Astaxanthin, one of the most powerful natural antioxidants, is used in high-value industries such as those of cosmetics, nutraceuticals, and food products derived from microalgae. In this study, Ettlia sp. mutants were generated by expressing two types of heterologous orange proteins, IbOr and IbOr-R96H, in which the 96th arginine of IbOr was substituted with histidine derived from Ipomoea batatas to enhance astaxanthin production. The Ett-IbOr-R96H mutant showed a 2.4-fold increase in β-carotene content compared to the wildtype (4.59 mg g−1DCW), reaching 10.82 mg g−1 under high-light conditions via two-phase cultivation. Under the stress treatment combination of high-light intensity and nitrogen deprivation, total carotenoid content increased to 17.24 mg L−1 and 21.94 mg L−1 in Ett-IbOr and Ett-IbOr-R96H, respectively. The astaxanthin and canthaxanthin contents in Ett-IbOr-R96H was 4.89 mg L−1 and 0.47 mg L−1, respectively, which were 1.8- and 1.5-fold higher, respectively, than those in Ett-IbOr. Additionally, photosynthetic efficiency (Fv/Fm) recovered in Ett-IbOr-R96H under dual-stress conditions compared to the wildtype while reactive oxygen species levels decreased throughout the cultivation period. Our findings suggest that the heterologous IbOr expression in Ettlia sp. may be an effective approach for enhancing the production of ketocarotenoids and improving stress resistance for industrial applications.

CRISPR/Cas-based technologies have revolutionized biology, offering a wide range of gene editing and engineering applications due to their diverse enzyme characteristics. Among the CRISPR/Cas nucleases, Cas9, and more recently, Cas12a (formerly known as Cpf1), have been employed in various gene editing applications in many eukaryotes, including the model green alga Chlamydomonas reinhardtii. To provide a comprehensive picture of their applicability in single-strand templated DNA repair and gene editing, we first mapped their target space by analysing the corresponding PAM frequencies, and then compared Cas9 and Cas12a activities by targeting overlapping regions at three independent loci in the Chlamydomonas genome. We identified 8 and 32 times more target sites for Cas9 compared to Cas12a within promoter regions and coding sequences, respectively. We found that Cas9 and Cas12a RNPs- co-delivered with ssODN repair templates- induced similar levels of total editing, achieving as much as 20–30 % in all viably recovered cells. Importantly, the level of precision editing was slightly higher for Cas12a. In contrast, Cas9 alone was able to induce more edits at the FKB12 locus than its Cas12a counterpart, overall making Cas9 the preferable enzyme for genome engineering among the currently available nucleases in C. reinhardtii.

Although, native microalgae (MA) grows in alkaline environments, there is a lack of information from previous studies on the separation of microalgae culture under alkaline coagulation-flocculation-sedimentation (CFS) conditions. This study evaluated the separation efficiency of tannin (TA), Moringa oleifera seed extract (MOSE) natural coagulants in comparison with Aluminum sulfate (AS) for harvesting MA grown in anaerobically digested sanitary wastewater in a pilot flat panel photobioreactor in outdoor environment. The aim was to establish a pathway for recovery of microalgae biomass and supernatant without pH control and save cost for pH adjustment chemicals, in alignment with the circular economy concepts. Total suspended solids (TSS), turbidity and MA concentration (optical density – OD) were monitored throughout the tests. Optimum dosages of TA (1100 mg L−1), MOSE (3000 mg L−1) and AS (320 mg L−1) determined from jar tests were evaluated after MA cultivation, with natural pH of 10.4 under CFS condition (Coagulation: velocity gradient (Gf) of 200 s−1 for 2 min, flocculation: Gf of 10 s−1 for 15 min and sedimentation: 10 min observation time). TA and AS presented similar high removal efficiencies for turbidity (≥ 95 %), OD (≥ 87 %) and TSS (≥ 62 %). However, TA recorded a good pH (7.6) for the supernatant compared to an unsatisfactorily low 5.2 for AS. TA presented the potential of harvesting MA biomass without prior pH control and without adversely impacting the medium's pH. This shows that biomass has a potential usage as a biofertiliser and as well the resultant supernatant is reusable for non-potable purposes.

