Optimization and mechanism analysis of photosynthetic EPA production in Nannochloropsis salina: Evaluating the effect of temperature and nitrogen concentrations.

Microalgae, recognized as sustainable and eco-friendly photosynthetic microorganisms, play a pivotal role in converting CO2 into value-added products. Among these, Nannochloropsis salina (Microchloropsis salina) stands out, particularly for its ability to produce eicosapentaenoic acid (EPA), a crucial omega-3 fatty acid with significant health benefits such as anti-inflammatory properties and cardiovascular health promotion. This study focused on optimizing the cultivation conditions of Nannochloropsis salina to maximize EPA production. We thoroughly investigated the effects of varying temperatures and nitrogen (NaNO3) concentrations on biomass, total lipid content, and EPA proportions. We successfully identified optimal conditions at an initial NaNO3 concentration of 1.28 g.L-1 and a temperature of 21 °C. This condition was further validated by response surface methodology, which resulted in the highest EPA productivity reported in batch systems (14.4 mg.L-1.day-1). Quantitative real-time PCR and transcriptomic analysis also demonstrated a positive correlation between specific gene expressions and enhanced EPA production. Through a comprehensive lipid analysis and photosynthetic pigment analysis, we deduced that the production of EPA in Nannochloropsis salina seemed to be produced by the remodeling of chloroplast membrane lipids. These findings provide crucial insights into how temperature and nutrient availability influence fatty acid composition in N. salina, offering valuable guidance for developing strategies to improve EPA production in various microalgae species.

Low-cost cultivation of Nannochloropsis oceanica in newly designed photobioreactors and its productivity trends in semi-continuous cultivation under inland outdoor conditions.

Most marine microalgae are typically cultivated in coastal areas due to challenges in inland cultivation. In this 185 days experiment, Nannochloropsis oceanica was semi-continuously cultivated inland using different photobioreactors (PBRs). The newly designed 700-liter (L) PBR exhibited tolerance to seasonal changes compared to the 150-L PBRs. The innovative in-situ oxygen release rate (ORR) measurement method results indicated that ORR was influenced by light intensity and temperature. The optimal temperature range for N. oceanica growth was 14-25 â„ƒ, demonstrated cold tolerance and lipid accumulation at low temperatures. The maximum lipid content in 700-L and 150-L PBRs was 29 % and 28 %, respectively. Based on the average biomass productivity, the price of N. oceanica was $11.89 kg-1 (or $3.35 kg-1 based on maximum biomass productivity), which is cheaper than the current market price of $20.19 kg-1. From results, smaller PBRs at the same hydro electricity price are more cost-effective.

Transcriptomics aids in uncovering the metabolic shifts and molecular machinery of Schizochytrium limacinum during biotransformation of hydrophobic substrates to docosahexaenoic acid.

BACKGROUND: Biotransformation of waste oil into value-added nutraceuticals provides a sustainable strategy. Thraustochytrids are heterotrophic marine protists and promising producers of omega (ω) fatty acids. Although the metabolic routes for the assimilation of hydrophilic carbon substrates such as glucose are known for these microbes, the mechanisms employed for the conversion of hydrophobic substrates are not well established. Here, thraustochytrid Schizochytrium limacinum SR21 was investigated for its ability to convert oils (commercial oils with varying fatty acid composition and waste cooking oil) into ω-3 fatty acid; docosahexaenoic acid (DHA). RESULTS: Within 72 h SR21 consumed ~ 90% of the oils resulting in enhanced biomass (7.5 g L- 1) which was 2-fold higher as compared to glucose. Statistical analysis highlights C16 fatty acids as important precursors of DHA biosynthesis. Transcriptomic data indicated the upregulation of multiple lipases, predicted to possess signal peptides for secretory, membrane-anchored and cytoplasmic localization. Additionally, transcripts encoding for mitochondrial and peroxisomal ß-oxidation along with acyl-carnitine transporters were abundant for oil substrates that allowed complete degradation of fatty acids to acetyl CoA. Further, low levels of oxidative biomarkers (H2O2, malondialdehyde) and antioxidants were determined for hydrophobic substrates, suggesting that SR21 efficiently mitigates the metabolic load and diverts the acetyl CoA towards energy generation and DHA accumulation. CONCLUSIONS: The findings of this study contribute to uncovering the route of assimilation of oil substrates by SR21. The thraustochytrid employs an intricate crosstalk among the extracellular and intracellular molecular machinery favoring energy generation. The conversion of hydrophobic substrates to DHA can be further improved using synthetic biology tools, thereby providing a unique platform for the sustainable recycling of waste oil substrates.

