Reactive oxygen species derived from NADPH oxidase as signaling molecules regulate fatty acids and astaxanthin accumulation in Chromochloris zofingiensis.

Abiotic stresses can increase the total fatty acid (TFA) and astaxanthin accumulation in microalgae. However, it remains unknown whether a unified signal transduction mechanism exists under different stresses. This study explored the link between nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) and the accumulation of fatty acids and astaxanthin in Chromochloris zofingiensis under three abiotic stresses. Results showed significant increases in fatty acid, astaxanthin, and ROS levels under nitrogen deficiency, phosphorus deficiency, and high-salinity stress. The introduction of the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the content of these components. This underscores the pivotal role of NADPH oxidase-derived ROS in the accumulation of fatty acid and astaxanthin under abiotic stress. Analysis of transcriptomes across three conditions following DPI addition revealed 1,445 shared differentially expressed genes (DEGs). Enrichment analysis revealed that biotin, betalain, thiamine, and glucosinolate may be important in stress responses. The heatmap demonstrated that DPI notably suppressed gene expression in the fatty acid and carotenoid biosynthesis pathways. Our findings underscore the pivotal role of NADPH oxidase-derived ROS in the accumulation of fatty acid and astaxanthin under abiotic stresses.

Regulatory mechanisms of autophagy on DHA and carotenoid accumulation in Crypthecodinium sp. SUN.

BACKGROUND: Autophagy is a crucial process of cellular self-destruction and component reutilization that can affect the accumulation of total fatty acids (TFAs) and carotenoids in microalgae. The regulatory effects of autophagy process in a docosahexaenoic acid (DHA) and carotenoids simultaneously producing microalga, Crypthecodinium sp. SUN, has not been studied. Thus, the autophagy inhibitor (3-methyladenine (MA)) and activator (rapamycin) were used to regulate autophagy in Crypthecodinium sp. SUN. RESULTS: The inhibition of autophagy by 3-MA was verified by transmission electron microscopy, with fewer autophagy vacuoles observed. Besides, 3-MA reduced the glucose absorption and intracellular acetyl-CoA level, which resulting in the decrease of TFA and DHA levels by 15.83 and 26.73% respectively; Surprisingly, 3-MA increased intracellular reactive oxygen species level but decreased the carotenoids level. Comparative transcriptome analysis showed that the downregulation of the glycolysis, pentose phosphate pathway and tricarboxylic acid cycle may underlie the decrease of acetyl-CoA, NADPH and ATP supply for fatty acid biosynthesis; the downregulation of PSY and HMGCR may underlie the decreased carotenoids level. In addition, the class I PI3K-AKT signaling pathway may be crucial for the regulation of carbon and energy metabolism. At last, rapamycin was used to activate autophagy, which significantly enhanced the cell growth and TFA level and eventually resulted in 1.70-fold increase in DHA content. CONCLUSIONS: Our findings indicate the mechanisms of autophagy in Crypthecodinium sp. SUN and highlight a way to manipulate cell metabolism by regulating autophagy. Overall, this study provides valuable insights to guide further research on autophagy-regulated TFA and carotenoids accumulation in Crypthecodinium sp. SUN.

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.

Salicylic acid enhances cell growth, fatty acid and astaxanthin production in heterotrophic Chromochloris zofingiensis without reactive oxygen species elevation.

BACKGROUND: The induction of lipid and astaxanthin accumulation in microalgae is often achieved through abiotic stress. However, this approach usually leads to oxidative stress, which results in relatively low growth rate. Phytohormones, as important small molecule signaling substances, not only affect the growth and metabolism of microalgae but also influence the intracellular reactive oxygen species level. This study aimed to screen phytohormones that could promote the fatty acids and astaxanthin yield of heterotrophic Chromochloris zofingiensis without causing oxidative damage, and further investigate the underlying mechanisms. RESULTS: In the present study, among all the selected phytohormones, the addition of exogenous salicylic acid (SA) could effectively promote cell growth along with the yield of total fatty acids (TFA) and astaxanthin in heterotrophic C. zofingiensis. Notably, the highest yields of TFA and astaxanthin were achieved at 100 µM SA, 43% and 97.2% higher compared with the control, respectively. Interestingly, the intracellular reactive oxygen species (ROS) levels, which are usually increased with elevated TFA content under abiotic stresses, were significantly decreased by SA treatment. Comparative transcriptome analysis unveiled significant alterations in overall carbon metabolism by SA. Specifically, the upregulation of fatty acid synthesis pathway, upregulation of ß-carotene-4-ketolase (BKT) in carotenoid synthesis aligned with biochemical findings. Weighted gene co-expression network analysis highlighted ABC transporters and GTF2B-like transcription factor as potential key regulators. CONCLUSION: This study found that salicylic acid can serve as an effective regulator to promote the celling growth and accumulation of fatty acids and astaxanthin in heterotrophic C. zofingiensis without ROS elevation, which provides a promising approach for heterotrophic production of TFA and astaxanthin without growth inhibition.

