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.     

高山被孢霉产花生四烯酸的代谢研究

研究了3个批次的高山被孢霉发酵产花生四烯酸(ARA)过程中,生物量、糖耗、氮耗、油脂含量和ARA含量的变化.结果表明,发酵7d的生物量(干重)为30.650 g/L,糖耗85.33％,氮耗78.81％,发酵液油脂含量17.142 g/L,ARA含量7.482 g/L.     

碳氮比（C/N）对高山被孢霉中花生四烯酸积累的影响

为探究氮源及碳氮比（C/N）对高山被孢霉（Mortierella alpina）中花生四烯酸（ARA）积累的影响,选取无机氮硝酸钠和有机氮尿素为氮源,分别于高、中、低C/N条件下（C/N为40∶ 1、20∶ 1和10∶ 1）进行M.alpina发酵实验。分析发酵过程中菌体生物量（DCW）、发酵液残C量、发酵液残N量的变化,并通过气相色谱质谱联用技术（GC-MS）检测菌体中脂肪酸组分及其含量的动态变化,同时对各分析指标间进行了Pearson相关性分析。结果表明,以硝酸钠为氮源,高C/N条件下所获DCW更高（6.42 g/L）,为低C/N条件下的2.3倍,且高C/N条件下ARA产量也更高（0.21 g/L）,高C/N条件下ARA产量与DCW和N消耗量呈显著正相关。以尿素为氮源,低C/N条件下获得较高的DCW（18.2 g/L）,但因低C/N条件下菌体中ARA含量较低,故ARA产量不高;且相同C/N条件下,DCW和ARA产量远高于以硝酸钠为氮源的;不同C/N条件下,ARA产量与DCW、C消耗量、N消耗量和总脂肪酸含量均呈显著正相关。综上所述,有机氮比无机氮更有利于M.alpina菌体的生长和ARA的积累,且尿素的中C/N条件更有利于提高ARA的产量（0.99 g/L）。     

高山被孢霉发酵生产花生四烯酸发酵条件优化

通过对一株高山被孢霉菌株（Mortierella alpina）发酵生产花生四烯酸（ARA）的摇瓶培养研究,以菌体干质量、出油率和ARA含量为标准,确定了其最佳发酵工艺条件。摇床实验确定的最佳培养条件为摇床转速180 r/min,培养基碳氮比为3.0,发酵温度为26 ℃,pH 值为6.2,发酵周期为5 d。在此发酵条件下,在10 L自动发酵罐进行放大发酵试验,菌体干质量、出油率和ARA含量分别高达45.4 g/L、24.1 g/L和49.6%。     

气升式反应器中高山被孢霉发酵生产花生四烯酸过程的动态温度控制研究

在利用高山被孢霉发酵生产花生四烯酸(Arachidonic Acid,ARA)过程中,考察了不同规模和培养环境下,单一以及动态温度控制对菌体生长和ARA合成的影响。结果表明:相对于机械搅拌罐,利用气升式反应器发酵生产ARA具有明显优势。同时,较高温度(25℃)有利于高山被孢霉菌体生长,而较低温度(16℃)有利于ARA合成。在此基础上进行动态控温发酵,即起始发酵温度25℃,72h后以每12h下降1.5℃至16℃,然后保持16℃至发酵结束。采用此控制策略,ARA产量达到4.7g/L,与单一温度(25℃)控制相比提高了38.2%。      

Image analysis of morphological change during arachidonic acid production by Mortierella alpina 1S-4

The changes in mycelial morphology during arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated using an image analysis system. Cultivation was performed in a 10-kl fermentor, and the culture broth was separated into two fractions by sieving (0.5 mm aperture size): the filament fraction (F-fraction, <0.5 mm), and the pellet fraction (P-fraction, >0.5 mm). The effect of the mycelial morphology in each fraction on AA production was analyzed. As a result, a product distribution in the culture broth wherein the AA content in the mycelia of the P-fraction was observed to be higher than that in the mycelia of the F-fraction throughout the cultivation. Morphological analysis of the P-fraction revealed that the hairy pellets became smooth because the mycelia on the pellet surface were shaved off; some pellets were broken and reduced in size. The shaved-off mycelia from the hairy pellets surface moved into the F-fraction and aggregated there. From the above findings, it was likely that the low AA content in the F-fraction was due to mycelial damage during the cultivation. In addition, the morphology of the hairy pellets was found to contribute to an increase in the viscosity of culture broth.     

Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.     

