Novel Radioresistant Alga of the Genus Coccomyxa

Abstract

The invention relates to novel algae of the genus Coccomyxa, in particular the algae of a new species called C-IR3-4C, and their use for capturing metals from aqueous media, and in particular from radioactive media.

Claims

1.-16. (canceled)

17. A method of capturing at least one element from an aqueous medium containing said element, the method comprising incubating in the aqueous medium a unicellular green alga of the genus Coccomyxa comprising, in the 18S ribosomal RNA-ITS1-5.8S, ribosomal RNA-ITS2-28S ribosomal RNA genes of having polynucleotide sequence at least 96% identity to the polynucleotide sequence of SEQ ID NO: 1, wherein the at least one element is selected from the group consisting of Cs, Ag, Co, Mn, Sr, Cu, Cr, Zn, Ni, Fe, Sb, U, Sc, Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, an actinide, a lanthanide, the .sup.14C radioisotope and the .sup.3H radioisotope.

18. The method of claim 17, wherein the unicellular green alga is the Coccomyxa strain deposited with the Culture Collection of Algae and Protozoa (CCAP) under deposit number CCAP 216/26.

19. The method of claim 17, wherein that element is selected from the group consisting of Sr and Cu.

20. The method of claim 17, wherein said aqueous medium is radioactive medium.

21. The method of claim 17, wherein said aqueous medium is nonradioactive medium.

22. The method of claim 20, wherein the element is a metal selected from the group consisting of Ag, Co, Cs, U, Mn, Cu and Sr, wherein said metal is in the form of a radioactive isotope, or in the form of a mixture of isotopes.

23. The method of claim 17, wherein said green algae are combined with at least one other microorganism and/or at least one multicellular plant.

24. The method of claim 17, wherein the growth of the unicellular green alga is controlled by regulating the illumination of said aqueous medium.

25. The method of claim 17, further comprising recovering said element from the alga.

26. The method of claim 17, wherein the pH of the aqueous medium is between about 1 and about 6.

27. The method of claim 17, wherein the aqueous medium is a polluted aqueous medium.

28. A method of depolluting a polluted aqueous medium containing at least one element, the method comprising incubating in the polluted aqueous medium a unicellular green alga of the genus Coccomyxa comprising, in the 18S ribosomal RNA-ITS1-5.8S, ribosomal RNA-ITS2-28S ribosomal RNA genes of having polynucleotide sequence at least 96% identity to the polynucleotide sequence of SEQ ID NO: 1, wherein the at least one element is selected from the group consisting of Cs, Ag, Co, Mn, Sr, Cu, Cr, Zn, Ni, Fe, Sb, an actinide, a lanthanide, the .sup.14C radioisotope and the .sup.3H radioisotope.

29. The method of claim 28, wherein said polluted aqueous medium is radioactive medium.

30. The method of claim 28, wherein the element is selected from the group consisting of Sr and Cu.

31. The method of claim 29, wherein said green alga is combined with at least one other radioresistant or radiotolerant microorganism and/or at least one radioresistant or radiotolerant multicellular plant.

32. The method of claim 30, wherein said green alga is combined with at least one other radioresistant or radiotolerant microorganism and/or at least one radioresistant or radiotolerant multicellular plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 depicts growth curves showing the change in the cell density of the algal population as a function of culture time.

[0055] FIG. 2 depicts the photosynthetic yield values of the microalgae as a function of different pH values. The microalgae can live in media of pH 1 to pH 9. The microalgae grown in these pH ranges retain a good physiological state in medium composed of demineralized water and that they retain a good physiological state whatever the pH.

[0056] FIG. 3 depicts shows the change in the cell density of the algal population as a function of pH over time in media of pH 1 to pH 9.

[0057] FIG. 4 depicts photographs of these microalgae observed, respectively, by photon microscopy (Zeiss Axioplan 2 binocular microscope, 1300 magnification) and by transmission electron microscopy (JEOL 1200EX and Philips CM 120). The microalgae contain a chloroplast (perhaps several) which contains chlorophyll and which is the site of photosynthesis. Other organelles, in particular the vacuole, occupy the rest of the cell.

[0058] FIG. 5 depicts the sequence of amplification products obtained by using DNA isolated from two independent cultures.

[0059] FIG. 6 depicts the phylogenetic tree obtained after alignment of the DNA sequences corresponding to the 18S rRNA (BLASTN). The bar indicates a substitution rate of 1% (0.01).

[0060] FIG. 7 depicts the percentage of mortality of Coccomyxa C-IR3-4C as a function of irradiation dose, measured three days after acute irradiation. The ionizing radiation dose that kills half the population is between 2 and 6 kGy.

[0061] FIG. 8 depicts changes in cell density (A) and photosynthetic yield (B) of Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.6 M rare earths at pH 6, optimal conditions for biological purification (n=3 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel. *=control samples not tested in parallel and not replicated. Values are presented as an indication of their relevance. The second sample was taken not after 6 days of exposure but after 5 days. The pH in pure water is 71.

[0062] FIG. 9 depicts the percentages of rare earths accumulated (%) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.6 M metal at pH 6, optimal conditions for biological purification (n=3 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel (meanstandard deviation).

[0063] FIG. 10 depicts changes in cell density (A) and photosynthetic yield (B) of Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M rare earths at pH 2, optimal conditions for metal uptake (n=5 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel. *=control samples not tested in parallel and not replicated. Values are presented as an indication of their relevance. The third sample was taken not after 7 days of exposure, but after 5 days. The pH in pure water is 71.

[0064] FIG. 11 depicts quantities of rare earths accumulated (mol/g of dry matter) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal at pH 2, optimal conditions for metal uptake (n=3 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel.

[0065] FIG. 12 depicts change in cell density (A) and photosynthetic yield (B) of Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal and at pH 1 or 2 (n=5 biological replicates). The two exposure conditions were tested in parallel. *=control samples not tested in parallel and not replicated. Values are presented as an indication of their relevance. The third sample was taken not after 7 days of exposure, but after 5 days. The pH in pure water is 7 1.

[0066] FIG. 13 depicts quantities of rare earths accumulated (arbitrary units) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal and pH 1 or 2 (n=3 biological replicates under the conditions 10.sup.2 M Gd.sup.3+ and pH 1; n=2 biological replicates under the conditions 10.sup.2 M Gd.sup.3+ and pH 2). The two exposure conditions were tested in parallel.

