Comparative Transcriptomic Analysis on the Effect of Sesamol on the Two-Stages Fermentation of Aurantiochytrium sp. for Enhancing DHA Accumulation.

Aurantiochytrium is a well-known long-chain polyunsaturated fatty acids (PUFAs) producer, especially docosahexaenoic acid (DHA). In order to reduce the cost or improve the productivity of DHA, many researchers are focusing on exploring the high-yield strain, reducing production costs, changing culture conditions, and other measures. In this study, DHA production was improved by a two-stage fermentation. In the first stage, efficient and cheap soybean powder was used instead of conventional peptone, and the optimization of fermentation conditions (optimal fermentation conditions: temperature 28.7 °C, salinity 10.7‱, nitrogen source concentration 1.01 g/L, and two-nitrogen ratio of yeast extract to soybean powder 2:1) based on response surface methodology resulted in a 1.68-fold increase in biomass concentration. In the second stage, the addition of 2.5 mM sesamol increased the production of fatty acid and DHA by 93.49% and 98.22%, respectively, as compared to the optimal culture condition with unadded sesamol. Transcriptome analyses revealed that the addition of sesamol resulted in the upregulation of some genes related to fatty acid synthesis and antioxidant enzymes in Aurantiochytrium. This research provides a low-cost and effective culture method for the commercial production of DHA by Aurantiochytrium sp.

Effect of algicidal compound Nω-acetylhistamine on physiological response and algal toxins in Heterosigma akashiwo.

The toxic alga Heterosigma akashiwo (Raphidophyceae) is known to form harmful algal blooms (HABs), which can have serious negative effects on the aquatic ecosystem and human life. Previous study has shown that Nω-acetylhistamine (N-AcH), an algicidal compound secreted by algicidal bacteria Bacillus sp. Strain B1, can inhibit the growth of H. akashiwo. In this study, the algicidal mechanism of N-AcH against H. akashiwo was explored, and the changes of toxicity of H. akashiwo treated with N-AcH were investigated. The algal inhibition rate was calculated by the optical density method, and the results showed that the growth inhibition rate of H. akashiwo was about 90% when treated in the medium with 40 µg/mL N-AcH at 96 h. After 72 h treatment, transmission electron microscopy (TEM) showed that the microstructure of H. akashiwo cell was seriously damaged at this concentration. The content of Chlorophyll a and Chlorophyll b decreased while malonaldehyde levels increased, and superoxide dismutase activity first increased and then decreased as well as soluble protein content. GC-MS revealed that the type and content of fatty acids cut down after 48 h and 96 h treatment. Hemolytic test, MTT assay, and micronucleus test all demonstrated the decrease in the toxicity of H. akashiwo treated with 40 µg/mL N-AcH. In brief, N-AcH mainly kills H. akashiwo cell through oxidative stress and can also reduce its toxicity, so it is a promising algicide with the dual functions of killing algae and inhibiting algal toxic effects.

Effect of water accommodated fractions of fuel oil on fixed carbon and nitrogen by microalgae: Implication by stable isotope analysis.

Effect of water accommodated fractions (WAF) of #180 fuel oil on fixed carbon and nitrogen in microalgae was studied by stable isotopes. Platymonas helgolandica, Heterosigma akashiwo and Nitzschia closterium were exposed to five WAF concentrations for 96 h. The δ13C value of microalgae was significantly lower than that of the control group, indicated that carbon was limited in the WAF concentrations. The δ13C value of microalgae appeared peak valley at 48 h in control group, corresponding to the enhanced capacity in carbon fixation during microalgae photosynthesis. The physiological acclimation capacity of microalgae was revealed by the occurrence time when the δ13C value was in peak valley, and thus the physiological acclimation capacity of microalgae decreased in the order of Nitzschia closterium > Heterosigma akashiwo > Platymonas helgolandica. Principal component analysis (PCA) were applied to the δ13C value in order to verify the "hormesis" phenomenon in microalgae. The δ13C value could discriminate between stimulatory effects at low doses and inhibitory effects at high doses. In addition, the present study also investigated the effect of the nitrogen on microalgae growth. Because microalgae could still absorb the NO3-N and release of NO2-N and NH4-N in present study, the nitrogen cycle in microalgae was in the equilibrium status. The δ15N value in microalgae exhibited no obvious change with the increasing of WAF concentrations at the same time. However, due to the enrichment of nitrogen, the δ15N value first increased gradually with the time and finally was stable. Overall, the fractionation of carbon and nitrogen stable isotopes illustrated that the effect of carbon on the growth of microalgae was more prominent than nitrogen. Stable isotopes was used to investigate the influence of WAF on fixed carbon and nitrogen in microalgae growth, providing a fundamental theoretical guidance for risk assessment of marine ecological environment.

