Influence of salinity, nitrogen and phosphorus concentrations on the physiological and biochemical characteristics of two Chlorophyceae isolated from Fez freshwater, Morocco.

Microalgae are widely exploited for numerous biotechnology applications, including biofuels. In this context, Chlamydomonas debaryana and Chlorococcum sp. were isolated from Fez freshwater (Morocco), and their growth and lipid and carbohydrate production were assessed at different concentrations of NaCl, NaNO3, and K2HPO4. The results indicate a small positive variation in growth parameters linked to nutrient enrichment, with no considerable variation in carbohydrate and lipid levels in both algae. Moreover, a negative variation was recorded at increased salinity and nutrient limitation, accompanied by lipid and carbohydrate accumulation. Chlorococcum sp. showed better adaptation to salt stress below 200 mM NaCl. Furthermore, its growth and biomass productivity were strongly reduced by nitrogen depletion, and its lipid production reached 47.64% DW at 3.52 mM NaNO3. As for Chlamydomonas debaryana, a substantial reduction in growth was induced by nutrient depletion, a maximal carbohydrate level was produced at less than 8.82 mM NaNO3 (40.59% DW). The effect of phosphorus was less significant. However, a concentration of 0.115 mM K2HPO4 increased lipid and carbohydrate content without compromising biomass productivity. The results suggest that growing the two Chlorophyceae under these conditions seems interesting for biofuel production, but the loss of biomass requires a more efficient strategy to maximize lipid and carbohydrate accumulation without loss of productivity.

Fossil-calibrated molecular clock data enable reconstruction of steps leading to differentiated multicellularity and anisogamy in the Volvocine algae.

BACKGROUND: Throughout its nearly four-billion-year history, life has undergone evolutionary transitions in which simpler subunits have become integrated to form a more complex whole. Many of these transitions opened the door to innovations that resulted in increased biodiversity and/or organismal efficiency. The evolution of multicellularity from unicellular forms represents one such transition, one that paved the way for cellular differentiation, including differentiation of male and female gametes. A useful model for studying the evolution of multicellularity and cellular differentiation is the volvocine algae, a clade of freshwater green algae whose members range from unicellular to colonial, from undifferentiated to completely differentiated, and whose gamete types can be isogamous, anisogamous, or oogamous. To better understand how multicellularity, differentiation, and gametes evolved in this group, we used comparative genomics and fossil data to establish a geologically calibrated roadmap of when these innovations occurred. RESULTS: Our ancestral-state reconstructions, show that multicellularity arose independently twice in the volvocine algae. Our chronograms indicate multicellularity evolved during the Carboniferous-Triassic periods in Goniaceae + Volvocaceae, and possibly as early as the Cretaceous in Tetrabaenaceae. Using divergence time estimates we inferred when, and in what order, specific developmental changes occurred that led to differentiated multicellularity and oogamy. We find that in the volvocine algae the temporal sequence of developmental changes leading to differentiated multicellularity is much as proposed by David Kirk, and that multicellularity is correlated with the acquisition of anisogamy and oogamy. Lastly, morphological, molecular, and divergence time data suggest the possibility of cryptic species in Tetrabaenaceae. CONCLUSIONS: Large molecular datasets and robust phylogenetic methods are bringing the evolutionary history of the volvocine algae more sharply into focus. Mounting evidence suggests that extant species in this group are the result of two independent origins of multicellularity and multiple independent origins of cell differentiation. Also, the origin of the Tetrabaenaceae-Goniaceae-Volvocaceae clade may be much older than previously thought. Finally, the possibility of cryptic species in the Tetrabaenaceae provides an exciting opportunity to study the recent divergence of lineages adapted to live in very different thermal environments.

Extraction and separation of astaxanthin with the help of pre-treatment of Haematococcus pluvialis microalgae biomass using aqueous two-phase systems based on deep eutectic solvents.