EXAMPLE 1

Isolation and Characterization of Coccomyxa C-IR3-4C

[0067] The microalga was collected from a spent nuclear fuel pool. The water contained in this pool has a pH between 6 and 7, conductivity of 1 to 2 S/cm, is in contact with ambient air and contains dissolved radioactive elements. Its temperature varies between 22 and 28 C. and is on average 25 C. The radiological activity in the pool is highly variable from weak to very strong, depending on the measuring points.

[0068] The presence of films of green organic matter was observed on the walls and various surfaces of this pool. Samples were taken, which when observed under a microscope showed that this was a unicellular green microalga.

Culture Conditions

[0069] The samples were stored and cultured in light, on nutrient agar, in a sterile environment, at a temperature of 20 to 23 C. The nutrient medium is Bold's Basal Medium (BBM, Sigma), pure or diluted in demineralized water. BBM is traditionally used to culture green algae. The culture medium has a pH of 6.4. Its composition is indicated in Table I below.

TABLE-US-00001 TABLE I Components in g/l BBM NaNO3 0.25 KH2PO4 0.175 K2HPO4 0.075 MgSO4, 7H2O 0.075 FeSO4, 7H2O 0.005 CaCl2, 2H2O 0.025 NaCl 0.025 Na2EDTA 0.01 KOH 0.006 H3BO4 12.86 MnCl2, 4H2O 1.81 ZnSO4, 7H2O 0.222 Na2MoO4, 2H2O 0.39 CuSO4, 5H2O 0.079 Co(NO3)2, 6H2O 0.049

[0070] The algae were placed in culture on solid BBM agar. Colonies were isolated and then diluted in order to spread the isolated cells over agar culture medium. This operation was repeated five times in order to obtain Coccomyxa strain C-IR3-4C, which was sequenced.

[0071] A sample of this culture was deposited according to the Budapest Treaty on 29 Jan. 2013 with the Culture Collection of Algae and Protozoa (CCAP), under number CCAP 216/26.

[0072] In liquid BBM, the Coccomyxa strain C-IR3-4C microalgae increased with an exponential growth phase. Microalgal growth was measured by counting the cell density of the algal population over time on three samples of algae cultured in BBM diluted 1:2, in an Infors Multitron incubator maintained at 211 C., with 100 rpm shaking and 9010 PAR continuous illumination. The count is made using a Malassez counting chamber under a microscope (X40 objective magnification). The growth curves are presented in FIG. 1, which shows the change in the cell density of the algal population as a function of culture time.

[0073] Coccomyxa strain C-IR3-4C microalgae are capable of living in liquid medium over a wide range of pH. The algae were cultivated in demineralized water the pH of which was adjusted to the target values by adding HCl or KOH. The cultures are grown in an Infors Multitron incubator maintained at 211 C., with 100 rpm shaking and 9010 PAR continuous illumination. The pH of the media is checked daily and readjusted as needed. The state of the algae as a function of the pH of the medium is evaluated by their photosynthetic yield, which is an indicator of the overall physiological state of the cells. Photosynthetic yield is measured using a PAM 103 fluorometer. FIG. 2 shows the photosynthetic yield values of the microalgae as a function of different pH values. It shows that they can live in media of pH 1 to pH 9 (pH range tested). FIG. 2 shows that the microalgae retain a good physiological state in medium composed of demineralized water and that they retain a good physiological state whatever the pH.

[0074] Coccomyxa strain C-IR3-4C microalgae are also capable of growing in liquid medium over a wide range of pH. FIG. 3 shows the change in the cell density of the algal population as a function of pH over time in media of pH 1 to pH 9. The culture conditions are identical to those of FIG. 2. The cell density count is made using a Malassez counting chamber under a microscope (40 magnification). To obtain rapid growth and high microalgae density, a pH range of 4 to 8 will be used.

Morphological and Biochemical Features

[0075] The isolated microalgae observed by photon microscopy and electron microscopy are unicellular, ellipsoidal and nucleated (FIG. 4). FIG. 4 are photographs of these microalgae observed, respectively, by photon microscopy (Zeiss Axioplan 2 binocular microscope, 1300 magnification) and by transmission electron microscopy (JEOL 1200EX and Philips CM 120). The observations with the Philips CM 120 TEM were carried out at the Technological Center for MicrostructuresClaude Bernard University Lyon 1). The microalgae contain a chloroplast (perhaps several) which contains chlorophyll and which is the site of photosynthesis. Other organelles, in particular the vacuole, occupy the rest of the cell.

[0076] The UV-visible absorption spectrum of this organism shows the presence of chlorophyll a (absorption peak at 663 nm), chlorophyll b (absorption peak at 647 nm) and carotenoids (absorption peak at 470 nm).

Amplification and Sequencing of Ribosomal DNA Genes

[0077] Total DNA of the C-IR3-4C microalga isolated as described above was extracted using the Wizard Genomic DNA Purification Kit (Promega).

[0078] The region of the genome covering the 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA (the first 500 bases) ribosomal DNA genes was amplified by PCR.

[0079] The primers used are EAF3: TCGACAATCTGGTTG ATCCTGCCAG (SEQ ID NO: 2) and ITS055R: CTCCTTGGTC CGTGTTTCAAGACGGG (SEQ ID NO: 3), traditionally used to amplify microalgal rRNA genes.

[0080] The amplification products obtained by using DNA isolated from two independent cultures were sequenced and are 3101 bases. The sequence of these amplification products is shown in FIG. 5 and in the sequence listing in the appendix under number SEQ ID NO: 1. This sequence represents the sequence of the genomic region covering the 18S rRNA-ITS1-5.85 rRNA-ITS2-28S rRNA (start) ribosomal DNA genes of the alga Coccomyxa C-IR3-4C. The portion corresponding to the 18S rRNA is underlined and italicized, the ITS1 is in bold, the 5.8S rRNA is underlined, the ITS2 is italicized, and the 28S rRNA is in bold and underlined.

[0081] The BLASTN algorithm (Altschul et al., Nucleic Acids Research, 25: 3389-3402, 1997) was used to search databases for ribosomal RNA gene sequences having maximum identity with the sequence of SEQ ID NO: 1. This search revealed that the species characterized as the most similar to microalga C-IR3-4C belong to the genus Coccomyxa.

