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Optimization of arachidonic acid production by fed-batch culture of Mortierella alpina based on dynamic analysis
Enzyme and Microbial Technology 38 (2006) 735–740
Optimization of arachidonic acid production by fed-batch culture of Mortierella alpina based on dynamic analysis Min Zhu, Long-Jiang Yu ∗ , Wei Li, Peng-Peng Zhou, Chun-Yan Li College of Life Science & Technology, Huazhong University of Science & Technology, 430074 Wuhan, People’s Republic of China Received 6 May 2005; received in revised form 25 July 2005; accepted 29 July 2005
Abstract 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. © 2005 Elsevier Inc. All rights reserved. Keywords: Arachidonic acid; Dynamic analysis; Fed-batch; Mortierella alpina; Substrate consumption
1. Introduction There has been increasing interest in the microbial production of lipid containing polyunsaturated fatty acids in the past decade. Arachidonic acid (AA, 5,8,11,14-ciseicosatetraenoic acid) is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes [1]. As a component of mature human milk, AA is necessary for the neurological and neurophysiological development of both term [2] and preterm infants [3]. Many expert organizations including FAO/WHO [4] recommended that AA should be supplied as a supplement in the infant formula. Animal viscera, e.g. porcine liver, are conventional sources of AA that contain very little AA. Therefore, production of AA by microorganisms as an alternative source is gaining interest. Some screening procedures were developed ∗
Corresponding author. Tel.: +86 27 87543633; fax: +86 27 87540184. E-mail address: [email protected] (L.-J. Yu).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.07.025
to obtain AA producing strains. Mortierella alpina, an oleaginous fungus, has been identified as a promising producer of polyunsaturated fatty acids, especially AA. Eroshin et al. [5] used aspirin-containing medium to select three Mortierella strains which produced more than 40% AA in fungal lipids. Low culture temperature was also used to isolate AA producing strains, most of which were M. alpina [6,7]. Sakuradani et al. [8] improved AA production by mutants of M. alpina with lower n-3 desaturation activity. In our previous study, a strain M. alpina M6 with good production of AA was obtained by screening at low temperature and examining triphenyltetrazolium chloride staining degree [9]. Culture conditions including temperature, pH, medium (carbon source, carbon/nitrogen ratio, etc.) and cultivation method for AA production were also extensively investigated [7,10,11]. To achieve good production, fed-batch culture is a common strategy in fermentation process [12]. Singh and Ward [10] applied a fed-batch culture system by supplying additional nutrients to basal medium and a high AA yield (9.1 g/L)
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was achieved in 8 days. However, dynamic analysis of products accumulation and substrates consumption in AA production process, which were the bases of fed-batch culture, has not yet been systematically investigated so far. Here, the time courses of fungal growth, lipids accumulation, AA accumulation and substrate (glucose, nitrate and protein) consumption were investigated in order to find out the dynamic characteristics of AA production and a fed-batch culture method was developed accordingly to obtain a higher AA yield.
2. Materials and methods 2.1. Microorganism M. alpina M6 was isolated from soil [9]. It was maintained on potato dextrose agar (PDA; glucose 20 g/L, potato extract 4 g/L and agar 17 g/L) slants at 4 ◦ C and transferred every 3 months. 2.2. Culture conditions The fungi grew on PDA (50 mL PDA in a 250 mL flask) at 25 ◦ C for a week, then 20 mL sterile water with some glass beads was poured into the flask and the mycelia and spores were suspended by shaking vigorously. The suspension was inoculated into 200 mL inoculum medium (5% glucose and 50% fresh soybean sprout extract) in a 500 mL flask, and then incubated at 25 ◦ C for 3 days with shaking at 150 rpm. Fifty percent soybean sprout extract was obtained by filtering the mixture of soybean sprout and water (1:2) which had boiled for 30 min. Then, 250 mL flasks containing 50 mL production medium were inoculated with this inoculum at a rate of 10% (v/v) and incubated at 25 ◦ C on a shaker at 150 rpm. Production medium consisted of (g/L): potassium nitrate, 3; protein, 4.5; K2 HPO4 ·3H2 O, 4; CaCl2 ·2H2 O, 0.05; MgSO4 ·7H2 O, 0.5 (pH 5.5). Initial concentrations of glucose were varied as described in the text.
