Microbial recovery of rare earth elements from various waste sources: a mini review with emphasis on microalgae.

Rare Earth Elements (REEs) are indispensable in contemporary technologies, influencing various aspects of our daily lives and environmental solutions. The escalating demand for REEs has led to increased exploitation, resulting in the generation of diverse REE-bearing solid and liquid wastes. Recognizing the potential of these wastes as secondary sources of REEs, researchers are exploring microbial solutions for their recovery. This mini review provides insights into the utilization of microorganisms, with a particular focus on microalgae, for recovering REEs from sources such as ores, electronic waste, and industrial effluents. The review outlines the principles and distinctions of bioleaching, biosorption, and bioaccumulation, offering a comparative analysis of their potential and limitations. Specific examples of microorganisms demonstrating efficacy in REE recovery are highlighted, accompanied by successful methods, including advanced techniques for enhancing microbial strains to achieve higher REE recovery. Moreover, the review explores the environmental implications of bio-recovery, discussing the potential of these methods to mitigate REE pollution. By emphasizing microalgae as promising biotechnological candidates for REE recovery, this mini review not only presents current advances but also illuminates prospects in sustainable REE resource management and environmental remediation.

Bio-removal of rare earth elements from hazardous industrial waste of CFL bulbs by the extremophile red alga Galdieria sulphuraria.

In recent decades, a shift has been seen in the use of light-emitting diodes over incandescent lights and compact fluorescent lamps (CFL), which eventually led to an increase in wastes of electrical equipment (WEE), especially fluorescent lamps (FLs) and CFL light bulbs. These widely used CFL lights, and their wastes are good sources of rare earth elements (REEs), which are desirable in almost every modern technology. Increased demand for REEs and their irregular supply have exerted pressure on us to seek alternative sources that may fulfill this demand in an eco-friendly manner. Bio-removal of wastes containing REEs, and their recycling may be a solution to this problem and could balance environmental and economic benefits. To address this problem, the current study focuses on the use of the extremophilic red alga, Galdieria sulphuraria, for bioaccumulation/removal of REEs from hazardous industrial wastes of CFL bulbs and the physiological response of a synchronized culture of G. sulphuraria. A CFL acid extract significantly affected growth, photosynthetic pigments, quantum yield, and cell cycle progression of this alga. A synchronous culture was able to efficiently accumulate REEs from a CFL acid extract and efficiency was increased by including two phytohormones, i.e., 6-Benzylaminopurine (BAP - Cytokinin family) and 1-Naphthaleneacetic acid (NAA - Auxin family).

Very long chain fatty acids.

Very long chain fatty acids (VLCFAs) are important components of various lipid classes in most organisms, from bacteria to higher plants and mammals, including humans. VLCFAs, or very long chain polyunsaturated fatty acids (VLCPUFAs), can be defined as fatty acids with 23 or more carbon atoms in the molecule. The main emphasis in this review is on the analysis of these acids, including obtaining standards from natural sources or their synthesis. Furthermore, the occurrence and analysis of these compounds in both lower (bacteria, invertebrates) and higher organisms (flowering plants or mammals) are discussed in detail. Attention is paid to their biosynthesis, especially the elongation of very long chain fatty acids protein (ELOVL4). This review deals with papers describing these very interesting compounds, whose chemical, biochemical and biological properties have not been fully explored.

Identification of potential plant species hyperaccumulating light rare earth elements (LREE) in a mining area in Minas Gerais, Brazil.

Phytoextraction of rare earth elements (REE) from contaminated soils has gained importance during the last few decades. The Poços de Caldas municipality in Brazil is known for its mineral richness, including large reserves of REE. In this study, we report light REE (La, Ce, Sm, Pr, and Nd) in soils and plants collected in an area. Composite soil samples and plant individuals were collected, and total concentrations of LREE in soils were determined by wavelength dispersive X-ray fluorescence (WDXRF). The plant available LREE concentrations in soils were estimated upon the acetic acid method (F1 fractions) of the stepwise sequential extraction procedure, together with plant content that was analysed by inductively coupled plasma mass spectrometry (ICP-MS). The total sum concentrations of tested LREE in soils varied from 5.6 up to 37.9 g kg-1, the bioavailable fraction was ca. 1%, and a linear relationship was found between them. The only exception was Sm, whose availability was lesser and did not show a linear relationship. The concentration of LREE in non-accumulator plants varied from 1.3-950 mg kg-1 for Ce, La 1.1-99 mg kg-1, Sm 0.04-9.31 mg kg-1, Pr 0.1-24.1 mg kg-1, and Nd 0.55-81 mg kg-1. The concentration of LREE among shoots did not show a linear relation either with the available fraction or total content. The screening also revealed Christella dentata (Forssk.) Brownsey & Jermy, Thelypteridaceae family, as a promising hyperaccumulator species. The concentrations of LREE among shoots of six individuals of this species were in the ranges from 115 to 1872 mg kg-1 for Ce, La 190-703 mg kg-1, Sm 9-48 mg kg-1, Pr 32-144 mg kg-1, and Nd 105-478 mg kg-1.

