Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Lipid biosynthesis in Cunninghamella bainieri 2A1 in N-limited and N-excess media

Lipid biosynthesis in Cunninghamella bainieri 2A1 in N-limited and N-excess media Ann Microbiol (2010) 60:615–622 DOI 10.1007/s13213-010-0096-2 ORIGINAL PAPER Lipid biosynthesis in Cunninghamella bainieri 2A1 in N-limited and N-excess media Ekhlass M. Taha & Othman Omar & Wan Mohtar Wan Yusoff & Aidil Abdul Hamid Received: 1 May 2010 /Accepted: 25 June 2010 /Published online: 28 July 2010 Springer-Verlag and the University of Milan 2010 . . Abstract Lipid biosynthesis and fatty acids composition Keywords Lipid biosynthesies Nitrogen-excess media . . of oleaginous zygomycetes, namely Cunninghamella Nitrogen-limited media Gamma linolenic acid bainieri 2A1, cultured in media with excess or limited NAD -ICDH enzyme nitrogen were quantitatively determined at different times of culture growth. Accumulation of lipids occurred even when the activity of NAD -ICDH (β-Nicotinamide Introduction adenine dinucleotide-isocitrate dehydrogenase) was still detectable in both media. In C. bainieri 2A1,under The biosynthesis of lipids such as triglycerides, phospholipids nitrogen limitation, the ratio of lipids was around 35%, and glycolipids by oleaginous microorganisms is well whereas in nitrogen excess medium (feeding media documented and might represent an alternative source of fats supplemented with ammonium tartarate), the lipid ratio and oils, a topic which has been widely studied (Fakas et al. decreased. The amount of this decrease depended on the 2006, 2008a, 2009;Ratledge 1982; Ratledge and Boulton level of ammonium tartarate in the media. The main 1985;Vaniet al. 1988). findings in this paper were that C. bainieri 2A1 has the Specifically, Cunninghamella echinulata strains have ability to accumulate lipid although nitrogen concentration been shown to accumulate lipid bodies rich in polyunsat- detected inside the media and that NAD-ICDH was active urated fatty acids (PUFA) including γ-linolenic acid (GLA) in all culture periods. These results proved that the strain (Gema et al. 2002; Chen and Chang 1996; Fakas et al. C. bainieri 2A1 has an alternative behavior in lipid 2007, 2008b, 2008c; Tao and Zhang 2007). Other micro- biosynthesis that differs from yeast. According to the old organisms producing oils containing a large amount of hypotheses, yeasts could not accumulate lipid more than PUFA, including Mortierella alpina, Mortierella isabellina, 10% when nitrogen was detected inside the media. Mucor circinelloides, Cryptococcus curvatus, Crypthecodi- Nitrogen-limited and excess media both contained the nium cohnii and Yarrowia lipolytica, have been widely same fatty acids (palmitic acid, stearic acid, olic acid, studied (Papanikolaou and Aggelis 2003; Papanikolaou et linoleic acid and γ-linolenic acid), but at different al. 2004a; Ratledge 2004; Szczesha-Antczak et al. 2006). concentrations. The C:N ratio was also studied and Lipid accumulation in these microbes is triggered by cell showed no effects on total lipid accumulation, but a exhausting nitrogen, but glucose continues to be assim- significant effect on γ-linolenic acid concentration. ilated as well as inhibition of ICDH activity within the mitochondrion. This leads to the accumulation of citrate, which istransportedintothe cytosoland cleavedto acetyl-CoA by ATP:citrate lyase (Papanikolaou et al. : : : E. M. Taha (*) O. Omar W. M. W. Yusoff A. A. Hamid 2004b; Ratledge 2002). The biochemical mechanism of School of Biosciences and Biotechnology , Faculty of Science lipid accumulation in oleaginous fungus seems to be more and Technology, University Kebangsaan Malaysia, complicated than that reported for yeasts ( Ratledge 1994; 43600 Bangi, Selangor, Malaysia Wynn et al. 2001). The characteristics of NAD -ICDH of e-mail: ekhlassch@yahoo.com 616 Ann Microbiol (2010) 60:615–622 oleaginous molds were shown to be distinct from those and homogenized, and lipids were extracted with chloroform/ described for oleaginous yeasts, as fungal NAD -ICDH was methanol (2:1, v/v) using the method of Folch et al. (1957). not absolutely dependent on cellular AMP for activation Total lipids were determined gravimetrically. Lipid fractions (Wynn et al. 2001). Synthesis of fatty acids in yeasts is were converted to methyl esters by using n-hexane to induced by decreased activity of the isocitrate dehydrogenase dissolve the oil and 1 M sodium methoxide to get methyl enzyme under diminished nitrogen levels in culture (Evans ester, and analyzed in a gas chromatograph shimadzu GC- and Ratledge 1984). The transition from biomass synthesis 2010 FID using packed column (DB-23) and flame to lipogenesis was regulated by NAD -ICDH (Makri et al. ionization detector (FID) at 250°C. The fatty acids compo- 2010) sition present in the sample was calculated based on the peak The nature and concentration of the nitrogen source used area of corresponding methyl ester against reference standard in the medium is an essential factor for regulation of FAME mixture. lipogenesis. Many reports have described various nitrogen sources employed for fungal fatty acid production (Certik Preparation of cell–free extracts and assays et al. 1993, 1999), and have also suggested that the initial for NAD -ICDH C:N ratio is important for lipid accumulation (Yong-Hong et al. 2006; Papanikolaou et al. 2004b). Harvested mycelia were washed using cold distilled water, In this study, the growth, lipid accumulation, types of and cell extracts for the determination of enzyme activities fatty acids present, and activity of ICDH were studied in C. were prepared by suspending and disrupting mycelia in an bainieri 2A1 cultured in nitrogen-limited (N-limited) or extraction buffer (Wynn 1998). The disrupted cell suspension nitrogen-excess (N-excess) media. Different concentrations was centrifuged at 6,000g for 15 min at 4°C and the of glucose and different levels of nitrogen were used. The supernatant was filtered through a Whatman No.1 filter paper. fatty acid composition of the cellular lipids was determined, The protein in the clear supernatant was measured according and the N-limited and N-excess media were compared with to the protocol of Bradford (1976), and NAD -ICDH (EC respect to lipid yield, biomass yield, and γ-linolenic acid 1.1.1.41) was assayed using the method described by concentration. Kornberg (1955); ΔA was measured at 340 nm and 30°C and changes were the result of reduction of NAD . Materials and methods Analysis of the culture supernatant Microorganism and culture conditions The glucose concentration in the culture medium was determined using a GOD test kit according to the The fungus Cunninghamella bainieri 2A1 was maintained on manufacturer's instructions. The ammonium concentration potato dextrose agar (PDA) plates at 30°C for 7 days. The in the culture filtrate was determined using the indophenol growth medium, Kendrick medium (Kendrick and Ratledge test (Chaney and Marbach 1962). 