高产花生四烯酸高山被孢霉的诱变育种研究

通过菌种选育获得具有优良产脂性能的菌株是实现微生物油脂工业化生产的重要前提。利用实验室前期构建的产油丝状真菌快速筛选技术,以前期筛选获得的一株性状优良的高山被孢霉TSM-3为出发菌株,通过常压室温等离子体(ARTP)诱变方法得到能够稳定遗传的高产突变菌株TSM-3-1,其油脂产量和花生四烯酸(AA)产量分别达到5.07 g/L和1.55 g/L,与出发菌株相比分别提高了47.81%和84.52%。将产油丝状真菌快速筛选技术与诱变育种相结合,提高了目标菌株筛选效率并强化了野生型菌株油脂合成能力,为产油丝状真菌菌种选育提供了新颖的研究思路及方法。     

被孢霉菌菌丝体在贮存过程中油脂的变化

报导了花生四烯酸生产菌被孢霉菌（Ｍｏｒｔｉｅｒｅｌａｓｐ．Ｍ１０）菌丝体在贮存过程中油脂的变化。研究结果表明，不同培养时间的菌丝体（３～７ｄ）在室温下贮存１５ｄ，菌丝体中总脂含量由１８％～３０％上升至３６％～４１％；菌丝体中ＡＡ含量由１．１％～２．６％上升至２．６％～３．７％。但菌丝体油脂中ＡＡ含量在贮存过程中根据不同培养时间显示出不同变化规律。不同贮存温度（５～２５℃）对菌丝体中ＡＡ含量没有显著影响。在ｐＨ为８．０时贮存更有利于菌丝体中花生四烯酸含量的提高。     

肉毒碱脂酰转移酶基因沉默对高山被孢霉脂质积累的影响

高山被孢霉是一种工业化生产多不饱和脂肪酸的产油真菌。通过分析高山被孢霉脂肪酸氧化途径中相关基因的转录水平,发现肉毒碱脂酰转移酶(carnitine acyl-transferase,CAT)基因与高山被孢霉脂肪酸氧化紧密相关,进一步利用RNA干扰技术实现在高山被孢霉中CAT基因的沉默,分析发现,在高山被孢霉重组菌中,CAT活性下调了33.3%,总脂肪酸积累量相比野生型菌株提高38.1%,表明通过抑制β氧化途径可以提高高山被孢霉的脂质积累量,这为推动高山被孢霉在脂质生物合成工业化方面的应用奠定了理论基础。     

Effect of Culture Variables on Mycelial Arachidonic acid Production by Mortierella alpina

Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.     

高山被孢霉生长和产脂的影响因素分析

为探究影响高山被孢霉生长和产脂的因素,分析不同初始葡萄糖质量浓度、活化次数、接种量、装液量、发酵时间、菌球打碎时间等条件下高山被孢霉生长和产脂特点。结果表明,初始葡萄糖质量浓度、装液量和发酵时间对菌株生长和产脂的影响最为显著,且初始葡萄糖质量浓度与花生四烯酸的产量呈负相关性。根据不同条件下菌株生长和产脂情况,得到高山被孢霉最佳培养条件为:菌株活化3次,用高速分散机8 000 r/min打碎20 s至均匀状态,按2%～3%(体积分数)接种量接入初始葡萄糖质量浓度为50 g/L、装液量为20%(体积分数)的发酵培养基中,200 r/min、28℃培养8. 5 d。     

利用被孢霉发酵生产花生四烯酸工艺配方优化研究

利用高山被孢霉Mortierella alpina I49-N18发酵生产花生四烯酸。优化筛选出最适于高山被孢霉斜面培养、种子摇瓶培养以及摇瓶发酵培养的配方,稳定的培养基配方为后续发酵生产提供保障。通过培养基配方单因子试验分别对碳源、氮源、无机盐、氨基酸、植物油等原料的合适添加量进行初步确定,然后设计正交实验进行验证。考察初始葡萄糖浓度在高山被孢霉发酵生产花生四烯酸过程中对脂肪酸组分的影响,分析脂肪酸主要组分在发酵过程中的变化趋势。考察结果显示发酵培养基中补充添加800 1000mg/kg磷酸盐,添加600 1000mg/kg谷氨酸钠盐、添加0.04%0.12%植物油可以有效提高发酵生产花生四烯酸的产量。通过发酵工艺放大,在200m3发酵罐规模的发酵生产中得到应用,发酵产量得到大幅度提升,培养7d发酵生产花生四烯酸(ARA)的产量达到10g/L以上。     

