Screening of diatoms for heterotrophic eicosapentaenoic acid production

Nine strains of diatoms (representing four species) were screened for their ability to produce eicosapentaenoic acid (EPA) when cultured heterotrophically on glucose. Four strains were able to produce EPA heterotrophically using glucose as its carbon and energy source. Of the four,Nitzschia laevis was the best EPA producer, yielding 0.017 g g^–1 dry cell weight.N. laevis was the only species tested which synthesised more EPA heterotrophically than photosynthetically. This study shows thatN. laevis is a potential source of EPA production using heterotrophic culture conditions with glucose as the carbon and energy substrate.Author for correspondence     

Application of statistically-based experimental designs for the optimization of eicosapentaenoic acid production by the diatom Nitzschia laevis

Statistically based experimental designs were applied to the optimization of medium components and environmental factors for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic conditions. First, the Plackett-Burman design was used to evaluate the effects of variables including medium components and environmental factors on cell growth and EPA production. Among these variables, NaCl, CaCl(2), PI metal solution, pH, and temperature were identified to have the significant effects (with confidence level > 90 %). Subsequently, the concentrations of NaCl, CaCl(2), PI metal solution as well as the values of pH and temperature were optimized using central composite design. The cell growth and EPA production were found to respectively correlate to NaCl, CaCl(2), pH, and temperature that could be represented by second-order polynomial models. The optimal values of the four parameters were determined by response surface and numerical analyses as 8 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 19.8 degrees C for cell dry weight (DW), and 14 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 18 degrees C for EPA production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy led to a DW of 9 g/L, an EPA yield of 280 mg/L and an EPA productivity of 28 mg/L/d, respectively, which were considerably higher than those obtained in the previous studies.     

Growth characteristics and eicosapentaenoic acid production by Nannochloropsis sp. in mixotrophic conditions

Nannochloropsis sp. was grown under mixotrophic conditions with 30 mM glucose as carbon source, reaching 507 mg dry wt l(-1) after 8 d. This was 1.4-fold of that obtained under photoautotrophic conditions. Under mixotrophic and photoautotrophic cultivations, the net photosynthetic rate of Nannochloropsis sp. did not change but the respiratory rate increased in the mixotrophic cultivation. The yield of eicosapentaenoic acid was 22 mg l(-1) in the mixotrophic cultivation and 20 mg l(-1) under the photoautotrophic cultivation.     

Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp. under mixotrophic conditions

Three species of microalgae able to produce eicosapentaenoic acid(EPA) were collected from brackish and sea water around Japan. The species were identified as Navicula saprophila, Rhodomonassalina and Nitzschia sp. EPA as a proportion of total fatty acids increased in the presence of acetic acid for Rhodomonas salina and Nitzschia sp. However, Navicula saprophila displayed the greatest productivity of EPA and the EPA content of its biomass was enhanced under mixotrophic conditions in the presence of acetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

Heterotrophic production of eicosapentaenoid acid by the diatom Nitzschia laevis: effects of silicate and glucose

The effects of silicate and glucose on growth and eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis were studied. By alternately altering the concentrations of silicate (2.7–64 mg l⁻¹) and glucose (1–40 g l⁻¹) in the medium, the highest cell dry weight (ca. 5.5 g l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest specific growth rate (ca. 0.65 day⁻¹) was obtained at a relatively low glucose concentration (5 g l⁻¹) and high silicate concentrations (32–64 mg l⁻¹). At glucose levels of 5 and 20 g l⁻¹, EPA content was higher with lower silicate concentrations (2.7 and 16 mg l⁻¹ silicate, respectively), while at a silicate level of 16 mg l⁻¹, higher glucose concentrations (20–40 g l⁻¹) facilitated EPA formation. The highest EPA yield (131 mg l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest EPA productivity (15.1 mg l⁻¹ day⁻¹) was obtained at 20 g l⁻¹ glucose and 64 mg l⁻¹ silicate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 218–224. Received 08 May 2000/ Accepted in revised form 21 July 2000     

