Évellin Do Espirito Santo

This study assessed the effects of aqueous extracts from turmeric (Curcuma longa), mint (Mentha sp.), grape peel (Vitis sp.), and mango peel (Mangifera indica) on the growth and total carotenoid content of the microalga Dunaliella salina. A two-stage cultivation strategy was employed, in which aqueous extracts prepared by decoction were added at 0.5% (v/v) either at the inoculation or after the exponential growth phase. Microalgal growth was monitored by direct cell counting, and total carotenoid content was determined spectrophotometrically in the harvested biomass. The chemical profiles of the extracts were characterised by HPLC-MS/MS, enabling the annotation of key phenolic and flavonoid compounds. Specific orange turmeric and mint formulations enhanced microalgal cell proliferation by up to 31.88% and 28.95%, respectively, compared to the control. However, despite the higher biomass, the total carotenoid content did not increase correspondingly; orange turmeric extract even led to reductions of 90.84 to 92.86% in carotenoid content. These findings suggest that exogenous antioxidants may suppress the oxidative stress signals required to trigger carotenoid biosynthesis in D. salina. Additionally, grape peel extracts prepared at higher temperatures promoted better algal growth than those obtained at lower temperatures. These findings highlight the dual role of plant extracts in modulating microalgal growth and secondary metabolism, demonstrating that antioxidant-rich extracts may be used to enhance biomass accumulation before the subsequent induction of stress-mediated carotenogenesis, thereby potentially improving biotechnological yields.

The discovery of a new extremophile alga, Chlamydomonas pacifica , provides an opportunity to expand on heterologous protein expression beyond the traditional Chlamydomonas reinhardtii. C. pacifica is a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown that C. reinhardtii can secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in the C. pacifica algae biomass. Graphical

Bioprospecting algae strains with tolerance to extreme conditions such as pH, temperature, salinity, and light is crucial for advancing biotechnology and environmental applications. However, traditional screening methods often involve significant costs and labor, restricting their accessibility and practical use. In this study, we developed and validated low-cost, high-throughput screening techniques, predominantly employing agar plates and liquid culture assays, to effectively differentiate tolerance levels among various algae strains. The methodologies were optimized using the model microalga Chlamydomonas reinhardtii and its closely related species Chlamydomonas incerta and the recently discovered extremophilic Chlamydomonas pacifica . We systematically evaluated the algae for tolerance to extremes by establishing precise gradients of pH (acidic to alkaline conditions), salinity (0 to 5 M NaCl), temperature (34–42°C), and light intensity (40 to 2977 μE·m⁻²·s⁻¹). Our results demonstrated that these cost-effective, agar plate-based methods effectively distinguished algae strains exhibiting superior tolerance to extreme environmental conditions. These screening techniques not only provided clear differentiation among the closely related strains but also delivered reproducible outcomes suitable for scaling up to larger bioprospecting efforts. Furthermore, the affordability and simplicity of these methods facilitate their implementation in resource-limited laboratories, thereby broadening participation in algae bioprospecting endeavors. This study highlights the potential of low-cost, accessible screening techniques to significantly enhance the discovery and characterization of algal strains with extreme traits. Ultimately, these methods support the development of robust algae-based resources, driving innovation in diverse industrial processes and environmental solutions. Graphical

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica , an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wildtype. However, CpaPSR1 increased lipid accumulation compared to wildtype in normal media despite causing no effect under nitrogen depravation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production. Graphical Created with BioRender.com .

Harnessing and controlling the localization of recombinant proteins is essential for advancing synthetic biology, industrial biotechnology, and drug delivery. This study presents a dual system for protein anchoring and controlled release in Chlamydomonas reinhardtii . Using truncated variants of the GP1 glycoprotein fused to the plastic-degrading enzyme PHL7, we identified the PPSPX motif as critical for anchoring proteins to the cell wall. Constructs with increased PPSPX content exhibited reduced secretion but enhanced anchoring, revealing key anchor-signal sites within GP1. To enable controlled release, we incorporated a pH-sensitive intein from derived from Mycobacterium tuberculosis RecA. Under acidic conditions (pH 5.5), this intein efficiently cleaved and released mCherry and PHL7 from the GP1 anchor. Fluorescence kinetics demonstrated significant mCherry release from the mCherry-intein-GP1 construct within 4 hours at pH 5.5, while release was minimal at pH 8.0 and negligible for the mCherry-GP1 control. Western blot analysis confirmed efficient cleavage, showing a lower band corresponding to free mCherry at pH 5.5 and no release at pH 8.0. This anchor-release strategy integrates glycomodules with pH-sensitive inteins to enable precise spatial and temporal protein control. The system offers broad utility for targeted drug delivery, environmental biosensing, and biocatalyst deployment. Overall, we establish a versatile framework for optimizing protein localization and environmentally triggered release in C. reinhardtii , with broad implications for proteomics, biofilm engineering, and scalable therapeutic delivery systems. Graphical

Chlamydomonas incerta , a genetically close relative of the model green alga Chlamydomonas reinhardtii , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii , C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii . For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta ’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology. GRAPHICAL

Taking into account the worrying scenario of water scarcity, it is essential to enable more efficient technologies for wastewater treatment. Wastewater may be treated by conventional biological processes that remove pathogenic organisms, particulate and soluble organic compounds, and other components. However, secondary effluents from treatment plants may still contain toxic elements or high concentrations of inorganic nutrients (mainly nitrogen and phosphorus), which enable the growth of photosynthetic microorganisms in water bodies, resulting in eutrophication. In this context, cultivation of photosynthetic microorganisms in secondary wastewater from sewage treatment allows the removal of nutrients from such wastewater, reducing the possibility of eutrophication. Moreover, microalgal biomass, produced in this tertiary wastewater treatment, may be harvested by different methods with the potential for different applications, such as fertilizer and biofuel.

The applications of microalgae biomass have been widely studied worldwide. The classical processes used in outdoor cultivations of microalgae, in closed or open photobioreactors, occur in the presence of bacteria. Understanding how communication between cells occurs through quorum sensing and evaluating co-cultures allows the production of microalgae and cyanobacteria to be positively impacted by bacteria, in order to guarantee safety and profitability in the production process. In addition, the definition of the effects that occur during an interaction, promotes insights to improve the production of biomolecules, and to develop innovative products. This review presents the interactions between microalgae and bacteria, including compounds exchanges and communication, and addresses the development of new pharmaceutical, cosmetic and food bioproducts from microalgae based on these evaluations, such as prebiotics, vegan skincare products, antimicrobial compounds, and culture media with animal free protein for producing vaccines and other biopharmaceutical products. The use of microalgae as raw biomass or in biotechnological platforms is in line with the fulfillment of the 2030 Agenda related to the Sustainable Development Goals (SDGs).