Advances in euglenoid genomics: unravelling the fascinating biology of a complex clade.

Euglenids have long been studied due to their unique physiology and versatile metabolism, providing underpinnings for much of our understanding of photosynthesis and biochemistry, and a growing opportunity in biotechnology. Until recently there has been a lack of genetic studies due to their large and complex genomes, but recently new technologies have begun to unveil their genetic capabilities. Whilst much research has focused on the model organism Euglena gracilis, other members of the euglenids have now started to receive due attention. Currently only poor nuclear genome assemblies of E. gracilis and Rhabdomonas costata are available, but there are many more plastid genome sequences and an increasing number of transcriptomes. As more assemblies become available, there are great opportunities to understand the fundamental biology of these organisms and to exploit them for biotechnology.

Strategies for producing high value small molecules in microalgae.

Eukaryotic microalgae are a diverse group of organisms that can be used for the sustainable production of a wide range of high value compounds, including lipids, flavours and dyes, bioplastics, and cosmetics. Optimising total biomass production often does not lead to optimal product yield and more sophisticated biphasic growth strategies are needed, introducing specific stresses to induce product synthesis. Genetic tools have been used to increase yields of natural products or to introduce new pathways to algae, and wider deployment of these tools offers promising routes for commercial production of high value compounds utilising minimal inputs.

Microalgae to biodiesel: A novel green conversion method for high-quality lipids recovery and in-situ transesterification to fatty acid methyl esters.

Greenhouse gases (GHGs) emissions due to increasing energy demand have raised the need to identify effective solutions to produce clean and renewable energy. Biotechnologies are an effective platform to attain green transition objectives, especially when synergically integrated to promote health and environmental protection. In this context, microalgae-based biotechnologies are considered among the most effective tools for treating gaseous effluents and simultaneously capturing carbon sources for further biomass valorisation. The production of biodiesel is regarded as a promising avenue for harnessing value from residual algal biomass. Nonetheless, the existing techniques for extracting lipids still face certain limitations, primarily centred around the cost-effectiveness of the process.This study is dedicated to developing and optimising an innovative and cost-efficient technique for extracting lipids from algal biomass produced during gaseous emissions treatment based on algal-bacterial biotechnology. This integrated treatment technology combines a bio-scrubber for degrading gaseous contaminants and a photobioreactor for capturing the produced CO2 within valuable algal biomass. The cultivated biomass is then processed with the process newly designed to extract lipids simultaneously transesterificated in fatty acid methyl esters (FAME) via In Situ Transesterification (IST) with a Kumagawa-type extractor. The results of this study demonstrated the potential application of the optimised method to overcome the gap to green transition. Energy production was obtained from residuals produced during the necessary treatment of gaseous emissions. Using hexane-methanol (v/v = 19:1) mixture in the presence KOH in Kumagawa extractor lipids were extracted with extraction yield higher than 12% and converted in fatty acid methyl esters. The process showed the enhanced extraction of lipids converted in bio-sourced fuels with circular economy approach, broadening the applicability of biotechnologies as sustainable tools for energy source diversification.

Breathing life into Mars: Terraforming and the pivotal role of algae in atmospheric genesis.

