Search Results (256)

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

The growing concern for environmental sustainability has led to an increased interest in biodegradable materials derived from renewable resources. This study explores the innovative use of residual biomass from the green photosynthetic microalga Chlamydomonas reinhardtii, left over after polysaccharide extraction, as a natural filler in the development of the compostable protein-based material SP-Milk^®. The microalgal biomass was characterized using Fourier transform infrared spectroscopy (FTIR) and UV-Visible Spectroscopy to assess its chemical and structural composition. Subsequently, it was incorporated into a biodegradable protein matrix, and the resulting biocomposites were evaluated for mechanical and thermal properties. The results demonstrate that the incorporation of algal filler improves the mechanical strength and elasticity of the material while reducing its glass transition temperature, highlighting its potential for use in sustainable applications as a possible substitute for conventional plastics. The biocomposite materials developed, based on the protein-based material SP-Milk^® and residual microalgal biomass, are environmentally friendly, contributing to the reduction in pollution and the risks associated with plastic accumulation. Thus, this study offers a simple, effective, and sustainable strategy for the valorization of microalgal biomass, enabling the production of biodegradable materials with enhanced mechanical performance, suitable for applications such as sustainable packaging within a circular economy framework. Full article

Ammonia is a critical compound for numerous industrial processes; however, the conventional methods for its production present substantial environmental challenges. Co-producing biofuels and ammonia from biomass through anaerobic digestion offers a promising alternative to address these concerns. This study presents a theoretical assessment of the co-production of biomethane and ammonia from microalgae and cyanobacteria, utilising water from abandoned mine and quarry pit-lakes—specifically focusing on the Alessandria district as a case study. The analysis is based on the average values reported in the literature for the anaerobic digestion of selected biomass types. The results highlight Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella spp., and Chlorella pyrenoidosa as the most promising species due to their superior yields of both ammonia and biomethane. This work aims to promote new opportunities for repurposing disused mining pit-lakes, contributing to the development of sustainable pathways for the integrated production of biofuels and ammonia. In this context, exploring integrated biorefinery systems within a bio-based economy represents an auspicious direction for future research, potentially enhancing the process efficiency and reducing costs. Full article

The environment is undergoing a constant incorporation of new pollutants, which must be eliminated to avoid toxicity problems. Amoxicillin (AMX) is a widely used antibiotic today, and for this reason, it reaches natural media with the consequent environmental risk. Biosorption is an effective and environmentally friendly solution which can be used for the removal of AMX. In the present study, the properties of the living biomass of the microalga Chlamydomonas reinhardtii were studied to determine the capacity of this biomass to remove AMX. This biomass has demonstrated to have good qualities to remove AMX with a maximum capacity of 12.72 ± 0.57 mg g⁻¹. Light was an important factor in increasing the removal capacity of this living biomass by 35.2%. Although this antibiotic underwent spontaneous degradation (unaffected by light), the presence of the biomass increased the amount removed and the removal rate. The amount removed by this biomass in the presence of light was always higher than the amount lost by spontaneous degradation. The kinetics that best adjusted was pseudo-second order. Maximum removal was obtained at pH 6. A point of zero charge and Fourier transform infrared spectrometry were used to characterize the biomass and study the process. Full article

Microalgae interact with mineral particles in an aqueous environment, yet how clay minerals affect physiological processes in algal cells remains unexplored. In this study, we compared the effects of palygorskite (Pal) and montmorillonite (Mt), which respectively represent fibrous and layered clay minerals, on the physiological processes of Chlamydomonas reinhardtii. It was observed that C. reinhardtii responded differently to the treatments of Pal and Mt. The Pal particles bound tightly to and even inserted themselves into cells, resulting in a significant decrease in cell numbers from 27.35 to 21.02 × 10⁷ mL⁻¹. However, Mt was only loosely attached to the cell surface. The photosynthesis in the algal cells was greatly inhibited by Pal, with the rETR_max significantly reduced from 103.80 to 56.67 μmol electrons m⁻²s⁻¹ and the downregulation of IF2CP, psbH and OHP1, which are key genes involved in photosynthesis. In addition, Pal reduced the quantities of proteins and polysaccharides in extracellular polymeric substances (EPSs) and the P uptake by C. reinhardtii when the P level in the culture was 3.15 mg/L. However, no significant changes were found regarding the above EPS components or the amount of P in algal cells upon the addition of Mt. Together, the impacts of fibrous Pal on C. reinhardtii was more profound than those of layered Mt. Full article

