Enhanced arachidonic acid production using a bioreactor culture of Mortierella alpina with a combined organic nitrogen source
© The Author(s) 2016
Received: 7 June 2016
Accepted: 8 September 2016
Published: 14 September 2016
The organic nitrogen source is one of the key factors affecting Mortierella alpina cell growth as well as arachidonic acid (ARA) production. The aim of the present work is to achieve an optimized recipe of organic nitrogen source for ARA production by M. alpina by testing four organic nitrogen sources.
In the flasks, the results showed yeast extract or corn steep liquor were the most suitable sole nitrogen sources for biomass and ARA yield. In the bioreactor, a biomass of 17.5 g L−1 and an ARA yield of 2.7 g L−1 were achieved when using sole yeast extract, while cell autolysis was induced when using sole corn steep liquor; When using the combined nitrogen source with corn steep liquor and yeast extract at a 3:7 weight ratio, the biomass and ARA yield were significantly improved to 37 and 7.8 g L−1, respectively.
This work evaluated whether using a mix of organic nitrogen sources could improve ARA production when scaling up from a flask to a bioreactor culture. The 3:7 ratio of corn steep liquor to yeast extract was quite favourable for large-scale ARA production, and as a result, this combination has great potential for improving fungal cultures.
Arachidonic acid (all-cis-5, 8, 11, 14-eicosatetraenoic acid, ARA) is classified as a polyunsaturated fatty acid and is an important fatty acid for human nutrition. It is a direct precursor of several key eicosanoid hormones, which are important biological regulators. Owing to its unique biological properties, ARA has been widely used for a variety of applications in medicine, pharmacology, cosmetics, the food industry, agriculture, and other fields (Birch et al. 2000; Dedyukhina et al. 2011).
Traditionally, ARA was extracted from animal livers, adrenal glands, and egg yolk. These feedstock are readily available; they only contain small amounts of ARA. Consequently, these feedstock cannot be able to meet the amazing rising market demand for ARA. There has recently been an increased interest in using microorganisms to produce ARA (Ratledge 2004; Eroshin et al. 2000). As an alternative source containing a high intracellular content of ARA, the filamentous fungus, Mortierella alpina, has been extensively studied for ARA production at various fermentation scales (Peng et al. 2010; Vali et al. 2003). The previous studies reported that cell growth, total fatty acid and ARA yields were influenced by many factors, such as cell morphology, carbon and nitrogen sources, mineral supplementation, and the dissolved oxygen concentration (Higashiyama et al. 2002; Jang et al. 2005). Among these parameters, nitrogen source was reported to be of critical importance in the cultivation of M. alpina. However, not all nitrogen sources enhance cell growth and ARA production equally well (Lu et al. 2011; Totani et al. 2000). Compared with inorganic nitrogen sources, organic nitrogen sources showed higher levels of cell growth, total fatty acids and ARA production. Replacement of sodium nitrate by corn steep liquor (CSL) significantly improved ARA production by M. alpina ATCC 32222 (Higashiyama et al. 2004). Nisha and Venkateswaran (2011) also studied the influence of various nitrogen sources on both the biomass and the production of ARA by M. alpina. They showed that yeast extract (YE) produced the maximum biomass (6.8 g L−1) and highest ARA content in lipids (35.28 %), peptone produced the highest lipid content in biomass (42.0 %), and the urea produced the lowest ARA content in lipids (12.06 %). The previous studies also reported that natural nitrogen sources had a marked effect on the morphology of M. alpina in submerged culture (Park et al. 2001) and that the morphology of the cells had an effect on cellular enzymes and secondary metabolites. They found that the morphology of M. alpina cells changed from pelleted shape to filamentous form. It was suggested that the fluffy pellet morphology was more suitable for ARA production than the smooth pellet or filamentous morphology.
The diversity of available organic nitrogen sources is important for cell growth and ARA production, so it is necessary to further study the effect of different nitrogen sources on biomass as well as on the ARA yield for M. alpina. Furthermore, few studies have focused on adopting a combination of organic nitrogen sources to M. alpina cultivation. The aim of this work is to achieve an optimized recipe of organic nitrogen source for ARA production by M. alpina.
Microorganism and cultivation conditions
Mortierella alpina LU166 used in this study was a UV mutation of ATCC 3222, which was obtained from the American Type Culture Collection (ATCC, Rockville, USA). It was maintained on potato dextrose agar (PDA) slants at 4 °C and transferred monthly.
