Stimulating EPA Biosynthesis in Microchloropsis salina through Cultivation with Selected Myxobacterial Culture Supernatants

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

Climate change and eutrophication are among the main stressors of shallow freshwater ecosystems, and their effects on phytoplankton community structure and primary production have been studied extensively. However, their combined effects on the algal production of polyunsaturated fatty acids (PUFA), specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are currently unresolved. Moreover, the proximate reasons for changes in phytoplankton EPA and DHA concentrations are unclear, i.e., the relative importance of ecological (changes in the community composition) vs. ecophysiological (within taxa changes in EPA and DHA levels) factors.We investigated the responses of phytoplankton EPA and DHA concentrations to warming (IPCC climate scenario) and nutrient additions in mesocosms which had been run continuously at varying temperature and nutrient levels for 15 years prior to this study. Nutrient treatment had a significant effect on phytoplankton EPA and DHA concentrations and about 59 % of the variation in EPA and DHA concentrations could be explained by changes in the phytoplankton community structure. Increased biomass of diatoms corresponded with high EPA and DHA concentrations, while cyanobacteria/chlorophyte dominated mesocosm had low EPA and DHA concentrations. Warming had only a marginal effect on the EPA and DHA concentrations in these mesocosms. However, a significant interaction was observed with warming and N:P ratio.Our findings indicate that direct nutrient/temperature effects on algal physiology and PUFA metabolism were negligible and the changes in EPA and DHA concentrations were mostly related to the phytoplankton community structure and biomass. These results also imply that in shallow temperate lakes eutrophication, leading to increased dominance of cyanobacteria, will probably be a greater threat to phytoplankton EPA and DHA production than warming. EPA and DHA are nutritionally important for upper trophic level consumers and decreased production may impair secondary production.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are long chain polyunsaturated fatty acids (LC-PUFA) belonging to the omega-3 fatty acids, which have demonstrated to be nutritionally important for the development, cardiovascular and brain function of higher eukaryotes. Exponential growth of the omega-3 fatty acids market has dramatically reduced the stocks of traditional sources for those fatty acids that are typically derived from small fatty fish. Microalgae are the major provider of LC-PUFAs in the marine ecosystem; hence research has turned towards commercial land-based cultivation of algae with high nutritional content. Heterotrophic culture systems of microalgae are already being used commercially, in particular for the production of DHA as a food supplement. However, production has high setup and operational costs, and faces limitations for scale-up as contamination of these heterotrophic cultivation systems cannot be easily controlled. Autotrophic production systems may allow further expansion of the use of microalgae as LC-PUFA biofactories, and therefore EPA obtained from marine microalgae can form the basis for sustainable industrial production, when optimal culturing conditions specifically stimulate the synthesis of these bio-products. The proposition was made at the start of this thesis, that optimal environmental cultivation conditions for EPA production in marine microalgae can be promoted under certain conditions and optimized to achieve higher yields. Among others, two potential EPA-producing microalgae were further investigated based on initial production data using unoptimized standard laboratory conditions. These include the microalga Nannochloropsis sp. BR2, originally isolated from the Brisbane River, Australia, as well as the marine microalga Tetraselmis sp. M8, that was first isolated from a rock pool at Australia’s Sunshine Coast. In the present study, it was found that the highest EPA content can be achieved for Nannochloropsis sp. when conditions were optimal for growth and high biomass productivity. For example, a well-controlled pH or the use of additional CO2 during Nannochloropsis sp. cultivation led to the highest overall EPA productivity, with EPA proportions of up to 40% of total isolated fatty acids. However, the total EPA contents per dry weight in this alga can be higher when stimulated by external stress factors, but these typically slow down growth. For Tetraselmis sp. M8, optimal EPA productivity was achieved with progressive nutrient stress. LC-PUFA biosynthesis in this alga correlated well with gene expression and differed according to nutrient and growth phase in this microalgal strain. However, no single stress factor was identified that specifically induced only EPA biosynthesis. In conclusion, this study demonstrates that improved EPA production can be achieved for autotrophic microalgae. LC-PUFAs, including EPA were highest in relation to total fatty acids during optimal growth phases, suggesting that they are required in larger amounts under these conditions for cellular functions, such as membrane assembly. Stress conditions, such as nutrient deprivation and UV-C light exposure can lead to higher total amounts of EPA, most likely to repair cellular damage, but overall EPA productivities were highest during exponential growth for the high yielding Nannochloropsis sp. BR2 strain. Large-scale cultivation of autotrophic microalgae should be developed to determine whether additional optimization measures can be identified to enable more sustainable supplies of omega-3 fatty acids via land-based grown autotrophic microalgae.