产油丝状真菌高山被孢霉中蝶呤类化合物的提取及检测

建立了高效液相色谱-荧光检测法同时测定高山被孢霉体内新蝶呤、蝶呤和生物蝶呤的方法。高山被孢霉在液氮破壁、酸性条件下经碘-碘化钾溶液氧化1h、滴加抗坏血酸还原液后,体内的四氢生物蝶呤及其前体二氢蝶呤三磷酸盐和6-丙酮酰四氢生物蝶呤分别被氧化成相应的氧化产物新蝶呤、蝶呤、生物蝶呤。经离子交换树脂纯化后,利用高效液相色谱进一步分离得到了氧化产物蝶呤、新蝶呤和生物蝶呤,并采用电喷雾质谱对定性结果进行确认。色谱柱为Inertsil ODS-3C₁₈（5μm,150mm×4.6mm）,用10mmol/L的磷酸氢二钠（pH6.0）作为流动相,流速为1.2mL/min,荧光检测器激发波长为350nm,吸收波长为450nm。检测出的新蝶呤、生物蝶呤和蝶呤的检出限依次是:0.003、0.002、0.005μg/mL。本方法能快速并准确的检测各类蝶呤类化合物,为利用高山被孢霉发酵生产四氢生物蝶呤奠定一定基础。      

碳氮源及碳氮比对BAA中温α-淀粉酶发酵的影响研究

研究了BAA中温α-淀粉酶生产过程中,碳、氮源种类及碳氮比对发酵产酶的影响,结果表明,采用以40%玉米粉和60%玉米淀粉作碳源,以标准棉籽饼粉为氮源,碳氮源质量比为14：5,添加万分之四的进口专用发酵消泡剂,同时采用补料工艺,在最佳发酵工艺条件下,发酵酶活力突破900U/mL,平均832U/mL,较优化前的600U/mL平均提高38%。     

响应面法优化深黄被孢霉发酵生产花生四烯酸的培养基条件

为了降低深黄被孢霉YZ-124生产花生四烯酸的成本,研究了不同添加量的玉米黄浆水对发酵的影响,与葡萄糖培养基相比,在发酵培养基中添加一定量的玉米黄浆水对发酵产量无显著影响。在单因素实验的基础上,利用Design Expert设计了响应面实验,研究了葡萄糖浓度、不同添加量的玉米黄浆水和初始pH对花生四烯酸产量的影响。结果表明,最佳的培养基条件是葡萄糖浓度为90g/L、添加体积分数为25%的玉米黄浆水、初始pH6时,花生四烯酸（ARA）产量达到最大,为3.11g/L。      

响应面法优化深黄被孢霉发酵生产花生四烯酸的培养基条件

为了降低深黄被孢霉YZ-124生产花生四烯酸的成本,研究了不同添加量的玉米黄浆水对发酵的影响,与葡萄糖培养基相比,在发酵培养基中添加一定量的玉米黄浆水对发酵产量无显著影响.在单因素实验的基础上,利用Design Expert设计了响应面实验,研究了葡萄糖浓度、不同添加量的玉米黄浆水和初始pH对花生四烯酸产量的影响.结果表明,最佳的培养基条件是葡萄糖浓度为90g/L、添加体积分数为25％的玉米黄浆水、初始pH6时,花生四烯酸(ARA)产量达到最大,为3.11g/L.     

Effect of carbon source on the mycelial growth and exo-biopolymer production by submerged culture of Paecilomyces japonica

A significant difference was observed in the production pattern of exo-biopolymer and broth rheology during submerged culture of Paecilomyces japonica when two efficient carbon sources (maltose and sucrose) were employed. The maximum concentration of biopolymers in maltose medium (30 g/l) was higher than that in sucrose medium (25 g/l), whereas maximum mycelial concentration displayed an inverse result (i.e., 25 g/l in sucrose medium and 20 g/l in maltose medium). The broth rheology and morphology of P. japonica were also markedly different between the two culture media.     

Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH,temperature and carbon source,nitrogen source,metal ions and oil supplementation

A locally isolated (Perryvale, Alta., Canada) fungal genus, Mortierella ramanniana var. ramanniana, was evaluated as a potential industrial producer of γ-linolenic acid (GLA). Six growth variables (pH, temperature, carbon source, nitrogen source, and metal ions and oil supplementation) were systematically manipulated. The results indicated that the GLA production for this particular strain, could be maximized by using a basal growth medium consisting of 5% dextrose and 1% yeast extract, supplemented with 5 mg/L Mn²⁺ with incubation at 20 °C. The lipid yield under optimum conditions was 54.2% of the total dry biomass and consisted of 84.3% unsaturated fatty acids. The yield of GLA per gram biomass represented 13.3% of the total lipid content, slightly higher than that of the typical evening primrose oil GLA yield. The study establishes this variety as an effective intermediate fungal source of GLA.