Dual stress factors adaptive evolution for high EPA production in Schizochytrium sp. and metabolomics mechanism analysis.

Eicosapentaenoic acid (EPA) is a vital É·-3 polyunsaturated fatty acid (PUFA) for human body with various physiological functions. In this study, we proposed an adaptive evolutionary strategy based on high-temperature and high-oxygen two-factor stress to increase the EPA production capacity of Schizochytrium. High-temperature stress was used to increase EPA yield, and high oxygen was implemented to continuously stimulate cell growth and lipid accumulation. The biomass and EPA production of ALE-D50 reached 35.33 g/L and 1.54 g/L, which were 43.85% and 71.11% higher than that of the original strain, respectively. Lower in vivo reactive oxygen species levels indicated that the evolved strain possessed stronger antioxidant activity. Liquid chromatography-mass spectrometry metabolomics showed that enhanced glucose consumption and glycolysis metabolism, as well as a weakened tricarboxylic acid cycle and reduced amino acid metabolic tributaries in the evolved strain, might be associated with increased growth and EPA synthesis. Finally, the lipid production and EPA production in a fed-batch fermentation were further increased to 48.93 g/L and 3.55 g/L, improving by 54.30% and 90.86%, respectively. This study provides a novel pathway for promoting EPA biosynthesis in Schizochytrium.

Turning waste into treasure: A new direction for low-cost production of lipid chemicals from Thraustochytrids.

Thraustochytrids are marine microorganisms known for their fast growth and ability to store lipids, making them useful for producing polyunsaturated fatty acids (PUFAs), biodiesel, squalene, and carotenoids. However, the high cost of production, mainly due to expensive fermentation components, limits their wider use. A significant challenge in this context is the need to balance production costs with the value of the end products. This review focuses on integrating the efficient utilization of waste with Thraustochytrids fermentation, including the economic substitution of carbon sources, nitrogen sources, and fermentation water. This approach aligns with the 3Rs principles (reduction, recycling, and reuse). Furthermore, it emphasizes the role of Thraustochytrids in converting waste into lipid chemicals and promoting sustainable circular production models. The aim of this review is to emphasize the value of Thraustochytrids in converting waste into treasure, providing precise cost reduction strategies for future commercial production.

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Endogenous virophages are active and mitigate giant virus infection in the marine protist Cafeteria burkhardae.

Endogenous viral elements (EVEs) are common genetic passengers in various protists. Some EVEs represent viral fossils, whereas others are still active. The marine heterotrophic flagellate Cafeteria burkhardae contains several EVE types related to the virophage mavirus, a small DNA virus that parasitizes the lytic giant virus CroV. We hypothesized that endogenous virophages may act as an antiviral defense system in protists, but no protective effect of virophages in wild host populations has been shown so far. Here, we tested the activity of virophage EVEs and studied their impact on giant virus replication. We found that endogenous mavirus-like elements (EMALEs) from globally distributed Cafeteria populations produced infectious virus particles specifically in response to CroV infection. However, reactivation was stochastic, often inefficient, and poorly reproducible. Interestingly, only one of eight EMALE types responded to CroV infection, implying that other EMALEs may be linked to different giant viruses. We isolated and cloned several reactivated virophages and characterized their particles, genomes, and infection dynamics. All tested virophages inhibited the production of CroV during coinfection, thereby preventing lysis of the host cultures in a dose-dependent manner. Comparative genomics of different C. burkhardae strains revealed that inducible EMALEs are common and are not linked to specific geographic locations. We demonstrate that naturally occurring virophage EVEs reactivate upon giant virus infection, thus providing a striking example that eukaryotic EVEs can become active under specific conditions. Moreover, our results support the hypothesis that virophages can act as an adaptive antiviral defense system in protists.