Ascorbate (Asc) is an important antioxidant that also participates in various biological processes in plants such as hormone metabolism, stress response and signaling pathways. Asc is also a vital vitamin for human health and enriching its content through biofortification is a desirable objective. Therefore, reliable in situ methods for assessing Asc levels are essential. However, most of the existing fluorescent probes for Asc detection are limited to liquid samples, such as human sera or plant extract, or require sophisticated techniques and equipment for in-cell detection, such as photo-induced electron transfer or time-gated luminescence microscopy. Moreover, many of these probes are not cell wall permeable and cannot be used in plants or algal cells. In this article, we introduce a reaction-based, Asc probe – AP-Cyan, that can efficiently and qualitatively detect Asc in various microalgal cultures, including Chlamydomonas reinhardtii, Chlorella sorokianiana and Parachlorella kessleri. The probe is simple to use and produces fast results that can be observed with standard fluorescence microscopes with basic blue, green and red filters. The probe has an emission range (λem = 488 nm) that does not overlap with chlorophyll autofluorescence, making it suitable for algal cells. Thus, our probe offers a simple and powerful method to detect Asc in microalgal cells.

Aquaculture of the red seaweed genus Asparagopsis is rapidly expanding due to its potential to mitigate enteric methane emissions from cattle rumen. These developments include sea-based culture of gametophytes and land-based culture of tetrasporophytes. Successful establishment of land-based aquaculture relies on providing species-specific optimal conditions, including stocking density of the culture. However, an understanding of the gene expression across the Asparagopsis holobiont at different stocking densities is lacking. Here, we applied a meta-transcriptomic approach to investigate the gene expression of Asparagopsis taxiformis tetrasporophytes and their associated microbiome at different stocking densities ranging from 0.18 g FW L−1 to 5 g FW L−1. Overall, the specific growth rate of A. taxiformis decreased as stocking density increased (from 10.7 % day−1 to −0.2 % day−1). The highest density was characterised by downregulation of photosynthesis-related genes and upregulation of some putative defense-related genes in the seaweed. The microbial transcript abundance was positively correlated to stocking density, with prominent activity of Actinomycetes as seaweed growth rate slowed. Additionally, several microbial virulence factors such as OmpA were upregulated in the highest seaweed density. Altogether, our transcriptomic data suggested the onset of senescence in A. taxiformis cultured at the highest stocking density, although the appearance of the filaments was visibly unaffected. This “hidden” senescence was accompanied by a shift to virulence in the associated microbiome. The meta-transcriptome could therefore be used as a tool to monitor system health for commercial Asparagopsis farms. This work also demonstrates the potential of employing omics to assess optimal culture conditions of any seaweed species.

Egyptian seaweeds contain diverse bioactive compounds, but our understanding of their antimicrobial potential, particularly against multidrug-resistant (MDR) human pathogens, is still limited and underestimated. This study screened the antimicrobial potential of ethanolic extracts of Caulerpa racemosa, Dictyopteris polypodioides, and Padina pavonica against various MDR bacterial and fungal strains using broth microdilution technique. Antibiofilm assay of the seaweed extracts against Pseudomonas aeruginosa clinical isolates was also investigated. Our findings showed that the minimum inhibitory concentrations (MICs) of the tested seaweed extracts ranged between 62 and 500 μg mL−1. P. pavonica displayed the most biofilm inhibition percentages against P. aeruginosa clinical isolate by 31.2 % and 62.5 % for BIC50 and BIC90, respectively, as comparing to D. polypodioides and C. racemosa. Liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS) metabolic profiling of P. pavonica ethanolic extract was characterized. Twenty-seven metabolites were identified and checked for their inhibitory biofilm formation based on molecular docking simulation on LasR protein-specific quorum sensing. Interestingly, squalene (4), hydroperoxy-24 vinyl-24 cholesterol (18), fucoxanthol (21), flavoxanthin (22), and fucoxanthin (23) exhibited adequate interaction energies and formed significant interactions inside the LasR active site, supporting them to possess potent antibiofilm properties. Overall, P. pavonica can be recommended as powerful agent of antibiofilm preparation.