Changes in glycosyl inositol phosphoceramides during the cell cycle of the red alga Galdieria sulphuraria.

Sphingolipids are significant component of plant-cell plasma membranes, as well as algal membranes, and mediate various biological processes. One of these processes is the change in lipid content during the cell cycle. This change is key to understanding cell viability and proliferation. There are relatively few papers describing highly glycosylated glycosyl inositol phosphorylceramide (GIPC) due to problems associated with the extractability of GIPCs and their analysis, especially in algae. After alkaline hydrolysis of total lipids from the red alga Galdieria sulphuraria, GIPCs were measured by high-resolution tandem mass spectrometry and fragmentation of precursor ions in an Orbitrap mass spectrometer in order to elucidate the structures of molecular species. Fragmentation experiments such as tandem mass spectrometry in the negative ion mode were performed to determine both the ceramide group and polar head structures. Measurement of mass spectra in the negative regime was possible because the phosphate group stabilizes negative molecular ions [M-H]-. ANALYSIS: of GIPCs at various stages of the cell cycle provided information on their abundance. It was found that, depending on the phases of the cell cycle, in particular during division, the uptake of all three components of GIPC, i.e., long-chain amino alcohols, fatty acids, and polar heads, changes. Structural modifications of the polar headgroup significantly increased the number of molecular species. Analysis demonstrated a convex characteristic for molecular species with only one saccharide (hexose or hexuronic acid) as the polar head. For two carbohydrates, the course of Hex-HexA was linear, while for HexA-HexA it was concave. The same was true for GIPC with three and four monosaccharides.

Supra-Optimal Temperature: An Efficient Approach for Overaccumulation of Starch in the Green Alga Parachlorella kessleri.

Green algae are fast-growing microorganisms that are considered promising for the production of starch and neutral lipids, and the chlorococcal green alga Parachlorella kessleri is a favorable model, as it can produce both starch and neutral lipids. P. kessleri commonly divides into more than two daughter cells by a specific mechanism-multiple fission. Here, we used synchronized cultures of the alga to study the effects of supra-optimal temperature. Synchronized cultures were grown at optimal (30 °C) and supra-optimal (40 °C) temperatures and incident light intensities of 110 and 500 µmol photons m-2 s-1. The time course of cell reproduction (DNA replication, cellular division), growth (total RNA, protein, cell dry matter, cell size), and synthesis of energy reserves (net starch, neutral lipid) was studied. At 40 °C, cell reproduction was arrested, but growth and accumulation of energy reserves continued; this led to the production of giant cells enriched in protein, starch, and neutral lipids. Furthermore, we examined whether the increased temperature could alleviate the effects of deuterated water on Parachlorella kessleri growth and division; results show that supra-optimal temperature can be used in algal biotechnology for the production of protein, (deuterated) starch, and neutral lipids.

Growth under Different Trophic Regimes and Synchronization of the Red Microalga Galdieria sulphuraria.

The extremophilic unicellular red microalga Galdieria sulphuraria (Cyanidiophyceae) is able to grow autotrophically, or mixo- and heterotrophically with 1% glycerol as a carbon source. The alga divides by multiple fission into more than two cells within one cell cycle. The optimal conditions of light, temperature and pH (500 µmol photons m-2 s-1, 40 °C, and pH 3; respectively) for the strain Galdieria sulphuraria (Galdieri) Merola 002 were determined as a basis for synchronization experiments. For synchronization, the specific light/dark cycle, 16/8 h was identified as the precondition for investigating the cell cycle. The alga was successfully synchronized and the cell cycle was evaluated. G. sulphuraria attained two commitment points with midpoints at 10 and 13 h of the cell cycle, leading to two nuclear divisions, followed subsequently by division into four daughter cells. The daughter cells stayed in the mother cell wall until the beginning of the next light phase, when they were released. Accumulation of glycogen throughout the cell cycle was also described. The findings presented here bring a new contribution to our general understanding of the cell cycle in cyanidialean red algae, and specifically of the biotechnologically important species G. sulphuraria.