1992) contained (g/L): glucose, 30; (NH ) C H O , 4 2 4 4 6 1; KH PO ,7; Na HPO ,2; MgSO H O, 1.5; yeast extract, Reproducibility of data 2 4 2 4 4 2 · · · 1.5; CaCl 2H O, 0.1; FeCl 6H O, 0.008; ZnSO 7H O, 2 2 3 2 4 2 · · 0.0001; CuSO 5H O, 0.001; Co(NO ) 6H O, 0.0001 and All experiments were carried out at least three times, all 4 2 3 2 MnSO 5H O, 0.0001. The PH was adjusted to 6. After data presented are reproducible. One-way ANOVA of 4 2 sterilization (121°C for 15 min), the media were inoculated triplicate data revealed a significant difference in lipid ratio with the spore suspension at a final concentration of 1 x 10 between the first and third days, but no significant spores/ml, which was produced by growing the strain on difference between the second and fourth days. PDA for 7 days at 30°C. All cultivation experiments were performed in 500-ml Erlenmeyer flasks containing 200 ml of the above medium, incubated in a rotary shaker at 200 rpm Results and discussion and 30°C. Growth and accumulation of reserve lipid in N-limited media: Analytical methods The kinetics of growth and lipid accumulation in C. Total biomass was harvested by vacuum filtration bainieri 2A1 were investigated in N-limited batch cultures through a Whatman No. 1 filter, washed with distilled with different concentrations of glucose including 30 g/L water, frozen at –20°C, freeze-dried for 24 h, and then (Fig. 1; Table 1). At the early stage of culture, the biomass gravimetrically determined. The dry biomass was disrupted accumulated as the culture period increased (Fig. 1). Lipid Ann Microbiol (2010) 60:615–622 617 Fig. 1 Evaluation of total bio- 14 1.2 12 40 35 300 mass (g/L ♦), lipid in total bio- mass % (wt/wt ■), reduced 12 30 1.0 10 250 glucose (g/L, ×), nitrogen (g/L, *), and γ-linoleic acid (GLA %) 10 25 (wt/wt ●)levelsaswell as 0.8 8 200 isocitrate dehydrogenase activity 8 20 (ICDH nmol/min mg,) during 0.6 6 20 150 growth of C. bainieri 2A1 on 6 15 N-limited media 0.4 4 100 4 10 0.2 2 50 2 5 0 0.0 0 0 0 0 0 24 46 72 96 120 Time h Biomass g/l Nitrogen concentration (g/l) Lipid % (wt/wt) GLA % (wt/wt lipid) Glucose concentration (g/l) Specific activity ICDH (nmol/min.mg) yield increased quickly from the first day of cultivation to and lipid yield accumulation was the fastest process. The the third day, where this might have been due to GLA percentage did not increase. During the lipid expenditure of nitrogen sources, resulting in the use of accumulation stage, the lipid yield reached a maximum on glucose to synthesize lipids. Other reasons for phenomena the fourth day and then began to decrease with time. In observed during this culture period include the closed contrast, Papanikolaou et al. (2004a) reported that the nature of the batch culture system, the abundance of maximum lipid level was achieved after 310–400 h. nutritional substrates, and limited metabolic materials at Another study in 2002 showed that the maximum lipid the initial stage of culture. C. bainieri 2A1 quickly entered level was achieved after 250 h, and that this strain C. the logarithmic phase after a short period of retention and echinulata cultivated in medium with a C:N ratio >100 accelerated growth, where it began to make use of nitrogen accumulated more than 35% of cellular lipid with a GLA sources and other nutritional substrates to reproduce. content greater than 11% (Gema et al. 2002), while our During this period, a great amount of biomass accumulated study achieved the same results with C:N ratio 42 and after (5.2 g/L). After the nitrogen sources were expended (at 96 h of cultivation. about 24 h after inoculation), carbon sources in the culture The hypothesis was that oleaginous microorganisms liquid were used. ICDH enzyme activity was present at all degrade AMP in low-nitrogen conditions in order to release culture times from the first (129 nmol/min mg) until the nitrogen, thereby allowing synthesis of cellular materials. fifth day (119 nmol/min mg), despite the depletion of The 'energy charge' ratio should thus be increased inside the nitrogen in the media. cell, resulting in inhibition of ICDH and accumulation of Our findings with respect to the C. bainieri 2A1 growth intracellular citric acid. Acetyl-CoA would then be pro- curve were in agreement with those of Tao and Zhang duced by citric acid cleavage via the ATP:citrate lyase (2007), who showed that Cunninghamella echinulata reaction (Botham and Ratledge 1979; Moreton 1988). Our mainly used glucose to synthesize lipids, and accumulated results related to NAD -ICDH show that ICDH in C. lipids until they reached a maximum on the fourth day. The bainieri 2A1 appeared in high activity during the period of same study also showed that, in the growing stage, biomass growth, and it appears that this enzyme was not implicated Table 1 Growth of C. bainieri 2A1 in N-limited media C/N Glc (g/L) X (g/L) Y Y L/X (% wt/wt) Pro X (mg/ml h) Pro L(mg/ml h) x/Glc L/Glc 42 30 9.4 0.37 0.13 35 0.09 0.035 69 50 10.6 0.28 0.097 34 0.14 0.05 97 70 11.6 0.23 0.085 36 0.12 0.043 Maximum L/X was achieved after 72–96 h. Culture conditions: initial pH 6 and temperature 30°C X total biomass,Y total biomass yield on glucose consumed, Y reserve lipid yield on glucose consumed, L/X maximum ratio of lipid x/Glc L/Glc accumulated, Pro X productivity of biomass, Pro L productivity of reserve lipid GLA % (wt/wt lipid) Nitrogen concentration (g/l) Biomass g/l Lipid % (wt/wt ) Glucose concentration (g/l) Specific activity ICDH (nmol/min.mg) 618 Ann Microbiol (2010) 60:615–622 in lipid biosynthesis and that this activity may be due to the to yeast and to other studies on fungi (Papanikolaou et al. implication of NAD -ICDH in different pathways, as, for 2004b), who demonstrated that the restriction of ICDH example, it can be suggested to have a role in maintaining activity in the mycelium of C. echinulata and M. isabellina complete TCA cycle operation under conditions of suffi- strains induced lipid biosynthesis. This was accompanied by cient isocitrate supply. Also, participation of two ICDH in a considerable decrease of respiration rate, and it is mitochondria and one ICDH in cytosol in interconversions remarkable that, in C. echinulata, the activity of NAD- between isocitrate and oxoglutarate represents a flexible ICDH was maintained at high levels considerably later after mechanism in which the ICDH enzymes are involved in the nitrogen depletion in the medium, explaining the biosynthesis maintenance of the metabolic balance between reduced and of lipid-free material biosynthesis for an extensive period of oxidized pyridine nucleotides (Igamberdiev and Gardestrom time and the simultaneous low reserve lipid accumulation 2003). Generally, NAD -ICDH was inhibited by two key inside the mycelia. These results in some way agreed with our metabolites, citrate and the ratio of NAD /NADH, which results in cases of build-up of lipid-free material. Nitrogen, again illustrates the strategic position of this enzyme in indispensable for lipid-free material synthesis, may be cellular