The metabolic mechanisms of Cd-induced hormesis in photosynthetic microalgae, Chromochloris zofingiensis.

Cadmium, an environmental pollutant, is highly toxic and resistant to degradation. It exhibits toxicity at elevated doses but triggers excitatory effects at low doses, a phenomenon referred to as hormesis. Microalgae, as primary producers in aquatic ecosystems, demonstrate hormesis induced by cadmium, though the specific mechanisms are not yet fully understood. Consequently, we examined the hormesis of cadmium in Chromochloris zofingiensis. A minimal Cd2+ concentration (0.05 mg L-1) prompted cell proliferation, whereas higher concentrations (2.50 mg L-1) inhibited growth. The group exposed to higher doses exhibited increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Contrastingly, the group exposed to low doses exhibited a moderate antioxidant response without significantly increasing ROS. This implies that increased levels of antioxidative components counteract excessive ROS, maintaining cellular redox balance and promoting growth under conditions of low Cd2+. Validation experiments have established that NADPH oxidase-derived ROS primarily coordinates the hormesis effect in microalgae. Comparative transcriptome analysis has proved the involvement of antioxidant systems and photosynthesis in regulating hormesis. Notably, Aurora A kinases consistently displayed varying expression levels across all Cd2+ treatments, and their role in microalgal hormesis was confirmed through validation with SNS-314 mesylate. This study unveils the intricate regulatory mechanisms of Cd-induced hormesis in C. zofingiensis, with implications for environmental remediation and industrial microalgae applications.

Low and high temperatures promote docosahexaenoic acid accumulation in Crypthecodinium sp. SUN by regulating the polyunsaturated fatty acid synthase pathway and the expression of saturated fatty acid preferred diacylglycerol acyltransferases.

Low (15 °C) and high (35 °C) temperatures significantly increased DHA as a percentage of total fatty acids (TFAs) to 43.6 % and 40.46 %, respectively (1.28- and 1.18-fold of that at 25 °C, respectively). The incompleteness of the FAS pathway indicates that DHA synthesis does not occur via this pathway. Meanwhile, Comparative transcriptome analysis showed that the PUFA synthase pathway might be responsible for DHA synthesis in C. sp. SUN. Additionally, the three diacylglycerol acyltransferases all had a substrate preference for saturated fatty acid (SFA)-CoA, which also contributed to the decreased SFA and increased DHA at both low and high temperatures. Additionally, WGCNA analysis identifies key regulatory genes that may be involved in temperature-regulated DHA proportion. The findings of this study indicate the mechanisms of temperature-regulated DHA accumulation in C. sp. SUN and shed light on the manipulation of DHA proportion by changes in temperature.

Mechanisms of Sodium-Acetate-Induced DHA Accumulation in a DHA-Producing Microalga, Crypthecodinium sp. SUN.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that is critical for the intelligence and visual development of infants. Crypthecodinium is the first microalga approved by the Food and Drug Administration for DHA production, but its relatively high intracellular starch content restricts fatty acid accumulation. In this study, different carbon sources, including glucose (G), sodium acetate (S) and mixed carbon (M), were used to investigate the regulatory mechanisms of intracellular organic carbon distribution in Crypthecodinium sp. SUN. Results show that glucose favored cell growth and starch accumulation. Sodium acetate limited glucose utilization and starch accumulation but caused a significant increase in total fatty acid (TFA) accumulation and the DHA percentage. Thus, the DHA content in the S group was highest among three groups and reached a maximum (10.65% of DW) at 96 h that was 2.92-fold and 2.24-fold of that in the G and M groups, respectively. Comparative transcriptome analysis showed that rather than the expression of key genes in fatty acids biosynthesis, increased intracellular acetyl-CoA content appeared to be the key regulatory factor for TFA accumulation. Additionally, metabolome analysis showed that the accumulated DHA-rich metabolites of lipid biosynthesis might be the reason for the higher TFA content and DHA percentage of the S group. The present study provides valuable insights to guide further research in DHA production.

