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.

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.

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.

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.

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.

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.

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.

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.

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.

Complex role of titanium dioxide nanoparticles in the trophic transfer of arsenic from Nannochloropsis maritima to Artemia salina nauplii.

Increasing concern has been focused on the potential risks associated with the trophic transfer to aquatic organisms of ambient contaminants in the presence of titanium dioxide nanoparticles (nano-TiO2). This study investigated the influence of nano-TiO2 on the trophic transfer of arsenic (As) from the microalgae Nannochloropsis maritima to the brine shrimp Artemia salina nauplii. We found that nano-TiO2 could significantly facilitate As sorption on N. maritima within an exposure period of 24 h, and this sorption subsequently led to higher As trophic transfer from the algae to A. salina according to trophic transfer factors (TTFAs+nano-TiO2 > TTFAs). However, after 48 h of depuration, the retention of As in A. salina fed As-nano-TiO2-contaminated algae was even lower than that in A. salina fed As-contaminated algae at the same exposure concentrations. This result indicates that the increased food chain transfer of As in the presence of nano-TiO2 can be explained by adsorption of As onto nano-TiO2 in contaminated food (algae), but the bioavailability of As in A. salina is reduced after the introduction of nanoparticles. Although the stress enzyme activities of superoxide dismutase (SOD) and acetylcholinesterase (AChE) in A. salina at a lower As concentration treatment in the presence of nano-TiO2 were not significantly changed, they increased with higher exposure concentrations of As with or without nano-TiO2. Our study highlighted the complex role of nanomaterials in the transfer of ambient contaminants via trophic chains and the potential of nano-TiO2 to reduce the bioavailability of As via trophic transfer to saltwater zooplankton.

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.

Selection of Schizochytrium limacinum mutants based on butanol tolerance

Background: Mutation breeding is one of the most important routes to achieving high docosahexaenoic acid (DHA) productivity using Schizochytrium. However, few selection strategies have been reported that aim to generate a high DHA content in Schizochytrium lipids. Results: First, culture temperature altered the butanol tolerance of Schizochytrium limacinum B4D1. Second, S. limacinum E8 was obtained by selecting mutants with high butanol tolerance. This mutant exhibited a 17.97% lower proportion of DHA than the parent strain S. limacinum B4D1. Third, a negative selection strategy was designed in which S. limacinum F6, a mutant with poor butanol tolerance, was obtained. The proportion of DHA in S. limacinum F6 was 11.22% higher than that of parent strain S. limacinum B4D1. Finally, the performances of S. limacinum B4D1, E8 and F6 were compared. These three strains had different fatty acid profiles, but there was no statistical difference in their biomasses and lipid yields. Conclusion: It was feasible to identified the relative DHA content of S. limacinum mutants based on their butanol tolerance.

Effects of butanol on high value product production in Schizochytrium limacinum B4D1.

Schizochytrium is a microalgae-like fungus and is widely used for producing docosahexaenoic acid (DHA). It is also a promising source of squalene and carotenoids. However, few fermentation strategies are available in enhancing squalene and carotenoid content in Schizochytrium. This study showed that butanol addition had multiple effects on Schizochytrium limacinum B4D1. First, butanol addition altered the lipid content of cells. Second, 6g/L of butanol decreased the proportion of DHA by nearly 40%. Third, the squalene content increased 31-fold in the presence of 6g/L butanol. Finally, cells accumulated more carotenoids upon butanol addition. Specifically, when cells were treated with 8g/L butanol, the astaxanthin content increased to 245 times than that of the untreated control. These results are helpful for the commercial exploitation of Schizochytrium in producing squalene and carotenoids.

Results from a round robin test for the ecotoxicological evaluation of construction products using two leaching tests and an aquatic test battery.