metabolism. Citrate is a weak inhibitor but, as it provided by AMP-desaminase reaction, while ICDH activity accumulates in the mitochondria, it could feed forward to should be unaffected by the reduction of AMP. Indeed, it was increase its own production due to the equilibrium of found that NAD -ICDH of some oleaginous zygomycetes aconites. NAD, on the other hand, is very strongly inhibitory. was active even at high energy charge ratios (Certik et al. There are some indications that the catalysis by NAD - 1999;Wynnet al. 2001), whereas in the cases of oleaginous ICDH is not reversible and that it is highly inhibitory with yeasts it was not (Botham and Ratledge 1979). the ratio of NAD /NADH. In contrast, the NADP-ICDH The effect of glucose concentrations on the growth of C. catalyzed reaction is easily reversible. This may be bainieri 2A1 and lipid accumulation in N-limited media connected with different enzyme functions in isocitrate were also investigated (Table 1) with specific respect to metabolism (Evans and Ratledge 1985). This enzyme was biomass, lipid content of biomass, ,Y (total biomass x/Glc studied widely in yeast as such changes in Rhodosporidium yield on glucose consumed), Y (reserve lipid yield on L/Glc toruloides CBS 14 would result in rapid inactivation of glucose consumed) productivity for lipids, and productivity NAD :ICDH, thus indicating why this enzyme occupies for biomass. Different C:N ratios were achieved by varying such a strategic position in the sequence of events leading to the concentration of glucose (30, 50, 70 g/L) while fixing lipid accumulation (Evans and Ratledge 1985). The recent the concentrations of the nitrogen sources, which consisted research on yeast by Makri et al. (2010)also proved that of yeast extract (1.5 g/L) and ammonium tartrate (1 g/L). NAD -ICDH activity was gradually decreased during tran- Maximum lipid accumulation occurred at C:N 42, although sition from biomass production to lipogenic phase and it should be emphasized that yeast extracts containing further to citric acid production phase. In contrast, the significant quantities of N (8.9%) were included in the C:N activity of NADP -ICDH that was essentially located in the ratio calculation, as were contributions from ammonium cytoplasm, especially during both lipogenic and citric acid tartrate. Higher Y and Y values were also observed X/Glc L/Glc production phases, remained unchanged during the growth at C:N 42. There were no significant differences among cycle. During biomass production phase, glycerol was different C:N ratio with respect to the lipid percentage. essentially converted into fat-free cellular material, and when Oleaginous organisms usually do not accumulate lipids ammonium nitrogen was depleted, some quantities of storage to any great extent in media with initial C:N ratios <20; the lipid were then synthesized during the lipogenic phase. This optimum ratio for any organism will probably be between data again suggest that transition from biomass synthesis to 30 and 80 (Moreton 1988). Previous studies showed that lipogenesis and then to citric acid synthesis was regulated by the lipid content of C. echinulata grown with various NAD -ICDH. The stringent control of citrate metabolism at concentrations of the carbon source (soluble starch, 3–12%) the level of ICDH therefore provides the necessary first step decreased with increasing starch concentration and C:N in the accumulation of lipid by oleaginous yeasts. This ratio throughout the experimental range (Chen and Chang phenomena cannot be applied to our outcome as in C. 1996). Maximum lipid accumulation and GLA yield both bainieri 2A1 our data show high activity of ICDH in the occurred at 10% soluble starch concentration, where this growth phase. This activity may be due to the involvement was equivalent to an initial C:N ratio of 35. The previous of ICDH in other pathways or may be due to the build-up of study was somewhat in agreement with our results showing lipid-free material which is produced until the stationary no effect of carbon concentration on reserve lipids in total phase. The phenomena of C. bainieri 2A1 growth in Bach biomass. The maximum Y and Y were observed at x/Glc L/Glc culture seems to be different from other species in the C:N ratios (42) of 0.37 and 0.13, respectively, while the biomass production and lipogenic phases. The two phases best productivity was observed at C:N ratios (69) of 0.14 come together at the same time and this result is in contrast and 0.05, respectively. Another study in 2004 demonstrated Ann Microbiol (2010) 60:615–622 619 Fig. 2 Evaluation of total bio- 10 18 20 300 mass (g/L ♦), lipid in total biomass % (wt/wt ■), GLA% (wt/wt ●), 8 16 and isocitrate dehydrogenase activity (NAD -ICDH nmol/min 12 200 mg,) during growth of C. bainieri 6 12 2A1 in N-excess media with thrice daily feeding with ammo- 10 150 nium tartrate (1 g/L ) 4 8 6 100 2 4 0 0 0 0 0 24 48 72 96 120 Time h Biomass (g/l) GLA% (wt/wt lipid) Lipid % ( wt/wt ) Specific activity ICDH(nmol/min.mg) that the initial C:N ratio is important for lipid accumulation three nitrogen concentrations with respect to biomass, lipid (Papanikolaou et al. 2004b). Specifically, in C. echinulata percentage, lipid yield, and biomass yield. N-excess media cultivated in multiple limited media, when the C:N ratio with different levels of nitrogen showed more biomass was increased from 83.5 to 133.5, lipid content in C. yield than N-limited media. However, the lipid yield in N- echinulata subsequently increased from 36 to 47%. excess media with different levels of nitrogen was less than Papanikolaou et al. (2004b) also found that, at a high C:N that observed in N-limited media. Despite the effects of ratio varying from 150 to 340, lipid productivity increased excess nitrogen on microorganisms, the lipid yield when from 0.05 to 0.07 g/h. the nitrogen level was held between 0.5–1 g/L was around The role of the C:N ratio in lipid accumulation has been 15%. This finding disagrees with the old hypothesis shown to depend on the microorganism and the fermenta- suggesting that microorganisms can only accumulate lipids tion liquid medium (Vani et al. 1988). During growth-phase in N-limited media. However, to date, few studies have batch fermentation with an initial C:N ratio of 17, the lipid focused on N-concentration. Certik et al. (1999) used yield of the cells increased from 0.25 to 0.48 at the end of different concentrations of nitrogen (NaNO ) at the begin- fed-batch mode, when the initial C:N was 30. When the C: ning of culture in modified Czapek–Dox media. On the N was further increased to 40 in fed-batch mode, the lipid second day of cultivation, the percent lipid accumulation yield decreased to 0.33. Another study of yeast reported was between 5 and 10% when using 1.2 g/L NaNO and that the optimum C:N ratio was 420, but at this ratio, biomass and lipid production was very low (Yong-Hong et al. 1.2 2006). 31 31 1.0 Growth and accumulation of reserve lipids in N-excess media 24 24 0.8 Growth and lipid accumulation were studied by feeding 0.6 17 17 cultures with ammonium tartrate and by controlling the ammonium tartrate concentration (Fig. 2); cultures were fed 0.4 thrice daily with ammonium tartrate (1 g/L). 7 7 With three times daily feeding, biomass was (13 g/L) 0.2 Nitrogen concentration (g/l) and lipid accumulation was (16%). Nitrogen concentration Glucose concentration (g/l) 0.0 0 was between 0.4 and 0.6 g/L in the first days, and glucose 0 7 17 24 24 31 31 41 41 48 48 55 55 65 65 72 72 79 79 89 89 96 96 103 103 113 113120 120 gradually decreased (Fig. 3). Time h Based on the above findings, we focused more on Fig. 3 Evaluation of reduced glucose (g/L ♦) and nitrogen concen- nitrogen level by controlling the nitrogen concentration tration before and after feeding with ammonium tartrate (g/L) during (0.2–0.8, 0.5–1 and 0.8–1 g/L) for 2 days of culture C. bainieri 2A1 growth in N-excess media with thrice daily feeding with ammonium tartrate (1 g/L) (Table 2). There were significant differences among the GLA % (wt/wt lipid) Biomass (g/l) Nitrogen concentration (g/l) Lipid % (wt/wt ) Glucose concentration (g/l) Specific activity ICDH (nmol/min.mg) 620 Ann Microbiol (2010) 60:615–622 Table 2 Growth of C. bainieri 2A1 in N-excess media of increasing nitrogen concentration resulting in increased biomass production but decreased oil accumulation, the Nitrogen X (g/L) Y Y L/X Pro X Pro L x/Glc L/Glc only difference being that, even when we controlled the level (g/L) (% wt/wt) (mg/ml h) (mg/ml h) level of nitrogen, the microbes still accumulate lipid but not 0.2-0.8 8.3 0.55 0.12 21 0.17 0.039 the same as in limited media. The glucose concentration 0.5-1 9.3 0.6 0.09 15 0.19 0.03 may divert the carbon source to either lipid-free cellular 0.8-1 10 0.7 0.08 11 0.2 0.02 mass production or storage lipid synthesis. However, when the nitrogen source has no effect on glucose uptake, the C: X total biomass,Y total biomass yield on glucose consumed, Y x/Glc L/Glc N is an important factor affecting lipogenesis, which means reserve lipid yield on glucose consumed, L/X maximum ratio of lipid that when carbon uptake rate was high, lipid accumulation accumulated, Pro X productivity of biomass, Pro L productivity of reserve lipid during 48 h of culture occurred, even in the presence of relative high amounts of nitrogen in the growth medium (Fakas et al. 2008c). From around 15% when using 0.4 g/L NaNO . On the fourth day this explanation, we can conclude that, when C. bainieri of cultivation, the percent lipid accumulation was around 2A1 is cultured in N-excess media, glucose uptake was 10% when using 1.2 g/L NaNO and around 20% when high, so lipid accumulation occurred, even in the presence using 0.4 g/L. NaNO levels and NAD ICDH activity of nitrogen. increased concomitantly with NaNO concentration. This There have been insufficient previous studies of N- study thus indicated that lipid accumulation in C. echinulata excess media to explain the phenomena observed in the is indirectly correlated with nitrogen concentration. present study since all previous studies have used N-limited In contrast, in C. echinulata cultivated on hydrolyzed media. The hypotheses for N-limited media were that tomato waste (Fakas et al. 2007), the lipid content increased oleaginous species could accumulate more than 10% lipids to up to 25% of the biomass during the growth and and that they would then degrade AMP in order to release lipogenic phases. Although approximately 0.4 g/L of additional nitrogen from ammonium. This would result in organic nitrogen were measured in the growth environment, an increased energy charge ratio inside the cell, resulting in lipogenesis still commenced, indicating that this nitrogen inhibition of ICDH and intra-cellular citric acid accumula- could not be assimilated. Acid treatment for hydrolyzed tion. Acetyl-CoA would then be produced by citric acid tomato waste (TWH) oil yield reached around 40% at the cleavage via the ATP:citrate lyase reaction (Holdsworth and highest nitrogen concentration utilized (Fakas et al. 2008b), Ratledge 1988; Holdsworth et al. 1988). However, in the the main trend observation being that increasing nitrogen present study, the microorganisms accumulated lipids to concentration increased biomass production but decreased around 15% despite the presence of excess nitrogen (0.5– oil accumulation, except for growth on TWH. Our 1 g/L). Thus, further studies are required to explain lipid observation agreed with (Fakas et al. (2008b) in the case production in N-excess media. Table 3 Fatty acid composition C:N Culture time (h) Fatty acid concentration (wt/wt) in total mycelium lipids of total lipid produced by C. bainieri 2A1 in N-limited media Δ9 Δ9,12 Δ6,9,12 C16:0 C18:0 C18:1 C18:2 C18:3 C24:0 42 24 18.8 10.0 40.2 14.6 8.6 2.5 48 19.3 10.5 40.11 13.3 9.2 3.1 72 17.8 10.4 39.6 15.0 9.6 3.2 96 16.6 8.4 41.11 15.4 11.0 3.1 120 16.3 8.3 40.4 16.1 11.2 3.2 69 24 18 27.1 31.2 9.4 4.5 4.3 48 19.3 23.6 33.3 9.7 5 4.1 72 18.1 23.6 33.8 9.7 5.5 4.3 69 17.4 22.4 34.3 9.9 6.4 4.5 120 18.9 18.7 36.4 10.4 6.5 4.0 97 24 19.2 24.6 33.4 8.8 5.1 3.7 48 18.3 18.9 37.6 8.8 6.0 5.3 72 19.2 16.2 38.6 10.9 6.9 3.4 96 18.6 16.4 39.0 10.2 6.7 4.1 Culture conditions: initial pH 6, temperature 30°C and C:N ratios 120 18.4 15.2 39.6 10.2 7.0 4.3 42, 69 and 97 Ann Microbiol (2010) 60:615–622 621 Table 4 Fatty acid composition of total lipid produced by C. bainieri 2A1 in N-excess media Culture Fatty acid concentration (wt/wt) in total mycelium lipid time (h) Δ9 Δ9,12 Δ6,9,12 C16:0 C18:0 C18:1 C18:2 C18:3 C24:0 24 15.5 16.0 41.3 9.2 6.4 4.1 48 15.4 18.4 37.8 11.3 6.5 4.9 72 15.9 15.5 37.4 12.6 6.3 6.4 96 15.8 14.9 37.1 15.6 6.9 4.9 120 14.7 12.6 37.1 15.2 7.8 6.8 Fig. 4 GLA (g/L) in the reserve lipid versus lipid-free materials determine (g/L) in N-limited media (■) and N-excess media (♦) Culture conditions: initial pH 6, temperature 30°C, glucose, 30 g/L, and thrice daily feeding with ammonium tartrate (1 g/L) Conclusion Fatty acid composition of total lipid produced by C. bainieri 2A1 Lipid biosynthesis in C. bainieri 2A1 occurred in N- limited media at C:N ratios of 42, 69, and 93, and activity In C. bainieri 2A1 cultivated on N-limited media at C:N 42, of NAD - ICDH at C:N 42 appeared in high activity Δ9 the principal cellular fatty acid was oleic acid ( C18), the during the period of growth; this activity may be due to concentration of which remained constant during growth. In the build-up of lipid-free material which is produced until contrast, the concentrations of palmitic (C16:0) and stearic the stationary phase,. The lipid percentage was around (C18:0) acids slightly decreased and that of linolenic 35% and GLA was around 11%. However, there were no Δ9,12 ( C18:3) acid slightly increased (Table 3). In the first significant differences among the three types of C:N ratio phase of growth, γ-linolenic acid GLA content was elevated in total reserve lipids, whereas there were significant during lipid accumulation. Our results demonstrate