Polyamines play a pivotal role in regulating the growth and metabolic adaptation of microalgae, yet their integrative regulatory roles remain underexplored. This review advances a comprehensive perspective of microalgae growth, integrating polyamine dynamics, amino acid metabolism, and redox balance. Polyamines (putrescine, spermidine, and spermine) biology in microalgae, particularly Chlamydomonas reinhardtii, is reviewed, exploring their critical function in modulating cell cycle progression, enzymatic activity, and stress responses through nucleic acid stabilization, protein synthesis regulation, and post-translational modifications. This review explores how the exogenous supplementation of polyamines modifies their intracellular dynamics, affecting growth phases and metabolic transitions, highlighting the complex regulation of internal pools of these molecules. Comparative analyses with Chlorella ohadii and Scenedesmus obliquus indicated species-specific responses to polyamine fluctuations, linking putrescine and spermine levels to important tunable metabolic shifts and fast growth phenotypes in phototrophic conditions. The integration of multi-omic approaches and computational modeling has already provided novel insights into polyamine-mediated growth regulation, highlighting their potential in optimizing microalgae biomass production for biotechnological applications. In addition, genomic-based modeling approaches have revealed target genes and cellular compartments as bottlenecks for the enhancement of microalgae growth, including mitochondria and transporters. System-based analyses have evidenced the overlap of the polyamines biosynthetic pathway with amino acids (especially arginine) metabolism and Nitric Oxide (NO) generation. Further association of the H₂O₂ production with polyamines metabolism reveals novel insights into microalgae growth, combining the role of the H₂O₂/NO rate regulation with the appropriate balance of the mitochondria and chloroplast functionality. System-level analysis of cell growth metabolism would, therefore, be beneficial to the understanding of the regulatory networks governing this phenotype, fostering metabolic engineering strategies to enhance growth, stress resilience, and lipid accumulation in microalgae. This review consolidates current knowledge and proposes future research directions to unravel the complex interplay of polyamines in microalgal physiology, opening new paths for the optimization of biomass production and biotechnological applications. Full article

Molybdenum (Mo) is a vital micronutrient for nearly all living organisms, serving as a cofactor for molybdoenzymes that catalyze essential redox reactions in nitrogen metabolism. Among these enzymes, nitrate reductase plays a crucial role in nitrate assimilation. Maintaining Mo homeostasis—including uptake, storage, and utilization—is critical to avoid both deficiency and toxicity. Our research focuses on uncovering novel molecular components involved in Mo homeostasis, particularly in connection with nitrate assimilation, using Chlamydomonas reinhardtii, a model green microalga. To achieve this, we generated more than 5000 Chlamydomonas transformants through insertional mutagenesis using a paromomycin resistance cassette (AphVIII) and screened them for altered growth on nitrate and under different Mo concentrations. We identified four strains showing altered growth patterns when using nitrate as a nitrogen source or exhibiting increased sensitivity or resistance to Mo. The genomic alterations in these strains were identified. Notably, both a Mo-resistant and a Mo-sensitive transformant had disruptions in the genes that encoded ABC-type transport proteins, indicating a potential role for these proteins in Mo transport. Additionally, two strains were unable to grow on nitrate. One of them had a mutation in the CNX7, a gene involved in Mo cofactor biosynthesis, while the other had a mutation in BAT1, an amino acid transporter. The BAT1 mutant represents an interesting case study, as this gene has not previously been associated with nitrate metabolism. These findings enhance our understanding of Mo and nitrate homeostasis mechanisms and open new paths for engineering microalgae with improved nitrogen assimilation. Full article

Microalgae have evolved a diverse carotenoid profile, enabling efficient light harvesting and photoprotection. Previous studies have demonstrated the feasibility of genome editing in the green algal model species Chlamydomonas reinhardtii, leading to significant modifications in carotenoid accumulation. By overexpressing a fully redesigned β-carotene ketolase (bkt), the metabolic pathway of C. reinhardtii was successfully redirected toward astaxanthin biosynthesis, a high-value ketocarotenoid with exceptional antioxidant properties, naturally found in only a few microalgal species. In this study, a tailor-made double knockout targeting lycopene ε-cyclase (LCYE) and zeaxanthin epoxidase (ZEP) was introduced as a background for bkt expression to ensure higher substrate availability for bkt enzyme. The increased zeaxanthin availability resulted in a 2-fold increase in ketocarotenoid accumulation compared to the previously engineered bkt1 or bkt5 strain in the UVM4 background. Specifically, the best Δzl-bkt-expressing lines reached 2.84 mg/L under low light and 2.58 mg/L under high light, compared to 1.74 mg/L and 1.26 mg/L, respectively, in UVM4-bkt strains. These findings highlight the potential of rationally designed microalgal host strains, developed through genome editing, for biotechnological applications and high-value compound production. Full article