[0082] The comparison of the sequences corresponding to the RNA of the small ribosomal subunit (18S rRNA) of microalga C-IR3-4C (1807 bp) and of other Coccomyxa species listed in the databases was carried out by multiple sequence alignment using the BLAST algorithm. Table II below presents the results of this sequence comparison. The sequences of the other species can be accessed in the GenBank database, and the corresponding accession numbers are also indicated in Table II.

TABLE-US-00002 TABLE II Total Query Max Accession Description score coverage ident HQ317304.1 Coccomyxa rayssiae isolate UTEX273 small 3290 99% 99% subunit ribosomal RNA gene, partial sequence FN298927.1 Coccomyxa sp. CCAP 216/24 18S rRNA 3269 98% 99% gene (partial), ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene (partial), strain CCAP 216/24 FN298926.1 Pseudococcomyxa simplex 18S rRNA gene 3264 98% 99% (partial), ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene (partial), strain SAG 216-9a HE586518.1 Choricystis sp. GSE4G genomic DNA 3262 98% 99% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain GSE4G FR865679.1 Chlorella saccharophila genomic DNA 3262 98% 99% containing 18S rRNA gene, ITS1, culture collection CCAP 211/60 FN597598.1 Coccomyxa chodatii SAG 216-2 3254 98% 99% FJ946891.1 Trebouxiophyceae sp. VPL5-6 18S 3234 97% 99% ribosomal RNA gene, partial sequence FJ648514.1 Pseudococcomyxa simplex strain UTEX 274 3229 97% 99% 18S ribosomal RNA gene, complete sequence FN597599.1 Coccomyxa peltigerae SAG 216-5 3221 98% 99% HE586504.1 Pseudococcomyxa simplex genomic DNA 3195 96% 99% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain CAUP H 102 HE586513.1 Coccomyxa sp. KN-2011-E4 genomic DNA 3181 96% 99% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain E4 FN298928.1 Coccomyxa sp. CCAP 211/97 18S rRNA 3181 98% 99% gene (partial), ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene (partial), strain CCAP 211/97 AB742451.1 Coccomyxa sp. KGU-D001 gene for 18S 3158 95% 99% ribosomal RNA, partial sequence HE586512.1 Coccomyxa sp. KN-2011-C15 genomic DNA 3129 97% 99% containing 18S rRNA gene, strain C15 FR850476.1 Coccomyxa actinabiotis 18S rRNA, ITS1, 3126 98% 99% 5.8S rRNA, ITS2 (CCAP 216-25) HE586505.1 Pseudococcomyxa simplex genomic DNA 3109 96% 99% containing 18S rRNA gene, strain CAUP H 103 HE586514.1 Coccomyxa sp. KN-2011-T2 genomic DNA 3103 97% 98% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain T2 AY422078.1 Paradoxia multiseta 18S small subunit 3081 93% 99% ribosomal RNA gene, partial sequence AM981206.1 Coccomyxa sp. CPCC 508 18S rRNA gene, 3077 99% 98% strain CPCC 508 AJ302939.1 Coccomyxa sp. SAG 2325 18S rRNA gene, 3070 99% 98% culture collection SAG: 2325 AM167525.1 Coccomyxa glaronensis 18S rRNA gene, 3061 99% 97% strain CCALA 306 HE586507.1 Ellipsoidion sp. UTEX B SNO113 genomic 3059 98% 98% DNA containing 18S rRNA gene, strain UTEX B SNO113 HE586519.1 Monodus sp. CR2-4 genomic DNA 3055 98% 98% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain CR2-4 FR865588.1 Chlamydomonas bipapillata genomic DNA 3053 98% 98% containing 18S rRNA gene, ITS1, culture collection CCAP 11/47 EU282454.1 Paradoxia sp. 294-GA206 18S ribosomal 3044 93% 99% RNA gene, partial sequence GQ122371.1 Trebouxiophyceae sp. KMMCC FC-10 18S 3042 92% 99% ribosomal RNA gene, partial sequence HE586506.1 Monodus sp. UTEX B SNO83 genomic DNA 3035 98% 97% containing 18S rRNA gene, ITS1 and 5.8S rRNA gene, strain UTEX B SNO83 HE586509.1 Coccomyxa sp. KN-2011-C10 genomic DNA 3022 97% 98% containing 18S rRNA gene, strain C10 JQ946088.1 Coccomyxa sp. XDL-2012 18S ribosomal 3014 92% 99% RNA gene, partial sequence EU127471.1 Coccomyxa sp. Flensburg fjord 2 18S 3014 98% 97% ribosomal RNA gene, partial sequence FJ648513.1 Coccomyxa mucigena strain SAG 216-4 2998 97% 97% 18S ribosomal RNA gene, complete sequence HE586508.1 Coccomyxa sp. KN-2011-C4 genomic DNA 2964 96% 97% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain C4 EU127470.1 Coccomyxa sp. Flensburg fjord 1 18S 2953 97% 97% ribosomal RNA gene, partial sequence AY195970.1 Choricystis sp. AS 5-1 18S ribosomal RNA 2931 99% 96% gene, partial sequence AY197620.1 Chlorella sp. Mary 9/21 BT-10w 18S 2913 99% 96% ribosomal RNA gene, partial sequence AB080308.1 Chlorella vulgaris gene for 18S rRNA, partial 2896 99% 96% sequence EF106784.1 Chlamydomonas sp. CCMP 681 2235 86% 93%

[0083] This sequence comparison shows that the species the most similar to strain C-IR3-4C are Coccomyxa rayssiae strain UTEX273, Coccomyxa strain CCAP 216/24, Pseudococcomyxa simplex strain SAG 216-9a, Coccomyxa chodatii strain SAG 216-2, Pseudococcomyxa simplex strain UTEX 274, Coccomyxa peltigerae strain SAG 216-5, Pseudococcomyxa simplex strain CAUP H 102, Coccomyxa strain KN-2011-E4, Coccomyxa strain CCAP 211/97, as well as other strains belonging to the genus of Coccomyxae with 99% sequence identity, then other strains still belonging to the genus of Coccomyxae such as Coccomyxa strain CPCC 508 or Coccomyxa strain SAG 2325 with 98% sequence identity, then other Coccomyxae such as Coccomyxa glaronensis strain CCALA 306, Coccomyxa sp. Flensburg fjord 2 or Coccomyxa mucigena strain SAG 216-4 with 97% sequence identity.