Characterization of Growth and Cell Cycle Events Affected by Light Intensity in the Green Alga Parachlorella kessleri: A New Model for Cell Cycle Research.

Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m-2s-1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.

Starch Production in Chlamydomonas reinhardtii through Supraoptimal Temperature in a Pilot-Scale Photobioreactor.

An increase in temperature can have a profound effect on the cell cycle and cell division in green algae, whereas growth and the synthesis of energy storage compounds are less influenced. In Chlamydomonas reinhardtii, laboratory experiments have shown that exposure to a supraoptimal temperature (39 °C) causes a complete block of nuclear and cellular division accompanied by an increased accumulation of starch. In this work we explore the potential of supraoptimal temperature as a method to promote starch production in C. reinhardtii in a pilot-scale photobioreactor. The method was successfully applied and resulted in an almost 3-fold increase in the starch content of C. reinhardtii dry matter. Moreover, a maximum starch content at the supraoptimal temperature was reached within 1-2 days, compared with 5 days for the control culture at the optimal temperature (30 °C). Therefore, supraoptimal temperature treatment promotes rapid starch accumulation and suggests a viable alternative to other starch-inducing methods, such as nutrient depletion. Nevertheless, technical challenges, such as bioreactor design and light availability within the culture, still need to be dealt with.

Plasmalogens - Ubiquitous molecules occurring widely, from anaerobic bacteria to humans.

Plasmalogens are a group of lipids mainly found in the cell membranes. They occur in anaerobic bacteria and in some protozoa, invertebrates and vertebrates, including humans. Their occurrence in plants and fungi is controversial. They can protect cells from damage by reactive oxygen species, protect other phospholipids or lipoprotein particles against oxidative stress, and have been implicated as signaling molecules and modulators of membrane dynamics. Biosynthesis in anaerobic and aerobic organisms occurs by different pathways, and the main biosynthetic pathway in anaerobic bacteria was clarified only this year (2021). Many different analytical techniques have been used for plasmalogen analysis, some of which are detailed below. These can be divided into two groups: shotgun lipidomics, or electrospray ionization mass spectrometry in combination with high performance liquid chromatography (LC-MS). The advantages and limitations of both techniques are discussed here, using examples from anaerobic bacteria to specialized mammalian (human) organs.

Detailed structural characterization of cardiolipins from various biological sources using a complex analytical strategy comprising fractionation, hydrolysis and chiral chromatography.

Cardiolipins (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol) (CLs) are widespread in many organisms, from bacteria to higher green plants and mammals. CLs were observed in Gram-positive bacterium of the genus Kocuria, brewer's yeast Saccharomyces, the green alga Chlamydomonas, spinach and beef heart. A mixture of molecular species of CLs was obtained from total lipids by hydrophilic interaction liquid chromatography (HILIC), and these were further separated and identified by reversed phase LC/MS with negative tandem electrospray ionization. The majority of CLs molecular species from each organism were cleaved using phospholipase C from Bacillus cereus. This phospholipase cleaves CLs into 1,2-diglycerols and phosphatidylglycerol 3-phosphates, which were then separated. After CLs cleavage, diacylglycerols such as sn-1,2-diacyl-3-acetyl-glycerols (i.e., triacylglycerols) were separated and identified by chiral chromatography/MS-positive tandem ESI. Significant differences in the composition of the molecular species between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of CLs were found in all organisms tested. Molecular species of CLs that contained four different fatty acids were identified in all five samples, and CLs containing very long chain fatty acids were identified in yeast. In addition, CLs containing both enantiomers (at the sn-2 carbon) were present in the bacterium tested. These findings were further supported by data already published in GenBank where, in the same family - Micrococcaceae - both enzymes responsible for chirality in the sn-2 position, glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases, were present.

The biosynthesis of phospholipids is linked to the cell cycle in a model eukaryote.

