This study investigated the technical feasibility of using electrogermination to activate dormant cysts as an inoculum for subsequent 14-d photosynthetic astaxanthin production in Haematococcus lacustris. Electrotreatment affected the cell viability, surface charge, and morphology of H. lacustris cysts. At an optimal voltage of 2 V for 60 min, the cyst germination rate peaked at 44.6 % after 1 d, representing a 2.2-fold increase compared with that of the untreated control. Notably, electrogermination significantly enhanced both the astaxanthin content (44.9 mg/g cell) and productivity (13.2 mg/L/d) after 14 d of photobioreactor cultivation, corresponding to 1.7- and 1.5-fold increases compared with those in control, respectively. However, excessive electrotreatment, particularly at voltages exceeding 2 V or for durations beyond 60 min, did not enhance the astaxanthin production capability of H. lacustris. Proper optimization of renewable electrogermination can enable sustainable algal biorefinery to produce multiple bioactive products without compromising cell viability and astaxanthin productivity.

Astaxanthin has 550 times more antioxidant activity than vitamin E, so it can scavenge free radicals in vivo and improve body immunity. However, the poor stability of astaxanthin becomes a bottleneck problem that limits its application. Herein, Haematococcus pluvialis (H. pluvialis) as a raw material was used to extract astaxanthin, and the optimal extraction conditions included the extraction solvent (EA:EtOH = 1:6, v/v), extraction temperature (60 °C), and extraction time (70 min). The extracted astaxanthin was then loaded using lecithin to form corresponding liposomes via the ethanol injection method. The results showed that the particle size and zeta potential of the prepared liposomes were 105.8 ± 1.2 nm and -38.0 ± 1.7 mV, respectively, and the encapsulation efficiency of astaxanthin in liposomes was 88.83%. More importantly, the stability of astaxanthin was significantly improved after being embedded in the prepared liposomes.

This work explores astaxanthin (AXT), a valuable xanthophyll ketocarotenoid pigment with significant health benefits and diverse applications across various industries. It discusses the prevalence of synthetic AXT, and the development of natural-based alternatives derived from microorganisms such as microalgae, bacteria, and yeast. The chapter examines the potential of microbial AXT production, highlighting the advantages and challenges associated with natural AXT. Key microorganisms like Haematococcus pluvialis, Paracoccus carotinifaciens, and Phaffia rhodozyma are emphasized for their role in commercially producing this valuable ketocarotenoid. The narrative covers the complexities and opportunities in microbial AXT production, from cell structure implications to downstream processing strategies. Additionally, the chapter addresses current applications, commercialization trends, and market dynamics of natural microbial AXT, emphasizing the importance of cost-effective production, regulatory compliance, and technological advancements to reduce the market cost of the final product. As demand for natural microbial-based AXT rises, this chapter envisions a future where research, innovation, and collaboration drive sustainable and competitive microbial AXT production, fostering growth in this dynamic market.

Microalgae play an important role in aquatic ecosystems, but the widespread presence of micro- and nano-plastics (MNPs) poses significant threats to them. Haematococcus pluvialis is well-known for its ability to produce the antioxidant astaxanthin when it experiences stress from environmental conditions. Here we examined the effects of polystyrene nanoplastics (PS-NPs) at concentrations of 0.1, 1, and 10 mg/L on H. pluvialis over an 18-day period. Our results show that PS-NPs caused a significant, dose-dependent inhibition of H. pluvialis growth and a reduction in photosynthesis. Furthermore, PS-NPs severely damaged the morphology of H. pluvialis, leading to cell shrinkage, collapse, content release, and aggregation. Additionally, PS-NPs induced a dose-dependent increase in soluble protein content and a decrease in the production of extracellular polymeric substances. These findings indicate that PS-NPs has the potential to adversely affect both the physiology and morphology of H. pluvialis. An increase in reactive oxygen species and antioxidant enzyme activities was also observed, suggesting an oxidative stress response to PS-NPs exposure. Notably, the synthesis of astaxanthin, which is crucial for H. pluvialis\'s survival under stress, was significantly inhibited in a dose-dependent manner under strong light conditions, along with the down-regulation of genes involved in the astaxanthin biosynthesis pathway. This suggests that PS-NPs exposure reduces H. pluvialis\'s ability to survive under adverse conditions. This study enhances our understanding of the toxic effects of PS-NPs on microalgae and underscores the urgent need for measures to mitigate MNP pollution to protect aquatic ecosystems.

The extraction of astaxanthin from Haematococcus pluvialis involves the utilization of petroleum-derived organic solvents or supercritical CO2, beset by safety concerns, high costs, and environmental sustainability limitations. This study, in contrast, employed a method involving the adjustment of salt concentration, propylene glycol, and vegetable oil fraction to disrupt emulsion in aqueous cell lysates for facilitating the separation of astaxanthin. Under optimized conditions, an astaxanthin-containing oil with a content of 1.88% was obtained even with the use of wet biomass, and four rounds of consecutive extraction resulted in a cumulative recovery yield of 66.41%. This process produced astaxanthin-enriched soybean oil with 9.49 times improved antioxidant capacity that satisfies a requirement for health functional application. Omitting the solvent removal and drying processes, which consume tremendous energy, can reduce the production cost by 2.98 times compared to conventional methods. Consequently, this study suggests an effective technique for producing edible oil containing H. pluvialis-derived astaxanthin.