Investigation of carbon and energy metabolic mechanism of mixotrophy in Chromochloris zofingiensis.

BACKGROUND: Mixotrophy can confer a higher growth rate than the sum of photoautotrophy and heterotrophy in many microalgal species. Thus, it has been applied to biodiesel production and wastewater utilization. However, its carbon and energy metabolic mechanism is currently poorly understood. RESULTS: To elucidate underlying carbon and energy metabolic mechanism of mixotrophy, Chromochloris zofingiensis was employed in the present study. Photosynthesis and glucose metabolism were found to operate in a dynamic balance during mixotrophic cultivation, the enhancement of one led to the lowering of the other. Furthermore, compared with photoautotrophy, non-photochemical quenching and photorespiration, considered by many as energy dissipation processes, were significantly reduced under mixotrophy. Comparative transcriptome analysis suggested that the intermediates of glycolysis could directly enter the chloroplast and replace RuBisCO-fixed CO2 to provide carbon sources for chloroplast organic carbon metabolism under mixotrophy. Therefore, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped, allowing for more efficient utilization of photoreaction-derived energy. Besides, compared with heterotrophy, photoreaction-derived ATP reduced the need for TCA-derived ATP, so the glucose decomposition was reduced, which led to higher biomass yield on glucose. Based on these results, a mixotrophic metabolic mechanism was identified. CONCLUSIONS: Our results demonstrate that the intermediates of glycolysis could directly enter the chloroplast and replace RuBisCO-fixed CO2 to provide carbon for photosynthesis in mixotrophy. Therefore, the photosynthesis rate-limiting enzyme, RuBisCO, was skipped in mixotrophy, which could reduce energy waste of photosynthesis while promote cell growth. This finding provides a foundation for future studies on mixotrophic biomass production and photosynthetic metabolism.

DHA protects against monosodium urate-induced inflammation through modulation of oxidative stress.

Acute gouty inflammation could be triggered by phagocytosis of monosodium urate (MSU) by immune cells. This study investigated the protective effect and underlying mechanism of docosahexaenoic acid (DHA) on MSU-induced inflammation in vitro and in vivo. Results showed that DHA effectively inhibited MSU-induced expression and secretion of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in THP-1 cells. Intracellular reactive oxygen species (ROS) production triggered by MSU was alleviated by DHA treatment. Furthermore, DHA promoted the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), wherein Nrf2 further mediated the expression of multiple antioxidant enzymes such as, heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase-1 (NQO1) and catalase, which are closely related with redox homeostasis. DHA treatment also restored MSU-induced impairment of mitochondrial transmembrane potential. In addition, oral administration of DHA-rich microalgal oil to C57BL/6 mice effectively reduced the infiltration of neutrophils, and decreased the expression and secretion of inflammatory cytokines. Altogether, our results suggest that DHA or DHA-rich microalgal oil may be a promising natural agent for the prevention of MSU-induced inflammation and potentially acute gout at least partly by attenuating oxidative stress.

Light induces carotenoids accumulation in a heterotrophic docosahexaenoic acid producing microalga, Crypthecodinium sp. SUN.

In the present study, the effect of various light conditions on carotenoid accumulation in a novel heterotrophic microalga, Crypthecodinium sp. SUN was investigated. The results showed that C. sp. SUN mainly produced γ-carotene and ß-carotene. The total carotenoid content could reach to 12.8 mg g-1 dry weight under high light intensity (100 µmol m-2 s-1), which was >100-fold higher than that under dark condition. Besides, along with the light intensity increased, the ROS level in vivo was decreased at 48 h and 72 h. Further study showed that, light could efficiently promote the gene expression of PSY and LCYb, which explain the molecular mechanisms of carotenoids accumulation under light conditions. Meanwhile, slightly inhibited fatty acids accumulation could promote the carotenoids yield. The present work proposed that C. sp. SUN could be a potential carotenoid producer, and provided valuable insight for carotenoids biosynthesis.

Inhibition of autophagy modulates astaxanthin and total fatty acid biosynthesis in Chlorella zofingiensis under nitrogen starvation.

The present study showed that inhibition of autophagy significantly increased cellular levels of reactive oxygen species in Chlorella zofingiensis under nitrogen starvation. This was accompanied with increased expression of PSY, and enhanced accumulation of astaxanthin after 48h of cultivation. Nevertheless, the proportion of astaxanthin in secondary carotenoids remained unchanged. Meanwhile, the expression level of ACCase was also elevated in the 3-MA-treated cells compared to the control despite a >20% lower content of fatty acid in the former than the latter. This phenomenon might be due to inhibition of recycling of cellular components by 3-MA and suggests the potential involvement of post-transcriptional regulation in fatty acid biosynthesis. In summary, our work has been the first to report a potentially important role of autophagy in fatty acid and astaxanthin accumulation in C. zofingiensis under stress conditions. The findings might provide valuable insights to guide further research in this area.