Strategies for enhancing eicosapentaenoic acid production: From fermentation to metabolic engineering

The nitrogen (N) sources including nitrate, ammonium, urea, tryptone and yeast extract were optimized for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic cultures. First, nitrate and urea were found to be the preferred N sources for both cell growth and EPA content. The optimal concentrations of nitrate and urea for cell growth and EPA yield were 620 mg/l and 600 mg/l, respectively. Secondly, tryptone and yeast extract were respectively added to the medium and both of them were found to enhance EPA production compared with the control. To investigate the coeffects of tryptone and yeast extract, in the subsequent experiments, a central composite experimental design was used to optimize the two complex nitrogen sources. The results showed that the EPA production was influenced not only by tryptone or yeast extract, but also by their interactions. The cell dry weight (DW), EPA content and EPA yield could be described by the second-order polynomial equations with high confidence levels (>99%). Based on the surface plots of the responses, the optimal concentrations of tryptone and yeast extract were determined to be 1.6 g/l and 0.8 g/l, respectively. To verify the predicted models, the alga was grown in the medium with tryptone and yeast extract at optimal concentrations. The resulting DW, EPA content and EPA yield reached 6.48 g/l, 2.74 % (w/w), and 175 mg/l, respectively, which agreed with the predicted values, and were much higher than those obtained in the previous studies.

Large-scale photoautotrophic production of microalgae has the potential to provide a sustainable supply of omega-3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) for human and animal nutrition. This study presents a kinetic model for the EPA-producing microalga Phaeodactylum tricornutum in photoautotrophic conditions, with light and nitrogen being the growth limiting factors. The model was developed using a dataset obtained from bench-scale (5 L) cultures and was successfully validated against pilot-scale (50 L) cultures. This model is the first to predict the biomass and total fatty acid accumulation along with the EPA concentrations in the biomass and total fatty acid fraction for microalgae. The model was used to develop an optimized repeated-batch strategy; implementation of this led to increases in the biomass and EPA productivities of 50 and 20% respectively. This clearly indicates the potential of the model to be used as a tool in the design, optimization and scale-up of microalgal systems for EPA production.

Production of eicosapentaenoic acid by Vacuoliviride crystalliferum under 20% CO2 conditions

The effects of several nutritional factors on the growth and eicosapentaenoic acid (EPA) production of diatom Nitzschia laevis were studied. 4 LDM (quadrupled concentration of the nutrient salt) was the optimal concentration of nutrient salt for the growth and EPA production of N. laevis. The growth of N. laevis was inhibited when the glucose concentration was either lower than 10 gL−1 or higher than 15 gL−1. Both sodium nitrate and urea were good nitrogen sources for the growth and EPA production, while ammonium chloride seriously decreased the dry cell weight (DW) and the EPA content. Silicate seriously influenced the growth of N. laevis. The maximum DW of 2.34 gL−1 was obtained in the presence of 150 mgL−1 Na2SiO3·9H2O. The EPA content remained almost the same when the silicate concentration was lower than 150 mgL−1; however, higher silicate concentrations resulted in a steady decrease of EPA content. Low medium salinity (⩽29) did not seem to influence the DW of N. laevis, and high salinity resulted in a decrease of DW. The highest EPA content (4.08%) and yield (110 mgL−1) were observed at the salinity of 36 and 29, respectively.

Optimization of nitrogen sources for heterotrophic production of eicosapentaenoic acid by the diatom Nitzschia laevis

The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m-2s-1 ) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.

The production of polyunsaturated fatty acids by microalgae: from strain selection to product purification

A model for the prediction of eicosapentaenoic acid (EPA) productivity from Phaeodactylum tricornutum cultures that takes into account the existence of photolimitation and photoinhibition of growth under outdoor conditions is presented. The effects of the external irradiance on the culture surface, the average irradiance inside the culture, and the light regime at which the cells are exposed on pigments and EPA content are studied. The chlorophyll content decreases exponentially with the average irradiance, whereas the carotenoids content increases linearly with the external irradiance due to a higher extension of photoinhibition. A decrease in the fatty acid content of the biomass with irradiance on reactor surface is observed when photoinhibition becomes relevant. The average irradiance within the culture mainly influenced the fatty acid profile of the biomass. As the average irradiance becomes higher, percentages of saturated and monounsaturated fatty acids decrease, increasing the portion of EPA. By taking into account the different relationships among pigment and EPA content with the irradiance, the variation in EPA productivity over the year can be simulated as a function of average and external irradiance. For the two photobioreactors employed the maximum EPA productivity is attained in spring and fall (30 mg L(-1) day(-1) for tube diameter 0. 06 m and 50 mg L(-1) day(-1) for tube diameter 0.03 m). In winter, the biomass productivity is limited by low light availability although the EPA content is maximum. In summer, the biomass productivity is higher although the EPA content diminished by photoinhibition; the higher the dilution rate, the lower the minimum. Thus, the conditions that increase the biomass productivity and the polyunsaturated fatty acids content are in opposition, the optimum being reached by operating under photolimitation with high growth rates in order to produce a high proportion of polyunsaturated fatty acids.