The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. We tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure is important for progress through and perhaps even control of the cell cycle. Lipidomics (31P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote, Desmodesmus quadricauda. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G1 phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol (+100%, phosphatidylglycerol synthase increased 22×) over the G1/pre-replication phase boundary. The relative abundance of phosphatidylcholine fell by ~35% during the G1. N-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the de novo transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. This study shows for the first time that there are considerable changes in the biosynthesis of the three most abundant phospholipid classes in the normal cell cycle of D. quadricauda, by margins large enough to elicit changes to the physical properties of membranes.

Effect of nanomolar concentrations of lanthanum on Desmodesmus quadricauda cultivated under environmentally relevant conditions.

Toxicity of lanthanides is generally regarded as low, and they even have been suggested to be beneficial at low concentrations. This research was conducted to investigate effects of Lanthanum (La) on Desmodesmus quadricauda, a freshwater green microalga. The algal cultures were treated with nanomolar La concentrations under controlled environmentally relevant conditions. Intracellular localization of La was analyzed with µXRF tomography in frozen-hydrated samples. At sublethal concentration (128 nM) La was in hotspots inside the cells, while at lethal 1387 nM that led to release of other ions (K, Zn) from the cells, La filled most of the cells. La had no clear positive effects on growth or photosynthetic parameters, but increasing concentrations led to a dramatic decrease in cell counts. Chlorophyll fluorescence kinetic measurements showed that La led to the inhibition of photosynthesis. Maximal photochemical quantum yield of the PSII reaction center in dark-adapted state (Fv/Fm) decreased at > 4.3 nM La during the 2nd week of treatment. Minimum dark-adapted fluorescence quantum yield (F0) increased at > 13.5 nM La during the 2nd week of treatment except for control (0.2 nM La, baseline from chemicals) and 0.3 nM La. NPQ at the beginning of the actinic light phase showed significant increase for all the treatments. Metalloproteomics by HPLC-ICPMS showed that La binds to a >500 kDa soluble protein complex already in the sub-nM range of La treatments, in the low nM range to a small-sized (3 kDa) soluble peptide, and at >100 nM La additionally binds to a 1.5 kDa ligand.

Exploring Mycosporine-Like Amino Acids (MAAs) as Safe and Natural Protective Agents against UV-Induced Skin Damage.

Prolonged exposure to harmful ultraviolet radiation (UVR) can induce many chronic or acute skin disorders in humans. To protect themselves, many people have started to apply cosmetic products containing UV-screening chemicals alone or together with physical sunblocks, mainly based on titanium-dioxide (TiO2) or zinc-oxide (ZnO2). However, it has now been shown that the use of chemical and physical sunblocks is not safe for long-term application, so searches for the novel, natural UV-screening compounds derived from plants or bacteria are gaining attention. Certain photosynthetic organisms such as algae and cyanobacteria have evolved to cope with exposure to UVR by producing mycosporine-like amino acids (MAAs). These are promising substitutes for chemical sunscreens containing commercially available sunblock filters. The use of biopolymers such as chitosan for joining MAAs together or with MAA-Np (nanoparticles) conjugates will provide stability to MAAs similar to the mixing of chemical and physical sunscreens. This review critically describes UV-induced skin damage, problems associated with the use of chemical and physical sunscreens, cyanobacteria as a source of MAAs, the abundance of MAAs and their biotechnological applications. We also narrate the effectiveness and application of MAAs and MAA conjugates on skin cell lines.

Comparing Biochemical and Raman Microscopy Analyses of Starch, Lipids, Polyphosphate, and Guanine Pools during the Cell Cycle of Desmodesmus quadricauda.

Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.

Lipidomic analysis of diatoms cultivated with silica nanoparticles.

Polar lipids from the diatoms Diadesmis gallica and Navicula atomus were separated and their structures were determined using high resolution tandem MS HILIC-LC/ESI. This method allowed us to identify 34 classes of lipids, each containing dozens of molecular species, including regioisomers. The largest differences were found in two sulfur-containing lipids, sulfoquinovosyldiacylglycerol and phosphatidylsulfocholine caused probably by the remodeling of lipid species. These diatoms have been found to use several mechanisms to resolve growth in extreme environments, i.e. silica starvation. The presence of insoluble nano-SiO2 leads to the replacement of cellular phospholipids with sulfolipids. Regioisomer ratios also vary depending on the concentration of nano-SiO2 in the culture medium, i.e. the biosynthesis of polar lipids via the prokaryotic (plastidial) and/or eukaryotic (explastidial) pathways. Complex analyses of polar lipids using high resolution HILIC-LC/ESI-tandem, as used for diatoms, can also be used for other photosynthetic microorganisms.