the effect differences in GLA content. Statistical analysis of the lipid of the C:N ratio on fatty acid concentration. γ-linolenic acid profile demonstrated a significant difference between the GLA content reached 11.2% during lipid accumulation when first day of culture and the third day, whereas there were using C:N 42, but only reached 6.5% and 7.0% when using no significant differences from the second to the fifth C:N ratios of 69 and 97, respectively (Table 3). These findings days. In N-excess media, lipid biosynthesis still occurred indicate that the C:N ratio increases have effects contrary to even when N was still detected in the media at levels of those of GLA concentration. The fatty acid composition of N- 0.2–0.8, 0.5–1, and 0.8–1 g/L, where the lipid percentages excess media differed from that of N-limited media; palmitic were 21, 15, and 11, respectively. NAD -ICDHactivity acid (C16:0) remained almost constant during growth, and the was strongly detected on all the 5 days of culture. γ-linolenic acid GLA content remained constant (Table 4 ). A Microbial growth and production of reserve lipids were negative relationship was observed between ammonium not negatively affected by N-excess media and this may be tartrate level and γ-linolenic acid concentration (Table 5). due to the high uptake of glucose. Fatty acid composition A correlation between the quantity of GLA produced and was thus affected by the use of N-excess and N-limited the amount of lipid-free material was established in both media media, by different C:N ratios, and by different levels of types. Y wasdeterminedto be 0.22inN-limited media nitrogen. This result proved that the strain C. bainieri 2A1 GLA/Xf and 0.1 in N-excess media by linear regression analysis of has an alternative behavior in lipid biosynthesis that GLA produced and units of lipid free biomass (Fig. 4). differs from yeast. Table 5 Fatty acid composition Ammonium tartrate Culture time Fatty acid concentration (wt/wt) in total mycelium lipid of total lipid produced by C. (g/L) (h) bainieri 2A1 in N-excess media Δ9 Δ9,12 Δ6,9,12 C16:0 C18:0 C18:1 C18:2 C18:3 C24:0 0.2 -0.8 24 22.2 14.4 38.1 10.4 6.4 3 48 20.2 17.3 36.4 9.7 6.4 4 0.5-1 24 22.3 15.6 34.9 10.4 5.5 3.4 48 20.7 18.5 35 9.2 5.2 5.2 Culture conditions: initial pH 6, 0.8-1 24 20.6 20.9 31.5 11.3 4.5 5.3 temperature 30°C, glucose 30 g/L, and different levels of ammonium 48 20.3 19.5 33.6 11.4 4.7 5.0 tartrate 622 Ann Microbiol (2010) 60:615–622 Holdsworth JE, Veenhuis M, Ratledge C (1988) Enzyme activates in References oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids. J Gen Microbiol 134:2907–2915 Botham AP, Ratledge C (1979) A biochemical explanation for lipid Igamberdiev UA, Gardestrom P (2003) Regulation of NAD and NAD- accumulation in candida 107 and other oleaginous micro dependent isocitrate dehydrogenases by reduction levels of organism. J Gen Microbiol 114:361–375 pyridine nucleotides in mitochondria and cytosol of pea leaves. Bradford MM (1976) Arapid and sensitive method for the quantitation Biochim Biophys Acta: Bioenergetics 1606:117–125 of microgram quantities of protein utilizing the principle of Kendrich A, Ratiedge C (1992) Desaturation of polyunsaturated fatty protein dye-binding. Anal Biochem 72:248–254 acid in mucorcircinelloides and the inevolvement of a novel Certik M, Sajbidor J, Stredanska S (1993) Effect of carbon and membrane-bound malic enzyme. Eur J Microbiol 209:667–673 nitrogen sources on growth, lipid production and fatty acid Kornberg A (1955) Isocitrate dehydrogenase of yeast. Meth Enzymol composition of Mucormucedo F1384. Microbiol 74:7–15 1:705–7010 Certik M, Megova J, Horenitzky R (1999) Effect of nitrogen source Makri A, Fakas S, Aggelis G (2010) Metabolic activities of biotechno- on the activities of lipogenic enzymes in oleaginous fungus logical interest in Yarrowia lipolytica grown on glycerol in repated Cunninghamella echinulata. J Gen Appl Microbiol 45:289–293 bach cultures. Bioresour Technol 101:2351–2358 Chaney AL, Marbach EP (1962) Modified reagents for determination Moreton RS (1988) Physiology of lipid accumulation yeasts. In: of urea and ammonium. Clin Chem 8:130–132 Moreton RS (ed) Single cell oil. Longman, Essex, UK, pp 1–32 Chen H-C, Chang C-C (1996) Production of gamma-linoleic acid by Papanikolaou S, Aggelis G (2003) Modeling lipid accumulation and the fungus Cunninghamella echinulata CCRC 31840. Biotechnol degradation in Yarrowia lipolytic cultivated on industrial fats. Prog 12:338–341 Curr Microbiol 46:398–402 Evans TC, Ratledge C (1984) Effect of nitrogen source on lipid Papanikolaou S, Komaitis M, Aggelis G (2004a) Single cell oil (SCO) accumulation in oleaginous yeasts. J Gen Microbiol 130:1693–1704 production by Mortierella isabellina grown on high-sugar Evans TC, Ratledge C (1985) The role of mitochondrial NAD :isocitrate content media. Bioresour Technol 95:287–291 dehydrogenase in lipid accumulation by the oleaginous yeast Papanikolaou S, Sarantou S, Komaitis M, Aggelis G (2004b) Repression Rhodosporidium toruloides CBS14. Can J Microbiol 31:845–850 of reserve lipid turnover in Cunninghamella echinulata and Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, Mortierella isabellina cultivated in multiple-limited media. J Appl Aggelis G (2006) Lipid of Cunninghamella echinulata with Microbiol 97:867–875 emphasis of γ-linolenic acid distribution among lipid classes. Ratledge C (1982) Microbial oils and fats: an assessment of their Appl Microbiol Biotechnol 73:676–683 commercial potential. In: Ball JM (ed)Progress in industrial Fakas S, Galiotoupanayotou M, Papanikolaou S, Komaitis M, Aggelis microbiology 16. Elsevier, Oxford, pp 119–206 G (2007) Compositional shifts in lipid fraction during lipid Ratledge C (1994) Yeasts, moulds, algae and bacteria as sources of lipid. turnover in Cunninghamella echinulata. Enzyme Microb Technol In: Kamel BS, Kakuda Y (eds) Technological advances in improved 1321–1327 and alternative sources of lipids. Blackie, London, pp 235–291 Fakas S, Papapostolou I, Papanikolaous S, Georgiou DC, Aggelis G Ratledge C (2002) Regulation of lipid accumulation in oleaginous (2008a) Susceptibilit to peroxidation of the major mycelia lipids microorganisms. Biochem Soc Trans 30:1047–1050 of Cunninghamella echinulata. Eur J Lipid Sci Technol Ratledge C (2004) Fatty acid biosynthesis in microorganism being 110:1062–1067 used for single cell oil production. Biochimie 86:807–815 Fakas S, Certik M, Papanikolaou S, Aggelis G, Komaitis M, Galiotou- Ratledge C, Boulton AC (1985) Fat and oil. In: Murray Moo–Young (ed) Panayotou M (2008b) γ-Linolenic acid production by Cunning- Comprehensive biotechnology. Peramon, New York, pp 983–1003 hamella echinulata growing on complex organic nitrogen sources. Szczesha-Antczak M, Antczak T, Piotrowicz-Wasiak M, Rzyska M, Bioresour Technol 99:5986–5990 Binkowska N, Bielecki S (2006) Relationships between lipases Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, and lipids mycelia of two mucor strains. Enzyme Microb Technol