Chromochloris Zofingiensis Research Articles

Regulation Mechanism of Gibberellic Acid-3 for Astaxanthin Biosynthesis in Heterotrophic Growing Chromochloris zofingiensis.

Chromochloris zofingiensis has been proven as a potential resource for large-scale astaxanthin production, but little information on phytohormones for its growth and astaxanthin accumulation could be obtained. This study explored the impact of gibberellic acid-3 (GA3) on growth and astaxanthin biosynthesis in heterotrophic C. zofingiensis. After 6 days of cultivation with GA3, biomass and astaxanthin yields in 7.5 L fermenters reached 268.5 g·L-1 and 0.34 g·L-1, respectively, which were 6% and 89% higher than those in the control. GA3 changed transcription levels of genes linked to carbon metabolism, lipid metabolism, astaxanthin production, and ABC transporters. Genes related to astaxanthin biosynthesis, such as phytoene synthase (PSY), phytoene desaturase (PDS), beta-carotenoid hydroxylase (CHYb), and beta-carotenoid ketolase (BKT), were up-regulated under GA3 induction. The enhancement of carbon metabolism and lipid metabolism led to elevated consumption of substrates and generation of reducing power, thus facilitating astaxanthin biosynthesis. By using GA3 and arginine together, the astaxanthin yield increased to 0.39 g·L-1, which was 18% higher than that obtained under GA3 induction. It could be concluded that GA3 showed significant effects on astaxanthin biosynthesis. This research proposed novel feasible approaches to enhance astaxanthin production in heterotrophic C. zofingiensis.

Toxicological effects, absorption and biodegradation of bisphenols with different functional groups in Chromochloris zofingiensis

Bisphenols (BPs) are recognized as endocrine disrupting compounds and have garnered increasing attention due to their widespread utilization. However, the varying biological toxicities and underlying mechanisms of BPs with different functional groups remain unknown. In the present study, the toxic effects of four BPs (BPA, BPS, BPAF, and TBBPA) on a photosynthetic microalgae Chromochloris zofingiensis were compared. Results showed that halogen-containing BPs exhibited higher cellular uptake, leading to more severe oxidative stress, lower photosynthetic efficiency, and greater accumulation of starch and lipids. Specifically, TBBPA with bromine groups showed a greater toxicity than BPAF with fluorine groups, possibly due to the incomplete debromination in C. zofingiensis. Transcriptomic analysis revealed that halogen-containing BPs triggered greater number of differentially expressed genes (DEGs), and only 64 common DEGs were found among different BPs, indicating that the effects of BPs with different functional groups varied greatly. Genes involved in endocytosis, peroxisomes, and endoplasmic reticulum protein processing pathways were mostly upregulated across different BPs, while photosynthesis-related genes showed varied expression, possibly due to their distinct functional groups. Additionally, SIN3A, ZFP36L, CHMP, and ATF2 emerged as potential key regulatory genes. Overall, this study thoroughly explained how functional groups impact the toxicity and biodegradation of BPs in C. zofingiensis.

Establishment of an RNA-based transient expression system in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a unicellular green alga, is a prominent model for green biotechnology and for studying organelles’ function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii, including the 5’ UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46–1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae.

Insight into the regulation mechanism of light spectra on astaxanthin biosynthesis in mixotrophic Chromochloris zofingiensis

This study studied the effects of white light (WL), blue light (BL), and yellow-green light (YGL) on astaxanthin production in mixotrophically grown Chromochloris zofingiensis. The biomass yield achieved under BL was higher than that obtained under WL and YGL, respectively. Remarkably, compared with WL and YGL, the RuBisCO activity under BL was reduced, suggesting that the light wavelength could influence the RuBisCO activity. The results showed that BL was more suitable for astaxanthin accumulation than WL and YGL. When C. zofingiensis was grown under BL, the total yields of astaxanthin reached 0.2844 g·L−1 in 12 days, of which the biomass yield was 204.5 g·L−1 and astaxanthin content was 0.139 % of DW. The metabolomic study revealed that the central carbon metabolism and astaxanthin biosynthesis were enhanced, supporting fast cell growth and high astaxanthin contents under BL. Moreover, BL could enhance astaxanthin production by increasing the PSY, LCYb, CHYb, and BKT gene expression. This study provides effective strategies to facilitate astaxanthin production in C. zofingiensis.