The Martian environment, characterized by extreme aridity, frigid temperatures, and a lack of atmospheric oxygen, presents a formidable challenge for potential terraforming endeavors. This review article synthesizes current research on utilizing algae as biocatalysts in the proposed terraforming of Mars, assessing their capacity to facilitate Martian atmospheric conditions through photosynthetic bioengineering. We analyze the physiological and genetic traits of extremophile algae that equip them for survival in extreme habitats on Earth, which serve as analogs for Martian surface conditions. The potential for these organisms to mediate atmospheric change on Mars is evaluated, specifically their role in biogenic oxygen production and carbon dioxide sequestration. We discuss strategies for enhancing algal strains' resilience and metabolic efficiency, including genetic modification and the development of bioreactors for controlled growth in extraterrestrial environments. The integration of algal systems with existing mechanical and chemical terraforming proposals is also examined, proposing a synergistic approach for establishing a nascent Martian biosphere. Ethical and ecological considerations concerning introducing terrestrial life to extra-planetary bodies are critically appraised. This appraisal includes an examination of potential ecological feedback loops and inherent risks associated with biological terraforming. Biological terraforming is the theoretical process of deliberately altering a planet's atmosphere, temperature, and ecosystem to render it suitable for Earth-like life. The feasibility of a phased introduction of life, starting with microbial taxa and progressing to multicellular organisms, fosters a supportive atmosphere on Mars. By extending the frontier of biotechnological innovation into space, this work contributes to the foundational understanding necessary for one of humanity's most audacious goals-the terraforming of another planet.

Characterization and Biological Activities of the Ulvan Polysaccharide-Rich Fraction Obtained from Ulva rigida and Ulva pseudorotundata and Their Potential for Pharmaceutical Application.

Seaweed from the genus Ulva (Ulvales, Chlorophyta) has a worldwide distribution and represents a potential biomass source for biotechnological applications. In the present study, we investigated the ulvan polysaccharide-rich fraction (UPRF) isolated from two Ulva species (U. rigida and U. pseudorotundata), naturally occurring on the Spanish Mediterranean coast. Chemical characterization of UPRFs was performed in order to explore the polysaccharides' composition. Biological assessments of UPRFs were compared by antioxidant activity and in vitro toxicity tests in the human cell lines: HCT-116 (colon cancer), G-361 (malignant melanoma), U-937 (leukemia), and HaCaT cells (immortalized keratinocytes). Chemical analysis revealed that both UPRFs presented rhamnose as the major relative sugar constituent, followed by glucose in U. rigida and xylose in U. pseudorotundata. Both also presented glucuronic acid, galactose, ribose, and mannose as the remaining monosaccharides. Similar antioxidant activity was obtained, where we observed increased activity in response to increased polysaccharide concentrations. Both UPRFs presented moderate toxicity against HCT-116 cell lines and a selectivity index ≥ 3, suggesting a good potential for use in pharmaceutical products.

The synthetic future of algal genomes.

Algae are diverse organisms with significant biotechnological potential for resource circularity. Taking inspiration from fermentative microbes, engineering algal genomes holds promise to broadly expand their application ranges. Advances in genome sequencing with improvements in DNA synthesis and delivery techniques are enabling customized molecular tool development to confer advanced traits to algae. Efforts to redesign and rebuild entire genomes to create fit-for-purpose organisms currently being explored in heterotrophic prokaryotes and eukaryotic microbes could also be applied to photosynthetic algae. Future algal genome engineering will enhance yields of native products and permit the expression of complex biochemical pathways to produce novel metabolites from sustainable inputs. We present a historical perspective on advances in engineering algae, discuss the requisite genetic traits to enable algal genome optimization, take inspiration from whole-genome engineering efforts in other microbes for algal systems, and present candidate algal species in the context of these engineering goals.

Evaluation of selected tropical marine microalgal cultures for use in biophotovoltaic platforms.