The seed medium contained (g L−1): glucose 30, yeast extract 4, KH2PO4 2.5 and MgSO4–7H2O 0.3. Inocula were prepared in 250 mL Erlenmeyer flasks containing 50 mL seed medium. The culture was grown for 72 h at 28 °C in a rotary shaker set at 150 rpm. The fermentation medium consisted of (g L−1): glucose 60, yeast extract 10, glutamic acid 1, MgSO4–7H2O 0.1, KH2PO4 0.5, CaCO3 0.05, and vitamin concentrate 1 mL (0.5 mg vitamin B12, 0.5 mg vitamin H, 1 mg vitamin B1, dissolved in 1 L of water). The pH of the medium was adjusted to 6.0 before autoclaving at 121 °C for 15 min. Scale-up batch cultures were carried out in a 3.6 L bioreactor containing 2 L fermentation medium. The culture was maintained at 28 °C with 0.5 vvm aeration and 200 rpm agitation. The pH was maintained at 4–6 in the exponential growth phase and at 7–8 in the subsequent fermentation period by feeding HCl or NaOH automatically. The experimental results comprise the average of two or three parallel experiments, and the data given in this paper are representative.
The dinitrosalicylic acid method was used to assay the glucose concentration (Miller 1959). The biomass of fungal mycelia was harvested by filtration, washed with distilled water three times and then dried at 60 °C until the constant dry cell weight, (DCW) was obtained. The dried biomass was grinded into powder and used for extraction of the total lipids, which was determined gravimetrically. Fatty acid methyl esters (FAMEs) were prepared as follows: 5 mL 0.5 M KOH-methanol was added to a tube containing TLs. The tubes were heated in a water bath at 65 °C for 10 min, after then 5 mL 30 % BF3-ether was added. The tubes were then heated in a water bath at 65 °C for 30 min again, and then 5 mL hexane was added when the tubes cooled down to room temperature. It was settled for separation of two phases after adding 1 mL saturated sodium chloride solution for preventing emulsification. The upper phase containing FAMEs was applied to a gas chromatograph (Agilent GC 7890, USA) equipped with a 100 m × 0.25 mm capillary column (SP™-2560, USA). The column was increased from 140 to 240 °C at 3 °C/min and then maintained at 240 °C for further 30 min. The temperature of the injector and detector were both set at 260 °C, and nitrogen was used as the carrier gas at 20 cm/s. The quantity of ARA was estimated from the peak areas on the chromatogram using docosahexaenoic acid methyl ester as an internal standard.
The experimental design
Based on the literature (Lu et al. 2011; Nisha and Venkateswaran 2011), four types of organic nitrogen sources (YE, peptone, soybean meal, and CSL) were first tested individually in the flask culture at a concentration of 10 g L−1 to determine the optimal single nitrogen source. Then, the chosen optimal single nitrogen source was studied in the bioreactor culture to investigate its scale-up effect on ARA production. According to the results of the bioreactor culture using the sole nitrogen source, the proper nitrogen source mix based on the different weight ratios was tested for improving cell growth and ARA production from the flask culture to the bioreactor culture.
Results and discussion
Effect of a single nitrogen source on ARA production in the flask culture
Effects of nitrogen sources on DCW, total lipids, ARA yield and mycelial morphology of M. alpina
DCW (g L−1)
13.5 ± 0.15
6.5 ± 0.35
14.2 ± 0.23
15 ± 0.14
Residual glucose (g L−1)
5.3 ± 0.28
28.1 ± 1.3
9.4 ± 0.6
0.5 ± 0.24
Lipids (g L−1)
2.8 ± 0.25
1.2 ± 0.31
2.6 ± 0.45
3.9 ± 0.26
ARA (g L−1)
1.3 ± 0.13
0.06 ± 0.15
0.9 ± 0.22
1.9 ± 0.22
Lipid/DCW (w/w, %)
21.8 ± 0.53
16.2 ± 2.31
17.6 ± 0.42
26.4 ± 0.16
ARA/DCW (w/w, %)
10.1 ± 0.32
0.89 ± 1.62
6.4 ± 0.16
13.1 ± 0.42
ARA/lipid (w/w, %)
46.5 ± 0.13
5.3 ± 1.54
34.5 ± 0.12
49.1 ± 0.36
Effect of a single nitrogen source on ARA production in the bioreactor culture
When CSL was used as the sole nitrogen source in the bioreactor, the painful problem of cell autolysis occurred during the culturing process of M. alpina. It was found that the cell morphology was filamentous, which was unfavourable for ARA production. CSL, as one type of quick-acting nitrogen source, was consumed so fast that it may have disturbed the consumed C/N ratio balance. A proper consumed C/N ratio also influences cell growth and lipid accumulation in the culture of M. alpina (Koike et al. 2001). However, the results in Table 1 showed that when CSL was used as the sole nitrogen source in flask culture, biomass, lipid and ARA yields were the highest, especially the ARA content in lipid. These results indicated CSL was more favourable for ARA production. In view of the results of the flask culture and the bioreactor culture, the combined nitrogen sources of CSL + YE were considered for the following experiment.