硅藻变温发酵生产二十碳五烯酸的研究

本文考察不了同温度（10℃～30℃）对硅藻(Nitzschia laevis)合成二十碳五烯酸(EPA)的影响,对不同温度下的发酵过程进行动力学特性分析。在此基础上,提出了EPA合成的变温培养方法：延滞期及对数期初期温度控制在25℃,从对数期中期开始在20℃条件下进行培养。采用此变温培养进行发酵,EPA的含量和产量分别达到了6.00%和291.60 mg·L-1,较采用单一温度(25℃)发酵的最大值分别提高了24.07%和18.81%。     

硅藻发酵生产二十碳五烯酸基础培养基的选择研究

考察了不同培养基中硅藻(Nitzschia laevis)合成二十碳五烯酸(EPA)的情况,筛选出了EPA合成的基础培养基8LDM,即将LDM培养基中营养物质浓度提高7倍。采用此培养基进行发酵,EPA的产量达到了95.22mg/L,是采用f/2和LDM发酵最大值的4.24倍和4.36倍。     

Continuous cultivation of the diatom Nitzschia laevis for eicosapentaenoic acid production: physiological study and process optimization

The continuous cultures of the diatom Nitzschia laevis were performed at different dilution rates (D) and feed glucose concentrations (S(0)) to investigate cellular physiological responses and its production potential of eicosapentaenoic acid (EPA). Steady-state cell dry weight, residual glucose concentration, cell growth yield, specific glucose consumption rate, and fatty acid profiles were investigated within the range of D from 0.1 to 1.0 day(-1) (S(0) fixed at 20 g/L) and the range of S(0) from 5 to 35 g/L (D fixed at 0.3 day(-1)), respectively. The highest EPA productivity of 73 mg L(-1) day(-1) was obtained at D = 0.5 day(-1) and S(0) = 20 g/L. However, when the continuous culture achieved high productivities of EPA at certain dilution rates and feed glucose concentrations, glucose in the feed could not be consumed completely. Accordingly, the continuous culture was evaluated in terms of both EPA productivity (P) and glucose assimilation efficiency (E). The parameter eta, defined as the product of P and E, was used as an overall performance index. Since eta is a function of the two independent variables D and S(0), we employed a central composite design to optimize D and S(0) for the highest eta value. Based on the experimental results of the design, a second-order polynomial equation was established to represent the relationship between eta and D and S(0). The optimal values of D and S(0) were subsequently determined as 0.481 day(-1) and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, eta value reached 46.5 mg L(-1) day(-1), suggesting a considerably high efficiency of the continuous culture of N. laevis in terms of EPA production and glucose utilization.     

Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile

Mixotrophic growth of the eicosapentaenoic acid (EPA)producing diatom Phaeodactylum tricornutum UTEX640 was carried out in 1-L batch cultures under anexternal irradiance of 165 mol photons m^-2s^-1 by supplementing the inorganic culture mediumwith glycerol. The effect on the growth and the fattyacid profile was studied for different initialglycerol concentrations (0–0.1 M). The optimalglycerol concentration was 0.1 M.A lag phase was observed at high glycerolconcentrations. The present study also shows thatsuccessive additions of glycerol at 0.1M concentrationand using ammonium chloride as a nitrogen sourceremarkably increased the maximum biomass concentration(16.2 g L^-1) and maximum biomass productivity(61.5 mg L^-1 h^-1). These values wererespectively 9 and 8-fold higher than in thephotoautotrophically grown control. The level ofsaponifiable lipids in mixotrophically cultured cellswas significantly higher than in photoautotrophicallycultured cells and increased with the glycerolconcentration in the medium. The concentration ofstorage lipids, saturated and monounsaturated fattyacids, were enhanced but the EPA content did notchange significantly. The EPA content was around 2.2%of biomass dry weight. The maximum EPA yield was33.5 mg L^-1 d^-1 and was obtained in aculture containing 0.1 M glycerol, supplementedperiodically by ammonium chloride. This productivitywas 10-fold higher than the EPA productivity obtainedunder mixotrophic conditions.     

Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow

A local marine diatom, Nitzschia conspicua Grunow, was cultured in enriched synthetic seawater using flasks (agitated by magnetic stirring) and a 1.2 l fermenter. Lipids, fatty acids, proteins, carbohydrates and ash of the flask cultures were determined at various stages of growth (day 3, 5, 7, 10, 13, 15 and 17). The fermenter culture was harvested during the stationary phase for similar chemical analyses. N. conspicua attained a higher biomass concentration during the stationary phase when cultured in the fermenter (188 mg dry weight l^–1) than in flasks (140–151 mg dry weight l^–1). However, both systems showed similar specific growth rates based on chlorophyll-a concentration. Appreciable amounts of the essential fatty acids 20:4 (0.6–4.7% total fatty acids) and 20:5 (1.9–4.7% total fatty acids) are present in this diatom. Maximal amounts of these fatty acids were produced after 7 days' growth (i.e. 2 days after the end of the exponential phase). Lipids, fatty acids, proteins, carbohydrates and ash varied with culture age in N. conspicua.author for correspondence     

Production of eicosapentaenoic acid (EPA) in Monodus subterraneus grown in a helical tubular photobioreactor as affected by cell density and light intensity

The effect of cell density (1–4.5 g L^-1) and light intensity (44 and 82 mol m^-2 s^-1) on fatty acid composition andeicosapentaenoic acid (EPA, 20:5 3) production was studied ina semi-continuous culture of Monodus subterraneus grown in a helicaltubular photobioreactor (`Biocoil') under laboratory conditions. Under lowlight, the highest proportion of EPA (31.5% of total fatty acids) and EPAcontent (3.5% of dry weight), biomass productivity (1.3 g L^-124 h^-1) and EPA productivity (44 mg L^-1 24 h^-1)occurred at optimal cell density of about 1.7 g L^-1. Cell densityhad no effect on the total fatty acid (TFA) content and was maintained atca. 11% of dry weight. Under high light, the highest proportion ofEPA to fatty acids (31.8%), the total fatty acids content (13.4%) andEPA content (4.3% of dry weight) occurred at cell density of about 3.4gL^-1. But the highest biomass productivity (1.7 g L^-124 h^-1) and EPA productivity (56 mg L^-1 24 h^-1) wereobtained at a cell density of 1.6 and 2.6g L^-1, respectively. Ourresults suggest that manipulating the cell density and light intensity canmodify the composition of fatty acid and production of eicosapentaenoicacid (EPA) in M. subterraneus.     

Cryopreservation ofTetraselmis suecica cultured under different nutrients regimes

The commercially important member of the PrasinophyceaeTetraselmis suecica CCAP 66/4, which is widely used in the aquaculture industry, can be cryopreserved with levels of post-thaw viability in excess of 65%. The nutritional mode which the alga uses appears to be of secondary importance to the cryoprotectant and cooling protocol used, to maximise survival. Glycerol was the least cytotoxic and most protective of the cryoprotectants employed and a cooling rate of 5 °C min^-1 to -30 °C with a 10-minute equilibration, followed by plunging into liquid nitrogen was found to give the highest levels of post-thaw viability.     