Assessment of impact of α-Fe2 O3 and γ-Fe2 O3 nanoparticles on phytoplankton species Selenastrum capricornutum and Nannochloropsis oculata.

In this study, the impact of alpha-iron oxide (α-Fe2 O3 , 20-40 nm) and gamma iron oxide (γ-Fe2 O3 , 20-40 nm) nanoparticles (NPs) on phytoplankton species Selenastrum capricornutum and Nannochloropsis oculata was investigated Characterizations of the NPs were systematically carried out by TEM, dynamic light scattering, zeta potential, X-ray diffraction, SEM, and Fourier transformation infrared spectroscopy. Acute toxicity was tested between 0.2 and 50 mg/L for each NP for a period of 72 hours exposure. γ-Fe2 O3 NP inhibited development of N oculata at the rate of 54% in 0.2 mg/L group with a high mortality rate of up to 82%. α-Fe2 O3 NPs were less toxic that induced 97% mortality on N oculata at 10 mg/L suspensions. In contrast, α-Fe2 O3 NP inhibited growth of S capricornutum strongly (73%) in 0.2 mg/L group. γ-Fe2 O3 NPs showed similar growth inhibition (72%) on S capricornutum in 10 mg/L suspensions. Despite the differential effects, the results indicated acute toxicity of α-Fe2 O3 and γ-Fe2 O3 NPs on N oculata and S capricornutum.

The role of fluconazole in the regulation of fatty acid and unsaponifiable matter biosynthesis in Schizochytrium sp. MYA 1381.

BACKGROUND: Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and ß-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium. RESULTS: To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole's role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the ß-carotene production was increased by 53% at 96 h. The increase of squalene and ß-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium. CONCLUSION: This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

Toxicological Assessment of Intermediates in Natural Attenuation of p-Xylene to Marine Microalgae.

The toxic effects of p-xylene, 4-methylbenzyl alcohol, p-methyl benzaldehyde, and p-toluic acid on two marine microalgae (Phaeodactylum tricornutum Bohlin, and Skeletonema costatum) were investigated. p-Xylene was the most toxic to Pha. tricornutum with a 96 h EC50 value of 6.76 mg L-1. Based on the 96 h EC50 values for two microalgae, the toxicity of the four chemicals, in descending order, was: p-xylene, p-methyl benzaldehyde, 4-methylbenzyl alcohol, then p-toluic acid. The results showed that the toxicity of the transformed products of p-xylene was lower than that of p-xylene.

Enhancement of Schizochytrium DHA synthesis by plasma mutagenesis aided with malonic acid and zeocin screening.

Schizochytrium sp. accumulates valuable polyunsaturated fatty acid (PUFA), such as docosahexaenoic acid (DHA). In order to increase DHA synthesis in this microorganism, physical or chemical mutagenesis aided with powerful screening methods are still preferable, as its DHA synthetic pathway has not yet been clearly defined for gene manipulation. To breed this agglomerate microorganism of thick cell wall and rather large genome for increasing lipid content and DHA percentage, a novel strategy of atmospheric and room temperature plasma (ARTP) mutagenesis coupled with stepped malonic acid (MA) and zeocin resistance screening was developed. The final resulted mutant strain mz-17 was selected with 1.8-fold increased DHA production. Accompanied with supplementation of Fe2+ in shake flask cultivation, DHA production of 14.0 g/L on average was achieved. This work suggests that ARTP mutation combined with stepped MA and zeocin resistance screening is an efficient method of breeding Schizochytrium sp. of high DHA production, and might be applied on other microorganisms for obtaining higher desired PUFA products.

Antialgal compounds with antialgal activity against the common red tide microalgae from a green algae Ulva pertusa.

Nine antialgal active compounds, (i.e. trehalose (1), twenty-two methyl carbonate (2), (-)-dihydromenisdaurilide (3), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (4), isophytol (5), 8-hexadecenol (6), 17-hydroxyheptadecanoic acid (7), trans-asarone (8) and 2-amino-3-mercaptopropanoic acid (9)) were isolated from Ulva pertusa for the first time by sephadex LH-20 column chromatography, silica gel column chromatography and repeated preparative TLC. Except for compound 4, all compounds represented novel isolated molecules from marine macroalgae. Further, antialgal activities of these compounds against Amphidinium carterae, Heterosigma akashiwo, Karenia mikimitoi, Phaeocystis globosa, Prorocentrum donghaiense and Skeletonema costatum were investigated for the first time. Results showed these nine compounds have selectivity antialgal effects on all test red tide microalgae, and antialgal activities against red tide microalgae obviously enhanced with the increase of concentration of antialgal compounds. Based on this, EC50-96 h values of these nine compounds for six red tide microalgae were obtained for the first time. By analyzing and comparing EC50-96 h values, it has been determined that seven compounds (1, 3, 4, 6, 7, 8 and 9) showed the superior application potential than potassium dichromate or gossonorol and other six compounds as a characteristic antialgal agent against Heterosigma akashiwo, Karenia mikimitoi and Prorocentrum donghaiense. Overall this study has suggested that green algae Ulva pertusa is a new source of bioactive compounds with antialgal activity.