Effect of combined nitrogen sources on ARA production in the flask culture
Effect of combined nitrogen sources on ARA production in the bioreactor culture
Parameters of DCW, total lipids, ARA yield of M. alpina cultivated with different nitrogen source in the 3.6-L bioreactor fermentation
DCW (g L−1)
17.5 ± 0.47
37.0 ± 1.1
Lipids (g L−1)
6.0 ± 0.25
16.0 ± 0.54
ARA (g L−1)
2.7 ± 0.2
7.8 ± 0.4
Lipid/DCW (w/w, %)
34.3 ± 0.32
43.2 ± 0.63
ARA/DCW (w/w, %)
15.4 ± 0.28
21.1 ± 0.52
CSL is a nitrogen source that contains abundance of soluble proteins micronutrients, both of which can be used rapidly by microorganisms to promote cell growth. YE would be accessed slowly. As a result, cells can be supplied nutrition continually, which in turn helps prevent cell autolysis, a phenomenon caused mainly by a C/N ratio imbalance. As presented in Figs. 1 and 3, the cell growth reached 22 g L−1 at 48 h when using CSL + YE, which was higher than the highest value of 17.5 g L−1 obtained at 96 h using YE only. Furthermore, at 48 h, 59 % of total biomass had been produced in CSL + YE culture, while only 40 % of total biomass had been obtained in YE culture. The time courses of the lipid and ARA accumulation show that the lipid and ARA reached their highest concentrations at 144 h in CSL + YE culture, which was 12 h earlier than when peak concentrations reached in YE culture. This result indicated that CSL + YE culture can shorten cultivation duration, which is favourable for large-scale production.
In the other culture, which had a CSL:YE ratio of 7:3, it was found that M. alpina grew well in the incipient stages of fermentation but the cells still underwent autolysis quickly on the 3rd day and onward (Figure omitted). It was concluded that the bioreactor provided a better environment for M. alpina growth than the culture flask. When the percentage of CSL was higher than YE in the medium, most of the nitrogen source was consumed rapidly and the C/N ratio was disturbed. Consequently, the cells underwent autolysis because of insufficient nutrition.
Comparison of ARA production of M. alpina reported in the literatures
Fermentor volume (L)
Culture time (d)
ARA yield(g L−1)
Eroshin et al. (2000)
Hwang et al. (2005)
Li et al. (1995)
Lu et al. (2011)
Nie et al. (2014)
Ji et al. (2014)
Li et al. (2015)
Zhu et al. (2006)
Singh and Ward (1997)
Hwang et al. (2005)
Jin et al. (2008)
The experimental results indicate that YE and CSL are favourable for the morphology of M. alpina LU 166 to form the fluffy circular pellets, which is linked to high biomass and ARA yields. And the combined nitrogen source containing 3 g L−1 CSL and 7 g L−1 YE was found to be the most suitable composition to avoid cell autolysis and promote high cell density and ARA production during scale-up of the culture in the bioreactor. The corresponding biomass and ARA yield are 37.0 and 7.8 g L−1, respectively. In addition, extensive efforts, including research on the mechanism of morphology formation and on fed-batch culture strategies (including glucose and nitrogen source feeding), are in progress to facilitate the large-scale industrial ARA production.
XPL and SYZ carried out the main experiments. CXC and XTL helped in the cultivation of the strain and fatty acids assay. XPL, SYZ and YHL are involved in the drafting and revision of the manuscript. YHL has given final approval of the version to be published. All authors read and approved the final manuscript.
This work was funded by grants from the Science and Technology Program of Xiamen, China (No. 3502Z20153005), and from the National High Technology Research and Development Program of China (No. 2014AA021701).
The authors declare that they have no competing interests.
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