Fig. 1. Time courses of glucose consumption by Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
quickly in the early days of cultivation and slowly in the late days of cultivation. When initial concentrations of glucose were 30 and 50 g/L, the glucose was almost used up in the 3rd and 4th day, respectively, the residual glucose concentrations were under 5 g/L. When initial concentration of glucose was 80 g/L, glucose was almost depleted after 12 days of cultivation and the residual glucose was 6.1 g/L. When the initial concentrations of glucose were 100 and 120 g/L, glucose was not depleted even after 15 days of cultivation and the residual glucose were 16.5 and 40 g/L, respectively. Dry biomass yield of M. alpina at different initial glucose concentrations is shown in Fig. 2. The fungi grew faster in the early days (i.e. the 2nd day) of cultivation when initial
2.3. Analytical methods The dinitrosalicylic acid (DNS) method was used to determine glucose concentrations [13]. Protein concentrations were assayed by Lowry’s method [14]. Nitrate concentrations were assayed by salicylic acid method [15]. The dry weight of biomass, total lipids and content of AA were determined as described in previous publications [9].
3. Results and discussion 3.1. Dynamic courses of glucose consumption by M. alpina and fungal growth at different initial glucose concentrations Glucose consumption by M. alpina at different initial glucose concentrations is shown in Fig. 1. Glucose was uptaken
Fig. 2. Time courses of dry biomass yield of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
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concentrations of glucose were 30 and 50 g/L than when they were 80, 100 and 120 g/L, and the higher the initial glucose concentrations were, the slower the fungi grew at the beginning. The fungal growth was related to the time of glucose depletion according to Figs. 1 and 2. When residual glucose concentration was under 10 g/L, fungi stopped growing. When initial concentrations were 30, 50 and 80 g/L, dry fungal biomass reached their peak at 3rd, 4th and 9th day, respectively, and then the biomass yield declined. When initial concentrations were 100 and 120 g/L, there was enough glucose in the medium for growth, so the biomass increased during the whole period of cultivation, faster in the early days and slower in the late days. Though fungi grew fast at low initial glucose concentrations at early days of cultivation, high biomass (22.5 g/L) was achieved when initial concentration of glucose was 120 g/L. 3.2. Dynamic courses of lipids accumulation by M. alpina at different initial glucose concentrations Time courses of lipids content in biomass at different initial glucose concentrations are presented in Fig. 3. When initial concentration of glucose was 30 g/L, the lipids content in biomass reached its peak at 5th day, and this value was obviously lower than other peak values of other initial glucose concentrations. It indicated that too low glucose concentration which was exhausted quickly and no carbon source for lipid synthesis led to small lipids accumulation. Lipids were still accumulated, even the growth reached peak according to Figs. 2 and 3. The lipids content in biomass reached peak at 5th day when initial concentrations of glucose were 30 and 50 g/L, later than what biomass did. When initial concentrations of glucose were 80, 100 and 120 g/L, lipids in biomass increased during the whole period of cultivation.
Fig. 3. Time courses of lipids in biomass of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
Fig. 4. Time courses of lipids yield of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
It was obvious that lipids were still accumulated for a period of time, even glucose was used up according to Figs. 1 and 3. Time courses of lipids yields at different initial glucose concentrations are presented in Fig. 4. The lipids yield was determined by biomass and lipids content in biomass. Lipids yield was obviously low when initial concentration of glucose was 30 g/L due to its significantly low lipids content in biomass and low biomass yield. The highest value of lipids yield (9.09 g/L) was achieved after 15 days of cultivation when initial glucose concentration was 120 g/L. 3.3. Dynamic courses of AA production by M. alpina at different initial glucose concentrations Time courses of AA content in lipids of M. alpina at different initial glucose concentrations are shown in Fig. 5. AA content in lipids kept increasing during the whole period of cultivation no matter what the initial glucose concentration was. The lower the initial glucose concentrations, the higher the AA in lipids. AA contents in lipids which increased significantly after cultivation of 5 days when initial glucose concentrations were 30 and 50 g/L indicated that the great increase of AA content in lipids occurred after the glucose was exhausted according to Figs. 5 and 1. When lipid content of dry biomass remained constant or decreased, AA content in lipids increased significantly due to the conversion of other fatty acids such as saturated and monounsaturated fatty acids (data not shown here). Time courses of AA yields of M. alpina at different initial glucose concentrations are shown in Fig. 6. AA yields were determined by biomass, lipids in biomass and AA in lipids. AA yields kept increasing during the whole cultivation time. When initial glucose concentration was 30 g/L, the final AA yield was low because of low biomass and low lipids in biomass in spite of high AA content in lipids. Maximum