A European round robin test according to ISO 5725-2 was conceptually prepared, realised, and evaluated. The aim was to determine the inter-laboratory variability of the overall process for the ecotoxicological characterization of construction products in eluates and bioassays. To this end, two construction products BAM-G1 (granulate) and HSR-2 (roof sealing sheet), both made of EPDM polymers (rubber), were selected. The granular construction product was eluted in a one stage batch test, the planar product in the Dynamic Surface Leaching test (DSLT). A total of 17 laboratories from 5 countries participated in the round robin test: Germany (12), Austria (2), Belgium (1), Czech Republic (1) and France (1). A test battery of four standardised ecotoxicity tests with algae, daphnia, luminescent bacteria and zebrafish eggs was used. As toxicity measures, EC50 and LID values were calculated. All tests, except the fish egg test, were basically able to demonstrate toxic effects and the level of toxicity. The reproducibility of test results depended on the test specimens and the test organisms. Generally, the variability of the EC50 or LID values increased with the overall level of toxicity. For the very toxic BAM-G1 eluate a relative high variability of CV = 73%-110% was observed for EC50 in all biotests, while for the less toxic HSR-2 eluate the reproducibility of EC50 varied with sensitivity: it was very good (CV = 9.3%) for the daphnia test with the lowest sensitivity, followed by the algae test (CV = 36.4%). The luminescent bacteria test, being the most sensitive bioassay for HSR-2 Eluate, showed the highest variability (CV = 74.8%). When considering the complex overall process the reproducibility of bioassays with eluates from construction products was acceptable.

Removal of harmful alga, Chattonella marina, by recyclable natural magnetic sphalerite.

Fish-killing harmful algal blooms (HABs) of Chattonella marina causes serious hazards and risks to fish farming and environment throughout the world. At present, it is necessary to explore cost-effective and recyclable materials for controlling C. marina blooms to reduce the cost and control the potential side effect to the environment. A novel earth-abundant natural magnetic sphalerite (NMS) for removing C. marina was systematically investigated, including the effect of NMS dosage, temperature, pH and salinity on algal removal efficiency. Algal cells could be rapidly removed by NMS (1-2g/L) through adsorption and physical interaction. The algal destruction process was enhanced under the following reaction conditions: temperature>25°C, salinity>30 ppt and pH value<7.5. The reusability of magnetic recycled NMS and effect of light irradiation on algal cell removal were also determined. NMS exhibited excellent stability after repeated algal cell removal, and the efficiency was further enhanced by light illumination. The current study suggested that using NMS to control C. marina blooms could be a novel promising strategy, which is cost-effective, stable, and easy for recycling.

Regulation of the Docosapentaenoic Acid/Docosahexaenoic Acid Ratio (DPA/DHA Ratio) in Schizochytrium limacinum B4D1.

Docosapentaenoic acid/docosahexaenoic acid ratio (DPA/DHA ratio) in Schizochytrium was relatively stable. But ideally the ratio of DPA/DHA will vary according to the desired end use. This study reports several ways of modulating the DPA/DHA ratio. Incubation times changed the DPA/DHA ratio, and changes in this ratio were associated with the variations in the saturated fatty acid (SFAs) content. Propionic acid sharply increased the SFAs content in lipids, dramatically decreased the even-chain SFAs content, and reduced the DPA/DHA ratio. Pentanoic acid (C5:0) and heptanoic acid (C7:0) had similar effects as propionic acid, whereas butyric acid (C4:0), hexanoic acid (C6:0), and octanoic acid (C8:0) did not change the fatty acid profile and the DPA/DHA ratio. Transcription analyses show that ß-oxidation might be responsible for this phenomenon. Iodoacetamide upregulated polyunsaturated fatty acid (PUFA) synthase genes, reduced the DHA content, and improved the DPA content, causing the DPA/DHA ratio to increase. These results present new insights into the regulation of the DPA/DHA ratio.

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase.

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4°C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120Gy yielded more DHA compared with cells from 40Gy, 80Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4°C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27g/Lh and 30% from 21 to 27g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress.