Novel context-specific genome-scale modelling explores the potential of triacylglycerol production by Chlamydomonas reinhardtii.

Gene expression data of cell cultures is commonly measured in biological and medical studies to understand cellular decision-making in various conditions. Metabolism, affected but not solely determined by the expression, is much more difficult to measure experimentally. Finding a reliable method to predict cell metabolism for expression data will greatly benefit metabolic engineering. We have developed a novel pipeline, OVERLAY, that can explore cellular fluxomics from expression data using only a high-quality genome-scale metabolic model. This is done through two main steps: first, construct a protein-constrained metabolic model (PC-model) by integrating protein and enzyme information into the metabolic model (M-model). Secondly, overlay the expression data onto the PC-model using a novel two-step nonconvex and convex optimization formulation, resulting in a context-specific PC-model with optionally calibrated rate constants. The resulting model computes proteomes and intracellular flux states that are consistent with the measured transcriptomes. Therefore, it provides detailed cellular insights that are difficult to glean individually from the omic data or M-model alone. We apply the OVERLAY to interpret triacylglycerol (TAG) overproduction by Chlamydomonas reinhardtii, using time-course RNA-Seq data. We show that OVERLAY can compute C. reinhardtii metabolism under nitrogen deprivation and metabolic shifts after an acetate boost. OVERLAY can also suggest possible 'bottleneck' proteins that need to be overexpressed to increase the TAG accumulation rate, as well as discuss other TAG-overproduction strategies.

Scaling-up and proteomic analysis reveals photosynthetic and metabolic insights toward prolonged H2 photoproduction in Chlamydomonas hpm91 mutant lacking proton gradient regulation 5 (PGR5).

Clean and sustainable H2 production is crucial to a carbon-neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not large-scalable because of lacking desirable strains with both optimal H2 productivity and sufficient knowledge of underlying molecular mechanism. We hereby carried out extensive and in-depth investigations of H2 photoproduction of hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5) toward its up-scaling and fundamental mechanism issues. We show that hpm91 is at least 100-fold scalable (up to 10 L) with continuous H2 collection of 7287 ml H2/10L-HPBR in averagely 26 days under sulfur deprivation. Also, we show that hpm91 is robust and active during sustained H2 photoproduction, most likely due to decreased intracellular ROS relative to wild type. Moreover, we obtained quantitative proteomic profiles of wild type and hpm91 at four representing time points of H2 evolution, leading to 2229 and 1350 differentially expressed proteins, respectively. Compared to wild type, major proteome alterations of hpm91 include not only core subunits of photosystems and those related to anti-oxidative responses but also essential proteins in photosynthetic antenna, C/N metabolic balance, and sulfur assimilation toward both cysteine biosynthesis and sulfation of metabolites during sulfur-deprived H2 production. These results reveal not only new insights of cellular and molecular basis of enhanced H2 production in hpm91 but also provide additional candidate gene targets and modules for further genetic modifications and/or in artificial photosynthesis mimics toward basic and applied research aiming at advancing solar-H2 technology.

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a "plant-like" algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins.

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II.

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼ 320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼ 65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼ 78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼ 60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.

Physiological and transcriptomic analysis reveals the toxicological mechanisms of polystyrene micro- and nano-plastics in Chlamydomonas reinhardtii.

With the accumulation of plastic waste in the environment, the toxicity of micro- and nano-plastics (MNPs) to microalgae has attracted increasing attention. However, the underlying toxic mechanisms of MNPs remain to be elucidated. In this study, we synthesized micro- and nano-scale of polystyrene MNPs (PS MNPs) to investigate their toxicity and toxic mechanisms in Chlamydomonas reinhardtii. We found that PS MNPs significantly inhibit the production of photosynthetic pigments and increase soluble protein content. The detailed analysis of results shows that both materials affect photosynthetic efficiency by damaging the donor side, reaction center, and electron transfer of photosystem II. Moreover, compared to PS MPs, PS NPs have a greater negative impact on algal cells. Analyzing the transcriptome of cells suggests that the most sensitive metabolic pathways in response to PS MNPs involve oxidative phosphorylation, biosynthesis of secondary metabolites, and photosynthesis. Especially, genes related to photosynthesis and oxidative phosphorylation showed significant changes in expression after exposure to PS MNPs. This study provided molecular-level insights into the toxic mechanisms of PS MNPs on microalgae.