[0084] These high identity scores obtained for Coccomyxa strain C-IR3-4C compared with the genus Coccomyxa are close to those obtained after comparison of the sequences of Coccomyxae between one another (96-99%) and far from the score obtained for the sequence comparison with a unicellular microalga belonging to another genus (Chlamydomonas sp. CCMP681 (EF106784.1), 93% identity). This indicates that strain CCAP 216/26 is a member of the genus Coccomyxa.

[0085] Furthermore, the comparison of the ITS region sequences of strain C-IR3-4C with those of other Coccomyxae was also performed. Table III below presents the results of this sequence comparison of the ITS1-5.8S rRNA-ITS2 regions. The sequences of the other species can be accessed in the GenBank database, and the corresponding accession numbers are also indicated in Table III.

TABLE-US-00003 TABLE III Total Query Max Accession Description score coverage ident AY293967.1 Coccomyxa solarinae var. saccatae internal 1035 99% 95% transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence AY293966.1 Coccomyxa solarinae var. bisporae internal 1030 99% 95% transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence AY293965.1 Coccomyxa solarinae var. croceae internal 1009 99% 94% transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence HE586513.1 Coccomyxa sp. KN-2011-E4 genomic DNA 1002 100% 95% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain E4 HE586545.1 Coccomyxa sp. KN-2011-E5 genomic DNA 985 99% 94% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain E5 HE586551.1 Coccomyxa sp. KN-2011-T5 genomic DNA 939 93% 94% containing ITS1, 5.8S rRNA gene and ITS2, strain T5 AY293964.1 Coccomyxa peltigerae var. variolosae 910 94% 94% internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence HQ335215.1 Coccomyxa sp. S2 internal transcribed 745 83% 92% spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence HQ335216.1 Coccomyxa sp. S10 internal transcribed 745 83% 92% spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence FN298926.1 Pseudococcomyxa simplex 18S rRNA gene 673 92% 95% (partial), ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene (partial), strain SAG 216-9a HE586504.1 Pseudococcomyxa simplex genomic DNA 668 92% 94% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain CAUP H 102 FN298927.1 Coccomyxa sp. CCAP 216/24 18S rRNA 651 100% 88% gene (partial), ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene (partial), strain CCAP 216/24 AY328524.1 Coccomyxa rayssiae strain SAG 216-8 18S 640 92% 94% ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence HE586526.1 Pseudococcomyxa sp. KN-2011-A1 genomic 568 90% 91% DNA containing ITS1, 5.8S rRNA gene and ITS2, intragenomic variability copy B, strain A1 HE586525.1 Pseudococcomyxa sp. KN-2011-A1 genomic 566 90% 91% DNA containing ITS1, 5.8S rRNA gene and ITS2, intragenomic variability copy A, strain A1 AY293968.1 Coccomyxa chodatii internal transcribed 559 92% 90% spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence FR850476.1 Coccomyxa actinabiotis rRNA 376 59% 88% ITS1-5.8S-ITS2-28S AY328522.1 Coccomyxa peltigerae strain SAG 216-5 366 31% 94% 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence HE586515.1 Coccomyxa sp. KN-2011-T3 genomic DNA 158 23% 83% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain T3 HE586550.1 Coccomyxa sp. KN-2011-T1 genomic DNA 158 23% 83% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain T1 HE586514.1 Coccomyxa sp. KN-2011-T2 genomic DNA 128 15% 86% containing 18S rRNA gene, ITS1, 5.8S rRNA gene and ITS2, strain T2

[0086] This sequence comparison shows that the sequence of strain C-IR3-4C has 83% to 95% identity with the other Coccomyxae strains referenced, of the same order as the scores obtained for the comparison of the ITS region of the referenced Coccomyxae with one another (78% to 99%), and very far from that obtained for the comparison with other genera (about 30% to 50%).

[0087] It is thus a Coccomyxa species different from all those referenced heretofore. These results indicate, in fact, that the microalga isolated is a member of the genus Coccomyxa, but that its DNA differs sufficiently from that of the other species of Coccomyxae listed in the databases, in particular in terms of the ITS1 and ITS2 DNA, so as to be deemed a novel species, which is named herein Coccomyxa C-IR3-4C.

[0088] FIG. 6 presents the phylogenetic tree obtained after alignment of the DNA sequences corresponding to the 18S rRNA (BLASTN). The bar indicates a substitution rate of 1% (0.01).

EXAMPLE 2

Coccomyxa C-IR3-4C Resistance to Ionizing Radiation

[0089] The resistance to ionizing radiation of Coccomyxa C-IR3-4C algae was tested by exposing them to various doses of gamma radiation from decaying spent fuel. Post-irradiation mortality was determined by vital staining (neutral red).

[0090] FIG. 7 shows the percentage of mortality of Coccomyxa C-IR3-4C as a function of irradiation dose, measured three days after acute irradiation. The ionizing radiation dose that kills half the population is between 2 and 6 kGy.

EXAMPLE 3

Decontamination of Water from Nuclear Plants by Means of Coccomyxa C-IR3-4C

[0091] Water with a composition typical of that of water from a spent fuel pool was brought into contact with Coccomyxae C-IR3-4C for 24 hours, under illumination. A fresh 100 mg mass of Coccomyxae C-IR3-4C algae first cultivated in liquid BBM and rinsed beforehand three times with Milli-Q water is brought into contact with 50 ml of water initially containing the beta-emitting radionuclides .sup.3H (200,000 Bq/l) and .sup.14C (1000 Bq/l) and the gamma-emitting radionuclides listed in Table IV below.

TABLE-US-00004 TABLE IV Radionuclide .sup.54Mn .sup.60Co .sup.110mAg .sup.137Cs .sup.238U Activity (Bq/l) 9 42 23 67 21

[0092] The -spectrometry assay of the water and the algae 24 hours post-contact shows that 81% of they activity of the water is removed.

[0093] The percentage of -emitting radionuclides bound by the algae in 24 hours is indicated in Table V below.