除草剂精喹禾灵用于硅藻过量产生二十碳五烯酸

硅藻itzschia laevis是EPA很好的替代来源。除了对硅藻进行高密度培养外,EPA的产量还能利用除草剂来提高。本文研究了除草剂精喹禾灵对硅藻的生长和产EPA的影响。DMSO作为除草剂的溶剂,会对硅藻的生长造成抑制,DMSO在培养基中的添加量最好不要超过0.2%。除草剂能对硅藻的细胞形态造成损害,随着除草剂浓度的增加,硅藻的产量减低了,但脂质和EPA的含量提高了。当除草剂浓度为0.1mmol/L时,EPA的含量从3.00%增加到3.58%,提高了19.3%,EPA占总脂肪酸的含量也由25.15%提高到了32.88%。实验表明,除草剂精喹禾灵能促进硅藻EPA的积累,因而在筛选过量产生EPA微藻方面有重要用途。     

Optimization of eicosapentaenoic acid production byPhaeodactylum tricornutumin the flat panel airlift (FPA) reactor

Biomass and eicosapentaenoic acid (EPA) productivities were investigated in a flat panel airlift loop reactor ideally mixed by static mixers. Growth with ammonium, urea and nitrate as nitrogen source were performed at different aeration rates. Cultures grew on ammonium but the decay of pH strongly inhibited biomass increase. On urea biomass productivity reached 2.35 g L^–1d^–1at an aeration rate of 0.66 vvm (24 h light per day, 1000 mol photon m^–2s^–1). Aeration rates between 0.33 vvm and 0.66 vvm and maximal productivities on urea were linearly dependent. Productivity on nitrate never exceeded 1.37 g L^–1d^–1. In the range of maximum productivity photosynthesis efficiency of 10.6% was reached at low irradiance (250 mol photon m^–2s^–1). Photosynthesis efficiency decreased to 4.8% at 1000 mol photon m^–2s^–1. At these high irradiances the flat panel airlift reactor showed a 35% higher volume productivity than the bubble column. At continuous culture conditions the influence of CO₂concentration in the supply air was tested. Highest productivities were reached at 1.25% (v/v) CO₂where the continuous culture yielded 1.04 g L^–1d^–1(16 h light per day, 1000 mol photon m^–2s^–1). The average EPA content amounted to 5.0% of cell dry weight, that resulted in EPA productivities of 52 mg L^–1d^–1(continuous culture, 16 h light per day) or 118 mg L^–1d^–1(batch culture, 24 h light per day).     

Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture

Production of astaxanthin by sequential heterotrophic-photoautotrophiccultivation of a green alga, Haematococcus pluvialis was investigated.This involved cultivating the cells heterotrophically to high cellconcentration, followed by illumination of the culture for astaxanthinaccumulation. The optimum pH and temperature for heterotrophic biomassproduction were 8 and 25 ^°C, respectively. There was no significantdifference in the specific growth rate of the cells when acetateconcentration was varied between 10 mM and 30 mM. However, cellgrowth was inhibited at higher acetate concentrations. A pH stat methodwas then used for fed-batch heterotrophic culture, using acetate as theorganic carbon source. A cell concentration of 7 g L^-1 wasobtained. Higher cell concentration could not be obtained because the cellschanged from vegetative to cyst forms during the heterotrophic cultivation.However, by using repeated fed-batch processes, the cells could bemaintained in the vegetative form, leading to more than two times increasein cell number output rate. When the vegetative cells were transferred tophotoautotrophic phase, there was a sharp decrease in the cell number andonly very few cells encysted and accumulated astaxanthin. On the otherhand, when the shift from heterotrophic to photoautotrophic condition wasdone when most of the cells had encysted, there was still a decrease in cellnumber but astaxanthin accumulation was very high. The astaxanthinconcentration (114 mg L^-1) and productivity (4.4 mg L^-1d^-1) obtained by this sequential heterotrophic-photoautotrophiccultivation method are very high compared to the data in the literature.     