Effects of high-pressure homogenization and ultrasound on the composition, structure, and physicochemical properties of proteins extracted from Nannochloropsis Oceania.

This study examined the effects of high-pressure homogenization (HPH) and ultrasonication pre-treatment on the structural and physicochemical properties of proteins extracted from defatted Nannochloropsis Oceania biomass (DNOB). HPH treatment was found to enhance the solubility of protein extracted from DNOB compared to ultrasound, where samples pretreated with three passes (3P) of HPH exhibited lower solubility than two passes (2P). The morphology of extracted samples was visualized by scanning electron microscopy, which HPH pre-treatment, especially with more passes, were able to breakdown DNOB into fragments. Alternatively, more holes were displayed on the surface of the extracts pretreated with ultrasound especially when higher amplitude applied. The particle size of extracts from HPH3P (129.5 µm) significant dropped from HPH2P (314.25 µm), where samples pretreated with ultrasound at 20 % amplitude (US20) also decreased in particle size compared to 40 % amplitude (US40), from 115.25 µm to 78.22 µm. Protein flexibility of DNOB extracts were enhanced by both HPH2P and HPH3P but decreased for ultrasound samples. ß-sheets were found to be the most abundant protein secondary structure for all samples, where samples treated with HPH3P contained the highest percentage of ß-sheets (72 %) than control, HPH2P, ultrasonication at 20 and 40 % amplitude (52-62 %). The high percentage of ß-sheets found in HPH3P sample also contributed to its outstanding emulsifying properties which stood out among all, especially at concentrations over 1 mg/ml. Results obtained from this study helped to direct the application of DNOB extracts as functional food ingredient for future food innovation.

Taxonomic difference in marine bloom-forming phytoplanktonic species affects the dynamics of both bloom-responding prokaryotes and prokaryotic viruses.

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promote marine carbon biogeochemical cycling. Although prokaryotic viruses presumably affect this process, their dynamics during blooms are not fully understood. Here, we investigated the effects of taxonomic difference in bloom-forming phytoplankton on prokaryotes and their viruses. We analyzed the dynamics of coastal prokaryotic communities and viruses under the addition of dissolved intracellular fractions from taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF), using microcosm experiments. Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF or HIF, indicating that taxonomic difference in bloom-forming phytoplankton promotes distinct dynamics of not only the prokaryotic community but also prokaryotic viruses. Furthermore, combining our microcosm experiments with publicly available environmental data mining, we identified both known and novel possible host-virus pairs. In particular, the growth of prokaryotes associating with phytoplanktonic organic matter, such as Bacteroidetes (Polaribacter and NS9 marine group), Vibrio spp., and Rhodobacteriales (Nereida and Planktomarina), was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that changes in bloom-forming species can be followed by an increase in a specific group of prokaryotes and their viruses and that elucidating these tripartite relationships among specific phytoplankton, prokaryotes, and prokaryotic viruses improves our understanding of coastal biogeochemical cycling in blooms.IMPORTANCEThe primary production during marine phytoplankton bloom and the consumption of the produced organic matter by heterotrophic prokaryotes significantly contribute to coastal biogeochemical cycles. While the activities of those heterotrophic prokaryotes are presumably affected by viral infection, the dynamics of their viruses during blooms are not fully understood. In this study, we experimentally demonstrated that intracellular fractions of taxonomically distinct bloom-forming phytoplankton species, the diatom Chaetoceros sp. and the raphidophycean alga Heterosigma akashiwo, promoted the growth of taxonomically different prokaryotes and prokaryotic viruses. Based on their dynamics and predicted hosts of those viruses, we succeeded in detecting already-known and novel possible host-virus pairs associating with either phytoplankton species. Altogether, we propose that the succession of bloom-forming phytoplankton would change the composition of the abundant prokaryotes, resulting in an increase in their viruses. These changes in viral composition, depending on bloom-forming species, would alter the dynamics and metabolism of prokaryotes, affecting biogeochemical cycling in blooms.