In this study, the bioelectrical power generation potential of four tropical marine microalgal strains native to Malaysia was investigated using BPV platforms. Chlorella UMACC 258 produced the highest power density (0.108 mW m-2), followed by Halamphora subtropica UMACC 370 (0.090 mW m-2), Synechococcus UMACC 371 (0.065 mW m-2) and Parachlorella UMACC 245 (0.017 mW m-2). The chlorophyll-a (chl-a) content was examined to have a linear positive relationship with the power density (p < 0.05). The photosynthetic performance of strains was studied using the pulse-amplitude modulation (PAM) fluorometer; parameters measured include the following: maximum quantum efficiency (Fv/Fm), alpha (α), maximum relative electron transport rate (rETRmax), photo-adaptive index (Ek) and non-photochemical quenching (NPQ). The Fv/Fm values of all strains, except Synechococcus UMACC 371, ranged between 0.37 and 0.50 during exponential and stationary growth phases, suggesting their general health during those periods. The low Fv/Fm value of Synechococcus UMACC 371 was possibly caused by the presence of background fluorescence from phycobilisomes or phycobiliproteins. Electrochemical studies via cyclic voltammetry (CV) suggest the presence of electrochemically active proteins on the cellular surface of strains on the carbon anode of the BPV platform, while morphological studies via field emission scanning electron microscope (FESEM) imaging verify the biocompatibility of the biofilms on the carbon anode. KEY POINTS: • Maximum power output of 0.108 mW m-2 is recorded by Chlorella UMACC 258 • There is a positive correlation between chl-a content and power output • Proven biocompatibility between biofilms and carbon anode sans exogenous mediators.

Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

Cultivation of the polyextremophile Cyanidioschyzon merolae 10D during summer conditions on the coast of the Red Sea and its adaptation to hypersaline sea water.

The west coast of the Arabian Peninsula borders the Red Sea, a water body which maintains high average temperatures and increased salinity compared to other seas or oceans. This geography has many resources which could be used to support algal biotechnology efforts in bio-resource circularity. However, summer conditions in this region may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiophyceae are a class of polyextremophilic red algae that natively inhabit acidic hot springs. C. merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at elevated temperatures and acidic pH. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, ß-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme coastal desert summer environments.

Membrane inlet-ion mobility spectrometry with automatic spectra evaluation as online monitoring tool for the process control of microalgae cultivation.

The cultivation of algae either in open raceway ponds or in closed bioreactors could allow the renewable production of biomass for food, pharmaceutical, cosmetic, or chemical industries. Optimal cultivation conditions are however required to ensure that the production of these compounds is both efficient and economical. Therefore, high-frequency analytical measurements are required to allow timely process control and to detect possible disturbances during algae growth. Such analytical methods are only available to a limited extent. Therefore, we introduced a method for monitoring algae release volatile organic compounds (VOCs) in the headspace above a bioreactor in real time. This method is based on ion mobility spectrometry (IMS) in combination with a membrane inlet (MI). The unique feature of IMS is that complete spectra are detected in real time instead of sum signals. These spectral patterns produced in the ion mobility spectrum were evaluated automatically via principal component analysis (PCA). The detected peak patterns are characteristic for the respective algae culture; allow the assignment of the individual growth phases and reflect the influence of experimental parameters. These results allow for the first time a continuous monitoring of the algae cultivation and thus an early detection of possible disturbances in the biotechnological process.

Isolation and characterization of algicidal bacteria from freshwater aquatic environments in China.

Algicidal bacteria can inhibit the growth of algae or lyse algal cells, thus playing roles in shaping aquatic microbial communities and maintaining the functions of aquatic ecosystems. Nevertheless, our understanding of their diversities and distributions remains limited. In this study, we collected water samples from 17 freshwater sites in 14 cities in China and screened a total of 77 algicidal bacterial strains using several prokaryotic cyanobacteria and eukaryotic algae as target strains. According to their target-specificities, these strains were classified into three subgroups, cyanobacterial algicidal bacteria, algal algicidal bacteria, and broad-target algicidal bacteria, each displaying distinctive compositions and geographical distribution patterns. They are assigned to Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes bacterial phyla, of which Pseudomonas and Bacillus are the most abundant gram-negative and gram-positive genus, respectively. A number of bacterial strains, such as Inhella inkyongensis and Massilia eburnean, are suggested as new algicidal bacteria. The diverse taxonomies, algal-inhibiting abilities and distributions of these isolates have suggested that there are rich algicidal bacterial resources in these aquatic environments. Our results provide new microbial resources for algal-bacterial interaction studies, and shed new insights into how algicidal bacteria can be used in the control of harmful algal blooms, as well as in algal biotechnology.