Transcriptome analysis reveals the molecular mechanism of differences in growth between photoautotrophy and heterotrophy in Chlamydomonas reinhardtii.

Background: The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing light and CO2, and heterotrophically utilizing acetate. The physiological and biochemical responses of autotrophy and heterotrophy are different in C. reinhardtii. However, there is no complete understanding of the molecular physiology between autotrophy and heterotrophy. Therefore, we performed biochemical, molecular and transcriptome analysis of C. reinhardtii between autotrophy and heterotrophy. Results: The cell growth characterization demonstrated that heterotrophic cell had enhanced growth rates, and autotrophic cell accumulated more chlorophyll. The transcriptome data showed that a total of 2,970 differentially expressed genes (DEGs) were identified from photoautotrophy 12h (P12h) to heterotrophy 12h (H12h). The DEGs were involved in photosynthesis, the tricarboxylic acid cycle (TCA), pyruvate and oxidative phosphorylation metabolisms. Moreover, the results of qRT-PCR revealed that the relative expression levels of malate dehydrogenase (MDH), succinate dehydrogenase (SDH), ATP synthase (ATPase), and starch synthase (SSS) were increased significantly from P12h and H12h. The protein activity of NAD-malate dehydrogenase (NAD-MDH) and succinate dehydrogenase (SDH) were significantly higher in the H12h group. Conclusion: The above results indicated that the high growth rate observed in heterotrophic cell may be the effects of environmental or genetic regulation of photosynthesis. Therefore, the identification of novel candidate genes in heterotrophy will contribute to the development of microalga strains with higher growth capacity and better performance for biomass production.

Exploring cell aggregation as a defense strategy against perchlorate stress in Chlamydomonas reinhardtii through multi-omics analysis.

Perchlorate (ClO4-) is a type of novel, widely distributed, and persistent inorganic pollutant. However, the impacts of perchlorate on freshwater algae remain unclear. In this study, the response and defense mechanisms of microalgae (Chlamydomonas reinhardtii) under perchlorate stress were investigated by integrating physiological and biochemical monitoring, transcriptomics, and metabolomics. Weighted gene co-expression network analysis (WGCNA) of transcriptome data was used to analyze the relationship between genes and phenotype and screen the key pathways. C. reinhardtii exhibited aggregate behavior when exposed to 100- and 200-mM perchlorate but was restored to its unicellular lifestyle when transferred to fresh medium. WGCNA results found that the "carbohydrate metabolism" and "lipid metabolism" pathways were closely related to cell aggregation phenotype. The differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) of these pathways were upregulated, indicating that the lipid and carbohydrate metabolisms were enhanced in aggregated cells. Additionally, most genes and metabolites related to phytohormone abscisic acid (ABA) biosynthesis and the mitogen-activated protein kinase (MAPK) signaling pathway were significantly upregulated, indicating their crucial roles in the signal transmission of aggregated cells. Meanwhile, in aggregated cells, extracellular polymeric substances (EPS) and lipid contents increased, photosynthesis activity decreased, and the antioxidant system was activated. These characteristics contributed to C. reinhardtii's improved resistance to perchlorate stress. Above results demonstrated that cell aggregation behavior was the principal defense strategy of C. reinhardtii against perchlorate. Overall, this study sheds new light on the impact mechanisms of perchlorate to aquatic microalgae and provides multi-omics insights into the research of multicellular-like aggregation as an adaptation strategy to abiotic stress. These results are beneficial for assessing the risk of perchlorate in aquatic environments.

Structure of Photosystem I Supercomplex Isolated from a Chlamydomonas reinhardtii Cytochrome b6f Temperature-Sensitive Mutant.

The unicellular green alga, Chlamydomonas reinhardtii, has been widely used as a model system to study photosynthesis. Its possibility to generate and analyze specific mutants has made it an excellent tool for mechanistic and biogenesis studies. Using negative selection of ultraviolet (UV) irradiation-mutated cells, we isolated a mutant (TSP9) with a single amino acid mutation in the Rieske protein of the cytochrome b6f complex. The W143R mutation in the petC gene resulted in total loss of cytochrome b6f complex function at the non-permissive temperature of 37 °C and recovery at the permissive temperature of 25 °C. We then isolated photosystem I (PSI) and photosystem II (PSII) supercomplexes from cells grown at the non-permissive temperature and determined the PSI structure with high-resolution cryogenic electron microscopy. There were several structural alterations compared with the structures obtained from wild-type cells. Our structural data suggest that the mutant responded by excluding the Lhca2, Lhca9, PsaL, and PsaH subunits. This structural alteration prevents state two transition, where LHCII migrates from PSII to bind to the PSI complex. We propose this as a possible response mechanism triggered by the TSP9 phenotype at the non-permissive temperature.