Aggelis G (2008c) Organic nitrogen of tomato waste hydrolysate 39:1214–1222 enhances glucose uptake and lipid accumulation in Cunning- Tao J-YL, Zhang X-Y (2007) Growth low of gamma-inoleic acid hamella echinulata. J Appl Microbiol 105:1364–5072 production by Cunninghamella echinulata. J US-China Med Sci Fakas S, Makri A, Mavromati M, Tselepi M, Aggelis G (2009) Fatty 1:55–60 acid composition in lipid fraction lengthwise the mycelium of Vani S, Sharma CD, Bhagat SD, Saini VS, Adhikari DK (1988) Lipid Mortierella isabellina and lipid production by solid state and fatty acid biosynthesis by Rhodotorula minuta. JAOCS fermentation. Bioresour Technol 100:6118–6120 75:501–505 Folch J, Lees M, Sloane–Stanley GH (1957) Simple method for the Wynn PJ (1998) Microorganisms as sources of nutritionally important isolation and purification of total lipids from animal tissues. J polyunsaturated fatty acid. Forum Appl Biotechnol 1:1215–1222 Biol Chem 226:497–509 Wynn PJ, Hamid AA, Li Y, Ratledge C (2001) Biochemical events Gema H, Kavadia A, Dimou D, Tsagou V, Komaitis M, Aggelis G leading to the diversion of carbon into storage lipids in the (2002) Production of γ- linolenic acid by Cunninghamella oleaginous fungi Mucor circinelloides and Mortierella alpine. echinulata cultivated on glucose and orange peel. Appl Microbiol Microbiology 147:2857–2864 Biotechnol 58:303–307 Yong–Hong Li, BO Liu, ZHAO Zong–Bao, AB Feng Wa (2006) Holdsworth EJ, Ratledge C (1988) Lipid turnover in oleaginous Optimization of culture condition for lipid production by yeasts. J Gen Microbiol 134:339–346 Rhodosporidium toruloides. Chin J Biotechnol 22:650–656 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Lipid biosynthesis in Cunninghamella bainieri 2A1 in N-limited and N-excess media

Loading next page...
 
/lp/springer-journals/lipid-biosynthesis-in-cunninghamella-bainieri-2a1-in-n-limited-and-n-Iqs2b0gdLu

References (97)

Publisher
Springer Journals
Copyright
Copyright © 2010 by The Author(s)
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-010-0096-2
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2010) 60:615–622 DOI 10.1007/s13213-010-0096-2 ORIGINAL PAPER Lipid biosynthesis in Cunninghamella bainieri 2A1 in N-limited and N-excess media Ekhlass M. Taha & Othman Omar & Wan Mohtar Wan Yusoff & Aidil Abdul Hamid Received: 1 May 2010 /Accepted: 25 June 2010 /Published online: 28 July 2010 Springer-Verlag and the University of Milan 2010 . . Abstract Lipid biosynthesis and fatty acids composition Keywords Lipid biosynthesies Nitrogen-excess media . . of oleaginous zygomycetes, namely Cunninghamella Nitrogen-limited media Gamma linolenic acid bainieri 2A1, cultured in media with excess or limited NAD -ICDH enzyme nitrogen were quantitatively determined at different times of culture growth. Accumulation of lipids occurred even when the activity of NAD -ICDH (β-Nicotinamide Introduction adenine dinucleotide-isocitrate dehydrogenase) was still detectable in both media. In C. bainieri 2A1,under The biosynthesis of lipids such as triglycerides, phospholipids nitrogen limitation, the ratio of lipids was around 35%, and glycolipids by oleaginous microorganisms is well whereas in nitrogen excess medium (feeding media documented and might represent an alternative source of fats supplemented with ammonium tartarate), the lipid ratio and oils, a topic which has been widely studied (Fakas et al. decreased. The amount of this decrease depended on the 2006, 2008a, 2009;Ratledge 1982; Ratledge and Boulton level of ammonium tartarate in the media. The main 1985;Vaniet al. 1988). findings in this paper were that C. bainieri 2A1 has the Specifically, Cunninghamella echinulata strains have ability to accumulate lipid although nitrogen concentration been shown to accumulate lipid bodies rich in polyunsat- detected inside the media and that NAD-ICDH was active urated fatty acids (PUFA) including γ-linolenic acid (GLA) in all culture periods. These results proved that the strain (Gema et al. 2002; Chen and Chang 1996; Fakas et al. C. bainieri 2A1 has an alternative behavior in lipid 2007, 2008b, 2008c; Tao and Zhang 2007). Other micro- biosynthesis that differs from yeast. According to the old organisms producing oils containing a large amount of hypotheses, yeasts could not accumulate lipid more than PUFA, including Mortierella alpina, Mortierella isabellina, 10% when nitrogen was detected inside the media. Mucor circinelloides, Cryptococcus curvatus, Crypthecodi- Nitrogen-limited and excess media both contained the nium cohnii and Yarrowia lipolytica, have been widely same fatty acids (palmitic acid, stearic acid, olic acid, studied (Papanikolaou and Aggelis 2003; Papanikolaou et linoleic acid and γ-linolenic acid), but at different al. 2004a; Ratledge 2004; Szczesha-Antczak et al. 2006). concentrations. The C:N ratio was also studied and Lipid accumulation in these microbes is triggered by cell showed no effects on total lipid accumulation, but a exhausting nitrogen, but glucose continues to be assim- significant effect on γ-linolenic acid concentration. ilated as well as inhibition of ICDH activity within the mitochondrion. This leads to the accumulation of citrate, which istransportedintothe cytosoland cleavedto acetyl-CoA by ATP:citrate lyase (Papanikolaou et al. : : : E. M. Taha (*) O. Omar W. M. W. Yusoff A. A. Hamid 2004b; Ratledge 2002). The biochemical mechanism of School of Biosciences and Biotechnology , Faculty of Science lipid accumulation in oleaginous fungus seems to be more and Technology, University Kebangsaan Malaysia, complicated than that reported for yeasts ( Ratledge 1994; 43600 Bangi, Selangor, Malaysia Wynn et al. 2001). The characteristics of NAD -ICDH of e-mail: ekhlassch@yahoo.com 616 Ann Microbiol (2010) 60:615–622 oleaginous molds were shown to be distinct from those and homogenized, and lipids were extracted with chloroform/ described for oleaginous yeasts, as fungal NAD -ICDH was methanol (2:1, v/v) using the method of Folch et al. (1957). not absolutely dependent on cellular AMP for activation Total lipids were determined gravimetrically. Lipid fractions (Wynn et al. 2001). Synthesis of fatty acids in yeasts is were converted to methyl esters by using n-hexane to induced by decreased activity of the isocitrate dehydrogenase dissolve the oil and 1 M sodium methoxide to get methyl enzyme under diminished nitrogen levels in culture (Evans ester, and analyzed in a gas chromatograph shimadzu GC- and Ratledge 1984). The transition from biomass synthesis 2010 FID using packed column (DB-23) and flame to lipogenesis was regulated by NAD -ICDH (Makri et al. ionization detector (FID) at 250°C. The fatty acids compo- 2010) sition present in the sample was calculated based on the peak The nature and concentration of the nitrogen source used area of corresponding methyl ester against reference standard in the medium is an essential factor for regulation of FAME mixture. lipogenesis. Many reports have described various nitrogen sources employed for fungal fatty