The use of marine microalgae in microbial fuel cells, photosynthetic microbial fuel cells and biophotovoltaic platforms for bioelectricity generation.

Algal green energy has emerged as an alternative to conventional energy production using fossil fuels. Microbial fuel cells (MFCs), photosynthetic microbial fuel cells (PMFCs) and biophotovoltaic (BPV) platforms have been developed to utilize microalgae for bioelectricity generation, wastewater treatment and biomass production. There remains a lack of research on marine microalgae in these systems, so to the best of our knowledge, all information on their integration in these systems have been gathered in this review, and are used to compare with the interesting studies on freshwater microalgae. The performance of the systems is extremely reliant on the microalgae species and/or microbial community used, the size of the bio-electrochemical cell, and electrode material and distance used. The mean was calculated for each system, PMFC has the highest average maximum power density of 344 mW/m2, followed by MFC (179 mW/m2) and BPV (58.9 mW/m2). In addition, the advantages and disadvantages of each system are highlighted. Although all three systems face the issue of low power outputs, the integration of a suitable energy harvester could potentially increase power efficiency and make them applicable for lower power applications.

Combinatorial Engineering Enables Photoautotrophic Growth in High Cell Density Phosphite-Buffered Media to Support Engineered Chlamydomonas reinhardtii Bio-Production Concepts.

Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome modification and transgene expression are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we used combinatorial chloroplast and nuclear genome engineering to augment the metabolism of the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as sole sources of phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combined previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products from green microalgae.

Crossing and selection of Chlamydomonas reinhardtii strains for biotechnological glycolate production.

As an alternative to chemical building blocks derived from algal biomass, the excretion of glycolate has been proposed. This process has been observed in green algae such as Chlamydomonas reinhardtii as a product of the photorespiratory pathway. Photorespiration generally occurs at low CO2 and high O2 concentrations, through the key enzyme RubisCO initiating the pathway via oxygenation of 1.5-ribulose-bisphosphate. In wild-type strains, photorespiration is usually suppressed in favour of carboxylation due to the cellular carbon concentrating mechanisms (CCMs) controlling the internal CO2 concentration. Additionally, newly produced glycolate is directly metabolized in the C2 cycle. Therefore, both the CCMs and the C2 cycle are the key elements which limit the glycolate production in wild-type cells. Using conventional crossing techniques, we have developed Chlamydomonas reinhardtii double mutants deficient in these two key pathways to direct carbon flux to glycolate excretion. Under aeration with ambient air, the double mutant D6 showed a significant and stable glycolate production when compared to the non-producing wild type. Interestingly, this mutant can act as a carbon sink by fixing atmospheric CO2 into glycolate without requiring any additional CO2 supply. Thus, the double-mutant strain D6 can be used as a photocatalyst to produce chemical building blocks and as a future platform for algal-based biotechnology. KEY POINTS: • Chlamydomonas reinhardtii cia5 gyd double mutants were developed by sexual crossing • The double mutation eliminates the need for an inhibitor in glycolate production • The strain D6 produces significant amounts of glycolate with ambient air only.

Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome.

Eukaryotic green microalgae represent a sustainable, photosynthetic biotechnology platform for generating high-value products. The model green alga Chlamydomonas reinhardtii has already been used to generate high value bioproducts such as recombinant proteins and terpenoids. However, low, unstable, and variable nuclear transgene expression has limited the ease and speed of metabolic engineering and recombinant protein expression in this system. Here, novel genetic devices for transgene expression in C. reinhardtii have been developed by identifying cis-regulatory DNA elements capable of driving high transgene expression in C. reinhardtii promoters using de novo motif discovery informatics approaches. Thirteen putative motifs were synthesized as concatemers, linked to a common minimal basal promoter, and assayed for their activity to drive expression of a yellow fluorescent protein reporter gene. Following transformation of the vectors into C. reinhardtii by electroporation, in vivo measurements of yellow fluorescent protein expression by flow cytometry revealed that five of the DNA motifs analyzed displayed significantly higher reporter expression compared to the basal promoter control. Two of the concatemerized motifs, despite being much smaller minimal cis-regulatory elements, drove reporter expression at levels approaching that of the conventionally-used AR1 promoter. This analysis provides insight into C. reinhardtii promoter structure and gene regulation, and provides a new toolbox of cis-regulatory elements that can be used to drive transgene expression at a variety of expression levels.