Not so dangerous? PET microplastics toxicity on freshwater microalgae and cyanobacteria.

Microalgae and cyanobacteria are among the most important primary producers and are responsible for the production of 50-80% of the oxygen on Earth. They can be significantly affected by plastic pollution, as the vast majority of plastic waste ends up in rivers and then the oceans. This research focuses on green microalgae Chlorella vulgaris (C. vulgaris), Chlamydomonas reinhardtii (C. reinhardtii), filamentous cyanobacterium Limnospira (Arthrospira) maxima (L.(A.) maxima) and how they are affected by environmentally relevant PET-MPs (polyethylene-terephtalate microplastics). Manufactured PET-MPs have asymmetric shape, size between 3 and 7 µm and were used in concentrations ranging from 5 mg/L to 80 mg/L. The highest inhibitory rate of growth was found in C. reinhardtii (-24%). Concentration-dependent changes in chlorophyll a composition were found in C. vulgaris and C. reinhardtii, not in L. (A.) maxima. Furthermore, cell damage was detected in all three organisms by CRYO-SEM (shriveling, cell wall disruption), but the cyanobacterium was the least damaged. A PET-fingerprint was detected on the surface of all tested organisms using FTIR, indicating the adherence of PET-MPs. The highest rate of PET-MPs adsorption was detected in L. (A.) maxima. Specifically, characteristic spectra were observed at ∼721, 850, 1100, 1275, 1342, and 1715 cm-1 which are specific for functional groups of PET-MPs. Nitrogen and carbon content significantly increased in L. (A.) maxima under exposure to 80 mg/L due to the PET-MPs adherence and mechanical stress. In all three tested organisms, weak exposure-related ROS generation was detected. In general, cyanobacteria seem to be more resistant to the effects of MPs. However, organisms in the aquatic environment are exposed to MPs over a longer time scale, so it is important to use the present findings for further longer-term experiments on environmentally relevant organisms.

Site-specific gene knock-in and bacterial phytase gene expression in Chlamydomonas reinhardtii via Cas9 RNP-mediated HDR.

In the present study, we applied the HDR (homology-directed DNA repair) CRISPR-Cas9-mediated knock-in system to accurately insert an optimized foreign bacterial phytase gene at a specific site of the nitrate reductase (NR) gene (exon 2) to achieve homologous recombination with the stability of the transgene and reduce insertion site effects or gene silencing. To this end, we successfully knocked-in the targeted NR gene of Chlamydomonas reinhardtii using the bacterial phytase gene cassette through direct delivery of the CRISPR/Cas9 system as the ribonucleoprotein (RNP) complex consisting of Cas9 protein and the specific single guide RNAs (sgRNAs). The NR insertion site editing was confirmed by PCR and sequencing of the transgene positive clones. Moreover, 24 clones with correct editing were obtained, where the phytase gene cassette was located in exon 2 of the NR gene, and the editing efficiency was determined to be 14.81%. Additionally, site-specific gene expression was analyzed and confirmed using RT-qPCR. Cultivation of the positive knocked-in colonies on the selective media during 10 generations indicated the stability of the correct editing without gene silencing or negative insertion site effects. Our results demonstrated that CRISPR-Cas9-mediated knock-in could be applied for nuclear expression of the heterologous gene of interest, and also confirmed its efficacy as an effective tool for site-specific gene knock-in, avoiding nuclear positional effects and gene silencing in C. reinhardtii. These findings could also provide a new perspective on the advantageous application of RNP-CRISPR/Cas9 gene-editing to accelerate the commercial production of complex recombinant proteins in the food-grade organism "C. reinhardtii".

Salt induced oxidative stress alters physiological, biochemical and metabolomic responses of green microalga Chlamydomonas reinhardtii.

