was a consequence of physiological adaptation. On the other hand, the evolution of toxin cell quota showed a pattern attributable to neutral mutations because the final variances were significantly higher than those measured at the start of the experiment. It has been hypothesized that harmful algal blooms will increase under the future scenario of global change. Although this study might be considered an oversimplification of the reality, it can be hypothesized that toxic blooms will increase but no predictions can be advanced about toxicity.

Adaptation of microalgae to lindane: a new approach for bioremediation.

Lindane is especially worrisome because its persistence in aquatic ecosystems, tendency to bioaccumulation and toxicity. We studied the adaptation of freshwater cyanobacteria and microalgae to resist lindane using an experimental model to distinguish if lindane-resistant cells had their origin in random spontaneous pre-selective mutations (which occur prior to the lindane exposure), or if lindane-resistant cells arose by a mechanism of physiological acclimation during the exposure to the selective agent. Although further research is needed to determine the different mechanisms contributing to the bio-elimination of lindane, this study, however, provides an approach to the bioremediation abilities of the lindane-resistant cells. Wild type strains of the experimental organisms were exposed to increasing lindane levels to estimate lethal concentrations. Growth of wild-type cells was completely inhibited at 5mg/L concentration of lindane. However, after further incubation in lindane for several weeks, occasionally the growth of rare lindane-resistant cells was found. A fluctuation analysis demonstrated that lindane-resistant cells arise only by rare spontaneous mutations that occur randomly prior to exposure to lindane (lindane-resistance did not occur as a result of physiological mechanisms). The rate of mutation from lindane sensitivity to resistance was between 1.48 × 10(-5) and 2.35 × 10(-7) mutations per cell per generation. Lindane-resistant mutants exhibited a diminished fitness in the absence of lindane, but only these variants were able to grow at lindane concentrations higher than 5mg/L (until concentrations as high as 40 mg/L). Lindane-resistant mutants may be maintained in uncontaminated waters as the result of a balance between new resistant mutants arising from spontaneous mutation and resistant cells eliminated by natural selection waters via clone selection. The lindane-resistant cells were also used to test the potential of microalgae to remove lindane. Three concentrations (4, 15 and 40 mg/L) were chosen as a model. In these exposures the lindane-resistant cells showed a great capacity to remove lindane (until 99% lindane was eliminated). Apparently, bioremediation based on lindane-resistant cells could be a great opportunity for cleaning up of lindane- and other chlorinated organics-polluted habitats.

Burkholderia ferrariae sp. nov., isolated from an iron ore in Brazil.

A Gram-negative, non-spore-forming bacterial strain with the ability to solubilize highly insoluble phosphatic minerals was isolated from a high-phosphorous iron ore from Minas Gerais State, Brazil. This strain, designated FeGl01(T), was subjected to a polyphasic taxonomic investigation. Comparative 16S rRNA gene sequence analysis indicated that it formed a distinct phylogenetic lineage within the genus Burkholderia together with several other species of the genus, e.g. Burkholderia sacchari, Burkholderia tropica and Burkholderia unamae. Partial nucleotide sequencing and analysis of the recA gene roughly corroborated the phylogenetic position of strain FeGl01(T) within the genus Burkholderia. The chemotaxonomic properties of strain FeGl01(T), such as ubiquinone Q-8 as the predominant quinone system and C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c and C(19 : 0)omega8c cyclo as the major fatty acids, were also consistent with its classification within the genus Burkholderia. DNA-DNA hybridization experiments between strain FeGl01(T) and the type strains of B. unamae, B. sacchari and B. tropica yielded reassociation values of 40 % or lower, which, together with qualitative and quantitative differences in fatty acid composition and with differences in several phenotypic traits, support the separation of the new isolate from the phylogenetically most closely related species. Therefore, it is suggested that strain FeGl01(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia ferrariae sp. nov. is proposed. The type strain is FeGl01(T) (=LMG 23612(T)=CECT 7171(T)=DSM 18251(T)).