Enhanced fatty acid storage combined with the multi-factor optimization of fermentation for high-level production of docosahexaenoic acid in Schizochytrium sp.

Schizochytrium sp. hasreceived much attention for itsability to synthesize and accumulate high-level docosahexaenoic acid (DHA), which can reach nearly 40 % of total fatty acids. In this study, the titer of DHA in Schizochytrium sp. was successfully improved by enhancing DHA storage through overexpressing the diacylglycerol acyltransferase (ScDGAT2C) gene, as well as optimizing the supply of precursors and cofactors required for DHA synthesis by response surface methodology. Notably, malic acid, citric acid, and biotin showed synergistic and time-dependent effects on DHA accumulation. The maximum lipid and DHA titers of the engineered Schizochytrium sp. strain reached 84.28 ± 1.02 g/L and 42.23 ± 0.69 g/L, respectively, with the optimal concentration combination (1.62 g/L malic acid + 0.37 g/L citric acid + 8.28 mg/L biotin) were added 48 h after inoculation. This study provides an effective strategy for improving lipid and DHA production in Schizochytrium sp.

Concentration-dependent dual roles of proanthocyanidins on oxidative stress and docosahexaenoic acid production in Schizochytrium sp. ATCC 20888.

Antioxidant addition is an effective strategy to achieve docosahexaenoic acid (DHA) overproduction in oleaginous microorganisms. Nevertheless, antioxidants like phenolic compounds sometimes exert pro-oxidant activity. In this work, effects of proanthocyanidins (PAs) on fermentation performance and oxidative stress in Schizochytrium sp. were investigated. Low PAs addition (5 mg/L) reduced reactive oxygen species and enhanced lipogenic enzymes activities and NADPH, resulting in significant increase in lipid (20.3 g/L) by 33.6 % and DHA yield (9.8 g/L) by 53.4 %. In contrast, high PAs addition (500 mg/L) exerted pro-oxidant effects, aggravated oxidative damage and lipid peroxidation, leading to sharp decrease in biomass (21.3 g/L) by 35.1 %, lipid (8.2 g/L) by 46.0 %, and DHA (2.9 g/L) by 54.8 %. Therefore, the antioxidant concentration is especially crucial in DHA production. This study is the first to report concentration-dependant dual roles of PAs in oxidative stress and DHA production in Schizochytrium sp., providing new insights into microbial DHA production.

Transcriptome analysis of the harmful alga Heterosigma akashiwo under a 24-hour light-dark cycle.

The photoperiod, which is defined as the period of time within a 24-hour time frame that light is available, is an important environmental regulator of several physiological processes in phytoplankton, including harmful bloom-forming phytoplankton. The ichthyotoxic raphidophyte Heterosigma akashiwo is a globally distributed bloom-forming phytoplankton. Despite extensive studies on the ecological impact of H. akashiwo, the influence of the photoperiod on crucial biological processes of this species remains unclear. In this study, gene expression in H. akashiwo was analyzed over a 24-hour light-dark (14:10) treatment period. Approximately 36 % of unigenes in H. akashiwo were differentially expressed during this 24-hour treatment period, which is indicative of their involvement in the response to light-dark variation. Notably, the number of differentially expressed genes exhibited an initial increase followed by a subsequent decrease as the sampling time progressed (T0 vs. other time points). Unigenes associated with photosynthesis and photoprotection reached their peak expression levels after 2-4 h of illumination (T12-T14). In contrast, the expression of unigenes associated with DNA replication peaked at the starting point of the dark period (T0). Furthermore, although several unigenes annotated to photoreceptors displayed potential diel periodicity, genes from various photoreceptor families (such as phytochrome and cryptochrome) showed unique expression patterns. Collectively, our findings offer novel perspectives on the response of H. akashiwo to the light-dark cycle, serving as a valuable resource for investigating the physiology and ecology of this species.

Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica.

OBJECTIVE: Currently, there is lack of a consistent and highly enriched source for docosapentaenoic acid (n-3 DPA, C22:5), and this work report the isolation of microorganism that naturally produces n-3 DPA. RESULTS: In this work, we screened microorganisms in our culture collections with the goal to isolate a strain with high levels of n-3 DPA. We isolated a strain of Sphaeroforma arctica that produces up to 11% n-3 DPA in total fatty acid and has a high n-3 DPA to DHA/EPA ratio. The cell growth of the isolated strain was characterized using microscopy imaging and flow cytometer technologies to confirm the coenocytic pattern of cell divisions previously described in S. arctica. Our novel isolate of S. arctica grew more robustly and produced significantly more n-3 DPA compared to previously isolated and described strains indicating the uniqueness of the discovered strain. CONCLUSION: Overall, this work reports a first isolate n-3 DPA producing microorganism and establishes the foundation for future strain improvement and elucidation of the physiological function of this LC-PUFA for human nutrition and health.

Labyrinthulomycetes thrives in organic matter-rich waters with ecological partitioning in the Pearl River Estuary.

Labyrinthulomycetes play an important role in marine biogeochemical cycles, but their diversity, distribution patterns, and key regulatory factors remain unclear. This study measured the abundance and diversity of Labyrinthulomycetes in the Pearl River Estuary (PRE) to understand its distribution pattern and relationship with environmental and biological factors. The abundance of Labyrinthulomycetes ranged from 24 to 500 cells·mL-1, with an average of 144.37 ± 94.65 cells·mL-1, and its community composition showed obvious ecological partitioning in the PRE. The results of statistical analysis indicated that CDOM, salinity, and chlorophyll a contributed significantly (P < 0.01) to the community composition, explaining 46.59%, 11.34%, and 4.38% of the variance, respectively. The Labyrinthulomycetes distribution pattern combined with the niches of dominant species was revealed; low-salinity species mainly use terrigenous organic matter occupied dominant positions in the upper estuary and showed the highest abundance; moderate-salinity species that can use phytoplankton-derived resources thrived in the middle estuary; and seawater species dominated the lower estuary with the highest diversity but the lowest abundance. In addition, the results of phylogenetic tree analysis indicated that the existence of a novel lineage, and further study on the diversity and ecological functions of Labyrinthulomycetes is needed.IMPORTANCELabyrinthulomycetes play important roles in organic matter remineralization, carbon sinks, and food webs. However, the true diversity of Labyrinthulomycetes is still unclear due to limitations in isolation and culture methods. In addition, previous studies on their relationship with environmental factors are inconsistent and even contradictory, and it is speculated that their community composition may have spatial heterogeneity along the environmental gradient. In this study, the distribution pattern and key regulators of Labyrinthulomycetes in the PRE were revealed. Combining the niche of dominant species, it is suggested that salinity determines the spatial differences in Labyrinthulomycetes diversity, and the resources of substrate (terrestrial input or phytoplankton-derived) determine the dominant species, and its abundance is mainly determined by organic matter concentrations. Our study provided new information on the Labyrinthulomycetes diversity and verified the spatial heterogeneity of Labyrinthulomycetes community composition, providing reliable explanations for the inconsistencies in previous studies.

Quantum dot-based light conversion strategy for customized cultivation of microalgae.

