Arachidonic Acid Yield Research Articles

Enhancement of Arachidonic Acid Production by Mortierella isabellina Through Protoplast Regeneration Mutagenesis

A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast regeneration mutagenesis. The results showed that a commixture enzyme (cellulase and glusulase) at the concentration of 4%, enzymolysis temperature at 30°C and enzymolysis time on 7.5 h were the optimal conditions, in which the lethality of M. isabellina spores was 78.4%. After mutagenesis and re-screenings, M. isabellina mutant Y-69 was obtained. GC analysis showed that the yield of arachidonic acid by Y-69 (2.92 g·L−1) was 3.56 times higher than that of the wild-type strain (0.82 g · L−1). Pass generation tests showed that the properties of Y-69 by mutation were readily inherited.

Optimization of Biomass and Arachidonic Acid Production by Aureispira maritima Using Response Surface Methodology

AbstractStatistically based experimental designs, based on the Plackett–Burman protocol, were applied to the optimization of biomass and arachidonic acid (ARA) production in Aureispira maritima shake‐flask cultures. Tryptone and culture temperature were identified to have a significant effect on biomass production, whereas ARA production was only affected significantly by the pH and agitation rate. These four factors were subsequently optimized using response surface methodology. The validity of the optimum conditions was verified by separate experiments in which biomass and ARA yield were increased 4.02‐fold (2.05 g l−1) and 3.59‐fold (21.50 mg g−1), respectively, in 3‐day fermentations. Under non‐optimized culture conditions the corresponding values were 0.51 g l−1 and 5.99 mg g−1, respectively. The results suggest that A. maritima might be a potential strain for further large scale investigations to determine whether this bacterium might be suitable for commercial production of ARA. To our knowledge, this is the first report of the statistically optimization of biomass and ARA production from the marine gliding bacterium A. maritima.

Genome shuffling amplifies the carbon source spectrum and improves arachidonic acid production in Diasporangium sp.

The aim of this study was to develop a new fungal strain that simultaneously amplifies the carbon source spectrum and increases arachidonic acid (AA) productivity using genome shuffling between Diasporangium sp. and inactive Aspergillus niger. Through three rounds of genome shuffling, one of the stable daughter strains (F1) acquired the ability to produce arachidonic acid and utilize various carbon sources. Compared to the parental Diasporangium sp., which could only use four out of eight carbon sources tested, F1 could utilize all eight carbon sources. During fermentation with CMC-Na as the carbon source, F1 was able to obtain 30.16% of lipid effectively whereas the parental Diasporangium sp. was not able to grow at all. When glucose was used as the carbon source, the CMCase activity of F1 was 879.36 U, 298.23% higher than that of the parental Diasporangium sp. Under optimized fermentation conditions in a 5-L fermentation container, the AA yield of F1 reached 0.81 g/l, 94.78% higher than that of the parental generation. These results indicate that inter-kingdom genome shuffling can be used successfully in eukaryotic microorganisms and that it can effectively improve the production of desired metabolites within a short period of time. The findings of this study may be useful for extending the application of genome shuffling in eukaryotic microbial breeding.

Enhancing arachidonic acid production by Mortierella alpina ME-1 using improved mycelium aging technology

Traditional mycelium aging technology was improved to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1. Filtration step was skipped and additional carbon and nitrogen sources were fed during aging. The levels of the significant factors (time, temperature, ethanol, and KNO(3)) affecting ARA production during improved aging process were also optimized by applying response surface methodology (RSM), and the maximum ARA yield of 19.02 g/l was achieved in a 5 l fermentor at 5.6 days, temperature 13.7 degrees C, ethanol 42.44 g/l, and KNO(3) 2.62 g/l. This yield was 1.55 times higher than that of traditional aging technology. The improved mycelium aging technology is considered to be a useful strategy for enhancing ARA production.

Effects of illumination and nitrogen starvation on accumulation of arachidonic acid by the microalga Parietochloris incisa

Parietochloris incisa is a unicellular freshwater green alga capable of accumulating high amounts of the valuable long-chain polyunsaturated arachidonic acid (AA) in triacylglycerols (TAG) of cytoplasmic oil bodies. To find the cultivation conditions providing maximum AA yield, the effects of illumination and N-availability on the dry weight (DW), chlorophyll, carotenoid, and AA content were studied. Under nitrogen starvation, TAG accounted for over 30% of dry weight (DW) and the AA content became as high as about 55% of total fatty acids. For biomass accumulation, light intensity of ca 400 μE m−2 s−1 was found to be optimal for growing P. incisa on a complete medium. Lower light intensities (or a higher cell density of inoculum) resulted in a higher AA yield when the alga was cultivated on nitrogen-free media. In the absence of nitrogen, algal cells were unable to cope with high illumination and suffered from photooxidative damage, whereas the nutrientsufficient culture survived under such illumination conditions, probably due to accumulation of carotenoids. Nitrogen-deprived P. incisa cells displayed elevated sensitivity to light.