The microalgae Haematococcus pluvialis are the main source of the natural antioxidant astaxanthin. However, the effective extraction of astaxanthin from these microalgae remains a significant challenge due to the rigid, non-hydrolyzable cell walls. Energy savings and high-efficiency cell disruption are essential steps in the recovery of the antioxidant astaxanthin from the cysts of H. pluvialis. In the present study, H. pluvialis microalgae were first cultured in Bold's Basal medium under certain conditions to reach the maximum biomass concentration, and then light shock was applied for astaxanthin accumulation. The cells were initially green and oval, with two flagella. As the induction time increases, the motile cells lose their flagellum and become red cysts with thick cell walls. Pre-treatment of aqueous two-phase systems based on deep eutectic solvents was used to decompose the cell wall. These systems included dipotassium hydrogen phosphate salt, water, and two types of deep eutectic solvents (choline chloride-urea and choline chloride-glucose). The results of pre-treatment of Haematococcus cells by the studied systems showed that intact, healthy cysts were significantly ruptured, disrupted, and facilitated the release of cytoplasmic components, thus facilitating the subsequent separation of astaxanthin by liquid-liquid extraction. The system containing the deep eutectic solvent of choline chloride-urea was the most effective system for cell wall degradation, which resulted in the highest ability to extract astaxanthin. More than 99% of astaxanthin was extracted from Haematococcus under mild conditions (35% deep eutectic solvent, 30% dipotassium hydrogen phosphate at 50 °C, pH = 7.5, followed by liquid-liquid extraction at 25 °C). The present study shows that the pre-treatment of two-phase systems based on deep eutectic solvent and, thus, liquid-liquid extraction is an efficient and environmentally friendly process to improve astaxanthin from the microalgae H. pluvialis.

Rosetta gen. nov. (Chlorophyta): Resolving the identity of red snow algal rosettes.

Thick-walled rosette-like snow algae were long thought to be a life stage of various other species of snow algae. Rosette-like cells have not been cultured, but by manually isolating cells from 38 field samples in southern British Columbia, we assigned a variety of rosette morphologies to DNA sequence. Phylogenetic analysis of Rubisco large-subunit (rbcL) gene, ribosomal internal transcribed spacer 2 (ITS2) rRNA region, and 18S rRNA gene revealed that the rosette-like cells form a new clade within the phylogroup Chloromonadinia. Based on these data, we designate a new genus, Rosetta, which comprises five novel species: R. castellata, R. floranivea, R. stellaria, R. rubriterra, and R. papavera. In a survey of 762 snow samples from British Columbia, we observed R. floranivea exclusively on snow overlying high-elevation glaciers, whereas R. castellata was observed at lower elevations, near the tree line. The other three species were rarely observed. Spherical red cells enveloped in a thin translucent sac were conspecific with Rosetta, possibly a developmental stage. These results highlight the unexplored diversity among snow algae and emphasize the utility of single-cell isolation to advance the centuries-old problem of disentangling life stages and cryptic species.

Microfluidic impedance cytometry with flat-end cylindrical electrodes for accurate and fast analysis of marine microalgae.

Marine microalgae play an increasingly significant role in addressing the issues of environmental monitoring and disease treatment, making the analysis of marine microalgae at the single-cell level an essential technique. For this, we put forward accurate and fast microfluidic impedance cytometry to analyze microalgal cells by assembling two cylindrical electrodes and microchannels to form a three-dimensional detection zone. Firstly, we established a mathematical model of microalgal cell detection based on Maxwell's mixture theory and numerically investigated the effects of the electrode gap, microalgal positions, and ion concentrations of the solution on detection to optimize detection conditions. Secondly, 80 µm stainless steel wires were used to construct flat-ended cylindrical electrodes and were then inserted into two collinear channels fabricated using standard photolithography techniques to form a spatially uniform electric field to promote the detection throughput and sensitivity. Thirdly, based on the validation of this method, we measured the impedance of living Euglena and Haematococcus pluvialis to study parametric influences, including ion concentration, cell density and electrode gap. The throughput of this method was also investigated, which reached 1800 cells per s in the detection of Haematococcus pluvialis. Fourthly, we analyzed live and dead Euglena to prove the ability of this method to detect the physiological status of cells and obtained impedances of 124.3 Ω and 31.0 Ω with proportions of 15.9% and 84.1%, respectively. Finally, this method was engineered for the analysis of marine microalgae, measuring living Euglena with an impedance of 159.61 Ω accounting for 3.9%, dead Euglena with an impedance of 36.43 Ω accounting for 10.1% and Oocystis sp. with an impedance of 55.00 Ω accounting for about 81.0%. This method could provide a reliable tool to analyze marine microalgae for monitoring the marine environment and treatment of diseases owing to its outstanding advantages of low cost, high throughput and high corrosion resistance.