TABLE-US-00005 TABLE V Radionuclide .sup.54Mn .sup.60Co .sup.110mAg .sup.137Cs .sup.238U Binding % 52 45 100 95 100

[0094] All or virtually all of the .sup.110mAg, the .sup.137Cs and the .sup.238U and half of the .sup.54Mn and the .sup.60Co were purified from the water.

EXAMPLE 4

Decontamination of Storage Pool Water by Means of Coccomyxa C-IR3-4C via In Situ Action

[0095] The Coccomyxae C-IR3-4C microalgae present in a spent nuclear fuel pool decontaminate the radionuclides present in said pool. Said pool is filled with water of pH between 6 and 7, of conductivity between 1 and 2 S/cm and of mean temperature 25 C., and contains radioactive metal elements due to the materials stored therein. It is in contact with ambient air and is illuminated by neon lighting.

[0096] Under these conditions, Coccomyxa C-IR3-4C is able of colonize the storage racks within the pool and can live there and reproduce there for years.

[0097] Counts taken by spectrometry made it possible to determine the total activity and the nature and the activity of each emitter concentrated by about 1 g of fresh algal mass and are given in Table VI below. The activity of the microalgae is defined in this table as the ratio of the activity (Bq) of 1 g of fresh algal mass to the activity of 1 ml of the water in which the algae live.

TABLE-US-00006 TABLE VI Radionuclide Activity of the microalgae/Activity of the water .sup.60Co 67 .sup.66Cu 167 .sup.92Sr 7.5 .sup.110mAg 62 .sup.134Cs 17 .sup.137Cs 8030 Total 8380

[0098] These results show that the algae are up to 10.sup.4 times more active than the water in which they live, and thus that they in fact concentrated the radioelements .sup.60Co, .sup.66Cu, 92Sr, .sup.110mAg, .sup.134Cs and .sup.137Cs.

[0099] The alga Coccomyxa C-IR3-4C has a cesium-137 (.sup.137Cs) concentration factor, defined as the ratio of the concentration of Cs bound by the algae, in atoms/g of fresh matter, to the concentration of Cs in the water, in atoms/ml, of about 20,000.

EXAMPLE 5

Decontamination of Water from Nuclear Plants by Means of Coccomyxa Chodatii

[0100] The determination of the ionizing-radiation resistance of Coccomyxa chodatii (strain SAG 216/2) algae shows that the ionizing radiation dose that kills half the population is about 1.5 kGy (Rivasseau et al., 2013, cited above). The ionizing-radiation resistance LD.sub.50 of the algae is generally between 30 and 1200 Gy (IAEA, 1976, cited above).

[0101] It appears that the radiation tolerance of microalgae belonging to the genus Coccomyxa is higher than those of algae belonging to other genera, with exceptional resistance by Coccomyxa actinabiotis, in the same way that all bacteria belonging to the genus Deinococcus have high radiation tolerance.

[0102] In order to determine whether Coccomyxa chodatii shared the element uptake capacity of Coccomyxa actinabiotis and Coccomyxa C-IR3-4C, water with a composition typical of that of water from a spent nuclear fuel pool was brought into contact with Coccomyxae chodatii for 24 hours, under illumination. A fresh 110 mg mass of Coccomyxae chodatii algae first cultivated in liquid BBM and rinsed beforehand three times with Milli-Q water is brought into contact with 50 ml of water initially containing the beta-emitting radionuclides .sup.3H (200,000 Bq/l) and .sup.14C (1000 Bq/l) and the gamma-emitting radionuclides listed in Table IV above.

[0103] The assay of the water and the algae 24 hours post-contact shows that 80% of the activity of the water is removed.

[0104] The percentage of -emitting radionuclides bound by the algae in 24 hours is indicated in Table VII below.

TABLE-US-00007 TABLE VII Radionuclide .sup.54Mn .sup.60Co .sup.110mAg .sup.137Cs .sup.238U Binding % 100 54 100 94 100

[0105] All or virtually all of the .sup.110mAg, the .sup.54Mn, the .sup.137Cs and the .sup.238U and half of the .sup.60Co were purified from the water.

EXAMPLE 6

Control of the Proliferation or the Removal of the Microalgae

[0106] Microalgae are photosynthetic. They need light to carry out photosynthesis and to produce their organic matter. In the various very low-nutrient media and effluents from nuclear power stations, their growth can thus be controlled via illumination. In order for them to grow at a given spot, it suffices to provide them with light. Their growth can also be controlled by providing them with light allowing little or no photosynthesis, for example with a yellow-green inactinic lamp.

[0107] The water can be filtered in order to capture the algae suspended therein, and thereby to control their growth.

[0108] A chemical method such as oxidation, for example with hydrogen peroxide, can be used to remove them completely. Five milliliters of 20 g/l hydrogen peroxide is added to medium containing 20 ml of microalgal cell suspension, i.e., a final H.sub.2O.sub.2 concentration of 4 g/l. After 1 day, the culture contains aggregates of brown/white matter and its green color has disappeared. At the end of 1 week, nothing can be observed under the microscope.

[0109] The breakdown of the algae by hydrogen peroxide is thus fast, gradual and total, leaving no organic matter residue.

[0110] This solution can also be used to clean radioactive parts transferred from one medium to another and to avoid any algal contamination of the new medium, or to clean the walls of empty pools.

EXAMPLE 7

Bioprocesses Employing Rare Earth Uptake by Coccomyxa CCAP 216/26

[0111] This example refers to FIGS. 8 to 13, the captions for which are presented below:

[0112] FIG. 8: Change in a) cell density and b) photosynthetic yield of Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.6 M rare earths at pH 6, optimal conditions for biological purification (n=3 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel. *=control samples not tested in parallel and not replicated. These values are presented as an indication of their relevance. The second sample was taken not after 6 days of exposure but after 5 days. The pH in pure water is 71.

[0113] FIG. 9: Percentages of rare earths accumulated (%) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.6 M metal at pH 6, optimal conditions for biological purification (n=3 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel (meanstandard deviation).

[0114] FIG. 10: Change in a) cell density and b) photosynthetic yield of Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M rare earths at pH 2, optimal conditions for metal uptake (n=5 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel. *=control samples not tested in parallel and not replicated. These values are presented as an indication of their relevance. The third sample was taken not after 7 days of exposure, but after 5 days. The pH in pure water is 71.