The possible regulatory effects of C2H2 zinc finger proteins, which are important transcription factors, on intertidal seaweed responses to abiotic stress are unclear. This study was conducted to comprehensively analyze the C2H2 gene family of a representative intertidal seaweed species (Pyropia haitanensis) and clarify its genomic characteristics and biological functions. A total of 107 PhC2H2 zinc finger protein-encoding genes distributed on five P. haitanensis chromosomes were identified and divided into three subgroups. The expression levels of 85, 61, 58, 45, and 41 PhC2H2 genes responded in the maturation of filaments, high-temperature, salt, low-irradiance, and dehydration stress, respectively. The PhC2H2 gene family was conserved during Porphyra evolution, with no indications of large-scale genome-wide replication events. On average, PhC2H2 genes had more transposable element (TE) insertions than Pyropia yezoensis and Porphyra umbilicalisC2H2 genes, suggesting that TE insertions may have been the main driver of PhC2H2 gene family expansion. A key gene (PhC2H2.94) screened following a quantitative trait locus analysis was significantly responsive to high-temperature stress and was associated with photosynthesis, peroxisomes, the ubiquitin proteasome pathway, and the endoplasmic reticulum-related protein processing pathway, which contribute to the stress tolerance of P. haitanensis. Additionally, PhC2H2.94 transgenic Chlamydomonas reinhardtii exhibited increased tolerance to heat stress. This study provides new insights and genetic resources for characterizing the molecular mechanism underlying intertidal seaweed responses to abiotic stresses and breeding stress-resistant macroalgae. Full article

Understanding triacylglycerol (TAG) metabolism is crucial for developing algae as a source of biodiesel. TAGs are the main reservoir of energy in most eukaryotes. The final, rate-limiting step in the formation of TAGs is catalyzed by 1,2-diacylglycerol acyltransferases (DGATs). In the green alga Chlamydomonas reinhardtii, DGAT3 is phylogenetically related to plant DGAT3 but unrelated to other DGATs from eukaryotes, such as DGAT1 and DGAT2. In this study, we described the conformational preferences and the lipid-binding features of the DGAT3 from C. reinhardtii. To characterize its conformational stability and structural features, we used several biophysical probes, namely, fluorescence, circular dichroism (CD), and differential scanning calorimetry (DSC). Our results showed that the protein was mainly disordered, containing a small population of folded conformations in a narrow pH range (pH 8 to 10). The conformational stability of the folded structure of DGAT3 was very low, as shown by urea or guanidinium denaturations. Thermal denaturation, followed by fluorescence or CD, as well as calorimetric denaturation, followed by DSC, did not yield any transition in the pH range where DGAT3 acquired a “native-like” conformation. Furthermore, we used two approaches to demonstrate the interaction of DGAT3 with lipid membranes at the pH at which it had acquired a “native-like” conformation. The first involved the measurement of anisotropy and fluorescence quenching of the protein. The second approach focused on examining possible modifications of the biophysical properties of lipids due to their interaction with DGAT3, through anisotropy measurements and leakage assays. Both methods produced consistent results, suggesting that DGAT3 preferentially interacted with negatively charged membranes. These results will allow the design of a more efficient and stable DGAT3, as well as an in-depth understanding of how the metabolism of TAGs is accomplished in C. reinhardtii. Full article

In recent times, microalgae have emerged as powerful hosts for biotechnological applications, ranging from the production of lipids and specialized metabolites (SMs) of pharmaceutical interest to biofuels, nutraceutical supplements, and more. SM synthesis through bioengineered pathways relies on the availability of aromatic amino acids (AAAs) as an essential precursor. AAAs, phenylalanine, tyrosine, and tryptophan are also the building blocks of proteins, maintaining the structural and functional integrity of cells. Hence, they are crucial intermediates linking the primary and specialized metabolism. The biosynthesis pathway of AAAs in microbes and plants has been studied for decades, but not much is known about microalgae. The allosteric control present in this pathway has been targeted for metabolic engineering in microbes. This review focuses on the biosynthesis of AAAs in eukaryotic microalgae and engineering techniques for enhanced production. All the putative genes involved in AAA pathways in the model microalgae Chlamydomonas reinhardtii and Phaeodactylum tricornutum are listed in this review. Full article