Effects of ZnWO4 nanoparticles on growth, photosynthesis, and biochemical parameters of the green microalga Raphidocelis subcapitata.

Nanoparticles have applications in many sectors in the society. ZnWO4 nanoparticles (ZnWO4-NPs) have potential in the fabrication of sensors, lasers, and batteries, and in environmental remediation. Thus, these NPs may reach aquatic ecosystems. However, we still do not know their effects on aquatic biota and, to our knowledge, this is the first study that evaluates the toxicity of ZnWO4-NPs in a eukaryotic organism. We evaluated the toxicity of ZnWO4-NPs on the green microalga Raphidocelis subcapitata for 96 h, in terms of growth, cell parameters, photosynthesis, and biochemical analysis. Results show that most of Zn was presented in its particulate form, with low amounts of Zn2+, resulting in toxicity at higher levels. The growth was affected from 8.4 mg L-1, with 96h-IC50 of 23.34 mg L-1. The chlorophyll a (Chl a) content increased at 30.2 mg L-1, while the fluorescence of Chl a (FL3-H) decreased at 15.2 mg L-1. We observed increased ROS levels at 44.4 mg L-1. Regarding photosynthesis, the NPs affected the oxygen evolving complex (OEC) and the efficiency of the photosystem II at 22.9 mg L-1. At 44.4 mg L-1 the qP decreased, indicating closure of reaction centers, probably affecting carbon assimilation, which explains the decay of carbohydrates. There was a decrease of qN (non-regulated energy dissipation, not used in photosynthesis), NPQ (regulated energy dissipation) and Y(NPQ) (regulated energy dissipation via heat), indicating damage to the photoprotection system; and an increase in Y(NO), which is the non-regulated energy dissipation via heat and fluorescence. The results showed that ZnWO4-NPs can affect the growth and physiological and biochemical parameters of the chlorophycean R. subcapitata. Microalgae are the base of aquatic food chains, the toxicity of emerging contaminants on microalgae can affect entire ecosystems. Therefore, our study can provide some help for better protection of aquatic ecosystems.

Non-destructive monitoring of microalgae biofilms.

Biofilm-based cultivation systems are emerging as a promising technology for microalgae production. However, efficient and non-invasive monitoring routines are still lacking. Here, a protocol to monitor microalgae biofilms based on reflectance indices (RIs) is proposed. This framework was developed using a rotating biofilm system for astaxanthin production by cultivating Haematococcus pluvialis on cotton carriers. Biofilm traits such as biomass, astaxanthin, and chlorophyll were characterized under different light and nutrient regimes. Reflectance spectra were collected to identify the spectral bands and the RIs that correlated the most with those biofilm traits. Robust linear models built on more than 170 spectra were selected and validated on an independent dataset. Astaxanthin content could be precisely predicted over a dynamic range from 0 to 4% of dry weight, regardless of the cultivation conditions. This study demonstrates the strength of reflectance spectroscopy as a non-invasive tool to improve the operational efficiency of microalgae biofilm-based technology.

Toxicological effects and mechanisms of lithium on growth, photosynthesis and antioxidant system in the freshwater microalga Chromochloris zofingiensis.

The growing prevalence of lithium (Li) batteries has drawn public attention to Li as an emerging pollutant. The present study investigates the toxicity of Li+ on Chromochloris zofingiensis, examining physiological, biochemical and omics aspects. Results reveal hormesis effects of Li+ on C. zofingiensis growth. At Li+ concentrations below 5 mg L-1, Li+ can enhance chlorophyll content, mitochondrial activity, and antioxidant capacity, leading to increased dry cell weight and cell number. Conversely, when it exceeded 10 mg L-1, Li+ can reduce chlorophyll content, induce oxidative stress, and disrupt chloroplast and mitochondria structure and function, ultimately impeding cell growth. In addition, under 50 mg L-1 Li+ stress, microalgae optimize absorbed light energy use (increasing Fv/Fm and E TR ) and respond to stress by up-regulating genes in starch and lipid biosynthesis pathways, promoting the accumulation of storage components. Weighted gene co-expression network analysis indicates that peptidylprolyl cis/trans isomerase, GTPase and L-ascorbate oxidase might be the key regulators in response to Li+ stress. This research marks the toxic effects and molecular mechanisms of Li+ on freshwater microalga, which would improve our understanding of Li's toxicology and contributing to the establishment of Li pollution standards.

