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 to1 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.     

Arachidonic acid production by Mortierella alpina grown on solid substrates

Mortierella alpina grown in solid state fermentations on cereal substrates gave up to 16% lipid in the final biomass. Arachidonic was at 50% of total fatty acids, with a yield of 36 mg/g of original substrate. Microbial lipid production was successfully scaled up to use 5-kg dry substrate batches.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding

Studies on the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have proceeded in various fields regarding health and dietary requirements in a search for novel and rich sources. Filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, ones reaching 20 g/L and containing 30–70% arachidonic acid as to the total fatty acids. Mutants derived from M. alpina 1S-4, defective in Δ5 and Δ6 desaturases, accumulate triacylglycerols rich in unique PUFAs, i.e., dihomo-γ-linolenic acid and Mead acid, respectively. Furthermore, various mutants derived from M. alpina 1S-4 have led to the production of oils containing n−1, n−3, n−4, n−6, n−7, and n−9 PUFAs. A variety of genes encoding fatty acid desaturases and elongases involved in PUFA biosynthesis in M. alpina 1S-4 has been isolated and characterized. Molecular breeding of M. alpina strains by means of manipulation of these genes facilitates improvement of PUFA productivity and elucidation of the functions of enzymes involved in PUFA biosynthesis.     

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.     

花生四烯酸产生菌高山被孢霉的高糖驯化研究

【目的】提高花生四烯酸(Arachidonic acid,ARA)产量,克服ARA产生菌高山被孢霉(Mortierella alpina)在长期的保存及使用过程中易受到外界条件影响发生退化,从而导致菌种耗糖量降低、影响菌种摄入营养的能力和不利于工业化生产的缺点。【方法】首先采用固体培养基驯化,将菌种逐级涂布于梯度高糖PDA平板(含糖量分别为2%、5%、7%、10%和15%)培养,挑选经固体驯化后能耐受10%高糖浓度平板的菌种,转接到两种含不同氮源的梯度高糖(含糖量分别为3%、4%、5%和6%)液体培养基中进行驯化,最后对驯化后的菌种进行2 L发酵罐放大实验。【结果】当培养基中以酵母粉为氮源时,驯化后菌体的最高耗糖量由3 g/(L.d)提高到12 g/(L.d);当培养基中以玉米浆为氮源时,驯化后菌体的最高耗糖量由7 g/(L.d)提高到12 g/(L.d)。摇瓶驯化实验结果表明以玉米浆为氮源驯化的菌种发酵效果较好,发酵罐实验结果显示菌体生物量为50 g/L,总油脂为18 g/L,目的产物ARA产量为8 g/L。相比未驯化之前的发酵结果,生物量和总油脂含量提高了近3倍,ARA产量提高了近4倍。【结论】经过高糖驯化,菌种的耗糖能力得到提高,生物量、总油脂及ARA的产量也都有所增加,从而可以使菌种在保存和使用过程中不易退化,保持稳定。     

Enhancing arachidonic acid production by <Emphasis Type="Italic">Mortierella alpina</Emphasis> 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 KNO₃) 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 °C, ethanol 42.44 g/l, and KNO₃ 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.     

Optimisation of culture conditions for production of eicosapentaenoic acid byMortierella elongate NRRL 5513

Summary WhenMortierella elongata NRRL 5513 was cultured in shake flasks at 25°C, mycelial growth reached a stationary phase at 48 h but maximum eicosapentaenoic acid (EPA) production was observed at 6 days. When incubated at 11°C, EPA production also continued to rise during the stationary phase of growth, reaching a maximum after 10 days. An initial culture pH of 6.1 was found to be optimum for EPA production. The effect of temperature on EPA production was dependent on medium constituents. In glucose and linseed oil supplemented media, optimum temperature for EPA production was 11 and 15°C respectively. A maximum EPA yield of 0.61 g/l was obtained in linseed oil (2%), yeast extract (0.5%) supplemented basal medium. Maximum EPA content as a percentage of lipids (15.12%) was observed when the latter medium was supplemented with 0.25% urea.     

Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222

Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO₃ with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days. Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Growth-Coupled Lipid Synthesis in Mortierella alpina LPM 301, a Producer of Arachidonic Acid

Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of intense lipid synthesis in the period of active mycelium growth. Under batch cultivation of this strain in glucose-containing media with potassium nitrate or urea, the bulk of lipids (28–35% of dry biomass) was produced at the end of the exponential growth phase and remained almost unaltered in the stationary phase. The ARA content of lipids comprised 42–50% at the beginning of the stationary phase and increased continuously after glucose depletion in the medium due to the turnover of intracellular fatty acids; by the end of fermentation (189–210 h), the amount of ARA reached 46–60% of the total fatty acids (16–19% of dry mycelium). Plausible regulatory mechanisms of the growth-coupled lipid synthesis in microorganisms are discussed.     