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Though maximum AA yield was obtained after cultivation of 15 days when initial glucose concentration was 100 g/L according to the above results, the fungi grew slowly at the early days of cultivation and it took a long time to obtain this value. Furthermore, the residual glucose concentration was high and the AA content in lipids was low. This represented a material waste and a potential pollution source that would make the downstream treatment difficult, and low AA
content in lipids would impair the final AA production. In order to obtain a high production of AA, a fed-batch cultivation method was developed to shorten the lag phase, enhance the uptake of glucose and increase the AA content in lipids. According to dynamic analysis, higher initial glucose concentration impaired fungal growth at early days of cultivation and low initial concentration of glucose was good to shorten the lag phase of fungal growth after inoculation. When residual glucose concentration was under 10 g/L, fungi stopped growing. Therefore, nutrition needed supplying to the medium to keep the fungi growing. Fifty grams per liter was suitable as initial glucose concentration according to the above results, for the fungi grew faster in the early days of cultivation and the fungal performance of growth, lipids and AA accumulation was better at this concentration than what at 30 g/L initial glucose concentration. To determine how much to feed and when to feed, time courses of substrates consumption including glucose, protein and potassium nitrate were investigated. The results are shown in Fig. 7. Glucose and nitrate were consumed fast, they were exhausted at the 4th day but the soy protein was consumed slowly, it was not used up until harvested. So the feeding medium contained glucose and nitrate. The concentrations of the feed medium were determined by their consumption rate. Glucose was consumed by 16.2 and 15.9 g/(L day), respectively, at the 2nd and 3rd days, and nitrate by 2.2 g/L at the 2nd day. Considering substrate consumption and fungal growth of fed-batch culture might be different from those of the batch culture, feed concentrations of glucose and nitrate presented in Table 1 were applied. Glucose and nitrate were supplemented to the medium at the 3rd, 4th and 5th days of cultivation. Because lipids were still accumulated for a period, even glucose was used up and a great increase of AA content in lipids occurred after the glucose was exhausted; therefore, the fungi kept growing till the 8th day to use up residual glucose.
Fig. 6. Time courses of AA yield of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L.
Fig. 7. Time courses of glucose, nitrate and protein consumption by Mortierella alpina at initial glucose concentration of 50 g/L. Symbols: () nitrate, () glucose and (䊉) protein. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
Fig. 5. Time courses of AA content in lipids of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
AA yield (4.83 g/L) was obtained after cultivation of 15 days when initial glucose concentration was 100 g/L. 3.4. A fed-batch cultivation method was developed according to dynamic analysis for high production of AA
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Table 1 Effect of fed-batch culture on AA production by Mortierella alpina Feed concentration Glucose (g/(L day))
Nitrate (g/(L day))
10 10 10 10 20 20 20 20 30 30 30 30
1 1.5 2 3 1 1.5 2 3 1 1.5 2 3
Residual glucose (g/L)
Biomass (g/L)
Lipid in biomass (%)
Lipids (g/L)
AA in lipids (%)
AA (g/L)
0 0 0 0 10.95 1.02 1.02 3.20 17.72 21.10 23.35 18.85
27.2 27.0 27.4 26.8 33.2 37.0 36.0 35.6 31.8 35.4 35.8 35.6
33.2 27.5 28.3 28.8 40.7 38.4 36.4 36.2 38.0 37.4 33.3 35.0
9.03 7.43 7.76 7.72 13.51 14.21 13.11 12.88 12.08 13.25 11.92 12.46
64.5 66.7 68.8 66.0 53.0 54.5 56.1 54.6 43.2 49.7 52.4 51.9
5.82 4.96 5.34 5.10 7.16 7.74 7.35 7.03 5.22 6.59 6.25 6.47
Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
Results showed that fed-batch culture was an effective way to improve AA yield. Supplementation of glucose and nitrate in the process of cultivation significantly increased the biomass, lipid content and AA level compared to batch culture. When feed concentration of glucose was 10 g/(L day), glucose needed for their growth was inefficient; therefore, no matter how much nitrate was fed, fungal growth was relatively poor. The AA contents in lipids were high because of glucose depletion. When feed concentration of glucose was 30 g/(L day), concentrations of residual sugar were high and so the AA contents in lipids were low. When feed concentration of glucose was 20 g/(L day), glucose supplemented just met the consumption requirement, dry biomass, lipids yield and AA yield were higher than that feed concentrations of glucose were 10 and 30 g/(L day). Here, different nitrate supplementation affected glucose uptaking and fungal growth. A high biomass (37.0 g/L), lipid (14.21 g/L) and AA yield (7.74 g/L) in 8 days were obtained, which were 1.65-, 1.51and 1.61-fold of maximum values of batch cultures, respectively.
4. Discussion Batch culture of M. alpina using glucose as a carbon source, nitrate and soy protein as combined nitrogen source was studied to analyze the dynamic characteristics of substrate consumption, fungal growth, lipid accumulation and AA production. It is generally known that there are two phases of lipids accumulation in oleaginous microorganisms in batch culture growth though Eroshin et al. [16] reported growth-coupled lipid synthesis in M. alpina LPM 301. The first phase is biomass growth, lipid synthesis occurs mainly in the second phase. Ratledge [17] suggested that the depletion of nitrogen in the growth medium evokes a decrease in the intracellular concentration of AMP, an activator of isocitrate dehydrogenase, and, as a consequence, an increase in the contents of citrate and isocitrate in the cells.