acid production (Certik Preparation of cell–free extracts and assays et al. 1993, 1999), and have also suggested that the initial for NAD -ICDH C:N ratio is important for lipid accumulation (Yong-Hong et al. 2006; Papanikolaou et al. 2004b). Harvested mycelia were washed using cold distilled water, In this study, the growth, lipid accumulation, types of and cell extracts for the determination of enzyme activities fatty acids present, and activity of ICDH were studied in C. were prepared by suspending and disrupting mycelia in an bainieri 2A1 cultured in nitrogen-limited (N-limited) or extraction buffer (Wynn 1998). The disrupted cell suspension nitrogen-excess (N-excess) media. Different concentrations was centrifuged at 6,000g for 15 min at 4°C and the of glucose and different levels of nitrogen were used. The supernatant was filtered through a Whatman No.1 filter paper. fatty acid composition of the cellular lipids was determined, The protein in the clear supernatant was measured according and the N-limited and N-excess media were compared with to the protocol of Bradford (1976), and NAD -ICDH (EC respect to lipid yield, biomass yield, and γ-linolenic acid 1.1.1.41) was assayed using the method described by concentration. Kornberg (1955); ΔA was measured at 340 nm and 30°C and changes were the result of reduction of NAD . Materials and methods Analysis of the culture supernatant Microorganism and culture conditions The glucose concentration in the culture medium was determined using a GOD test kit according to the The fungus Cunninghamella bainieri 2A1 was maintained on manufacturer's instructions. The ammonium concentration potato dextrose agar (PDA) plates at 30°C for 7 days. The in the culture filtrate was determined using the indophenol growth medium, Kendrick medium (Kendrick and Ratledge test (Chaney and Marbach 1962). 1992) contained (g/L): glucose, 30; (NH ) C H O , 4 2 4 4 6 1; KH PO ,7; Na HPO ,2; MgSO H O, 1.5; yeast extract, Reproducibility of data 2 4 2 4 4 2 · · · 1.5; CaCl 2H O, 0.1; FeCl 6H O, 0.008; ZnSO 7H O, 2 2 3 2 4 2 · · 0.0001; CuSO 5H O, 0.001; Co(NO ) 6H O, 0.0001 and All experiments were carried out at least three times, all 4 2 3 2 MnSO 5H O, 0.0001. The PH was adjusted to 6. After data presented are reproducible. One-way ANOVA of 4 2 sterilization (121°C for 15 min), the media were inoculated triplicate data revealed a significant difference in lipid ratio with the spore suspension at a final concentration of 1 x 10 between the first and third days, but no significant spores/ml, which was produced by growing the strain on difference between the second and fourth days. PDA for 7 days at 30°C. All cultivation experiments were performed in 500-ml Erlenmeyer flasks containing 200 ml of the above medium, incubated in a rotary shaker at 200 rpm Results and discussion and 30°C. Growth and accumulation of reserve lipid in N-limited media: Analytical methods The kinetics of growth and lipid accumulation in C. Total biomass was harvested by vacuum filtration bainieri 2A1 were investigated in N-limited batch cultures through a Whatman No. 1 filter, washed with distilled with different concentrations of glucose including 30 g/L water, frozen at –20°C, freeze-dried for 24 h, and then (Fig. 1; Table 1). At the early stage of culture, the biomass gravimetrically determined. The dry biomass was disrupted accumulated as the culture period increased (Fig. 1). Lipid Ann Microbiol (2010) 60:615–622 617 Fig. 1 Evaluation of total bio- 14 1.2 12 40 35 300 mass (g/L ♦), lipid in total bio- mass % (wt/wt ■), reduced 12 30 1.0 10 250 glucose (g/L, ×), nitrogen (g/L, *), and γ-linoleic acid (GLA %) 10 25 (wt/wt ●)levelsaswell as 0.8 8 200 isocitrate dehydrogenase activity 8 20 (ICDH nmol/min mg,) during 0.6 6 20 150 growth of C. bainieri 2A1 on 6 15 N-limited media 0.4 4 100 4 10 0.2 2 50 2 5 0 0.0 0 0 0 0 0 24 46 72 96 120 Time h Biomass g/l Nitrogen concentration (g/l) Lipid % (wt/wt) GLA % (wt/wt lipid) Glucose concentration (g/l) Specific activity ICDH (nmol/min.mg) yield increased quickly from the first day of cultivation to and lipid yield accumulation was the fastest process. The the third day, where this might have been due to GLA percentage did not increase. During the lipid expenditure of nitrogen sources, resulting in the use of accumulation stage, the lipid yield reached a maximum on glucose to synthesize lipids. Other reasons for phenomena the fourth day and then began to decrease with time. In observed during this culture period include the closed contrast, Papanikolaou et al. (2004a) reported that the nature of the batch culture system, the abundance of maximum lipid level was achieved after 310–400 h. nutritional substrates, and limited metabolic materials at Another study in 2002 showed that the maximum lipid the initial stage of culture. C. bainieri 2A1 quickly entered level was achieved after 250 h, and that this strain C. the logarithmic phase after a short period of retention and echinulata cultivated in medium with a C:N ratio >100 accelerated growth, where it began to make use of nitrogen accumulated more than 35% of cellular lipid with a GLA sources and other nutritional substrates to reproduce. content greater than 11% (Gema et al. 2002), while our During this period, a great amount of biomass accumulated study achieved the same results with C:N ratio 42 and after (5.2 g/L). After the nitrogen sources were expended (at 96 h of cultivation. about 24 h after inoculation), carbon sources in the culture The hypothesis was that oleaginous microorganisms liquid were used. ICDH enzyme activity was present at all degrade AMP in low-nitrogen conditions in order to release culture times from the first (129 nmol/min mg) until the nitrogen, thereby allowing synthesis of cellular materials. fifth day (119 nmol/min mg), despite the depletion of The 'energy charge' ratio should thus be increased inside the nitrogen in the media. cell, resulting in inhibition of ICDH and accumulation of Our findings with respect to the C. bainieri 2A1 growth intracellular citric acid. Acetyl-CoA would then be pro- curve were in agreement with those of Tao and Zhang duced by citric acid cleavage via the ATP:citrate lyase (2007), who showed that Cunninghamella echinulata reaction (Botham and Ratledge 1979; Moreton 1988). Our mainly used glucose to synthesize lipids, and accumulated results related to NAD -ICDH show that ICDH in C. lipids until they reached a maximum on the fourth day. The bainieri 2A1 appeared in high activity during the period of same study also showed that, in the growing stage, biomass growth, and it appears that this enzyme was not implicated Table 1 Growth of C. bainieri 2A1 in N-limited media C/N Glc (g/L) X (g/L) Y Y L/X (% wt/wt) Pro X (mg/ml h) Pro L(mg/ml h) x/Glc L/Glc 42 30 9.4 0.37 0.13 35 0.09 0.035 69 50 10.6 0.28 0.097 34 0.14 0.05 97 70 11.6 0.23 0.085 36 0.12 0.043 Maximum L/X was achieved after 72–96 h. Culture conditions: initial pH 6 and temperature 30°C X total biomass,Y total biomass yield on glucose