High-level production of arachidonic acid by fed-batch culture of <Emphasis Type="Italic"> Mortierella alpina</Emphasis> using NH<Subscript>4</Subscript>OH as a nitrogen source and pH control

Mortierella alpina was grown in a fed-batch culture using a 12-l jar fermenter with an initial 8-l working volume containing 20 g glucose l⁻¹ and 10 g corn-steep powder l⁻¹. Glucose was intermittently fed to give 32 g l⁻¹ at each time. The pH of culture was maintained using 14% (v/v) NH₄OH, which also acted as a nitrogen source. A final cell density of 72.5 g l⁻¹ was reached after 12.5 days with a content of arachidonic acid (ARA) at 18.8 g l⁻¹. These values were 4 and 1.8 times higher than the respective values in batch culture. Our results suggest that the combined feeding of glucose and NH₄⁺ to the growth of M. alpina could be applied for the industrial scale production of ARA.     

高山被孢霉ATCC 32222中细胞色素b5还原酶I的克隆、表达和功能鉴定

【目的】鉴定产油微生物高山被孢霉ATCC 32222中细胞色素b_5还原酶Ⅰ的功能。【方法】将高山被孢霉ATCC 32222中膜结合细胞色素b_5还原酶Ⅰ基因与人可溶性细胞色素b_5还原酶基因序列比对,去除该基因N端穿膜区域后,与人可溶性细胞色素b_5基因分别在大肠杆菌中异源表达;通过钴离子亲和层析、离子交换和分子排阻色谱等方法对表达产物进行纯化;以2,6-二氯靛酚钠(DCIP)为底物,测定细胞色素b_5还原酶Ⅰ的体外活性及其对NADH和NADPH的偏好性;在反应体系中存在NADH时,通过全波长扫描方法检测细胞色素b_5还原酶Ⅰ与细胞色素b_5的相互作用。【结果】高山被孢霉ATCC 32222中膜结合细胞色素b_5还原酶Ⅰ被成功可溶表达,经纯化后检测到体外活性:使用NADH时酶活为564.57 U,使用NADPH时为51.97 U;在NADH存在时,细胞色素b_5还原酶Ⅰ能够还原细胞色素b_5,其吸收峰从411 nm偏移至422 nm,并在521 nm和554 nm处吸光值增加。【结论】细胞色素b_5还原酶Ⅰ N端穿膜区域的去除增加了其可溶性,并保持了蛋白质活性;高山被孢霉ATCC 32222中细胞色素b_5还原酶Ⅰ基因编码的是一种NADH-细胞色素b_5还原酶,其在体外能与细胞色素b_5相互作用。     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.     

An isolation procedure for arachidonic acid producing Mortierella species

Malt extract agar and an incubation temperature of 5 °C were used to selectively isolate representatives of the genus Mortierella from soil. Fungi in a soil sample from mountain grassland able to grow under these conditions, amounted to a total of 2640 colony forming units per gram soil. Circa 94% of the total fungal isolates represented Mortierella subgenus Mortierella. The rest of the colony-forming units consisted of Mucor isolates (6.0%) and higher fungi (1.5%). All the Mortierella isolates produced arachidonic acid.     

Phytosterol biosynthesis pathway in Mortierella alpina

The Zygomycetes fungus Mortierella alpina was cultured to growth arrest to assess the phytosterol biosynthesis pathway in a less-advanced fungus. The mycelium was found to produce 13 sterols, but no ergosterol. The sterol fractions were purified to homogeneity by HPLC and their identifies determined by a combination of GC-MS and 1H NMR spectroscopy. The principal sterol of the mycelium was cholesta-5, 24-dienol (desmosterol) (83%), with lesser amounts of 24beta-methyl-cholesta-5,25(27)-dienol (codisterol) (2%), 24-methyldesmosterol (6%), 24(28)-methylene cholesterol (3%) and lanosterol (3%) and several other minor compounds (3%). The total sterol accounted for approximately 0.07% of the mycelial dry wt. Mycelium fed methionine-methyl-2H3 for 6 days, generated 3 2H-24-methyl(ene) sterols, [C28-2H2]24(28)-methylenecholesterol, [C28-2H3]24-methylcholesta-5,24-dienol and [C28-2H3]24beta-methyl-cholesta-5,25(27)-dienol. The formation of the 24-methyl sterols seems to be catalyzed by the direct methylation of a common Delta24-acceptor sterol thereby bypassing the intermediacy of an isomerization step for rearrangement of the Delta24(28)-bond to Delta25(25)-position as operates in Ascomycetes fungi and all plants.     