Algicidal effect of hybrid peptides as potential inhibitors of harmful algal blooms.

OBJECTIVES: To biochemically characterize synthetic peptides to control harmful algal blooms (HABs) that cause red tides in marine water ecosystems. RESULTS: We present an analysis of several short synthetic peptides and their efficacy as algicidal agents. By altering the amino acid composition of the peptides we addressed the mode of algicidal action and determine the optimal balance of cationic and hydrophobic content for killing. In a controlled setting, these synthetic peptides disrupted both plasma and chloroplast membranes of several species known to result in HABs. This disruption was a direct result of the hydrophobic and cationic content of the peptide. Furthermore, by using an anti-HAB bioassay in scallops, we determined that these peptides were algicidal without being cytotoxic to other marine organisms. CONCLUSIONS: These synthetic peptides may prove promising for general marine ecosystem remediation where HABs have become widespread and resulted in serious economic loss.

Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock.

Aquatic organisms are often exposed to dramatic changes in salinity in the environment. Despite decades of research, many questions related to molecular and physiological mechanisms mediating sensing and adaptation to salinity stress remain unanswered. Here, responses of Vaucheria erythrospora, a turgor-regulating xanthophycean alga from an estuarine habitat, have been investigated. The role of ion uptake in turgor regulation was studied using a single cell pressure probe, microelectrode ion flux estimation (MIFE) technique and membrane potential (Em ) measurements. Turgor recovery was inhibited by Gd(3+) , tetraethylammonium chloride (TEA), verapamil and orthovanadate. A NaCl-induced shock rapidly depolarized the plasma membrane while an isotonic sorbitol treatment hyperpolarized it. Turgor recovery was critically dependent on the presence of Na(+) but not K(+) and Cl(-) in the incubation media. Na(+) uptake was strongly decreased by amiloride and changes in net Na(+) and H(+) fluxes were oppositely directed. This suggests active uptake of Na(+) in V. erythrospora mediated by an antiport Na(+) /H(+) system, functioning in the direction opposite to that of the SOS1 exchanger in higher plants. The alga also retains K(+) efficiently when exposed to high NaCl concentrations. Overall, this study provides insights into mechanisms enabling V. erythrospora to regulate turgor via ion movements during hyperosmotic stress.

Metabolic switching: synergistic induction of carotenogenesis in the aerial microalga, Vischeria helvetica, under environmental stress conditions by inhibitors of fatty acid biosynthesis.

OBJECTIVES: To investigate the roles of fatty acid biosynthesis in carotenogenesis in the high-lipid accumulating aerial microalga Vischeria helvetica KGU-Y001, we cultured algal cells with fatty acid biosynthesis inhibitors. RESULTS: Under nitrogen-deficient, high-light (200 µmol photons m(-2) s(-1)) conditions, the alga accumulated 6.2 mg carotenoids g(-1) dry weight cells (DWC) after 1 week of culture. The total fatty acid content increased gradually, and reached 290 mg g(-1) DWC after 9 weeks. When algal cells were cultured with a fatty acid biosynthesis inhibitor (molinate) under nitrogen-deficient, high-light conditions for 1 week, carotenoid accumulation was synergistically increased to 2.4 times that in algal cells cultured without the inhibitor in nitrogen-deficient, low-light conditions (40 µmol photons m(-2) s(-1)). The synergistic induction of carotenogenesis was suppressed by an inhibitor of c-jun N-terminal kinase, a mitogen-activated protein kinase-like protein. CONCLUSION: In a commercial context, carotenoid production could be increased by using fatty acid biosynthesis inhibitors to redirect metabolic flux to carotenoid biosynthesis instead of fatty acid synthesis.

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum.

The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cmr), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Bler) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes.

Global searches for microalgae and aquatic plants that can eliminate radioactive cesium, iodine and strontium from the radio-polluted aquatic environment: a bioremediation strategy.