Comparison of toxic effects and underlying mechanisms of carbon quantum dots and CdSe quantum dots on Chromochloris zofingiensis from the chemical composition perspective

Quantum dots (QDs) are widely utilized semiconductor nanocrystal materials with both nanotoxicity and composition-related toxicity. To determine the toxicological impacts and underlying mechanisms of QDs with different compositions on microalgae, carbon QDs (CQDs) and CdSe QDs were used in the present study. Results showed that QDs composed of CdSe were more toxic than QDs composed of carbon, which inhibited cell growth, with reductions in chl b content, chlorophyll fluorescence parameters, and increases in lipids and starch (two major storage substances). In addition, CdSe QDs elevated reactive oxygen species (ROS), resulting in oxidative damage, while CQDs had little effect on antioxidants. Comparative transcriptome analysis showed that gene expression was accelerated by CdSe QDs, and there was a compensatory upregulation of porphyrin metabolism, potentially to support chlorophyll synthesis. In addition, an MYB transcription factor was predicted by weighted gene co-expression network analysis (WGCNA) to serve as regulator in nanoparticle toxicity, while glutathione peroxidase (GPX) and dual-specificity tyrosine phosphorylation regulated kinases 2/3/4 (DYRK2/3/4) may be key mediators of the composition-related toxicity of CdSe QDs. This study highlights the critical role of QDs’ composition in determining their impacts on aquatic microalgae, providing a theoretical reference for selecting appropriate QDs materials for various industrial applications.

Influence of different light wavelengths and carbon sources on energy utilization efficiency by Chromochloris zofingiensis for cell growth and carotenoid synthesis

Chromochloris zofingiensis was cultured with different carbon sources (carbonate/glucose/acetate/pyruvate) and light conditions (dark/blue/red) under heterotrophic, autotrophic, and mixotrophic conditions to evaluate its energy utilization efficiency in terms of biomass and carotenoid accumulation. Enhanced specific growth rates of 0.58 d−1 and 0.51 d−1 were observed under blue- and red-light mixotrophic conditions, respectively, which both exceeded the rate of 0.44 d−1 obtained under heterotrophic conditions using a glucose-supplemented medium. The glucose medium showed superior cell synthesis compared to other carbon sources owing to its high utilization. Astaxanthin and neoxanthin accumulation were enhanced under mixotrophic conditions with blue-light. The energy efficiency for the synthesis of new cells was evaluated from a thermodynamic perspective. The efficiency was 1.1 to 2.1 % under autotrophic conditions, while 5.2 to 100 % was observed under heterotrophic conditions. Efficiencies in between were observed under mixotrophic conditions.

Iron rescues glucose-mediated photosynthesis repression during lipid accumulation in the green alga Chromochloris zofingiensis

Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (−Fe). Cells grown in −Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.

Light Intensity Enhances the Lutein Production in Chromochloris zofingiensis Mutant LUT-4.

Chromochloris zofingiensis, a unicellular green alga, is a potential source of natural carotenoids. In this study, the mutant LUT-4 was acquired from the chemical mutagenesis pool of C. zofingiensis strain. The biomass yield and lutein content of LUT-4 reached 9.23 g·L-1, and 0.209% of dry weight (DW) on Day 3, which was 49.4%, and 33% higher than that of wild-type (WT), respectively. The biomass yields of LUT-4 under 100, 300, and 500 µmol/m2/s reached 8.4 g·L-1, 7.75 g·L-1, and 6.6 g·L-1, which was 10.4%, 21%, and 29.6% lower compared with the control, respectively. Under mixotrophic conditions, the lutein yields were significantly higher than that obtained in the control. The light intensity of 300 µmol/m2/s was optimal for lutein biosynthesis and the content of lutein reached 0.294% of DW on Day 3, which was 40.7% more than that of the control. When LUT-4 was grown under 300 µmol/m2/s, a significant increase in expression of genes implicated in lutein biosynthesis, including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene epsilon cyclase (LCYe) was observed. The changes in biochemical composition, Ace-CoA, pyruvate, isopentenyl pyrophosphate (IPP), and geranylgeranyl diphosphate (GGPP) contents during lutein biosynthesis were caused by utilization of organic carbon. It was thereby concluded that 300 µmol/m2/s was the optimal culture light intensity for the mutant LUT-4 to synthesize lutein. The results would be helpful for the large-scale production of lutein.

A comprehensive review on the heterotrophic production of bioactive compounds by microalgae.

Bioactive compounds derived from microalgae have garnered considerable attention as valuable resources for drugs, functional foods, and cosmetics. Among these compounds, photosynthetic pigments and polyunsaturated fatty acids (PUFAs) have gained increasing interest due to their numerous beneficial properties, including anti-oxidant, anti-viral, anti-bacterial, anti-fungal, anti-inflammatory, and anti-tumor effects. Several microalgae species have been identified as rich sources of bioactive compounds, including the Chlorophyceae Dunaliella and Haematococcus, the Bacillariophyta Phaeodactylum and Nitzschia, and the dinoflagellate Crypthecodinium cohnii. However, most of the reported microalgae species primarily grow through autotrophic mechanisms, resulting in low yields and high production costs of bioactive compounds. Consequently, the utilization of heterotrophic microalgae, such as Chromochloris zofingiensis and Nitzschia laevis, has shown significant advantages in the production of astaxanthin and eicosapentaenoic acid (EPA), respectively. These heterotrophic microalgae exhibit superior capabilities in synthesizing target compounds. This comprehensive review provides a thorough examination of the heterotrophic production of bioactive compounds by microalgae. It covers key aspects, including the metabolic pathways involved, the impact of cultivation conditions, and the practical applications of these compounds. The review discusses how heterotrophic cultivation strategies can be optimized to enhance bioactive compound yields, shedding light on the potential of microalgae as a valuable resource for high-value product development.