Citrate is converted by ATP:citrate lyase to oxaloacetate and acetyl-CoA; the latter is used for the synthesis of fatty acids. According to our dynamic analysis, fungal growth, lipids and AA accumulation were not synchronous and were related to the glucose concentration in the medium. AA production by M. alpina in our study could be divided into three phases in batch culture. The first phase was fungal growth coupled with lipids and AA production. In this phase, glucose supply was enough. Lipids were kept synthesizing in the second phase when glucose concentration was low (i.e. under 10 g/L); in this phase, mycelium stopped growing; lipids were accumulated for a period till glucose was used up and AA content in lipids increased continuously. Dynamic analysis showed that residual glucose was low then and the nitrogen sources were not depleted (nitrate was used up but soy protein remained). Therefore, the lipids accumulation by our strain in this case could not be explained by the assumption of Ratledge and coworkers as described above. Mycelium stopped growing mainly because of carbon source limitation and consequently inefficient energy source and carbon source needed for their growth. The third phase is AA synthesis. In this phase, mycelium stopped growing or even decreased, lipids content in biomass stopped increasing while AA content in lipids increased. A great increase of AA content in lipids occurred after the glucose was exhausted and lipids yield reached peak. Similar results that AA content in lipids produced by M. alpina increased significantly after glucose depletion were observed by Eroshin et al. [16]. AA content in lipids increased as a result of other fatty acids conversion. Saturated fatty acids such as 16:0 and 18:0 and monounsaturated fatty acid (18:1) decreased when AA increased. Why glucose depletion stimulated fatty acids conversion to AA and whether this conversion only occurred in M. alpina still remains unclear. In present study, results showed that low initial glucose concentration was beneficial for fungi growth, to shorten the lag phase after inoculation, indicating that higher osmotic pressure in the medium of higher initial glucose
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concentration prevents the growth of the fungi. It is necessary to develop fed-batch culture to avoid the repressive effects of high substrate concentration. Fed-batch culture is a common strategy in fermentation industry to avoid the repressive effects of high substrate concentration, to control the organisms’ growth rate and to achieve good production [12]. There are lots of examples that good production was obtained through this strategy, such as PHB production by Ralstonia eutropha [18], ergosterol production by Saccharomyces cerevisiae [19], etc. In the case of AA production, fed-batch method was used by supplementing soy flour, corn steep, corn oil and additional glucose to the basal medium each day after 3 days of fermentation to obtain the best AA yield that is published [10]. However, it would be difficult to carry out fed-batch culture without dynamic analysis of substrate consumption and product yielding. Therefore, in present study, dynamic analysis was investigated to give information about what to feed, how much to feed and when to feed in a fed-batch culture. Consequently, a better AA production of fed-batch culture was achieved based on characteristics of dynamic analysis compared to that of batch culture.
References [1] Gill I, Valivety R. Polyunsaturated fatty acids, Part l: occurrence, biological activities and application. Trends Biotechnol 1997;15:401–9. [2] Brick EE, Garfield S, Hoffman DR, Uauy R, Birch DG. A randomized controlled trial of early dietary supply of long chain polyunsaturated fatty acids and mental development in term infants. Dev Med Child Neurol 2000;42:174–81. [3] Bougle D, Denise P, Uimard F, Nouvelet A, Penniello MJ, Guillois B. Early neurological and neurophysiological development of the preterm infant and polyunsaturated fatty acids supply. Clin Neurophysiol 1999;110:1363–70. [4] FAO/WHO Expert Committee. The role of essential fatty acids in neural development: implications for perinatal nutrition. Am J Clin Nutr 1994;57(Suppl.):703s–10s.