Meta-analysis of transcriptomic profiles in Dunaliella tertiolecta reveals molecular pathway responses to different abiotic stresses.

Microalgae are photosynthetic organisms and a potential source of sustainable metabolite production. However, different stress conditions might affect the production of various metabolites. In this study, a meta-analysis of RNA-seq experiments in Dunaliella tertiolecta was evaluated to compare metabolite biosynthesis pathways in response to abiotic stress conditions such as high light, nitrogen deficiency and high salinity. Results showed downregulation of light reaction, photorespiration, tetrapyrrole and lipid-related pathways occurred under salt stress. Nitrogen deficiency mostly induced the microalgal responses of light reaction and photorespiration metabolism. Phosphoenol pyruvate carboxylase, phosphoglucose isomerase, bisphosphoglycerate mutase and glucose-6-phosphate-1-dehydrogenase (involved in central carbon metabolism) were commonly upregulated under salt, light and nitrogen stresses. Interestingly, the results indicated that the meta-genes (modules of genes strongly correlated) were located in a hub of stress-specific protein-protein interaction (PPI) network. Module enrichment of meta-genes PPI networks highlighted the cross-talk between photosynthesis, fatty acids, starch and sucrose metabolism under multiple stress conditions. Moreover, it was observed that the coordinated expression of the tetrapyrrole intermediated with meta-genes was involved in starch biosynthesis. Our results also showed that the pathways of vitamin B6 metabolism, methane metabolism, ribosome biogenesis and folate biosynthesis responded specifically to different stress factors. Since the results of this study revealed the main pathways underlying the abiotic stress, they might be applied in optimised metabolite production by the microalga Dunaliella in future studies. PRISMA check list was also included in the study.

Impact of macronutrients and salinity stress on biomass and biochemical constituents in Monoraphidium braunii to enhance biodiesel production.

Microalgal lipids are precursors to the production of biodiesel, as well as a source of valuable dietary components in the biotechnological industries. So, this study aimed to assess the effects of nutritional (nitrogen, and phosphorus) starvations and salinity stress (NaCl) on the biomass, lipid content, fatty acids profile, and predicted biodiesel properties of green microalga Monoraphidium braunii. The results showed that biomass, biomass productivity, and photosynthetic pigment contents (Chl. a, b, and carotenoids) of M. braunii were markedly decreased by nitrogen and phosphorus depletion and recorded the maximum values in cultures treated with full of N and P concentrations (control, 100%). These parameters were considerably increased at the low salinity level (up to 150 mM NaCl), while an increasing salinity level (up to 250 mM NaCl) reduces the biomass, its productivity, and pigment contents. Nutritional limitations and salt stress (NaCl) resulted in significantly enhanced accumulation of lipid and productivity of M. braunii, which represented more than twofold of the control. Furthermore, these conditions have enhanced the profile of fatty acid and biodiesel quality-related parameters. The current study exposed strategies to improve M. braunii lipid productivity for biodiesel production on a small scale in vitro in terms of fuel quality under low nutrients and salinity stress.

Growth of Scenedesmus obliquus in anaerobically digested swine wastewater from different cleaning processes for pollutants removal and biomass production.

