Supercritical Fluid Extraction of Rare Earth Elements from Nickel Metal Hydride Battery

Today’s world relies upon critical green technologies that are made of elements with unique properties irreplaceable by other materials. Such elements are classified under strategic materials; examples include rare earth elements that are in increasingly high demand but facing supply uncertainty and near zero recycling. For tackling the sustainability challenges associated with rare earth elements supply, new strategies have been initiated to mine these elements from secondary sources. Waste electrical and electronic equipment contain considerable amounts of rare earth elements; however, the current level of their recycling is less than 1%. Current recycling practices use either pyrometallurgy, which is energy intensive, or hydrometallurgy that rely on large volumes of acids and organic solvents, generating large volumes of environmentally unsafe residues. This study put emphasis on developing an innovative and sustainable process for the urban mining of rare earth elements from waste electrical and electronic equipment, in particular, a nickel metal hydride battery. The developed process relies on supercritical fluid extraction utilizing CO₂ as the solvent, which is inert, safe, and abundant. This process is very efficient in the sense that it is safe, runs at low temperature, and does not produce hazardous waste while recovering ∼90% of rare earth elements. Furthermore, we propose a mechanism for the supercritical fluid extraction of rare earth elements, where we considered a trivalent rare earth element state bonded with three tri-<i>n</i>-butyl phosphate molecules and three nitrates model for the extracted rare earth tri-<i>n</i>-butyl phosphate complex. The supercritical fluid extraction process has the double advantage of waste valorization without utilizing hazardous reagents, thus minimizing the negative impacts of process tailings

Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in <i>Pseudomonas</i>

<div><p>Microorganisms such as <i>Pseudomonas putida</i> play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in <i>Pseudomonas putida</i>, we measured differential protein abundance levels with MS-based spectral counting in <i>P. putida</i> S16 grown on nicotine or glycerol, a non-repressive carbon source. <i>In silico</i> analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (<i>spmA</i>), molybdopterin dehydrogenase (<i>spmB</i>), and a (2Fe-2S)-binding ferredoxin (<i>spmC</i>) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (<i>nicA</i>2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.</p></div

Validation of transcriptional regulation of differentially expressed proteins.

<p>RT-qPCR and semi-quantitative RT-PCR analysis of target gene transcripts produced in <i>P. putida</i> S16 grown with or without nicotine. <b>A</b>. mRNA expression levels of 9 target genes involved in nicotine degradation of <i>P. putida</i> S16 were estimated using RT-qPCR and the 2^ΔΔCT method. The 16S rRNA gene was used as the reference gene. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003923#s2" target="_blank">Results</a> presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. <b>B</b>. Semi-quantitative RT-PCR analysis of target gene transcripts produced in <i>P. putida</i> S16 grown with or without nicotine. The expression of 16 S rDNA was used as an internal control. Primers specific for target genes and 16 S rDNA were used to amplify fragments by RT-PCR.</p

A map of the circular chromosome of <i>P. putida</i> S16.

<p>The map illustrated the location of known genes, predicted coding regions, genome islands, and differentially expressed proteins. From the outside inwards, circle 1 and circle 2 indicate the protein in the clockwise and anti-clockwise direction that was differentially expressed (> = 3-fold changes and p-values<0.05) in nicotine medium and glycerol medium. The red lines indicate proteins up-expression in nicotine medium, and blue lines indicate proteins up-expression in glycerol medium. Circle 3 and circle 4 indicate the predicted CDSs in the clockwise and anti-clockwise directions, analyzed using the COG database (colors were assigned according to the color code of the COG functional classes); Circle 5 indicates the genome islands predicted in the S16 genome. The red line represents the biggest genome island in S16, in which the nicotine degradation cluster is located in the circle. Circle 6 indicates tRNAs; circle 7 and circle 8 indicate the value of GC skew (G−C/G+C) and percentage of GC content, respectively, with a 4000-bp window size and 2000-bp overlap.</p

Cell growth and resting cell reactions of strain S16 and the gene deletion mutant S16d<i>nicA1</i>.