The Fukushima 1 Nuclear Power Plant accident in March 2011 released an enormously high level of radionuclides into the environment, a total estimation of 6.3 × 10¹7 Bq represented by mainly radioactive Cs, Sr, and I. Because these radionuclides are biophilic, an urgent risk has arisen due to biological intake and subsequent food web contamination in the ecosystem. Thus, urgent elimination of radionuclides from the environment is necessary to prevent substantial radiopollution of organisms. In this study, we selected microalgae and aquatic plants that can efficiently eliminate these radionuclides from the environment. The ability of aquatic plants and algae was assessed by determining the elimination rate of radioactive Cs, Sr and I from culture medium and the accumulation capacity of radionuclides into single cells or whole bodies. Among 188 strains examined from microalgae, aquatic plants and unidentified algal species, we identified six, three and eight strains that can accumulate high levels of radioactive Cs, Sr and I from the medium, respectively. Notably, a novel eustigmatophycean unicellular algal strain, nak 9, showed the highest ability to eliminate radioactive Cs from the medium by cellular accumulation. Our results provide an important strategy for decreasing radiopollution in Fukushima area.

Phytohormone-induced changes in growth, physiology, and biochemistry of Aurantiochytrium sp. for sustainable bioproduction.

The study aimed to assess the effects of nine combinations of phytohormones, salicylic acid (SA), gibberellic acid (GA), and jasmonic acid (JA) on the growth, physiology, and biochemistry of Aurantiochytrium sp. Parameters like optical density (OD), biomass, protein content, hydrogen peroxide (H2O2), malondialdehyde (MDA), catalase activity (CAT), and gene expression (malic enzyme (ME) and acetyl-CoA carboxylase (ACCase)) were assessed at various cultivation stages (24, 48, 72, and 96 h). The research also analyzed fatty acid composition, unsaturated fatty acids (UFA), saturated fatty acids (SFA), and the UFA to SFA ratio (USS) to understand the biochemical changes induced by phytohormones. Results demonstrated that modifying phytohormone concentrations significantly affected the characteristics of the microalgae, particularly in correlation with different growth stages, emphasizing the necessity of precise control of phytohormone levels for optimizing cultivation conditions and enhancing bioactive compound production in Aurantiochytrium sp.

Toxic effect of p-chloroaniline and butyl acrylateon Nannochloropsis oculata based on water samples from two sea areas.

To compare the influence of water samples collected from various areas on toxic effect of HNS, we examined the toxic effect of two commonly found HNS: p-chloroaniline and butyl acrylate, on Nannochloropsis oculata cultured in seawater collected from Laizhou bay and Jiaozhou bay (China). The results showed that both p-chloroaniline and butyl acrylate had significant toxic effect on N. oculata cultured in both water samples. P-chloroaniline inhibited its net oxygenation rate and oxygen consumption rate. Butyl acrylate inhibited the net oxygenation rate whereas significantly stimulated oxygen consumption rate. Performance of N. oculata changed between two water samples under same level of p-chloroaniline and butyl acrylate. The net oxygenation rate of N. oculata cultured in the seawater from the Jiaozhou bay increased by 11.60 %, the oxygen consumption rate increased by 26.91 %, algae cell growth decreased by 16.83 %, compared to those from Laizhou bay. The Fv/Fm of N. oculata cultured in Jiaozhou bay was more significantly inhibited at 0.5 and 2.0 mg L-1 p-chloroaniline, while it was significantly inhibited at 5. 0 mg L-1 of butyl acrylate, compared to those from Laizhou bay. Moreover, the toxic effect of both HNS on net oxygenation rate and oxygen consumption rate were significantly attenuated as the concentration increased. The growth inhibition of microalgae cultured in two seawater samples was more evident at 0.5 and 5.0 mg L-1 p-chloroaniline than at 2.0 mg L-1 p-chloroaniline,and the growth inhibition of microalgae cultured in two seawater samples was more evident at 2.0 and 5.0 mg L-1 butyl acrylate than at 0.5 mg L-1 butyl acrylate. These results indicated that toxic effect of p-chloroaniline and butyl acrylate on the growth of N. oculata was influenced by the pollutants in the two water samples. Consequently, a corresponding research on water sample is required in advance to increase accuracy of future ecological risk assessment of HNS.

Physiological response and morphological changes of Heterosigma akashiwo to an algicidal compound prodigiosin.