Anaerobically digested swine wastewater (ASW) purification by microalgae provides a promising strategy for nutrients recovery, biomass production and CO2 capture. However, the characteristics of ASW from different cleaning processes vary greatly. At present, the cultivation of microalgae in ASW from different manure cleaning processes is rarely investigated and compared. That may bring uncertainty for microalgae growth using different ASW in large-scale application. Thus, the ASW from three cleaning processes were tested for cultivating microalgae, including manure dry collection (I), water flushing (II) and water submerging processes (III). The characteristics of ASW from three manure cleaning processes varied greatly such as nutrient and heavy metals levels. High concentration of ammonia and copper in ASW significantly inhibited microalgae growth. Fortunately, the supply of high CO2 (10%) effectively alleviated negative influences, ensuring microalgal growth at low dilution ratio. The characteristics of three ASW resulted in significant differences in microalgae growth and biomass components. The maximal biomass production in optimal diluted ASW-I, II and III reached 1.46 g L-1, 2.19 g L-1 and 2.47 g L-1, respectively. The removal of organic compounds, ammonia and phosphorus by optimal microalgae growth in diluted ASW-I, II and III was 50.6%/94.2%/64.7%, 63.7%/82.3%/57.6% and 83.2%/91.7%/59.7%, respectively. The culture in diluted ASW-I, II and III obtained the highest lipids production of 12.1 mg L-1·d-1, 16.5 mg L-1·d-1 and 19.4 mg L-1·d-1, respectively. The analysis of lipids compositions revealed that the proportion of saturated fatty acids accounted for 36.4%, 32.4% and 27.9 % in optimal diluted ASW-I, II and III, as ideal raw materials for biodiesel production.

The Biosynthesis of Astaxanthin Esters in Schizochytrium sp. is Mediated by a Bifunctional Diacylglycerol Acyltransferase.

Astaxanthin esters are a major form of astaxanthin found in nature. However, the exact mechanisms of the biosynthesis and storage of astaxanthin esters were previously unknown. We found that Schizochytrium sp. synthesized both astaxanthin and docosahexaenoic acid (DHA)-enriched lipids. The major type of astaxanthin produced was free astaxanthin along with astaxanthin-DHA monoester and other esterified forms. DHA accounted for 41.0% of the total fatty acids from astaxanthin monoesters. These compounds were deposited mainly in lipid droplets. The biosynthesis of the astaxanthin esters was mainly carried out by a novel diacylglycerol acyltransferase ScDGAT2-1, while ScDGAT2-2 was involved only in the production of triacylglycerol. We also identified astaxanthin ester synthases from the astaxanthin-producing algae Haematococcus pluvialis and Chromochloris zofingiensis, as well as a thraustochytrid Hondaea fermentalgiana with an unknown carotenoid profile. This investigation enlightens the application of thraustochytrids for the production of both DHA and astaxanthin and provides enzyme resources for the biosynthesis of astaxanthin esters in the engineered microbes.

Features of the microalga Raphidocelis subcapitata: physiology and applications.

The microalga Raphidocelis subcapitata was isolated from the Nitelva River (Norway) and subsequently deposited in the collection of the Norwegian Institute of Water Research as "Selenastrum capricornutum Printz". This freshwater microalga, also known as Pseudokirchneriella subcapitata, acquired much of its notoriety due to its high sensitivity to different chemical species, which makes it recommended by different international organizations for the assessment of ecotoxicity. However, outside this scope, R. subcapitata continues to be little explored. This review aims to shed light on a microalga that, despite its popularity, continues to be an "illustrious" unknown in many ways. Therefore, R. subcapitata taxonomy, phylogeny, shape, size/biovolume, cell ultra-structure, and reproduction are reviewed. The nutritional and cultural conditions, chronological aging, and maintenance and preservation of the alga are summarized and critically discussed. Applications of R. subcapitata, such as its use in aquatic toxicology (ecotoxicity assessment and elucidation of adverse toxic outcome pathways) are presented. Furthermore, the latest advances in the use of this alga in biotechnology, namely in the bioremediation of effluents and the production of value-added biomolecules and biofuels, are highlighted. To end, a perspective regarding the future exploitation of R. subcapitata potentialities, in a modern concept of biorefinery, is outlined. KEY POINTS: • An overview of alga phylogeny and physiology is critically reviewed. • Advances in alga nutrition, cultural conditions, and chronological aging are presented. • Its use in aquatic toxicology and biotechnology is highlighted.

Biomass productivity and characterization of Tetradesmus obliquus grown in a hybrid photobioreactor.