Microalgae are photosynthetic microorganisms with the potential to mitigate the atmospheric greenhouse effect by carbon fixation. However, their growth is typically limited by light availability. A wavelength converter utilizing red, blue, and green quantum dots (QDs) was developed to optimize light quality for enhancing microalgal production. The growth, lipid content, and eicosapentaenoic acid titer of Nannochloropsis increased by 11.2%, 9.5%, and 15.5% with red QDs, respectively. The biomass and triacylglycerol content of Phaeodactylum tricornutum increased by 8.6% and 35.0%, respectively. Simultaneously, biodiesel production was accelerated in Nannochloropsis (20.2%) and P. tricornutum (11.6%), and improved with increased cetane number and reduced iodine value. Furthermore, red QDs increased the growth and biomass accumulation of Nannochloropsis under low light, while green QDs shielded Nannochloropsis from photoinhibition under high light. This customizable QD-based methodology overcomes microalgal light limitations, demonstrating a universally applicable approach to improve microalgal cultivation and biochemical component production.

Prediction of Aureococcus anophageffens using machine learning and deep learning.

The recurrent brown tide phenomenon, attributed to Aureococcus anophagefferens (A. anophagefferens), constitutes a significant threat to the Qinhuangdao sea area in China, leading to pronounced ecological degradation and substantial economic losses. This study utilized machine learning and deep learning techniques to predict A. anophagefferens population density, aiming to elucidate the occurrence mechanism and influencing factors of brown tide. Specifically, Random Forest (RF) algorithm was utilized to impute missing water quality data, facilitating its direct application in subsequent algal population prediction models. The results revealed that all four models-RF, Support Vector Regression (SVR), Multilayer Perceptron (MLP), and Convolutional Neural Network (CNN)-exhibited high accuracy in predicting A. anophagefferens population densities, with R2 values exceeding 0.75. RF, in particular, showed exceptional accuracy and reliability, with an R2 value surpassing 0.8. Additionally, the study ascertained five critical factors influencing A. anophagefferens population density: ammonia nitrogen, pH, total nitrogen, temperature, and silicate.

Squalene production under oxygen limitation by Schizochytrium sp. S31 in different cultivation systems.

The triterpene squalene is widely used in the food, cosmetics and pharmaceutical industries due to its antioxidant, antistatic and anti-carcinogenic properties. It is usually obtained from the liver of deep sea sharks, which are facing extinction. Alternative production organisms are marine protists from the family Thraustochytriaceae, which produce and store large quantities of various lipids. Squalene accumulation in thraustochytrids is complex, as it is an intermediate in sterol biosynthesis. Its conversion to squalene 2,3-epoxide is the first step in sterol synthesis and is heavily oxygen dependent. Hence, the oxygen supply during cultivation was investigated in our study. In shake flask cultivations, a reduced oxygen supply led to increased squalene and decreased sterol contents and yields. Oxygen-limited conditions were applied to bioreactor scale, where squalene accumulation and growth of Schizochytrium sp. S31 was determined in batch, fed-batch and continuous cultivation. The highest dry matter (32.03 g/L) was obtained during fed-batch cultivation, whereas batch cultivation yielded the highest biomass productivity (0.2 g/L*h-1). Squalene accumulation benefited from keeping the microorganisms in the growth phase. Therefore, the highest squalene content of 39.67 ± 1.34 mg/g was achieved by continuous cultivation (D = 0.025 h-1) and the highest squalene yield of 1131 mg/L during fed-batch cultivation. Volumetric and specific squalene productivity both reached maxima in the continuous cultivation at D = 0.025 h-1 (6.94 ± 0.27 mg/L*h-1 and 1.00 ± 0.03 mg/g*h-1, respectively). Thus, the choice of a suitable cultivation method under oxygen-limiting conditions depends heavily on the process requirements. KEY POINTS: • Measurements of respiratory activity and backscatter light of thraustochytrids • Oxygen limitation increased squalene accumulation in Schizochytrium sp. S31 • Comparison of different cultivation methods under oxygen-limiting conditions.

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.