Cell Cycle Arrest by Supraoptimal Temperature in the Alga Chlamydomonas reinhardtii.

Temperature is one of the key factors affecting growth and division of algal cells. High temperature inhibits the cell cycle in Chlamydomonas reinhardtii. At 39 °C, nuclear and cellular divisions in synchronized cultures were blocked completely, while DNA replication was partly affected. In contrast, growth (cell volume, dry matter, total protein, and RNA) remained unaffected, and starch accumulated at very high levels. The cell cycle arrest could be removed by transfer to 30 °C, but a full recovery occurred only in cultures cultivated up to 14 h at 39 °C. Thereafter, individual cell cycle processes began to be affected in sequence; daughter cell release, cell division, and DNA replication. Cell cycle arrest was accompanied by high mitotic cyclindependent kinase activity that decreased after completion of nuclear and cellular division following transfer to 30 °C. Cell cycle arrest was, therefore, not caused by a lack of cyclin-dependent kinase activity but rather a blockage in downstream processes.

Rapid screening of very long-chain fatty acids from microorganisms.

The analysis of triacylglycerols and phospholipids - phosphatidylcholines allowed the use of shotgun lipidomics to identify very long-chain fatty acids and very long-chain polyunsaturated fatty acids in microalgae. These fatty acids were determined in triacylglycerols by positive electrospray ionization of neutral loss scans of different fatty acids, e.g. 24:0, 24:1ω9, 24:6ω3, 26:0, 26:1ω9, 28:0, 28:1ω9, 28:2ω6, and 28:8ω3. Likewise, very long-chain fatty acids in phosphatidylcholines were identified by negative electrospray ionization mass spectrometry in the selected ion-monitoring of the two most important ions (R1COO- and R2COO-). The limit of detection was determined at 10 nmol/L (∼11 pg/µL) in triacylglycerols and 8.6 nmoles/L (∼8 pg/µL) in phosphatidylcholines. The use of liquid chromatography-mass spectrometry is suitable for very long-chain polyunsaturated fatty acids with up to 8 double bonds due to the time of analysis as well as for reasons of lower thermal stability of polyunsaturated fatty acids towards saturated fatty acids, but gas chromatography-mass spectrometry is better suited for the analysis of saturated very long-chain fatty acids.

Response of the Green Alga Chlamydomonas reinhardtii to the DNA Damaging Agent Zeocin.

DNA damage is a ubiquitous threat endangering DNA integrity in all living organisms. Responses to DNA damage include, among others, induction of DNA repair and blocking of cell cycle progression in order to prevent transmission of damaged DNA to daughter cells. Here, we tested the effect of the antibiotic zeocin, inducing double stranded DNA breaks, on the cell cycle of synchronized cultures of the green alga Chlamydomonas reinhardtii. After zeocin application, DNA replication partially occurred but nuclear and cellular divisions were completely blocked. Application of zeocin combined with caffeine, known to alleviate DNA checkpoints, decreased cell viability significantly. This was probably caused by a partial overcoming of the cell cycle progression block in such cells, leading to aberrant cell divisions. The cell cycle block was accompanied by high steady state levels of mitotic cyclin-dependent kinase activity. The data indicate that DNA damage response in C. reinhardtii is connected to the cell cycle block, accompanied by increased and stabilized mitotic cyclin-dependent kinase activity.

Deuterium and its impact on living organisms.

The rare stable isotope of hydrogen, deuterium, has fascinated researchers since its discovery in the 1930s. Subsequent large-scale production of deuterium oxide, commonly known as heavy water, became a starting point for further research. Deuterium exhibits unique physicochemical properties as well as having the strongest kinetic isotope effects among all other elements. Moreover, a broad variety of morphological and physiological changes have been observed in deuterium-treated cells and organisms, including changes in fundamental processes such as cell division or energy metabolism. Even though our understanding of such alterations is still insufficient, it is evident that some of them make growth in a deuterium-enriched environment a challenging task. There seems to be certain species-specific limits to their tolerance to heavy water, where some organisms are unable to grow in heavy water whilst others have no difficulties. Although the effects of deuterium on living organisms are, in general, negative, some of its applications are of great biotechnological potential, as is the case of stable isotope-labelled compounds or deuterated drugs.