<p><b>A</b>. UV-scan analysis of nicotine degradation by resting cell of gene deletion mutant S16d<i>nicA1</i>. <b>B</b>. UV-scan analysis of nicotine degradation by resting cell of strain S16. <b>C</b>. Strain S16, gene deletion mutant S16d<i>nicA1</i>, and gene deletion mutant S16d<i>nicA2</i> grown on a nicotine plate containing nicotine as the sole carbon and nitrogen source, and on a LB plate containing kanamycin.</p

Characterzation of NicA2.

<p><b>A</b>. SDS-PAGE analysis of expressed NicA2 in <i>E. coli</i> BL21(DE3) on a 12.5% gel. Lane M, protein molecular weight marker (MBI); lane 1, cell extracts of <i>E. coli</i> containing plasmid pET-28a; lanes 2, cell extracts of <i>E. coli</i> (pET28a-<i>nicA2</i>) obtained 4 h after IPTG induction. <b>B</b>. UV spectrum for the conversion of nicotine by transformant pET28a-<i>nicA2</i>. <b>C</b>. Mass spectra of <i>N</i>-methylmyosmine (P) as determined by GC-MS analysis. <b>D</b>. TLC analysis of the products formed by incubation of nicotine and whole cells of transformant pET28a-<i>nicA2</i>. The qualitative analysis of nicotine and <i>N</i>-methylmyosmine (P) was performed by using analytical TLC according to published procedures <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003923#pgen.1003923-Wang2" target="_blank">[10]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003923#pgen.1003923-Tang2" target="_blank">[12]</a>. Nicotine and <i>N</i>-methylmyosmine (P) are indicated in the figure.</p

Cell growth and resting cell reactions of strain S16 and the gene deletion mutants.

<p><b>A</b>. Strain S16 and gene deletion mutants grown on a nicotine plate containing nicotine as the sole carbon and nitrogen source. <b>B</b>. Strain S16 and gene deletion mutants grown on LB plate containing kanamycin. <b>C</b>. Growth curves of S16 (•), S16d<i>pnao</i> (▾), and S16d-<i>nicA2</i> (▪) with nicotine as sole carbon and nitrogen sources. <b>D</b>. HPLC analysis of nicotine degradation by cell culture of <i>P. putida</i> S16 (•), S16d<i>pnao</i> (▾), and S16d<i>nicA2</i> (▪). <b>E</b>. HPLC analysis of nicotine degradation by resting cells of <i>P. putida</i> S16 (•), S16d<i>pnao</i> (▾), and S16d<i>nicA2</i> (▪). The values are means of three replicates, and the error bars indicate the standard deviations.</p

Expression level changes of proteins that were involved in nicotine degradation.

<p>The expressions of proteins Porin, Mfs, SpmC, SpmA, Sapd, Pnao, NicA2, HspA, HspB, and NicA1 were identified and detected by MS-based spectral counting. The plot shows expression level of various proteins in different media: red, nicotine medium; blue, glycerol medium. Bars indicate the average ± s.e.m of the normalized spectral counts (y axis).</p

Pyrrolidine pathway of nicotine catabolism in <i>P. putida</i> S16.

<p><b>A</b>. All complete steps in the catabolism of nicotine by <i>P. putida</i> S16. <b>B</b>. The genetic organization of a gene cluster involved in nicotine catabolism in <i>P. putida</i> S16 were shown. The arrows indicate the size and direction of transcription of each gene, <i>nicA2</i>, nicotine oxido-reductase gene; <i>pnao</i>, pseudooxynicotine amidase gene; <i>sapd</i>, DSP dehydrogenase gene; <i>spm</i>, SP monoxygenase gene; <i>mfs</i>, major facilitator superfamily gene; <i>porin</i>, porin gene; <i>hspB</i>, HSP monoxygenase gene; <i>iso</i>, maleate isomerase gene; <i>nfo</i>, NFM deformylase gene; <i>hpo</i>, DHP diooxygenase gene; ami, maleamate amidase gene. Genes are annotated following the color mode indicated. The two genes <i>porin</i> and <i>hspB</i> are approximate 15 kb apart from each other.</p