[5] Eroshin VK, Dedyukhina EG, Chistyakova TI, Zhelifonova VP, Kurtzman CP, Bothast RJ. Arachidonic-acid production by species of Mortierella. World J Microbiol Biotechnol 1996;12:91–6. [6] Botha A, Paul I, Roux C, Kock JLF, Coetzee DJ, Strauss T, et al. An isolation procedure for arachidonic acid producing Mortierella species. Antonie Van Leeuwenhoek 1999;75:253–6. [7] Chen HC, Chang CC, Chen CX. Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate. J Am Oil Chem Soc 1997;74:569–78. [8] Sakuradani E, Hirano Y, Kamada N, Nojiri M, Ogawa J, Shimizu S. Improvement of arachidonic acid production by mutants with lower n-3 desaturation activity derived from Mortierella alpina 1S-4. Appl Microbiol Biotechnol 2004;66(3):243–8. [9] Zhu M, Yu LJ, Liu Z, Xu HB. Isolating Mortierella alpina strains of high yield of arachidonic acid. Lett Appl Microbiol 2004;39:332–5. [10] Singh A, Ward OP. Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC32222. Appl Microbiol Biotechnol 1997;48:1–5. [11] Higashyama K, Yaguchi T, Akimoto K, Fujikawa S, Shimimu S. Effects of mineral addition on the growth morphology of and arachidonic acid production by Mortierella alpina 1S-4. J Am Oil Chem Soc 1998;75:1815–9. [12] Stanbury PF, Whitaker A, Hall SJ. Principles of fermentation technology. 2nd ed. Butterworth-Heinemann; 1995. p. 27–31 (an imprint of Elsevier Science). [13] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [14] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin–phenol reagents. J Biol Chem 1951;193:265–75. [15] Hecht U, Mohr H. Factors controlling nitrate and ammonium accumulation in mustard (Sinapis alba) seedlings. Physiol Plant 1990;78:379–87. [16] Eroshin VK, Dedyukhina EG, Satroutdinov AD, Chistyakova TI. Growth-coupled lipid synthesis in Mortierella alpina LPM 301 a producer of arachidonic acid. Microbiology 2002;71:169–72. [17] Ratledge C. Microbial lipids. In: Rehm HJ, Reed G, editors. Biotechnology, vol. 7. VCH: Weinheim; 1997. p. 133–97. [18] Patwardhan PR, Srivastava AK. Model-based fed-batch cultivation of R. eutropha for enhanced biopolymer production. Biochem Eng J 2004;20:21–8. [19] Tan T, Zhang M, Gao H. Ergosterol production by fed-batch fermentation of Saccharomyces cerevisiae. Enzyme Microb Technol 2003;33:366–70.
Optimization of arachidonic acid production by fed-batch culture of Mortierella alpina based on dynamic analysis Min Zhu, Long-Jiang Yu ∗ , Wei Li, Peng-Peng Zhou, Chun-Yan Li College of Life Science & Technology, Huazhong University of Science & Technology, 430074 Wuhan, People’s Republic of China Received 6 May 2005; received in revised form 25 July 2005; accepted 29 July 2005
Abstract 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. © 2005 Elsevier Inc. All rights reserved. Keywords: Arachidonic acid; Dynamic analysis; Fed-batch; Mortierella alpina; Substrate consumption
1. Introduction There has been increasing interest in the microbial production of lipid containing polyunsaturated fatty acids in the past decade. Arachidonic acid (AA, 5,8,11,14-ciseicosatetraenoic acid) is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes [1]. As a component of mature human milk, AA is necessary for the neurological and neurophysiological development of both term [2] and preterm infants [3]. Many expert organizations including FAO/WHO [4] recommended that AA should be supplied as a supplement in the infant formula. Animal viscera, e.g. porcine liver, are conventional sources of AA that contain very little AA. Therefore, production of AA by microorganisms as an alternative source is gaining interest. Some screening procedures were developed ∗
Corresponding author. Tel.: +86 27 87543633; fax: +86 27 87540184. E-mail address: [email protected] (L.-J. Yu).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.07.025
to obtain AA producing strains. Mortierella alpina, an oleaginous fungus, has been identified as a promising producer of polyunsaturated fatty acids, especially AA. Eroshin et al. [5] used aspirin-containing medium to select three Mortierella strains which produced more than 40% AA in fungal lipids. Low culture temperature was also used to isolate AA producing strains, most of which were M. alpina [6,7]. Sakuradani et al. [8] improved AA production by mutants of M. alpina with lower n-3 desaturation activity. In our previous study, a strain M. alpina M6 with good production of AA was obtained by screening at low temperature and examining triphenyltetrazolium chloride staining degree [9]. Culture conditions including temperature, pH, medium (carbon source, carbon/nitrogen ratio, etc.) and cultivation method for AA production were also extensively investigated [7,10,11]. To achieve good production, fed-batch culture is a common strategy in fermentation process [12]. Singh and Ward [10] applied a fed-batch culture system by supplying additional nutrients to basal medium and a high AA yield (9.1 g/L)
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was achieved in 8 days. However, dynamic analysis of products accumulation and substrates consumption in AA production process, which were the bases of fed-batch culture, has not yet been systematically investigated so far. Here, the time courses of fungal growth, lipids accumulation, AA accumulation and substrate (glucose, nitrate and protein) consumption were investigated in order to find out the dynamic characteristics of AA production and a fed-batch culture method was developed accordingly to obtain a higher AA yield.