Growth and fatty acid composition of Nannochloropsis sp. grown mixotrophically in fed-batch culture

The cell growth and eicosapentaenoic acid (EPA) yields of Nannochloropsis sp. were enhanced in the fed-batch cultures. With feeding glucose solution, the biomass reached 1.1 g dry wt l(-1) after 10 days' culture, which was 40% higher than that obtained in the batch culture (0.8 g dry wt l(-1)). With supplement of nitrate solution, the biomass reached 1 g dry wt l(-1), and reached the stationary phase 2 days earlier than the others. The maximum of biomass (1.2 g dry wt l(-1)) was obtained with the supplement of the mixture of glucose and nitrate solution. The EPA yields of Nannochloropsis sp. after 10 days' growth in the fed-batch cultures were 52 mg l(-1), 43 mg l(-1) and 56 mg l(-1) with, respectively, addition of nitrate, glucose and both together. In batch culture only 35 mg EPA l(-1) was obtained.     

Density-dependent patterns of thiamine and pigment production in the diatom Nitzschia microcephala

In the present study we investigate how intraspecific (density-dependent) competition for nutrients by the diatom Nitzschia microcephala affects the level of oxidative stress in the algal cells as well as their production of pigments and thiamine. N. microcephala was grown in three different densities until the stationary growth phase was reached. Throughout the experiment, growth rate was negatively related to cell density. Superoxide dismutase activity, protein thiol, and diatoxanthin concentrations indicated increasing oxidative stress with increasing cell density, which was most probably caused by nutrient depletion of the medium. Pigment contents per cell (except for diatoxanthin) decreased with increasing cell density. N. microcephala was able to synthesize thiamine and its thiamine content per cell increased in concert with cell density. In comparison, the dinoflagellate Amphidinium carterae was unable to synthesize thiamine. These results suggest that cells of N. microcephala subjected to higher competition and lower growth rates have a lower carotenoid content and a higher thiamine content. If such responses would occur in nature as well, eutrophication (higher cell densities) may alter the quality of microalgae as food items for higher trophic levels not only by species shifts in the phytoplankton, but also by changes in the cellular nutritional value within species.     

Oil and eicosapentaenoic acid production by the diatom Phaeodactylum tricornutum cultivated outdoors in Green Wall Panel (GWP®) reactors

Phaeodactylum tricornutum is a widely studied diatom and has been proposed as a source of oil and polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). Recent studies indicate that lipid accumulation occurs under nutritional stress. Aim of this research was to determine how changes in nitrogen availability affect productivity, oil yield, and fatty acid (FA) composition of P. tricornutum UTEX 640. After preliminary laboratory trials, outdoor experiments were carried out in 40‐L GWP® reactors under different nitrogen regimes in batch. Nitrogen replete cultures achieved the highest productivity of biomass (about 18 g m^?2 d^?1) and EPA (about 0.35 g m^?2 d^?1), whereas nitrogen‐starved cultures achieved the highest FA productivity (about 2.6 g m^?2 d^?1). The annual potential yield of P. tricornutum grown outdoors in GWP® reactors is 730 kg of EPA per hectare under nutrient‐replete conditions and 5,800 kg of FA per hectare under nitrogen starvation. Biotechnol. Bioeng. 2017;114: 2204–2210. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.     

除草剂精喹禾灵用于硅藻过量产生二十碳五烯酸

硅藻Nitzschia laevis是EPA很好的替代来源。除了对硅藻进行高密度培养外,EPA的产量还能利用除草剂来提高。本文研究了除草剂精喹禾灵对硅藻的生长和产EPA的影响。DMSO作为除草剂的溶剂,会对硅藻的生长造成抑制,DMSO在培养基中的添加量最好不要超过0.2%。除草剂能对硅藻的细胞形态造成损害,随着除草剂浓度的增加,硅藻的产量减低了,但脂质和EPA的含量提高了。当除草剂浓度为0.1mmol/L时,EPA的含量从3.00%增加到3.58%,提高了19.3%,EPA占总脂肪酸的含量也由25.15%提高到了32.88%。实验表明,除草剂精喹禾灵能促进硅藻EPA的积累,因而在筛选过量产生EPA微藻方面有重要用途。