Differential responses of the green microalga Chlorella zofingiensis to the starvation of various nutrients for oil and astaxanthin production.

Chlorella zofingiensis has been proposed as a potential producer of lipids and the high-value carotenoid astaxanthin. In this study, the responses of photoautotrophic C. zofingiensis with respect to growth, lipid profiles and astaxanthin accumulation were investigated upon the starvation of N (NS), P (PS) and S (SS). NS and SS stimulated triacylglycerol (TAG) accumulation, which reached 27% and 21% of dry weight (DW), respectively. Stresses also stimulated astaxanthin accumulation greatly, reaching 3.9 mg/g DW by NS. SS led to the highest TAG productivity (52.4 mg L-1 d-1) while NS gave rise to the highest astaxanthin productivity (0.624 mg L-1 d-1). In combination with transcriptional analysis, a working model for stress-associated TAG and astaxanthin biosynthesis was proposed. Taken together, these detailed data shed light on the elucidation of differential responses to nutrient stresses and may provide insights into future engineering of this promising alga for improving TAG and astaxanthin production.

Light enhanced the accumulation of total fatty acids (TFA) and docosahexaenoic acid (DHA) in a newly isolated heterotrophic microalga Crypthecodinium sp. SUN.

In the present study, light illumination was found to be efficient in elevating the total fatty acid content in a newly isolated heterotrophic microalga, Crypthecodinium sp. SUN. Under light illumination, the highest total fatty acid and DHA contents were achieved at 96h as 24.9% of dry weight and 82.8mgg-1 dry weight, respectively, which were equivalent to 1.46-fold and 1.68-fold of those under the dark conditions. The elevation of total fatty acid content was mainly contributed by an increase of neutral lipids at the expense of starches. Moreover, light was found to alter the cell metabolism and led to a higher specific growth rate, higher glucose consumption rate and lower non-motile cell percentage. This is the first report that light can promote the total fatty acids accumulation in Crypthecodinium without growth inhibition.

Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP).

In the present study, high light and nitrogen starvation with glucose-fed to the culture was found efficient to induce astaxanthin accumulation in Chlorella zofingiensis. Therefore, a two-step cultivation strategy including high biomass yield fermentation and outdoor induction with an energy-free RFP was conducted. During the fermentation, the highest cell density of 98.4gL-1 and astaxanthin yield of 73.3mgL-1 were achieved, which were higher than those so far reported in C. zofingiensis. During the outdoor induction, astaxanthin content was further increased by 1.5-fold leading to the highest astaxanthin productivity of 5.26mgL-1day-1 under an optimal dilution of 5-fold. Our work thus provided an effective two-step cultivation strategy for production of astaxanthin by C. zofingiensis.

Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01.

Shewanella xiamenensis BC01 (SXM) was isolated from sediment collected off Xiamen, China and was identified based on the phylogenetic tree of 16S rRNA sequences and the gyrB gene. This strain showed high activity in the decolorization of textile azo dyes, especially methyl orange, reactive red 198, and recalcitrant dye Congo red, decolorizing at rates of 96.2, 93.0, and 87.5%, respectively. SXM had the best performance for the specific decolorization rate (SDR) of azo dyes compared to Proteus hauseri ZMd44 and Aeromonas hydrophila NIU01 strains and had an SDR similar to Shewanella oneidensis MR-1 in Congo red decolorization. Luria-Bertani medium was the optimal culture medium for SXM, as it reached a density of 4.69 g-DCW L(-1) at 16 h. A mediator (manganese) significantly enhanced the biodegradation and flocculation of Congo red. Further analysis with UV-VIS, Fourier Transform Infrared spectroscopy, and Gas chromatography-mass spectrometry demonstrated that Congo red was cleaved at the azo bond, producing 4,4'-diamino-1,1'-biphenyl and 1,2'-diamino naphthalene 4-sulfonic acid. Finally, SEM results revealed that nanowires exist between the bacteria, indicating that SXM degradation of the azo dyes was coupled with electron transfer through the nanowires. The purpose of this work is to explore the utilization of a novel, dissimilatory manganese-reducing bacterium in the treatment of wastewater containing azo dyes.