2. Materials and methods 2.1. Microorganism M. alpina M6 was isolated from soil [9]. It was maintained on potato dextrose agar (PDA; glucose 20 g/L, potato extract 4 g/L and agar 17 g/L) slants at 4 ◦ C and transferred every 3 months. 2.2. Culture conditions The fungi grew on PDA (50 mL PDA in a 250 mL flask) at 25 ◦ C for a week, then 20 mL sterile water with some glass beads was poured into the flask and the mycelia and spores were suspended by shaking vigorously. The suspension was inoculated into 200 mL inoculum medium (5% glucose and 50% fresh soybean sprout extract) in a 500 mL flask, and then incubated at 25 ◦ C for 3 days with shaking at 150 rpm. Fifty percent soybean sprout extract was obtained by filtering the mixture of soybean sprout and water (1:2) which had boiled for 30 min. Then, 250 mL flasks containing 50 mL production medium were inoculated with this inoculum at a rate of 10% (v/v) and incubated at 25 ◦ C on a shaker at 150 rpm. Production medium consisted of (g/L): potassium nitrate, 3; protein, 4.5; K2 HPO4 ·3H2 O, 4; CaCl2 ·2H2 O, 0.05; MgSO4 ·7H2 O, 0.5 (pH 5.5). Initial concentrations of glucose were varied as described in the text.
Fig. 1. Time courses of glucose consumption by Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
quickly in the early days of cultivation and slowly in the late days of cultivation. When initial concentrations of glucose were 30 and 50 g/L, the glucose was almost used up in the 3rd and 4th day, respectively, the residual glucose concentrations were under 5 g/L. When initial concentration of glucose was 80 g/L, glucose was almost depleted after 12 days of cultivation and the residual glucose was 6.1 g/L. When the initial concentrations of glucose were 100 and 120 g/L, glucose was not depleted even after 15 days of cultivation and the residual glucose were 16.5 and 40 g/L, respectively. Dry biomass yield of M. alpina at different initial glucose concentrations is shown in Fig. 2. The fungi grew faster in the early days (i.e. the 2nd day) of cultivation when initial
2.3. Analytical methods The dinitrosalicylic acid (DNS) method was used to determine glucose concentrations [13]. Protein concentrations were assayed by Lowry’s method [14]. Nitrate concentrations were assayed by salicylic acid method [15]. The dry weight of biomass, total lipids and content of AA were determined as described in previous publications [9].
3. Results and discussion 3.1. Dynamic courses of glucose consumption by M. alpina and fungal growth at different initial glucose concentrations Glucose consumption by M. alpina at different initial glucose concentrations is shown in Fig. 1. Glucose was uptaken
Fig. 2. Time courses of dry biomass yield of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
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concentrations of glucose were 30 and 50 g/L than when they were 80, 100 and 120 g/L, and the higher the initial glucose concentrations were, the slower the fungi grew at the beginning. The fungal growth was related to the time of glucose depletion according to Figs. 1 and 2. When residual glucose concentration was under 10 g/L, fungi stopped growing. When initial concentrations were 30, 50 and 80 g/L, dry fungal biomass reached their peak at 3rd, 4th and 9th day, respectively, and then the biomass yield declined. When initial concentrations were 100 and 120 g/L, there was enough glucose in the medium for growth, so the biomass increased during the whole period of cultivation, faster in the early days and slower in the late days. Though fungi grew fast at low initial glucose concentrations at early days of cultivation, high biomass (22.5 g/L) was achieved when initial concentration of glucose was 120 g/L. 3.2. Dynamic courses of lipids accumulation by M. alpina at different initial glucose concentrations Time courses of lipids content in biomass at different initial glucose concentrations are presented in Fig. 3. When initial concentration of glucose was 30 g/L, the lipids content in biomass reached its peak at 5th day, and this value was obviously lower than other peak values of other initial glucose concentrations. It indicated that too low glucose concentration which was exhausted quickly and no carbon source for lipid synthesis led to small lipids accumulation. Lipids were still accumulated, even the growth reached peak according to Figs. 2 and 3. The lipids content in biomass reached peak at 5th day when initial concentrations of glucose were 30 and 50 g/L, later than what biomass did. When initial concentrations of glucose were 80, 100 and 120 g/L, lipids in biomass increased during the whole period of cultivation.