In this study, the effects of CO2 addition on the growth performance and biochemical composition of the green microalga Tetradesmus obliquus cultured in a hybrid algal production system (HAPS) were investigated. The HAPS combines the characteristics of tubular photobioreactors (towards a better carbon dioxide dissolution coefficient) with thin-layer cascade system (with a higher surface-to-volume ratio). Experimental batches were conducted with and without CO2 addition, and evaluated in terms of productivity and biomass characteristics (elemental composition, protein and lipid contents, pigments and fatty acids profiles). CO2 enrichment positively influenced productivity, and proteins, lipids, pigments and unsaturated fatty acids contents in biomass. The HAPS herein presented contributes to the optimization of microalgae cultures in open systems, since it allows, with a simple adaptation-a transit of the cultivation through a tubular portion where injection and dissolution of CO2 is efficient-to obtain in TLC systems, greater productivity and better-quality biomass.

Interaction between Haematococcus pluvialis microalgae and lead nitrate: lead adsorption from water.

Our study aims to investigate the response of the unicellular alga, Haematococcus pluvialis, to the toxicity of lead and propose a low-cost, highly efficient biological adsorbent for the purification of wastewater and lead-contaminated water. The first part examines the effects of lead toxicity on certain physiological indicators of this alga. In the second part, the potential of this alga in lead removal and its adsorption capacity was assessed. The alga was cultivated in a BG11 medium and treated with lead nitrate concentrations of 10, 50, and 200 mg/L during its exponential growth. The results showed that with an increase in lead concentration up to 200 mg/L, the growth rate, chlorophyll a, chlorophyll b, carotenoid and total protein content decreased, while malondialdehyde (MDA) content increased. The astaxanthin content slightly increased at the 10 mg/L but decreased at the 200 mg/L treatment. Maximum lead adsorption was observed at 98.69% under optimal conditions, including a pH of 6, an adsorbent dose of 1 g/L, a lead concentration of 25 mg/L, a temperature of 25 °C, and an exposure time of 120 min. The results of this study demonstrate that Haematococcus pluvialis has the potential for effective lead removal from aquatic environments.

While the influence of heavy metals on certain algae species has been explored, research on the impact of lead on Haematococcus pluvialis­a microalga of significant interest for astaxanthin production­remains uncharted territory. Therefore, understanding the impact of this heavy metal and the alga's metal absorption capabilities has profound implications for biotechnology and bioremediation applications. This study promotes H. pluvialis as an economically viable lead absorbent suitable for both industrial and domestic purposes.

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae, Chlamydomonadales).

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.

Toxicity of ZnFe-SO4 layered double hydroxide in Tetradesmus obliquus and evaluation of some physiological responses of the microalgae for stress management.

Layered double hydroxides (LDHs), regarding their physical and structural properties, have different and wide applications industry and their increasing use may raise ecological and human health concerns. However, the potential toxicity mechanisms of LDHs in different organisms are still unclear. In the present work, after synthesizing of ZnFe-SO4 LDH and studying of its characterization by XRD, FT-IR, SEM, EDX-mapping, TEM and Raman, its toxicity in Tetradesmus obliquus was evaluated. According to experimental results, the growth of the algae and content of photosynthetic pigments were significantly decreased after treatment with 100 mg/L of ZnFe-SO4 LDH. The high dose exposure to the LDH also inhibited the activity of SOD and POD enzymes, possibly due to the LDH- catalyzed reactive oxygen species production. In addition, lipid peroxidation and the content of phenolic compounds, as no-enzymatic antioxidants were increased by enhancement of the LDH concentration. The rise of phenol, flavonoids and MDA contents could be regarded as some manifestations and responses to the toxic effects of the contaminant in the algae cells. The results provided a better understanding of the undesirable effects and toxicity of LDHs in aquatic organisms.

Selecting a preculture strategy for improving biomass and astaxanthin productivity of Chromochloris zofingiensis.