Eicosapentaenoic acid (EPA) productivity from continuous cultures of the marine microalga Isochrysis galbana was studied, taking into account the irradiance on the reactor surface, that is, the photolimitation/photoinhibition regime to which the cells are exposed. Experiments were conducted under a wide variety of operating conditions. The dilution rate ranged from 0.005 h−1 to 0.040 h−1 at five external irradiances (820, 1620, 2050, 2450 and 3270 μmol photons m−2 s−1) covering photolimited to photoinhibited growth. Under these conditions, the specific growth rate (μ) was found to be the main factor influencing EPA content (ranging from 2.35% to 5.23% dryweight) and productivity (up to 0.88 mg l−1 h−1). The fatty acid content was not significantly affected by the external irradiance, but was influenced by the state of growth of the microalga, depending on whether the light regime was photolimiting or photoinhibiting. It might be suggested that light should no longer be considered an isolated factor affecting EPA synthesis, but an indirect influence through the photolimitation/photoinhibition regime and growth rate. At a given dilution rate, EPA content and biomass concentration are lower under photoinhibiting external irradiances than those corresponding to photolimiting conditions, and consequently EPA productivity decays. Since the effect of photoinhibition is less marked at high biomass concentration, a strategy to optimize EPA productivity from microalgal cultures could consist of reducing the dilution rate when the external irradiance increases above the phoinhibition threshold.

Cottonseed meal (CSM), a common agricultural by‐product, was used as a nutrient source for the production of eicosapentaenoic acid (EPA) by Pythium irregulare. CSM can support good cell growth performance, as can yeast extract (YE). In terms of the maximum EPA content and EPA yield, CSM is superior to YE. Low concentrations of CSM are beneficial to lipid synthesis, and high concentrations favor the EPA content. Utilizing response surface methodology (RSM) analysis, the optimum contents of glucose and CSM in the fermentation medium were determined to be 40.2 and 16.1 g/l, respectively. After 6 days of fermentation at 25 °C and optimal conditions, the EPA yield and productivity were 245.3 and 40.9 mg/l day, respectively. Particle size of CSM was found to affect the EPA production, and a finely ground CSM (100 mesh) was determined to be best for EPA production. The variation in the fatty acid content of total fatty acid (TFA) indicates that EPA was synthesized through the n‐6 route in P. irregulare and Δ12 desaturase was the key enzyme for EPA biosynthesis. Sodium carbonate was determined to be notably good at removing free gossypol attached to biomass. After fungal biomass from each flask had been harvested from Na2CO3‐supplemented medium, 1 % (w/v) Na2CO3 solution was used to wash the mycelia three times; free gossypol (FG) was not detected (detection limit 0.0018 %). This work provides a new approach using cottonseed meal to produce EPA through fungal fermentation.

BACKGROUND: Purification and characterization of an intracellular lipase produced by Rhizopus chinenesis cultured in solid-state fermentation was investigated. The potential application in concentrating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil by the pure enzyme was also studied. RESULTS: Through four successive purification steps, the enzyme was purified to homogeneity with an apparent molecular mass of 36 kDa. The lipase was active for pH between 7.0 and 9.0 and temperatures 20–45 °C. Lipase activity was slightly increased in the presence of Ca2+ and Mg2+, but strongly inhibited by Hg2+ and SDS. The pure enzyme was most active on medium chain p-nitrophenol esters, with the highest activity towards pNP-caprylate (C8). The enzyme is a non-specific lipase, because it cleaved not only the 1,3-positioned ester bonds but also the 2-positioned bond in triolein. High EPA (17.6%) and DHA (32.9%) contents were achieved using the pure lipase (100 U) within 10 h. CONCLUSION: The enzymatic activity of the lipase on a wide variety of substrates and its stability in the presence of some organic solvents suggest that the lipase should be investigated for a range of commercial applications. The pure lipase was proved to possess potential ability for the production and concentration of EPA and DHA from fish oil. Copyright © 2008 Society of Chemical Industry