Fig. 3. Time courses of lipids in biomass of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
Fig. 4. Time courses of lipids yield of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
It was obvious that lipids were still accumulated for a period of time, even glucose was used up according to Figs. 1 and 3. Time courses of lipids yields at different initial glucose concentrations are presented in Fig. 4. The lipids yield was determined by biomass and lipids content in biomass. Lipids yield was obviously low when initial concentration of glucose was 30 g/L due to its significantly low lipids content in biomass and low biomass yield. The highest value of lipids yield (9.09 g/L) was achieved after 15 days of cultivation when initial glucose concentration was 120 g/L. 3.3. Dynamic courses of AA production by M. alpina at different initial glucose concentrations Time courses of AA content in lipids of M. alpina at different initial glucose concentrations are shown in Fig. 5. AA content in lipids kept increasing during the whole period of cultivation no matter what the initial glucose concentration was. The lower the initial glucose concentrations, the higher the AA in lipids. AA contents in lipids which increased significantly after cultivation of 5 days when initial glucose concentrations were 30 and 50 g/L indicated that the great increase of AA content in lipids occurred after the glucose was exhausted according to Figs. 5 and 1. When lipid content of dry biomass remained constant or decreased, AA content in lipids increased significantly due to the conversion of other fatty acids such as saturated and monounsaturated fatty acids (data not shown here). Time courses of AA yields of M. alpina at different initial glucose concentrations are shown in Fig. 6. AA yields were determined by biomass, lipids in biomass and AA in lipids. AA yields kept increasing during the whole cultivation time. When initial glucose concentration was 30 g/L, the final AA yield was low because of low biomass and low lipids in biomass in spite of high AA content in lipids. Maximum
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Though maximum AA yield was obtained after cultivation of 15 days when initial glucose concentration was 100 g/L according to the above results, the fungi grew slowly at the early days of cultivation and it took a long time to obtain this value. Furthermore, the residual glucose concentration was high and the AA content in lipids was low. This represented a material waste and a potential pollution source that would make the downstream treatment difficult, and low AA
content in lipids would impair the final AA production. In order to obtain a high production of AA, a fed-batch cultivation method was developed to shorten the lag phase, enhance the uptake of glucose and increase the AA content in lipids. According to dynamic analysis, higher initial glucose concentration impaired fungal growth at early days of cultivation and low initial concentration of glucose was good to shorten the lag phase of fungal growth after inoculation. When residual glucose concentration was under 10 g/L, fungi stopped growing. Therefore, nutrition needed supplying to the medium to keep the fungi growing. Fifty grams per liter was suitable as initial glucose concentration according to the above results, for the fungi grew faster in the early days of cultivation and the fungal performance of growth, lipids and AA accumulation was better at this concentration than what at 30 g/L initial glucose concentration. To determine how much to feed and when to feed, time courses of substrates consumption including glucose, protein and potassium nitrate were investigated. The results are shown in Fig. 7. Glucose and nitrate were consumed fast, they were exhausted at the 4th day but the soy protein was consumed slowly, it was not used up until harvested. So the feeding medium contained glucose and nitrate. The concentrations of the feed medium were determined by their consumption rate. Glucose was consumed by 16.2 and 15.9 g/(L day), respectively, at the 2nd and 3rd days, and nitrate by 2.2 g/L at the 2nd day. Considering substrate consumption and fungal growth of fed-batch culture might be different from those of the batch culture, feed concentrations of glucose and nitrate presented in Table 1 were applied. Glucose and nitrate were supplemented to the medium at the 3rd, 4th and 5th days of cultivation. Because lipids were still accumulated for a period, even glucose was used up and a great increase of AA content in lipids occurred after the glucose was exhausted; therefore, the fungi kept growing till the 8th day to use up residual glucose.
Fig. 6. Time courses of AA yield of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L.
Fig. 7. Time courses of glucose, nitrate and protein consumption by Mortierella alpina at initial glucose concentration of 50 g/L. Symbols: () nitrate, () glucose and (䊉) protein. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
Fig. 5. Time courses of AA content in lipids of Mortierella alpina at different initial glucose concentrations. The initial glucose concentrations were: () 30 g/L, (䊉) 50 g/L, () 80 g/L, () 100 g/L and () 120 g/L. Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
AA yield (4.83 g/L) was obtained after cultivation of 15 days when initial glucose concentration was 100 g/L. 3.4. A fed-batch cultivation method was developed according to dynamic analysis for high production of AA
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Table 1 Effect of fed-batch culture on AA production by Mortierella alpina Feed concentration Glucose (g/(L day))
Nitrate (g/(L day))
10 10 10 10 20 20 20 20 30 30 30 30
1 1.5 2 3 1 1.5 2 3 1 1.5 2 3
Residual glucose (g/L)
Biomass (g/L)
Lipid in biomass (%)
Lipids (g/L)
AA in lipids (%)
AA (g/L)
0 0 0 0 10.95 1.02 1.02 3.20 17.72 21.10 23.35 18.85
27.2 27.0 27.4 26.8 33.2 37.0 36.0 35.6 31.8 35.4 35.8 35.6
33.2 27.5 28.3 28.8 40.7 38.4 36.4 36.2 38.0 37.4 33.3 35.0
9.03 7.43 7.76 7.72 13.51 14.21 13.11 12.88 12.08 13.25 11.92 12.46
64.5 66.7 68.8 66.0 53.0 54.5 56.1 54.6 43.2 49.7 52.4 51.9
5.82 4.96 5.34 5.10 7.16 7.74 7.35 7.03 5.22 6.59 6.25 6.47
Data are presented as means of duplicate; the degree of standard deviations was below ±5%.