Salinity is one of the most significant environmental factors limiting microalgal biomass productivity. In the present study, the model microalga Chlamydomonas reinhardtii (C. reinhardtii) was exposed to 200 mM NaCl for eight days to explore the physiological, biochemical and metabolomic changes. C. reinhradtii exhibited a significant decrease in growth rate, and Chl a and Chl b levels. 200 mM NaCl induced ROS generation in C. reinhardtii with increase in H2O2 content. This caused lipid peroxidation with increase in MDA levels. C. reinhardtii also exhibited an increase in carbohydrate and lipid accumulation under 200 mM NaCl conditions as storage molecules in cells to maintain microalgal survival. In addition, NaCl stress increased the content of carotenoids, polyphenols and osmoprotectant molecules such as proline. SOD and APX activities decreased, while ROS-scavenger enzymes (POD and CAT) decreased. Metabolomic response showed an accumulation of the major molecules implicated in membrane remodelling and stress resistance such oleic acid (40.29%), linolenic acid (19.29%), alkanes, alkenes and phytosterols. The present study indicates the physiological, biochemical and metabolomic responses of C. reinhardtii to salt stress.

Interaction Effect of EDTA, Salinity, and Oxide Nanoparticles on Alga Chlamydomonas reinhardtii and Chlamydomonas euryale.

The interaction effects of organic ligand ethylene diamine tetra-acetic acid (EDTA) and oxide nanoparticles (magnetite Fe3O4-NPs and copper CuO-NPs) were investigated during a 72 h period on two green algal species-Chlamydomonas reinhardtii under freshwater conditions and Chlamydomonas euryale under saltwater conditions. Fe3O4-NPs had larger agglomerates and very low solubility. CuO-NPs, having smaller agglomerates and higher solubility, were more toxic than Fe3O4-NPs in freshwater conditions for similar mass-based concentrations, especially at 72 h under 100 mg L-1. Furthermore, the effect of EDTA increased nanoparticle solubility, and the salinity caused a decrease in their solubility. Our results on C. euryale showed that the increase in salinity to 32 g L-1 caused the formation of larger nanoparticle agglomerates, leading to a decrease in the toxicity impact on algal cells. In addition, EDTA treatments induced a toxicity effect on both freshwater and saltwater Chlamydomonas species, by altering the nutrient uptake of algal cells. However, C. euryale was more resistant to EDTA toxicity than C. reinhardtii. Moreover, nanoparticle treatments caused a reduction in EDTA toxicity, especially for CuO-NPs. Therefore, the toxicity impact caused by these environmental factors should be considered in risk assessment for metallic nanoparticles.

A Simple and Non-destructive Method for Chlorophyll Quantification of Chlamydomonas Cultures Using Digital Image Analysis.

Growing interest in the use of microalgae as a sustainable feedstock to support a green, circular, bio-economy has led to intensive research and development initiatives aimed at increasing algal biomass production covering a wide range of scales. At the heart of this lies a common need for rapid and accurate methods to measure algal biomass concentrations. Surrogate analytical techniques based on chlorophyll content use solvent extraction methods for chlorophyll quantification, but these methods are destructive, time consuming and require careful disposal of the resultant solvent waste. Alternative non-destructive methods based on chlorophyll fluorescence require expensive equipment and are less suitable for multiple sampling of small cultures which need to be maintained under axenic growth conditions. A simple, inexpensive and non-destructive method to estimate chlorophyll concentration of microalgal cultures in situ from digital photographs using the RGB color model is presented. Green pixel intensity and chlorophyll a, b and total chlorophyll concentration, measured by conventional means, follow a strong linear relationship (R 2 = 0.985-0.988). In addition, the resulting standard curve was robust enough to accurately estimate chlorophyll concentration despite changes in sample volume, pH and low concentrations of bacterial contamination. In contrast, use of the same standard curve during nitrogen deprivation (causing the accumulation of neutral lipids) or in the presence of high quantities of bacterial contamination led to significant errors in chlorophyll estimation. The low requirement for equipment (i.e., a simple digital camera, available on smartphones) and widely available standard software for measuring pixel intensity make this method suitable for both laboratory and field-based work, particularly in situations where sample, qualified personnel and/or equipment is limited. By following the methods described here it should be possible to produce a standard curve for chlorophyll analysis in a wide range of testing conditions including different microalga cultures, culture vessel and photographic set up in any particular laboratory.