[0115] FIG. 11: Quantities of rare earths accumulated (mol/g of dry matter) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal at pH 2, optimal conditions for metal uptake (n=3 biological replicates). Four rare earths (Gd, Nd, Eu and Tb) and two chemical forms (cation and citrate complexes) were tested in parallel.

[0116] FIG. 12: Change in a) cell density and b) photosynthetic yield of Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal and at pH 1 or 2 (n=5 biological replicates). The two exposure conditions were tested in parallel. *=control samples not tested in parallel and not replicated. These values are presented as an indication of their relevance. The third sample was taken not after 7 days of exposure, but after 5 days. The pH in pure water is 71.

[0117] FIG. 13: Quantities of rare earths accumulated (arbitrary units) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal and pH 1 or 2 (n=3 biological replicates under the conditions 10.sup.2 M Gd.sup.3+ and pH 1; n=2 biological replicates under the conditions 10.sup.2 M Gd.sup.3+ and pH 2). The two exposure conditions were tested in parallel.

7.1. Experimental Protocols

[0118] 7.1.1. Materials and Methods

[0119] In order to observe sterile conditions while culturing the algae, the protocol was carried out under a laminar flow hood, working within the sterile field created by a burner flame. The materials and solutions used were sterilized beforehand in an autoclave. The culture containers were covered with aluminum foil so as to allow gas exchange. On the other hand, the experiments in which the algae were exposed to metals were carried out under nonsterile conditions.

[0120] Centrifugations were carried out in a Megafuge 16R centrifuge (Thermo Scientific) equipped with a TX-400 rotor (Thermo Scientific). Suitable adapters (part nos. 75003683 and 75003682, Thermo Scientific) were used in order to centrifuge samples contained in 15 or 50 ml Falcon disposable centrifuge tubes (part nos. 734-0451 and 734-0448, VWR).

[0121] The microalgae were observed using an Optiphot light microscope (Nikon) equipped with X20 and X40 magnification objectives. Cell density was measured by counting 12 l of algae suspension on an aluminum-coated Malassez counting chamber with 0.0025 mm.sup.2 mini squares, 0.200 mm in depth (part no. 0640630, Marienfeld). The samples were diluted beforehand, if needed, so as to count between 50 and 100 cells per square.

[0122] Photosynthetic yield was measured in the dark using a PAM-103 chlorophyll fluorometer (Walz).

[0123] Rare earths were assayed in the liquids and in the algae by mass spectrometry coupled to a plasma torch, using the Hewlett-Packard 4500 ICP-MS System. The protocols used and the isotopes selected are described in paragraph 7.1.4.

[0124] 7.1.2. Preparing the Algae

Subculturing in Liquid Medium

[0125] The suspended algae were collected by centrifugation under gentle conditions (100 g, 25 min, 4 C., acceleration and deceleration 2). The algae contained in the pellet were then washed by adding sterile Milli-Q water followed by centrifugation, before being suspended in fresh sterile culture medium, with an initial cell density of 5.Math.10.sup.6 cells/ml.

Culturing the Algae

[0126] Coccomyxa CCAP 216/26 algae were cultivated in a 2-liter photobioreactor, in 1.5 liters of sterile culture medium consisting of BBM (B5282, Sigma-Aldrich) diluted 1:2 in Milli-Q water (0.5X). Temperature and luminosity were kept constant at 221 C. and 9010 mol/m.sup.2/s, respectively. The cells were agitated and aerated by means of continuous air bubbling. The initial concentration of the algae is about 5.Math.10.sup.6 cells/ml. Fifteen milliliters of concentrated (50) BBM was added every 3 to 4 days as of the second week of culture in order to nourish the cells. The photobioreactor was used in batch mode and was refreshed each month. The algae were used once the maximum cell density, greater than 100.Math.10.sup.6 cells/ml, was reached (fed steady-state).

Collecting and Washing the Algae

[0127] After collection by centrifugation (100 g, 25 min, 4 C., acceleration and deceleration 2), the algae were washed three times by adding sterile Milli-Q water followed by centrifugation (100 g, 10 min, 4 C., acceleration and deceleration 2) and removal of the supernatant. The washed algae were then taken up in sterile Milli-Q water and placed in an Erlenmeyer flask in an Infors incubator in which the temperature and the luminosity are kept constant (221 C. and 9010 mol/m.sup.2/s) and aeration is provided by shaking (100 rpm).

Quantifying the Algae

[0128] Three 4 ml to 5 ml samples of the suspension of washed algae were filtered through pre-tared nitrocellulose filters of 1.2 m pore size (part no. 512-0267, VWR). The filters were dried for 30 minutes at 70 C. and then weighed to determine an apparent concentration of dry matter. Measuring this value made it possible, by proportionality, to project the effective concentration of dry matter of the suspension and consequently to dilute the latter so as to obtain a concentration of 0.500.1 g/l of dry matter. The dry matter concentration of the suspension after dilution was controlled by drying the three 4 ml to 5 ml samples at 100 C. for 24 hours, after which the residue was weighed.

[0129] 7.1.3. Algal Metal Uptake Experiments

Exposing the Algae to Metals

[0130] For each experiment, 30 ml of the suspension of washed algae was placed in 100 ml Erlenmeyer flasks. The concentration of the algae is 0.50.1 g/l of dry matter. The pH was then adjusted to its set point by adding KOH or concentrated HCl. The samples were placed in an Infors incubator overnight in order for the algae to adapt to the pH. At t.sub.0, 300 l of LnCl.sub.3 solution (Ln=the lanthanide studied), 100 times more concentrated than the desired final concentration, was added to the samples. The quantities added were verified by weighing. The pH was readjusted to its set point daily.

Physiological Monitoring

[0131] The physiological state of the algae was monitored by measuring cell density and photosynthetic yield.

Sampling the Supernatant

[0132] A 1.2 ml sample of the algae suspension was taken. The supernatant was separated from the algae by two successive centrifugations (100 g, 10 min, 4 C., acceleration and deceleration 3) (centrifugation, sampling the supernatant and then centrifugation of said supernatant). After diluting 1 ml of supernatant in 4 ml of 1.25% HNO.sub.3 (each dilution being verified by weighing), the resulting solution was kept at 4 C. to await a subsequent assay of the metals in solution by ICP-MS.