Chromochloris zofingiensis is a potential source of natural astaxanthin; however, its rapid growth and astaxanthin enrichment cannot be achieved simultaneously. This study established autotrophic, mixotrophic, and heterotrophic preculture patterns to assess their ameliorative effect on the C. zofingiensis heterotrophic growth state. In comparison, mixotrophic preculture (MP) exhibited the best improving effect on heterotrophic biomass concentration of C. zofingiensis (up to 121.5 g L-1) in a 20 L fermenter, reaching the global leading level. The astaxanthin productivity achieved 111 mg L-1 day-1, 7.4-fold higher than the best record. The transcriptome and 13C tracer-based metabolic flux analysis were used for mechanism inquiry. The results revealed that MP promoted carotenoid and lipid synthesis, and supported synthesis preference of low unsaturated fatty acids represented by C18:1 and C16:0. The MP group maintained the best astaxanthin productivity via mastering the balance between increasing glucose metabolism and inhibition of carotenoid synthesis. The MP strategy optimized the physiological state of C. zofingiensis and realized its heterotrophic high-density growth for an excellent astaxanthin yield on a pilot scale. This strategy exhibits great application potential in the microalgae-related industry. KEY POINTS: • Preculture strategies changed carbon flux and gene expression in C. zofingiensis • C. zofingiensis realized a high-density culture with MP and fed-batch culture (FBC) • Astaxanthin productivity achieved 0.111 g L-1 day-1 with MP and FBC.

Regulation of a novel DsGATA1 from Dunaliella salina on the synthesis of carotenoids under red light.

Dunaliella salina is a high-quality industrial effector for carotenoid production. The mechanism by which red light regulates carotenoid synthesis is still unclear. In this study, a transcription factor of DsGATA1 with a distinct structure was discovered in D. salina. The recognition motif of DsGATA1 was comparable to that of plant and fungal GATA, despite its evolutionary proximity to animal-derived GATA. The expression of DsGATA1 in D. salina was still noticeably decreased when exposed to red light. Analysis of physiological and biochemical transcriptomic data from overexpressed, interfering, and wild-type strains of DsGATA1 revealed that DsGATA1 acts as a global regulator of D. salina carotenoid synthesis. The upregulated genes in the CBP pathway by DsGATA1 were involved in its regulation of the synthesis of carotenoids. DsGATA1 also enhanced carotenoid accumulation under red light by affecting N metabolism. DsGATA1 was found to directly bind to the promoter of nitrate reductase to activate its expression, promoting D. salina nitrate uptake and accelerating biomass accumulation. DsGATA1 affected the expression of the genes encoding GOGAT, GDH, and ammonia transporter proteins. Moreover, our study revealed that the regulation of N metabolism by DsGATA1 led to the production of NO molecules that inhibited carotenoid synthesis. However, DsGATA1 significantly enhanced carotenoid synthesis by NO scavenger removal of NO. The D. salina carotenoid accumulation under red light was elevated by 46% in the presence of overexpression of DsGATA1 and NO scavenger. Nevertheless, our results indicated that DsGATA1 could be an important target for engineering carotenoid production. KEY POINTS: • DsGATA1 with a distinct structure and recognition motif was found in D. salina • DsGATA1 enhanced carotenoid production and biomass in D. salina under red light • DsGATA1 is involved in the regulation of N metabolism and carotenoid synthesis.

Astaxanthin from microalgae: A review on structure, biosynthesis, production strategies and application.

Astaxanthin is a red-colored secondary metabolite with excellent antioxidant properties, typically finds application as foods, feed, cosmetics, nutraceuticals, and medications. Astaxanthin is usually produced synthetically using chemicals and costs less as compared to the natural astaxanthin obtained from fish, shrimps, and microorganisms. Over the decades, astaxanthin has been naturally synthesized from Haematococcus pluvialis in commercial scales and remains exceptional, attributed to its higher bioactive properties as compared to synthetic astaxanthin. However, the production cost of algal astaxanthin is still high due to several bottlenecks prevailing in the upstream and downstream processes. To that end, the present study intends to review the recent trends and advancements in astaxanthin production from microalgae. The structure of astaxanthin, sources, production strategies of microalgal astaxanthin, and factors influencing the synthesis of microalgal astaxanthin were discussed while detailing the pathway involved in astaxanthin biosynthesis. The study also discusses the relevant downstream process used in commercial scales and details the applications of astaxanthin in various health related issues.