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.

Near telomere-to-telomere genome assemblies of two Chlorella species unveil the composition and evolution of centromeres in green algae

BackgroundCentromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown.ResultsWe constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and Chlorella pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated their centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromeric CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements, although they differed significantly in their composition. Species within the Chlorella genus display a more diverse centromere composition, with major constituents including members of the LTR/Copia, LINE/L1, and LINE/RTEX families. This is in contrast to green algae including Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, in which centromere composition instead has a pronounced single-element composition. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeric sequence evolves more rapidly than sequence in non-centromeric regions.ConclusionsThis study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.

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

BackgroundThe 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.ResultsIn 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 β-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.ConclusionThis 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.Graphical

Global transcriptome analysis identifies critical functional modules associated with multiple abiotic stress responses in microalgae Chromochloris zofingiensis.

In the current study, systems biology approach was applied to get a deep insight regarding the regulatory mechanisms of Chromochloris zofingiensis under overall stress conditions. Meta-analysis was performed using p-values combination of differentially expressed genes. To identify the informative models related to stress conditions, two distinct weighted gene co-expression networks were constructed and preservation analyses were performed using medianRankand Zsummary algorithms. Moreover, functional enrichment analysis of non-preserved modules was performed to shed light on the biological performance of underlying genes in the non-preserved modules. In the next step, the gene regulatory networks between top hub genes of non-preserved modules and transcription factors were inferred using ensemble of trees algorithm. Results showed that the power of beta = 7 was the best soft-thresholding value to ensure a scale-free network, leading to the determination of 12 co-expression modules with an average size of 128 genes. Preservation analysis showed that the connectivity pattern of the six modules including the blue, black, yellow, pink, greenyellow, and turquoise changed during stress condition which defined as non-preserved modules. Examples of enriched pathways in non-preserved modules were Oxidative phosphorylation", "Vitamin B6 metabolism", and "Arachidonic acid metabolism". Constructed regulatory network between identified TFs and top hub genes of non-preserved module such as Cz06g10250, Cz03g12130 showed that some specific TFs such as C3H and SQUAMOSA promoter binding protein (SBP) specifically regulates the specific hubs. The current findings add substantially to our understanding of the stress responsive underlying mechanism of C. zofingiensis for future studies and metabolite production programs.

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.

Effectiveness and associated mechanisms of a combination of biofilm attached cultivation and mixotrophy in promoting microalgal biomass

The effectiveness and associated mechanisms of the biofilm attached cultivation (BAC) under mixotrophy in promoting algal proliferation were investigated. Commercially valuable unicellular microalgae Chromochloris zofingiensis was first used in BAC. Compared with suspended cultivation, the results unequivocally demonstrated the growth benefits of C. zofingiensis cells under BAC with high biomass productivity of 8.53 g m−2 d−1. The physiological and transcriptomic data revealed that the augmented biomass yield was attributable to larger cell size, higher accumulation of chemical substances, significantly upregulated carbon fixation pathway, and greater energy supply efficiency. Here, BAC acts as a “cage” was proposed. Specifically, cells allocate less energy toward mobility, directing a higher share toward growth and production due to their immobilized lifestyle. These findings provide novel insights for optimizing cultivation strategies for commercially valuable algal species and offer a novel perspective from microalgae physiological on understanding higher biomass yield in BAC.

One-step pyrolysis-catalytic hydrogen production by the oleaginous microalga Chromochloris zofingiensis

In this study, one-step pyrolysis-catalytic hydrogen production was developed with the substance of a typical oleaginous microalga, Chromochloris zofingiensis, with Ni/Zr0.2Ce0.8O2 as the catalyst. Our results demonstrated that the formation of a ZrO2–CeO2 solid provided thermal stability to the catalyst and showed abundant active sites for catalysis. Moreover, 450 °C was the optimum temperature for hydrogen production by microalgae. At this temperature, hydrogen production reached 25.47 mmol g−1, with hydrogen accounting for 43.5 % of the total gas, comparable to results previously reported for “two-step” hydrogen production methos. Further studies showed that lipids within microalgal cells were the major substance for hydrogen production, whereas starch and protein had limited hydrogen-producing capabilities. Therefore, by increasing the lipid content to 33.1 % of cell dry weight through abiotic stress, the hydrogen yields significantly improved, reaching 47.71 mmol g−1, with hydrogen constituting 69.4 % of the total gas. In summary, our study proved that oleaginous microalgae were feasible feedstock for one-step pyrolysis-catalytic hydrogen production.