Sampling the Pellet

[0133] A 3 ml sample of the algae suspension was taken. The algae were collected by centrifugation (500 g, 10 min, 4 C., acceleration and deceleration 4). They were then washed once or twice by taking them up in 4 ml of Milli-Q water, centrifugation (500 g, 10 min, 4 C., acceleration and deceleration 4) and removal of the supernatant. The algae pellet was kept at 4 C. to await mineralization and an assay of the incorporated metals by ICP-MS.

[0134] 7.1.4. Metals Assay

Mineralization of the Algae

[0135] The algae were dry mineralized in 1.5 ml of aqua regia (65% HNO.sub.3/30% HCl, 2:1 v/v) at 180 C. The residue was taken up in 1 ml of 10% HNO3 (solution prepared by diluting commercially-available ultrapure 65% HNO.sub.3 solution) and then diluted 1:10 with sterile Milli-Q water. Each dilution was verified by weighing.

Dilution and Metals Assay

[0136] The solutions arising from the supernatant samples or from mineralization of the algae were diluted in 1% HNO.sub.3 so as to obtain a metal concentration within the standard range. These samples were assayed by ICP-MS by comparison with a standard range prepared using solutions provided by Analab. Each dilution was verified by weighing. The metal concentration was calculated as the mean of the measurements taken on the various isotopes.

[0137] The rare-earth stock solution used for the algae exposure was also assayed.

[0138] For each element analyzed, the standard range and the isotopes measured are summarized in the Table.

TABLE-US-00008 TABLE VIII ICP-MS analysis parameters Element Standard range Isotopes analyzed Neodymium 0.5 to 50 nM .sup.142Nd, .sup.144Nd and .sup.146Nd Europium 0.5 to 10 nM .sup.151Eu and .sup.153Eu Gadolinium 0.1 to 10 nM .sup.156Gd, .sup.158Gd and .sup.160Gd Terbium 0.2 to 20 nM .sup.159Tb

[0139] Ideally, the metals assay was carried out in the supernatants when the sample showed a high accumulation percentage and in the pellets when low accumulation percentages were observed.

[0140] 7.1.5. Preparation of Ln[citrate] complexes

[0141] Citrate complexes were prepared by placing an equimolar amount of metal and citric acid in solution in Milli-Q water. According to the thermodynamic data, the complex then forms spontaneously.

7.2. Binding of Four Rare Earths Gadolinium, Neodymium, Europium and Terbium in Free Form and Complexed Form by Coccomyxa CCAP 216/26 under Optimal Conditions for Biological Purification (10.sup.6 M Metal, pH 6)

[0142] 7.2.1. Experiments Performed

[0143] The accumulation of four rare earths (Gd, Nd, Eu and Tb) in two chemical forms (cation and citrate complex) by Coccomyxa CCAP 216/26 was tested under optimal conditions for accumulation percentage: 10.sup.6 M rare earths, high pH. The pH conditions and the initial concentrations were optimized using central composite experimental designs, applied to a pH range between pH 2 and pH 9 and a concentration range between 10.sup.2M and 10.sup.6 M metal. The optima thus obtained, identical for two elements (Gd and Nd) and two chemical forms (cation and citrate complex), were generalized.

[0144] The experimental designs revealed an optimal pH of 6 for the cations and 9 for the citrate complex. However, the effect of pH on accumulation of the complex is very low and accumulations at pH 4, 6 or 8 are not significantly different. Consequently, during the present experiment, all the tests were performed at pH 61. 10.sup.6 M rare earths.

[0145] The eight corresponding experiments were performed in parallel and were repeated three times over three weeks and with three different biomasses, in order to estimate intermediate reliability.

[0146] 7.2.2. Physiological Monitoring

[0147] The physiological state of the algae was monitored by measuring photosynthetic yield and cell density (FIG. 8). The algae remain in excellent physiological state: cell density increases and photosynthetic yield remains close to 60%, even after 6 days of exposure to rare earths under the present conditions.

[0148] 7.2.3. Monitoring the Accumulation of Metals

[0149] Accumulated metals were assayed in the supernatants in order to determine accumulation percentages (%). The values obtained are presented in Table IX and FIG. 9. [0150] The accumulation percentages are of the same order of magnitude for all the rare earths: between 90% and 95%. [0151] For a given element, there is no significant difference between the accumulation of the cation or the citrate complex after 24 hours in contact with the algae. [0152] Virtually all of the rare earth cations accumulated by the algae are captured in less than 1 hour under the exposure conditions 10.sup.6 M metal, pH 6. There is little or no variation thereafter, up to 24 hours of contact. [0153] At least 3 hours of contact are necessary for accumulation of rare earths in citrate complex form under the conditions 10.sup.6 M metal, pH 6. The accumulation percentage continues to increase slightly between 3 hours and 24 hours of contact.

TABLE-US-00009 TABLE IX Percentages of rare earths accumulated (%) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.6 M metal at pH 6, optimal conditions for biological purification (n = 3 biological replicates) Metal 0 1 h 2 h 3 h 24 h Cation Gd 0.0 88.3 3.3 89.8 2.9 90.1 2.6 91.2 3.6 Nd 0.0 93.0 5.0 93.4 4.7 93.8 4.4 93.9 5.3 Eu 0.0 92.1 5.0 92.3 5.2 92.4 4.7 91.8 5.8 Tb 0.0 91.6 5.8 92.3 5.3 92.3 4.6 91.8 5.3 Citrate Gd 0.0 82.4 6.1 86.6 4.9 88.5 4.2 91.9 4.2 complex Nd 0.0 89.7 3.5 91.8 2.3 92.7 1.7 95.0 0.5 Eu 0.0 84.1 3.6 87.3 3.2 88.5 3.2 91.0 4.8 Tb 0.0 81.7 4.8 86.5 3.8 88.2 3.9 92.4 3.8

7.3. Binding of Four Rare Earths Gadolinium, Neodymium, Europium and Terbium in Free Form and Complexed Form by Coccomvxa CCAP 216/26 Under Optimal Conditions for Metal Uptake (10.SUP.2 .M Metal, pH 2)

[0154] 7.3.1. Experiments Performed

[0155] The accumulation of four rare earths (Gd, Nd, Eu and Tb) in two chemical forms (cation and citrate complex) by Coccomyxa CCAP 216/26 was tested under optimal conditions for the quantities accumulated: 10.sup.2 M rare earths, pH 2.