KLa对高山被孢霉产花生四烯酸的影响

研究了体积溶氧系数(K1a)对被孢霉产生四烯酸的影响。结果表明在摇瓶水平放大培养中,K1a、被孢霉生长、花生四烯酸（AA）产量均降低。利用0.3％Tween20作为氧载体提高各级的K1a,可以较大幅度提高AA产量。     

Production of high yields of docosahexaenoic acid byThraustochytrium roseum ATCC 28210

Culture conditions for growth and docosahexaenoic acid (DHA) production byThraustochytrium roseum ATCC 28210 were investigated with a view to increasing DHA titers. A medium was formulated (Medium 6) which produced a biomass and DHA content of 10.4 g L^–1 and 1011 mg L^–1, respectively, in a 5-day incubation. A fed-batch culture system was also developed which achieved biomass and DHA titers of 17.1 g L^–1 and 2000 mg L^–1, respectively, in 12 days.     

高山被孢霉三阶段培养法生产花生四烯酸油脂

为提高高山被孢霉（Mortierella alpina）生物合成花生四烯酸油脂的生产效率,基于花生四烯酸油脂的积累机制,建立了一种三阶段培养法：第一阶段在全培养基中培养促进菌体生物量的快速积累,确定了60 g/L糖初始质量浓度时,最有利于生物量的快速积累;第二阶段在C源丰富而其他营养缺乏的条件下培养,促进油脂快速积累,对糖最佳初始浓度、接种时间、pH和培养时间进行了优化;第三阶段培养诱导油脂中花生四烯酸的高效积累,并确定此阶段培养时间为36 h时为最佳时间。实验结果表明：三阶段培养工艺条件下,菌体生物量、油脂量、花生四烯酸量分别为41.6、26.6和11.4 g/L,本研究相比传统分批发酵工艺在产率和花生四烯酸最终产量方面都有了显著提高。     

Morphological diversity ofMortierella alpina: Effect of consumed carbon to nitrogen ratio in flask culture

The influence of the consumed carbon to nitrogen (C/N) ratio on mycelial morphology was investigated in cultures ofMortierella alpina using shake flasks. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of the carbon and nitrogen sources was 50 g/L. The whole mycelia and filamentous mycelia exhibited no relationship with the consumed C/N ratio below a consumed C/N ratio of 20 in the presence of either excess carbon or excess nitrogen. However, when the consumed C/N ratio increased higher than 20, the mycelial sizes increased in proportion to the consumed C/N ratio. However, the area ratio of filamentous mycelia to total mycelia was found to be independent of the consumed C/N ratio, and remained constant at 0.82. In the case of a fixed consumed C/N ratio of 20, the whole mycelia and filamentous mycelia increased in proportion to the degree of the medium strength, yet the area ratio of filamentous mycelia to total mycelia remained unchanged at 0.76. Accordingly, these results show that fungal morphology and mycelial size are both affected by the ratio of carbon to nitrogen. The findings of the current study will be helpful in obtaining the efficient production of useful bioproducts from fungal cultures.     

Isolating Mortierella alpina strains of high yield of arachidonic acid

AIMS: To develop a fast isolation method for arachidonic acid-producing fungi of high yield. METHODS AND RESULTS: Relation between the staining degree of mycelia of Mortierella alpina stained with triphenyltetrazolium chloride (TTC) and arachidonic acid content in the fungal lipids was investigated. Results showed that staining degree of mycelia stained with TTC increased when arachidonic acid content in mycelia lipids increased. This finding was used to isolate strains of high arachidonic acid yield. Arachidonic acid producing fungi were selectively isolated from soil at a low temperature of 4 degrees C and the mycelia of these isolates were stained with TTC. CONCLUSIONS: The strain M. alpina M6 that had the highest staining degree had the highest arachidonic acid content (72.3%). The yield of arachidonic acid in this strain reached 4.82 g l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: A fast and effective method to isolate strains of high arachidonic acid yield was established according to the finding that staining degree of mycelia of M. alpina was positively correlated with arachidonic acid content in mycelia lipid.