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology.

In the future, algae biotechnology could play an important role in sustainable development, especially with regard to the production of valuable chemicals. Among the established laboratory strains with efficient transgene expression, there are none that have demonstrated the required robustness for industrial applications, which generally require growth at larger scale. Here, we created a robust and mating-competent cell line of the green microalga Chlamydomonas reinhardtii, which also possesses a high transgene expression capacity. This strain shows a comparably high resistance to shear stress by accumulating increased amounts of biomass under these conditions. As a proof-of-concept, a high phototrophic productivity of cadaverine from CO2 and nitrate was demonstrated by efficiently expressing a bacterial l-lysine decarboxylase. In contrast to other established strains, this novel chassis strain for phototrophic production schemes is equipped with the traits required for industrial applications, by combining mating-competence, cell wall-mediated robustness and high level transgene expression.

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting.

The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent nonhomologous end-joining (cNHEJ) and DNA polymerase theta (POLQ)-mediated end joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair (HDR)/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect HDR of the inactivated paromomycin-resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate HDR/gene inactivation. Our data suggest that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced HDR and random integration of transgenes, whereas KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in C. reinhardtii and can be used for future development of algal biotechnology.

Advances in Microalgal Research in Brazil

Abstract Microalgae research has attracted interest worldwide and in order to advance algal biotechnology in Brazil, government has been funding several projects. In the last 10 years, two main funds were provided by the National Council of Scientific and Technological Development (CNPq) agency to researchers in Brazil, who study the potential uses of microalgae for biomass, bioproducts and biofuels production. These funded projects addressed aspects of algal strain identification, development of algal cultivation techniques, designing photobioreactors and raceway ponds, modeling harvesting and dewatering process, maximizing biomass and oil productivities, characterizing chemical composition with different extractions systems and determining physiochemical properties of biodiesel. This review presents the state of art of algal research conducted by Brazilian institutions. Special attention is given to the recent progress on microalgal cultivation, high-value products extracted from microalgae and potential biofuels production. This review may serve as a policy instrument for planning next steps for algal research in Brazil as well as for attracting attention from international researchers who work with microalgae and would like to pursue a future partnership on algal research with Brazilian research institutions.

Emerging Technologies to Enable Sustainable Controlled Environment Agriculture in the Extreme Environments of Middle East-North Africa Coastal Regions.

Despite global shifts in attitudes toward sustainability and increasing awareness of human impact on the environment, projected population growth and climate change require technological adaptations to ensure food and resource security at a global scale. Although desert areas have long been proposed as ideal sites for solar electricity generation, only recently have efforts shifted toward development of specialized and regionally focused agriculture in these extreme environments. In coastal regions of the Middle East and North Africa (MENA), the most abundant resources are consistent intense sunlight and saline sea water. MENA coastal regions hold incredible untapped potential for agriculture driven by the combination of key emerging technologies in future greenhouse concepts: transparent infrared collecting solar panels and low energy salt water cooling. These technologies can be combined to create greenhouses that drive regionally relevant agriculture in this extreme environment, especially when the target crops are salt-tolerant plants and algal biomass. Future controlled environment agriculture concepts will not compete for municipal fresh water and can be readily integrated into local human/livestock/fisheries food chains. With strategic technological implementation, marginal lands in these environments could participate in production of biomass, sustainable energy generation, and the circular carbon economy. The goal of this perspective is to reframe the idea of these environments as extreme, to having incredible untapped development potential.