Characterization of a Chromochloris zofingiensis mutant with enhanced canthaxanthin accumulation

The green alga Chromochloris zofingiensis is able to accumulate value-added ketocarotenoids and represents an alternative producer with industrial production potential. To further understand the biosynthesis of ketocarotenoids, we generated mutants of C. zofingiensis and obtained two (czchy1 and czchy2) with profound changes in carotenoid profile. Both mutants showed a considerably higher level of canthaxanthin than the wild type (WT), with czchy1 being more significant. Clearly, among various stress conditions, czchy1 accumulated the highest level of canthaxanthin under the combination of high light and nitrogen deprivation (HL + ND), which was more than doubled of WT. Prolonged induction of HL + ND allowed canthaxanthin of the alga to reach 2.8 mg g−1 of dry weight, which could be further promoted by the addition of fulvic acid. czchy1 also had higher β-carotene level and lower levels of astaxanthin, lutein and zeaxanthin than WT. Molecular characterization of czchy1 revealed that the β-carotene hydroxylase (CHYb) gene possessed a point mutation and encoded an enzyme with attenuated hydroxylating activity. Besides, in czchy1 as compared to WT, CHYb was transcriptionally down-regulated, β-carotene hydroxylase (BKT1) and lycopene β-cyclase (LCYb) were transcriptionally up-regulated. Therefore, czchy1 likely adopted a multiple strategy combining pull, push and impairment of competing pathway towards boosting canthaxanthin accumulation.

From green to orange: The change in biochemical composition of phototrophic-mixotrophic Chromochloris zofingiensis in pilot-scale photobioreactors

Upscaling algal cultures from laboratory to pilot-scale provides important biological and technical insights for larger industrial applications. This is particularly important when screening novel algal species to enter the biochemical and nutraceutical markets. One such novel species is Chromochloris zofingiensis. This species has been identified as a potential industrial competitor to Haematococcus sp. for natural astaxanthin production, due to higher biomass concentrations and quantity of product per unit volume. To test the scalability potential C. zofingiensis was cultured using a phototrophic-mixotrophic approach in 65 L photobioreactors. The biomass increased from 0.05 to 0.51 g/L DW during a 15 day phototrophic phase. The subsequent mixotrophic phase induced carotenogenesis and turned the cells from green to orange. This altered the composition of the biomass and increased the biomass concentration from 0.65 to 5.13 g/L in 8 days. Astaxanthin is the primary compound of interest in this biomass and its concentration per culture increased during the mixotrophic phase; 0, 12.7, and 14.7 mgDW/L on days 0, 5, and 8, respectively. The highest protein, carbohydrate, and lipid concentrations per culture were recorded on day 5 (570, 1760, and 3715 mgDW/L, respectively). This study reports the largest scale use of mixotrophic cultivation of C. zofingiensis available in the literature where astaxanthin production was initiated using glucose (30 g/L), nitrogen deprivation, and a continuous photoperiod with an increased light intensity (245 μmol/m2/s). The findings demonstrate the potential for further scaling to obtain high biomass concentrations at industrial volumes for production of astaxanthin along with additional products as part of a biorefinery process.

Lipids and high-value astaxanthin produced in a cupboard by heterotrophic Chromochloris zofingiensis: Improvement of process efficiency

Cultivation of microalgae with high lipid content continues to be pursued as a potential biodiesel feedstock. Processing of microalgae biomass is not sufficiently cost-efficient to allow for wide-scale implementation. Efficient, low-cost processing strategies must be developed. In this study, heterotrophic metabolism was combined with attached cultivation for co-production of lipids and astaxanthin to simplify processing, improve efficiency, and mitigate cost. Microalgae were cultivated on agar media surface so that culture was distributed homogeneously with high solid content (40 %) and harvesting was possible with little manual effort. Operational parameters – initial glucose, inoculation ratio, cultivation time and temperature, and dry vs. wet extraction – were tested and optimized to maximize lipid production. Accordingly, room temperature incubation for 30 days in a dark cupboard with 40 g/L initial glucose, 4 % inoculum, and wet extraction of the harvested biomass yielded 8.2 g/L lipids and 6.5 mg/L astaxanthin. Many advantages are observed with agar-attached cultivation of microalgae, including no energy input required to support the incubation term, simple and manual harvesting of cells, and no dewatering or drying needed for the extraction of biomass. Based on the benefits when compared to traditional suspended or attached cultivation, agar-attached growth for lipids and astaxanthin co-production has the potential to greatly improve the process efficiency and thus, feasibility, of using microalgae as an alternative fuel source.