Extracellular vesicles (EVs) are nano-sized particles involved in intercellular communications that intrinsically possess many attributes as a modern drug delivery platform. Haematococcus pluvialis-derived EVs (HpEVs) can be potentially exploited as a high-value-added bioproduct during astaxanthin production. The encapsulation of HpEV cargo is a crucial key for the determination of their biological functions and therapeutic potentials. However, little is known about the composition of HpEVs, limiting insights into their biological properties and application characteristics. This study examined the protein composition of HpEVs from three growth phases of H. pluvialis grown under high light (350 µmol·m-2·s-1) and sodium acetate (45 mM) stresses. A total of 2038 proteins were identified, the majority of which were associated with biological processes including signal transduction, cell proliferation, cell metabolism, and the cell response to stress. Comparative analysis indicated that H. pluvialis cells sort variant proteins into HpEVs at different physiological states. It was revealed that HpEVs from the early growth stage of H. pluvialis contain more proteins associated with cellular functions involved in primary metabolite, cell division, and cellular energy metabolism, while HpEVs from the late growth stage of H. pluvialis were enriched in proteins involved in cell wall synthesis and secondary metabolism. This is the first study to report and compare the protein composition of HpEVs from different growth stages of H. pluvialis, providing important information on the development and production of functional microalgal-derived EVs.

Microalgae are considered as a potential source of bioactive compounds to be used in different fields including food and pharmaceutical industry. In this context, fatty acid esters of hydroxy-fatty acids (FAHFA) are emerging as a new class of compounds with anti-inflammatory and anti-diabetic properties. An existing gap in the field of algal research is the limited knowledge regarding the production of these compounds. Our research questions aimed to determine whether the microalga H. pluvialis can synthesize FAHFA and whether the production levels of these compounds are increased when cultivated in a CO2-rich environment. To answer these questions, we used a LC-QTOF/MS method for the characterization of FAHFA produced by H. pluvialis while an LC-MS/MS method was used for their quantitation. The cultivation conditions of H. pluvialis, which include the utilization of CO2, can result in a 10-50-fold increase in FAHFA production.

BACKGROUND: Microalgae-derived extracellular vesicles (EVs), which transfer their cargos to the extracellular environment to affect recipient cells, play important roles in microalgal growth and environmental adaptation. And, they are also considered as sustainable and renewable bioresources of delivery nanocarrier for bioactive molecules and/or artificial drug molecules. However, their molecular composition and functions remain poorly understood.
RESULTS: In this study, isolation, characterization, and functional verification of Haematococcus pluvialis-derived EVs (HpEVs) were performed. The results indicated that HpEVs with typical EV morphology and size were secreted by H. pluvialis cells during the whole period of growth and accumulated in the culture medium. Cellular uptake of HpEVs by H. pluvialis was confirmed, and their roles in regulation of growth and various physiological processes of the recipient cells were also characterized. The short-term inhibition of HpEV secretion results in the accumulation of functional cellular components of HpEVs, thereby altering the biological response of these cells at the molecular level. Meanwhile, continuously inhibiting the secretion of HpEVs negatively influenced growth, and fatty acid and astaxanthin accumulation in H. pluvialis. Small RNA high-throughput sequencing was further performed to determine the miRNA cargoes and compelling details in HpEVs in depth. Comparative analysis revealed commonalities and differences in miRNA species and expression levels in three stages of HpEVs. A total of 163 mature miRNAs were identified with a few unique miRNAs reveal the highest expression levels, and miRNA expression profile of the HpEVs exhibited a clear stage-specific pattern. Moreover, a total of 12 differentially expressed miRNAs were identified and their target genes were classified to cell cycle control, lipid transport and metabolism, secondary metabolites biosynthesis and so on.
CONCLUSIONS: It was therefore proposed that cargos of HpEVs, including miRNA constituents, were suggested potential roles in modulate cell physiological state of H. pluvialis. To summarize, this work uncovers the intercellular communication and metabolism regulation functions of HpEVs.

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.

The effect of Haematococcus pluvialis (HP) (0.25∼1.25 %) as a colorant during high moisture extrusion (50 %) on the texture and microstructural properties of soy protein-based high moisture meat analogs (HMMA) was evaluated. Furthermore, the stability of HP-induced meat like color of the HMMA as a function of light exposure, freeze/thawing, frozen storage and cooking temperature and duration was investigated. The addition of HP reduced the elasticity of HMMA but enhanced its hardness, chewiness, and resilience. HP addition at low levels promoted the flexible and disordered regions within the protein secondary structure while excessive HP addition was unfavorable for protein cross-linking. The optimal degree of texturization was achieved with 0.75 % HP. Sensory evaluations revealed that HMMA with 1 %HP had a color similar to fresh beef sirloin, while HMMA with 0.25 % HP had a color closer to fresh pork loin. Light exposure induced the greatest color loss of the meat analogs compared with the cooking and frozen storage. The a* value of HMMA containing 1.25 % HP decreased by 30 % during the 14 days of light exposure. Frozen storage at darkness efficiently preserved the meat-like color of the extrudates. Overall, HP was found as promising colorant for HMMA production but the storage condition of the extrudates should be carefully optimized.