Kinesin-2 from C. reinhardtii Is an Atypically Fast and Auto-inhibited Motor that Is Activated by Heterotrimerization for Intraflagellar Transport.

Construction and function of virtually all cilia require the universally conserved process of intraflagellar transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, on average, 10 kinesin-2 motors "line up" in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other's way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4] but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit fully activating FLA8/10 for IFT in vivo. Finally, we linked fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and both complexes spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors work mostly alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

Novel Insights Into N-Glycan Fucosylation and Core Xylosylation in C. reinhardtii.

Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical ß1,2-core xylose, α1,3-fucose residues, as well as plant atypical terminal ß1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact N-glycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.

Off-line and on-line optical monitoring of microalgal growth.

The growth of Chlamydomonas reinhardtii microalgae cultures was successfully monitored, using classic off-line optical techniques (optical density and fluorescence) and on-line analysis of digital images. In this study, we found that the chlorophyll fluorescence ratio F 685/F 740 has a linear correlation with the logarithmic concentration of microalgae. By using digital images, the biomass concentration correlated with the luminosity of the images through an exponential equation and the length of penetration of a super luminescent blue beam (λ = 440 nm) through an inversely proportional function. The outcomes of this study are useful to monitor both research and industrial microalgae cultures.

Controlling the Antimicrobial Action of Surface Modified Magnesium Hydroxide Nanoparticles.

Magnesium hydroxide nanoparticles (Mg(OH)2NPs) have recently attracted significant attention due to their wide applications as environmentally friendly antimicrobial nanomaterials, with potentially low toxicity and low fabrication cost. Here, we describe the synthesis and characterisation of a range of surface modified Mg(OH)2NPs, including particle size distribution, crystallite size, zeta potential, isoelectric point, X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), energy dispersive X-ray analysis (EDX), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). We explored the antimicrobial activity of the modified Mg(OH)2NPs on the microalgae (C. reinhardtii), yeast (S. cerevisiae) and Escherichia coli (E. coli). The viability of these cells was evaluated for various concentrations and exposure times with Mg(OH)2NPs. It was discovered that the antimicrobial activity of the uncoated Mg(OH)2NPs on the viability of C. reinhardtii occurred at considerably lower particle concentrations than for S. cerevisiae and E. coli. Our results indicate that the antimicrobial activity of polyelectrolyte-coated Mg(OH)2NPs alternates with their surface charge. The anionic nanoparticles (Mg(OH)2NPs/PSS) have much lower antibacterial activity than the cationic ones (Mg(OH)2NPs/PSS/PAH and uncoated Mg(OH)2NPs). These findings could be explained by the lower adhesion of the Mg(OH)2NPs/PSS to the cell wall, because of electrostatic repulsion and the enhanced particle-cell adhesion due to electrostatic attraction in the case of cationic Mg(OH)2NPs. The results can be potentially applied to control the cytotoxicity and the antimicrobial activity of other inorganic nanoparticles.

Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii is a key model organism for studying photosynthesis and oxidative stress in unicellular eukaryotes. Using a forward genetics approach, we have identified and characterized a mutant x32, which lacks a predicted protein named CGLD1 (Conserved in Green Lineage and Diatom 1) in GreenCut2, under normal and stress conditions. We show that loss of CGLD1 resulted in minimal photoautotrophic growth and PSII activity in the organism. We observed reduced amount of PSII complex and core subunits in the x32 mutant based on blue-native (BN)/PAGE and immunoblot analysis. Moreover, x32 exhibited increased sensitivity to high-light stress and altered tolerance to different reactive oxygenic species (ROS) stress treatments, i.e., decreased resistance to H2O2/or tert-Butyl hydroperoxide (t-BOOH) and increased tolerance to neutral red (NR) and rose bengal (RB) that induce the formation of singlet oxygen, respectively. Further analysis via quantitative real-time PCR (qRT-PCR) indicated that the increased singlet-oxygen tolerance of x32 was largely correlated with up-regulated gene expression of glutathione-S-transferases (GST). The phenotypical and physiological implications revealed from our experiments highlight the important roles of CGLD1 in maintaining structure and function of PSII as well as in protection of Chlamydomonas under photo-oxidative stress conditions.