[0156] 7.3.2. Physiological Monitoring

[0157] The physiological state of the algae was monitored by measuring photosynthetic yield and cell density (FIG. 10). Growth of the algae exposed to pH 2, in the presence or absence of rare earths, stops completely. Photosynthetic yield decreases slightly after 6 days of exposure to 10.sup.2 M rare earths at pH 2. It remains above 50% after 2 days of exposure, which testifies to the good viability of the algae during the first 48 hours.

[0158] 7.3.3. Monitoring the Accumulation of Metals

[0159] Metals were assayed in the algae pellets after mineralization with aqua regia in order to determine the quantities of rare earths accumulated (mol/g of dry matter). The values obtained are presented in Table X and FIG. 11.

[0160] On the whole, the quantities of rare earths accumulated are relatively similar. Whatever the element or chemical form studied, the amount of metal accumulated is comprised between 40 and 100 mol/g of dry matter after a contact time of 3 hours or 24 hours. Small differences are observed in the details. [0161] Under the exposure conditions 10.sup.2 M metal at pH 2, there is a significant difference in accumulation between the various chemical elements. Gadolinium and neodymium appear to be more easily captured than europium and terbium (101 and 78 mol/g of metal dry matter captured in 24 hours for Gd.sup.3+ and Nd.sup.3+, respectively; 43 and 39 mol/g of metal dry matter captured in 24 hours for Eu.sup.3+ and Tb.sup.3+, respectively). [0162] In the case of gadolinium and neodymium, accumulation of the cations appears to be slightly more efficient than that of the citrate complexes. The cation accumulation maxima are reached within 24 hours of contact. Whatever the state of the biomass, repeatability is good up to 24 hours (FIG. 11). At 48 hours of contact, toxicity can begin to set in depending on the state of the biomass. Citrate complexes accumulate quickly during the first 3 hours of contact, then the increase is more gradual up to 48 hours of exposure. [0163] In the case of europium and terbium, the difference in accumulation between the two chemical forms does not appear to be significantly different. The large majority of the metals captured by the algae are captured within only 3 hours. Little variation is visible thereafter.

TABLE-US-00010 TABLE X Quantities of rare earths accumulated (mol/g of dry matter) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal at pH 2, optimal conditions for metal uptake (n = 3 biological replicates). Metal 0 1 h 3 h 24 h 48 h Cation Gd 0.0 45.6 8.6 65.6 8.0 79.1 15.9 82.4 17.7 Nd 0.0 53.2 7.4 60.0 9.2 72.0 7.5 90.7 26.9 Eu 0.0 46.6 8.3 51.4 5.7 62.5 16.4 65.3 14.2 Tb 0.0 42.1 14.1 52.0 11.6 65.0 12.8 73.7 8.8 Citrate Gd 0.0 50.4 17.6 56.0 4.2 61.6 16.5 65.9 2.6 complex Nd 0.0 44.1 9.0 59.8 7.1 64.2 5.0 77.0 8.1 Eu 0.0 42.0 6.8 56.5 4.1 53.8 1.2 66.5 16.5 Tb 0.0 37.5 8.7 49.9 9.7 50.1 9.6 62.6 13.3

[0164] The use of the cationic form of the rare earths, more quickly captured by the algae in the case of certain elements and less expensive in terms of reagents, thus seems preferable under the optimal conditions for recovering metals.

7.4. Binding of the Gd.SUP.3+ Ion by Coccomyxa CCAP 216/26 Under Lower pH Conditions (pH 1)

[0165] 7.4.1. Experiments Performed

[0166] In order to test the binding of rare earths under conditions as similar as possible to the solutions used to dissolve urban waste, the accumulation of Gd.sup.3+ by Coccomyxa CCAP 216/26 was tested under pH conditions lower (pH 1) than those tested heretofore: [0167] 10.sup.2 M Gd.sup.3+ and pH 2 [0168] 10.sup.2 M Gd.sup.3+ and pH 1

[0169] The two experiments were performed in parallel.

[0170] 7.4.2. Physiological Monitoring

[0171] The physiological state of the algae was monitored by measuring photosynthetic yield and cell density (FIG. 12). Growth of the algae exposed to pH 1 and 2, in the presence and in the absence of rare earths, slows significantly. Photosynthetic yield is affected by the exposure conditions tested here, but the algae are still viable at 48 hours and 6 days during exposures to 10.sup.2 M Gd.sup.3+, pH 1. After 6 days of exposure, the photosynthetic yield of the algae exposed to 10.sup.2 M Gd.sup.3+ and pH 2 remain close to 50%. The photosynthetic yield of algae exposed to 10.sup.2 M and pH 1 falls below 20% in 6 days.

[0172] 7.4.3. Monitoring the Accumulation of Metals

[0173] The metals were assayed in the algae pellets after mineralization with aqua regia in order to determine the quantities of rare earths accumulated (mol/g of dry matter). The values obtained are presented in Table XI and FIG. 12.

[0174] The algae are able to accumulate Gd.sup.3+ even when they are exposed for 48 hours to very low pH (pH 1). The accumulated quantities are higher at pH 2 than at pH 1.

[0175] The highest accumulation is observed after the algae are exposed for 24 hours to 10.sup.2 M Gd.sup.3+ and pH 2. An amount close to 100 mol/g of dry matter is then captured by the algae. This condition remains the optimal condition for metal uptake.

TABLE-US-00011 TABLE XI Quantities of rare earths accumulated (arbitrary units) by Coccomyxa CCAP 216/26 microalgae exposed to 10.sup.2 M metal and pH 1 or 2 (n = 3 biological replicates under the conditions 10.sup.2 M Gd.sup.3+ and pH 1; n = 2 biological replicates under the conditions 10.sup.2 M Gd.sup.3+ and pH 2). Concentration pH 0 1 h 3 h 24 h 48 h 10.sup.2 M 1 0.0 34.6 11.6 33.8 15.1 41.2 14.2 65.9 31.0 10.sup.2 M 2 0.0 35.1 2.1 35.4 1.0 47.0 0.4 58.6 0.2