Harmful algal blooms (HABs) occur all over the world, producing severely negative effects on human life as well as on marine ecosystems. The algicidal compound, prodigiosin, secreted by algicidal bacteria Hahella sp. KA22 can lyse the harmful alga Heterosigma akashiwo. This study is aimed to investigate the algicidal mechanism of prodigiosin against H. akashiwo by detecting physiological and morphological responses of H. akashiwo to presence of prodigiosin. The results indicated that prodigiosin showed strong algicidal effects on H. akashiwo at the concentration of 3 µg/mL. Chlorophyll a and protein levels of the microalgae decreased significantly while malonaldehyde levels increased at this concentration. Contents of ascorbic acid and activities of superoxide dismutase and peroxidase increased fast with the quick decrease of the reactive oxygen species (ROS). For the 3 µg/mL prodigiosin treatment group, transcription of genes related to photosynthesis and respiration were significantly inhibited at 12 h while respiration related genes increased at 24 h. Collectively, the results indicated that prodigiosin could kill the microalgae by inducing ROS overproduction which could destroy the cell integrity and change the antioxidant system levels and functional gene expression. Our results demonstrated that prodigiosin is an effective algicide for the control of harmful algae.

Algicidal hydroxylated C18 unsaturated fatty acids from the red alga Tricleocarpa jejuensis: Identification, synthesis and biological activity.

Bioassay-guided separation of a methanol extract of Tricleocarpa jejuensis by monitoring algicidal activity against the red tide phytoplankton Chattonella antiqua led to the isolation of an active fraction consisting of a mixture of four isomeric compounds. The active compounds were identified as (E)-9-hydroxyoctadec-10-enoic acid (1), (E)-10-hydroxyoctadec-8-enoic acid (2), (E)-11-hydroxyoctadec-12-enoic acid (3) and (E)-12-hydroxyoctadec-10-enoic acid (4) by NMR, IR and mass spectral data. The structures were confirmed by comparison of the NMR and MS data with those of authentic samples of 1-4 obtained by unambiguous syntheses. Synthesized hydroxy acids 1-4 and related compounds were assessed for algicidal activity against C. antiqua and it was found that all of 1-4 had high activity (>80% mortality at 24 h) at a concentration of 20 µg/mL. A structure-activity relationship study using 11 related compounds revealed that the presence of the hydroxyl group is important for the activity and the double bond may be replaced with a triple bond.

Development of Aurantiochytrium limacinum SR21 cultivation using salt-rich waste feedstock for docosahexaenoic acid production and application of natural colourant in food product.

Microalgae biorefinery is presently receiving a lot of attention as driven by its production of high value-added products. In this study, an oleaginous microalga Aurantiochytrium limacinum SR21 was cultured for docosahexaenoic acid (DHA) production using 20% (w/v) of K2HPO4-waste feedstock to replace 0.005% (w/v) of KH2PO4 in the flask culture. DHA is an essential nutrient for human's brain functionalities. Collectively, the K2HPO4-waste feedstock with working concentration of 0.005% (w/v) in the cultivation prompted a higher lipid content (8.29%) and DHA production (128.81 mg.L-1). Moreover, natural plant pigment products containing stabilised betacyanins were utilised as natural red colourants for hard candy production. This study develops microalgal cultivation using salt-rich waste feedstock for a higher lipid and DHA content as well as application of natural colouring agents in food products.

Transcriptomic Mechanism of the Phytohormone 6-Benzylaminopurine (6-BAP) Stimulating Lipid and DHA Synthesis in Aurantiochytrium sp.

The phytohormone 6-benzylaminopurine (6-BAP) significantly improves lipid synthesis of oleaginous microorganisms with the great potential applied in lipid production. In the current study, the lipid and DHA productions in oleaginous Aurantiochytrium sp. were found to be improved by 48.7% and 55.3%, respectively, induced by 6-BAP treatments. Then, using high-throughput RNA-seq technology, the overall de novo assembly of the cDNA sequence data generated 53871 unigenes, and 15902 of these were annotated in at least one database. The comparative transcriptomic profiles of cells with and without 6-BAP treatments revealed that a total of 717 were differently expressed genes (DE), with 472 upregulated and 245 downregulated. Further annotation and categorization indicated that some DE genes were involved in pathways crucial to lipid and DHA productions, such as fatty acid synthesis, central carbon metabolism, transcriptional factor, signal transduction, and mevalonate pathway. A regulation mode of 6-BAP, in turn, perception and transduction of 6-BAP signal, transcription factor, expression regulations of the downstream genes, and metabolic changes, respectively, was put forward for the first time in the present study. This research illuminates the transcriptomic mechanism of phytohormone stimulation of lipid and DHA production in an oleaginous microorganism and provides the potential targets modified using genetic engineering for improving lipid and DHA productivity.

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.