A novel two-step fermentation process for improved arachidonic acid production by Mortierella alpina

A novel two-step fermentation process was developed to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1 in a 5 l fermentor. Agitation speed and aeration rate were adjusted from 180 to 40 rpm and from 0.6 to 1 vvm, respectively, after 5 days cultivation, to decrease physical damage to the mycelia and to extend the stationary phase. Moreover, 3% (w/v) and 2% (w/v) ethanol were fed after 5 and 7 days cultivation, respectively, to enhance ARA content of total lipid. Eventually, an ARA yield of 19.8 g/l was achieved, which was 1.7 times higher than that of a one-step fed-batch cultivation.

Metabolic flux analysis on arachidonic acid fermentation

The analysis of flux distributions in metabolic networks has become an important approach for understanding the fermentation characteristics of the process. A model of metabolic flux analysis of arachidonic acid (AA) synthesis in Mortierella alpina ME-1 was established and carbon flux distributions were estimated in different fermentation phases with different concentrations of N-source. During the exponential, decelerating and stationary phase, carbon fluxes to AA were 3.28%, 8.80% and 6.97%, respectively, with sufficient N-source broth based on the flux of glucose uptake, and those were increased to 3.95%, 19.21% and 39.29%, respectively, by regulating the shifts of carbon fluxes via fermentation with limited N-source broth and adding 0.05% NaNO3 at 96 h. Eventually AA yield was increased from 1.3 to 3.5 g·L−1. These results suggest a way to improve AA fermentation, that is, fermentation with limited N-source broth and adding low concentration N-source during the stationary phase.

Optimization of arachidonic acid production by fed-batch culture of Mortierella alpina based on dynamic analysis

Arachidonic acid is an essential fatty acid in human nutrition. Improvement of arachidonic acid production by fungus Mortierella alpina was investigated. The time courses of fungal biomass, lipid and arachidonic acid production with different initial glucose concentrations were examined to study dynamic characteristics of arachidonic acid production on a shaker flask scale. Results showed that low initial concentrations of glucose were good for fungal growth at the early days of cultivation. When residual glucose concentration was under 10 g/L, fungi stopped growing because of inefficient energy source and carbon source needed for their growth. Meanwhile, the lipids were still accumulated for a period even though fungi stop growing. A great increase of arachidonic acid content in lipids occurred after the glucose was exhausted. To obtain high arachidonic acid yield, a fed-batch process was developed according to the dynamic analysis. Glucose and nitrate were included in the feed medium. Low initial glucose concentration (50 g/L) and nitrate concentration (3 g/L) were used to shorten the lag phase of fungal growth. Twenty grams per liter per day glucose and 1.5 g/(L day) nitrate were fed to the medium at the 3rd, 4th and 5th days of cultivation. A good arachidonic acid yield (7.74 g/L) on the 8th day was obtained, which was a great improvement over that of batch cultures.

Effect of culture media and conditions on polyunsaturated fatty acids production by Mortierella alpina

To improve the polyunsaturated fatty acid (PUFA) production by Mortierella, culture media and conditions were investigated. M. alpina ATCC 32222 had the highest yield of arachidonic acid, γ-linolenic acid and linoleic acid among 11 test microbes. Soluble starch at 10% and the mixture of KNO 3 and yeast extract at 2:1 (w/w) was the best carbon and nitrogen sources for arachidonic acid and total PUFAs production, respectively. The optimal C/N ratio ranged from 5.1 to 9.0. Each gram of carbon produced 17.4 mg of linoleic acid, 17.0 mg of γ-linolenic acid, 103.0 mg of arachidonic acid and 194.2 mg of total PUFAs at 20 °C, while it yielded 21.4 mg of linoleic acid, 25.6 mg of γ-linolenic acid, 2.6 mg of γ-linolenic acid, 110.3 mg of arachidonic acid, 4.3 mg of eicosapentaenoic acid and 218.4 mg of total PUFAs at 12 °C. A high degree of unsaturation was found at low temperature incubation. Linseed oil supplementation (1%, w/v) increased the PUFAs production and each gram of carbon produced 403.4 mg of α-linolenic acid, 123.1 mg of arachidonic acid, 33.6 mg of eicosapentaenoic acid, 1.68 mg of docosahexaenoic acid and 943.2 mg of total PUFAs. From the optimization of culture media and conditions, PUFAs production increased from 30% to 5 times that was optimal for practical use.

Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation.

The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l).

An inexpensive medium for production of arachidonic acid by Mortierella alpina

The production of arachidonic acid was studied in the fungus Mortierella alpina using an inexpensive medium. Glucose derived from maize starch hydrolysate was the sole carbon source and defatted soybean meal and sodium nitrate were the nitrogen sources. Optimal arachidonic acid yield (1.47 g l(-1)) was observed at a glucose concentration of 100 g l(-1). Various treatments of defatted soybean meal to extract soluble nitrogen nutrients were evaluated. Alkali extract was the most effective for arachidonic acid production. A mixture of soybean alkali-extract protein and sodium nitrate was an excellent nitrogen source for fungal growth, lipid accumulation, and arachidonic acid production. A maximum yield of 1.87 g arachidonic acid l(-1) was obtained with a soybean protein concentration of 4.6 g l(-1) and a sodium nitrate concentration of 2.3 g l(-1).

Production of arachidonic acid byMortierella fungi

The growing interest in the application of arachidonic acid (ARA) in various fields of health and dietary requirements has elicited much attention on the industrial production of ARA-containing oil by the cultivation ofMortierella fungi. For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies including the investigation of morphology are important because ARA is accumulated in the mycelia, and thus cultivation with high biomass concentration is essential for obtaining a high ARA yield. Combining the results derived from various studies, a high ARA yield was attained in an industrial fermentor. These ARA production techniques are applicable to the production of other polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation technology especially in the field of fungal cultivation.

Effect of consumed carbon to nitrogen ratio of mycelial morphology and arachidonic acid production in cultures of mortierella alpina

The influence of the consumed carbon to nitrogen (C/N) ratio on arachidonic acid ( AA) production and mycelial morphology was investigated in cultures of Mortierella alpina using shake flasks and a fermentor. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of carbon and nitrogen sources was 50 g/ l. Cellular yield increased markedly at C/N ratios below 7; carbon utilization was switched from cellular growth to lipid biosynthesis in the C/N ratio range of 7–15; lipid biosynthesis was most active when the C/N ratio was in the range of 15–32. However, for C/N ratios higher than 15, the mycelial concentration decreased due to nitrogen limitation but the lipid yield still increased. In the presence of excess nitrogen, the biomass concentration depended on the amount of the nitrogen source, but the AA yield was inversely related to this. On the other hand, in the presence of excess carbon, the fatty acid concentration increased with carbon source concentration but the AA concentration remained constant. From the viewpoint of AA production, the optimum C/N ratio was in the range of 15 to 20 with a balance between the amounts of carbon and nitrogen sources. When an enriched medium was used at a fixed C/N ratio of 20, the cellular and AA concentrations were shown to be proportional to the total concentrations of carbon and nitrogen sources in both flasks and the fermentor. The whole pellet size and width of pellet annular regions did not change with increasing C/N ratio for C/N ratios below 20 in the flask cultures. However, when the C/N ratio was higher than 20, these sizes increased in proportion to the C/N ratio.

Effect of Consumed Carbon to Nitrogen Ratio on Mycelial Morphology and Arachidonic Acid Production in Cultures of Mortierella alpina.

The influence of the consumed carbon to nitrogen (C/N) ratio on arachidonic acid (AA) production and mycelial morphology was investigated in cultures of Mortierella alpina using shake flasks and a fermentor. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of carbon and nitrogen sources was 50 g/l. Cellular yield increased markedly at C/N ratios below 7; carbon utilization was switched from cellular growth to lipid biosynthesis in the C/N ratio range of 7-15; lipid biosynthesis was most active when the C/N ratio was in the range of 15-32. However, for C/N ratios higher than 15, the mycelial concentration decreased due to nitrogen limitation but the lipid yield still increased. In the presence of excess nitrogen, the biomass concentration depended on the amount of the nitrogen source, but the AA yield was inversely related to this. On the other hand, in the presence of excess carbon, the fatty acid concentration increased with carbon source concentration but the AA concentration remained constant. From the viewpoint of AA production, the optimum C/N ratio was in the range of 15 to 20 with a balance between the amounts of carbon and nitrogen sources. When an enriched medium was used at a fixed C/N ratio of 20, the cellular and AA concentrations were shown to be proportional to the total concentrations of carbon and nitrogen sources in both flasks and the fermentor. The whole pellet size and width of pellet annular regions did not change with increasing C/N ratio for C/N ratios below 20 in the flask cultures. However, when the C/N ratio was higher than 20, these sizes increased in proportion to the C/N ratio.

Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4.

Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g(-1) glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate qD(FA/B)) of 9.5%. Our results also indicate that a qD(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid.

Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4.

Arachidonic acid (AA) production by Mortierella alpina 1S-4 was investigated using a 50-L fermentor. In order to optimize the dissolved oxygen (DO) concentration and to investigate the effect of DO on morphology, cultivation was carried out under constant DO at various levels in the range of 3-50 ppm. To maintain a DO concentration above 7 ppm, two methods, i.e., the oxygen-enrichment (OE) method (experimental range, 25-90% oxygen gas supplied) and the pressurization (PR) method (experimental range, 180-380 kPa headspace pressure), were used. As a result, the optimum DO concentration range was found to be 10-15 ppm. In this optimum DO concentration range, the AA yield was enhanced about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in the AA productivity between the OE and PR methods. When the DO concentration was maintained at 20-50 ppm using the OE method, the morphology changed from filaments to pellets, and the AA yield decreased drastically because of stress due to the limited mass transfer through the pellet wall. When the DO concentration was maintained at 15-20 ppm using the PR method, the morphology did not change, and the AA yield decreased gradually.

Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of mortierella alpina

The effects of nitrogen source on arachidonic acid (AA) production and morphological changes during the culture of Mortierella alpina were investigated using an image analysis system. When yeast extract, gluten meal, or corn steep liquor was used, a circular pellet morphology was obtained. However, when Pharmamedia, fish meal, or soybean meal was used, M. alpina formed radial filamentous mycelia. The radial filamentous area in the case of soybean meal was 75% of the total mycelial area. In a jar fermentor culture, M. alpina morphology varied with the cultivation period: (i) at 0–6 h culture, the inoculated pellet-like mycelia were adapted to the new environment, (ii) at 6 h-1 d culture, filamentous mycelia grew exponentially which yielded a feather-like morphology, (iii) at 1–2 d culture, the filamentous mycelia became disentangled as a result of the mechanical agitation; consequently, the proportion of filamentous mycelia was increased, (iv) at 2–4 d culture, mycelia showed stationary growth, but the AA concentration increased rapidly, and (v) at 4–6 d culture, hyphae grew thick radially with the AA concentration continuing to increase gradually. In the case of the cultures with feather-like morphology obtained using soybean meal, the AA yield was 0.14 g/g dry cell weight, which was two times higher than that in cultures grown using yeast extract. These results suggest that the feather-like morphology of culture of M. alpina is suitable for AA production.

Effects of mineral addition on the growth morphology of and arachidonic acid production by Mortierella alpina 1S‐4

AbstractThe culture conditions for arachidonic acid (AA) production by Mortierella alpina 1S‐4 were investigated by means of a morphological study with the aims of obtaining a high AA yield and scale‐up. In a 50‐L jar fermentor study, a medium containing 3.1% soy flour and 1.8% glucose with 0.3% KH2PO4, 0.1% Na2SO4, 0.05% CaCl2·2H2O and 0.05% MgCl2·6H2O was found to be optimum. The AA yield reached 9.8 g/L/7 d, and the major morphology was small pellets (1–2 mm). However, in the case of the only addition of KH2PO4, the major morphology was filaments. The apparent viscosity increased to 2240 cp, thereby requiring a high agitation speed to maintain adequate oxygen tension, which caused mycelial damage due to shear stress and therefore a decrease in the AA yield. When a medium with Na2SO4, CaCl2, and MgCl2 was used, the major morphology was large pellets (2–3 mm), and mass transfer limitation through the pellet wall caused a decrease in the AA yield. Based on these results, a scale‐up study was carried out under the optimal conditions described above. An AA yield of 10.9 g/L/8 d was obtained in a 10‐kL industrial fermentor, and the major morphology was small pellets.

Enhancement of arachidonic acid production by Mortierella alpina 1S‐4

AbstractThe effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S‐4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10‐L jar‐fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH2PO4, 0.1% Na2SO4, 0.05% CaCl2·2H2O, and 0.05% MgCl2·6H2O was found to enhance the AA yield 1.7‐fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35‐fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH2PO4 addition, and that the above‐mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.

Effect of mustard meal on the production of arachidonic acid by Mortierella elongata SC‐208

AbstractA strain of Mortierella elongata SC‐208 that was isolated from soil of a mustardseed extraction plant can produce arachidonic acid in significant amounts, and it was grown in three different media, one of which contained 0.5% deoiled mustard meal. The arachidonic acid content in the lipid part of dry mycelium (23.2 g/L) was as high as 33% w/w from the medium that contained mustard meal, and the overall yield of arachidonic acid was 0.49 g/L. The arachidonic acid contents in the phospholipid fraction and the triglyceride fraction were 39.5 and 30.2%, respectively.