Alkaline environments such as alkaline lands, lakes, and industrial wastewater are not conducive to the growth of plants and microorganisms due to high pH and salinity. ChbZIP1 is a bZIP family transcription factor isolated from an alkaliphilic microalgae (Chlorella sp. BLD). Previous studies have demonstrated its ability to enhance alkaline tolerance in Arabidopsis thaliana. However, the potential of ChbZIP1 to confer similar alkaline tolerance in other microalgae remains unclear, and the specific mechanisms are not fully understood. The analysis of cellular physiological and biochemical indicators revealed that the ChbZIP1 transformants exhibited enhanced photosynthetic activity, increased lipid accumulation, and reduced fatty acid unsaturation. Genes associated with cellular reactive oxygen species (ROS) detoxification were found to be upregulated, and a corresponding increase in antioxidant enzyme activity was detected. In addition, the relative abundance of intracellular ROS and malondialdehyde (MDA) was significantly lower in the transformants. In summary, our research indicates that ChbZIP1 enhances the tolerance of Chlamydomonas reinhardtii to alkaline environments through several mechanisms, including the repair of damaged photosynthesis, increased lipid accumulation, improved fatty acid unsaturation, and enhanced antioxidant enzyme activity. This study aims to contribute to a more comprehensive understanding of the mechanisms underlying alkalinity tolerance in microalgae and offers new insights and theoretical foundations for the utilization of microalgae in alkaline environments. Full article

The green unicellular algae Chlamydomonas reinhardtii contains 12–13 carbonic anhydrases (CAs). For a long time, the two closely related α-CAs of the periplasmic membrane CAH1 and CAH2 were considered to be the CAs with the highest CO₂ hydration activity. The recombinant protein α-CA CAH3 (rCAH3) from the thylakoid lumen obtained in the present study showed more than three times higher activity compared to CAH1 and more than 11 times higher compared to previous studies with rCAH3. Long-term sustainability of the enzyme was observed at alkaline pH (>8), with maintenance of half of its activity at 4 °C for up to 50 days. Thermostability of rCAH3 indicated the retention of the activity at 20 °C for one hour at pH 9–10 with its ~50% decrease at pH 6–7. However, the residual activity of rCAH3 after incubation at an extremely high temperature (75 °C) for 15 min led to the formation of the double-hump graph with maxima at pH 6 and 9. The enzyme demonstrated high sensitivity to ethoxyzolamide and acetazolamide at nM concentrations, to Zn²⁺ and Cu²⁺ cations at 1 mM concentrations, and L-cysteine was able to completely inhibit CA activity of rCAH3 through reduction of sulfhydryl groups. Esterase activity of rCAH3 was well detected with values comparable to those of bovine CAII, but with a maximum at pH 8 instead of pH 9, which is usual for bovine CAII. The results indicated that CAH3 may be the most active CA of C. reinhardtii and that its role in the photosynthetic apparatus function could have been underestimated in previous works. Full article

Stress on the Endoplasmic reticulum (ER) can severely disrupt cellular function by impairing protein folding and post-translational modifications, thereby leading to the accumulation of poor-quality proteins. However, research on its impact on photosynthesis remains limited. In this study, we investigated the impact of ER stress on the photosynthetic efficiency of Chlamydomonas reinhardtii using pharmacological inducers, tunicamycin (TM) and brefeldin A (BFA), which specifically target the ER. Our measurements of photosynthetic parameters showed that these ER stress-inducing compounds caused a significant decline in photosynthetic efficiency. A proteomic analysis confirmed that TM and BFA effectively induce ER stress, as evidenced by the upregulation of ER stress-related proteins. Furthermore, we observed a widespread downregulation of photosynthesis-related proteins, which is consistent with the results obtained from our measurements of photosynthetic parameters. These findings suggest that the stress on ER has a profound impact on chloroplast function, disrupting photosynthetic processes. This study highlights the critical interdependence between the ER and chloroplasts, and it underscores the broader implications of ER stress on the cellular metabolism and energy efficiency of photosynthetic organisms. Full article

Heavy metals, such as Pb(II), Cr(III), and Cd(II), are frequently discharged into the environment, resulting in a threat for the health of living organisms. Microalgae represent an ecological method for the removal of these pollutants. The capture capability of active and dried Chlamydomonas reinhardtii towards both mono-ionic and multi-ionic solutions is evaluated to study the bioremediation in low-polluted wastewaters. Several characterization techniques have been performed to study morphology, composition, and the interaction between the metals and the microalgal functional groups of the wall. The effect of the operating conditions, such as contact time and pH, is assessed; an increased contact time of 4 days did not influence the adsorption yields of Pb and Cr, but it had a negative effect on Cd adsorption, while the impact of pH was mainly affecting the chemical speciation, rather than the adsorption capability. The microalga showed a larger affinity for Pb and Cd, respectively, 80 and 76% (w/w) of adsorption, while only the 65% (w/w) of the total Cr was adsorbed, suggesting a preferential biosorption. Full article