Fucus vesiculosus is a rich source of bioactive substances with many biochemical functions that provide it a variety of biological effects. Over the years, significant research efforts have been made to extract bioactive compounds by applying different methodologies for various applications. There are several solvents used for the extraction of natural products since the choice of solvent must be based primarily on the characteristics of the matrices and the properties of the molecular classes to be obtained. Therefore, the aim of this study was to investigate the efficiency of different conventional solvents to maximize the yield of polyphenol and flavonoid content as well as the antioxidant, antimicrobial and anti-inflammatory capacity. The different extracts of F. vesiculosus were analyzed for the Total Polyphenol Content (TPC) and the Total Flavonoid Content (TFC). As well the antioxidant, antimicrobial, and anti-inflammatory capacities were evaluated. The results concerning the content of bioactive molecules disclosed that the extraction carried out with the methanol (50 %) was the one that gave the highest yield in both polyphenol (2.27 ± 0.17 mg GAE/ 50 mg of sample) and flavonoid content (187.12 ± 12.86 mg CE/50 mg of sample) compared to acetone and ethanol extracts. Regarding the functional properties, the results obtained disclose that the extract of F. vesiculosus had a high antioxidant capacity (90 % inhibition of radical scavenging activity). Additionally, the growth inhibition assay disclosed that F. vesiculosus can reduce significantly (p < 0.05) the growth of E. coli F18+, in particular when the alga is extracted with methanol and acetone. As well, a concentration of 1 mg/ml of F. vesiculosus inhibits the protein denaturation by 60 %, highlighting a potential anti-inflammatory activity. In conclusion, this study discloses the richness of bioactive molecules in F. vesiculosus and the resulting functional properties, highlighting also the power of methanol as extraction solvent.

The optimal temperature and irradiance to maximise oxygen production were 39.3 °C and 512.5 μmol photons·m−2·s−1, respectively. These values were obtained by photorespirometry, which is a quick method to measure the photosynthetic and respiration rates of microalgae at a laboratory scale. With these conditions, the global oxygen production rate of S. almeriensis was 246.23 mgoxygen·gbiomass−1·h−1. When the culture temperature was controlled at 39.3 °C for 1 h per day, the daily oxygen production capacity of S. almeriensis increased from 3129.5 mgoxygen·gbiomass−1 to 3778.5 mgoxygen·gbiomass−1. However, keeping the temperature at 39.3 °C for a longer time caused a damage to the photosynthetic apparatus. This was validated using laboratory-scale bubble columns. The damage was reversible when heating the cells for <2 h, but keeping the temperature of the culture at 39.3 °C for 3 h led to an irreversible damage and a 6 % decrease in the photosynthetic performance. Controlling the overheating of microalgal cultures is crucial to maximise growth. In addition, the duration of the exposure to high temperatures should also be included into growth and taken in consideration.

A significant increase in atmospheric greenhouse gases over the last century has led to the development of several methods and technologies to remove carbon dioxide (CO2). Microorganisms produce carbonate minerals through the natural mineralization of CO2; however, the feasibility of this process remains in research. This work aimed to study the cultivation of different microalgae under alkaline pH to maintain their potential for carbon mitigation. According to the results, the highest carbonic anhydrase activity has been reached (4.64 mg/g) for Chlamydomonas reinhardtii at a pH of 10. C. reinhardtii, at a pH of 9.5, yielded the highest chlorophyll content (23.58 mg/g), while Spirulina at a pH of 8.5 produced the highest biomass (882.9 mg/L). Also, a positive correlation existed between pH and lipid content for C. reinhardtii. Spirulina, however, exhibits the opposite effect. According to a principal component analysis, there is an opposite relationship between pH and carbonic anhydrase (CA) activity for C. reinhardtii and filamentous-type cyanobacteria from Salda Lake. The following order of the suitability of the microalgae species for high carbon capture is determined by the Analytic hierarchy process method: Spirulina>C. reinhardtii > Chlorella vulgaris > Coccus-type cyanobacteria > Filamentous-type cyanobacteria from Salda Lake. Additionally, this study provided important results regarding the cyanobacteria species isolated from an alkaline lake, Lake Salda. This would contribute to future studies of carbon capture and carbon mitigation mechanisms.