Results showed that fed-batch culture was an effective way to improve AA yield. Supplementation of glucose and nitrate in the process of cultivation significantly increased the biomass, lipid content and AA level compared to batch culture. When feed concentration of glucose was 10 g/(L day), glucose needed for their growth was inefficient; therefore, no matter how much nitrate was fed, fungal growth was relatively poor. The AA contents in lipids were high because of glucose depletion. When feed concentration of glucose was 30 g/(L day), concentrations of residual sugar were high and so the AA contents in lipids were low. When feed concentration of glucose was 20 g/(L day), glucose supplemented just met the consumption requirement, dry biomass, lipids yield and AA yield were higher than that feed concentrations of glucose were 10 and 30 g/(L day). Here, different nitrate supplementation affected glucose uptaking and fungal growth. A high biomass (37.0 g/L), lipid (14.21 g/L) and AA yield (7.74 g/L) in 8 days were obtained, which were 1.65-, 1.51and 1.61-fold of maximum values of batch cultures, respectively.
4. Discussion Batch culture of M. alpina using glucose as a carbon source, nitrate and soy protein as combined nitrogen source was studied to analyze the dynamic characteristics of substrate consumption, fungal growth, lipid accumulation and AA production. It is generally known that there are two phases of lipids accumulation in oleaginous microorganisms in batch culture growth though Eroshin et al. [16] reported growth-coupled lipid synthesis in M. alpina LPM 301. The first phase is biomass growth, lipid synthesis occurs mainly in the second phase. Ratledge [17] suggested that the depletion of nitrogen in the growth medium evokes a decrease in the intracellular concentration of AMP, an activator of isocitrate dehydrogenase, and, as a consequence, an increase in the contents of citrate and isocitrate in the cells.
Citrate is converted by ATP:citrate lyase to oxaloacetate and acetyl-CoA; the latter is used for the synthesis of fatty acids. According to our dynamic analysis, fungal growth, lipids and AA accumulation were not synchronous and were related to the glucose concentration in the medium. AA production by M. alpina in our study could be divided into three phases in batch culture. The first phase was fungal growth coupled with lipids and AA production. In this phase, glucose supply was enough. Lipids were kept synthesizing in the second phase when glucose concentration was low (i.e. under 10 g/L); in this phase, mycelium stopped growing; lipids were accumulated for a period till glucose was used up and AA content in lipids increased continuously. Dynamic analysis showed that residual glucose was low then and the nitrogen sources were not depleted (nitrate was used up but soy protein remained). Therefore, the lipids accumulation by our strain in this case could not be explained by the assumption of Ratledge and coworkers as described above. Mycelium stopped growing mainly because of carbon source limitation and consequently inefficient energy source and carbon source needed for their growth. The third phase is AA synthesis. In this phase, mycelium stopped growing or even decreased, lipids content in biomass stopped increasing while AA content in lipids increased. A great increase of AA content in lipids occurred after the glucose was exhausted and lipids yield reached peak. Similar results that AA content in lipids produced by M. alpina increased significantly after glucose depletion were observed by Eroshin et al. [16]. AA content in lipids increased as a result of other fatty acids conversion. Saturated fatty acids such as 16:0 and 18:0 and monounsaturated fatty acid (18:1) decreased when AA increased. Why glucose depletion stimulated fatty acids conversion to AA and whether this conversion only occurred in M. alpina still remains unclear. In present study, results showed that low initial glucose concentration was beneficial for fungi growth, to shorten the lag phase after inoculation, indicating that higher osmotic pressure in the medium of higher initial glucose
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concentration prevents the growth of the fungi. It is necessary to develop fed-batch culture to avoid the repressive effects of high substrate concentration. Fed-batch culture is a common strategy in fermentation industry to avoid the repressive effects of high substrate concentration, to control the organisms’ growth rate and to achieve good production [12]. There are lots of examples that good production was obtained through this strategy, such as PHB production by Ralstonia eutropha [18], ergosterol production by Saccharomyces cerevisiae [19], etc. In the case of AA production, fed-batch method was used by supplementing soy flour, corn steep, corn oil and additional glucose to the basal medium each day after 3 days of fermentation to obtain the best AA yield that is published [10]. However, it would be difficult to carry out fed-batch culture without dynamic analysis of substrate consumption and product yielding. Therefore, in present study, dynamic analysis was investigated to give information about what to feed, how much to feed and when to feed in a fed-batch culture. Consequently, a better AA production of fed-batch culture was achieved based on characteristics of dynamic analysis compared to that of batch culture.
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