consumed, Y reserve lipid yield on glucose consumed, L/X maximum ratio of lipid x/Glc L/Glc accumulated, Pro X productivity of biomass, Pro L productivity of reserve lipid GLA % (wt/wt lipid) Nitrogen concentration (g/l) Biomass g/l Lipid % (wt/wt ) Glucose concentration (g/l) Specific activity ICDH (nmol/min.mg) 618 Ann Microbiol (2010) 60:615–622 in lipid biosynthesis and that this activity may be due to the to yeast and to other studies on fungi (Papanikolaou et al. implication of NAD -ICDH in different pathways, as, for 2004b), who demonstrated that the restriction of ICDH example, it can be suggested to have a role in maintaining activity in the mycelium of C. echinulata and M. isabellina complete TCA cycle operation under conditions of suffi- strains induced lipid biosynthesis. This was accompanied by cient isocitrate supply. Also, participation of two ICDH in a considerable decrease of respiration rate, and it is mitochondria and one ICDH in cytosol in interconversions remarkable that, in C. echinulata, the activity of NAD- between isocitrate and oxoglutarate represents a flexible ICDH was maintained at high levels considerably later after mechanism in which the ICDH enzymes are involved in the nitrogen depletion in the medium, explaining the biosynthesis maintenance of the metabolic balance between reduced and of lipid-free material biosynthesis for an extensive period of oxidized pyridine nucleotides (Igamberdiev and Gardestrom time and the simultaneous low reserve lipid accumulation 2003). Generally, NAD -ICDH was inhibited by two key inside the mycelia. These results in some way agreed with our metabolites, citrate and the ratio of NAD /NADH, which results in cases of build-up of lipid-free material. Nitrogen, again illustrates the strategic position of this enzyme in indispensable for lipid-free material synthesis, may be cellular metabolism. Citrate is a weak inhibitor but, as it provided by AMP-desaminase reaction, while ICDH activity accumulates in the mitochondria, it could feed forward to should be unaffected by the reduction of AMP. Indeed, it was increase its own production due to the equilibrium of found that NAD -ICDH of some oleaginous zygomycetes aconites. NAD, on the other hand, is very strongly inhibitory. was active even at high energy charge ratios (Certik et al. There are some indications that the catalysis by NAD - 1999;Wynnet al. 2001), whereas in the cases of oleaginous ICDH is not reversible and that it is highly inhibitory with yeasts it was not (Botham and Ratledge 1979). the ratio of NAD /NADH. In contrast, the NADP-ICDH The effect of glucose concentrations on the growth of C. catalyzed reaction is easily reversible. This may be bainieri 2A1 and lipid accumulation in N-limited media connected with different enzyme functions in isocitrate were also investigated (Table 1) with specific respect to metabolism (Evans and Ratledge 1985). This enzyme was biomass, lipid content of biomass, ,Y (total biomass x/Glc studied widely in yeast as such changes in Rhodosporidium yield on glucose consumed), Y (reserve lipid yield on L/Glc toruloides CBS 14 would result in rapid inactivation of glucose consumed) productivity for lipids, and productivity NAD :ICDH, thus indicating why this enzyme occupies for biomass. Different C:N ratios were achieved by varying such a strategic position in the sequence of events leading to the concentration of glucose (30, 50, 70 g/L) while fixing lipid accumulation (Evans and Ratledge 1985). The recent the concentrations of the nitrogen sources, which consisted research on yeast by Makri et al. (2010)also proved that of yeast extract (1.5 g/L) and ammonium tartrate (1 g/L). NAD -ICDH activity was gradually decreased during tran- Maximum lipid accumulation occurred at C:N 42, although sition from biomass production to lipogenic phase and it should be emphasized that yeast extracts containing further to citric acid production phase. In contrast, the significant quantities of N (8.9%) were included in the C:N activity of NADP -ICDH that was essentially located in the ratio calculation, as were contributions from ammonium cytoplasm, especially during both lipogenic and citric acid tartrate. Higher Y and Y values were also observed X/Glc L/Glc production phases, remained unchanged during the growth at C:N 42. There were no significant differences among cycle. During biomass production phase, glycerol was different C:N ratio with respect to the lipid percentage. essentially converted into fat-free cellular material, and when Oleaginous organisms usually do not accumulate lipids ammonium nitrogen was depleted, some quantities of storage to any great extent in media with initial C:N ratios <20; the lipid were then synthesized during the lipogenic phase. This optimum ratio for any organism will probably be between data again suggest that transition from biomass synthesis to 30 and 80 (Moreton 1988). Previous studies showed that lipogenesis and then to citric acid synthesis was regulated by the lipid content of C. echinulata grown with various NAD -ICDH. The stringent control of citrate metabolism at concentrations of the carbon source (soluble starch, 3–12%) the level of ICDH therefore provides the necessary first step decreased with increasing starch concentration and C:N in the accumulation of lipid by oleaginous yeasts. This ratio throughout the experimental range (Chen and Chang phenomena cannot be applied to our outcome as in C. 1996). Maximum lipid accumulation and GLA yield both bainieri 2A1 our data show high activity of ICDH in the occurred at 10% soluble starch concentration, where this growth phase. This activity may be due to the involvement was equivalent to an initial C:N ratio of 35. The previous of ICDH in other pathways or may be due to the build-up of study was somewhat in agreement with our results showing lipid-free material which is produced until the stationary no effect of carbon concentration on reserve lipids in total phase. The phenomena of C. bainieri 2A1 growth in Bach biomass. The maximum Y and Y were observed at x/Glc L/Glc culture seems to be different from other species in the C:N ratios (42) of 0.37 and 0.13, respectively, while the biomass production and lipogenic phases. The two phases best productivity was observed at C:N ratios (69) of 0.14 come together at the same time and this result is in contrast and 0.05, respectively. Another study in 2004 demonstrated Ann Microbiol (2010) 60:615–622 619 Fig. 2 Evaluation of total bio- 10 18 20 300 mass (g/L ♦), lipid in total biomass % (wt/wt ■), GLA% (wt/wt ●), 8 16 and isocitrate dehydrogenase activity (NAD -ICDH nmol/min 12 200 mg,) during growth of C. bainieri 6 12 2A1 in N-excess media with thrice daily feeding with ammo- 10 150 nium tartrate (1 g/L ) 4 8 6 100 2 4 0 0 0 0 0 24 48 72 96 120 Time h Biomass (g/l) GLA% (wt/wt lipid) Lipid % ( wt/wt ) Specific activity ICDH(nmol/min.mg) that the initial C:N ratio is important for lipid accumulation three nitrogen concentrations with respect to biomass, lipid (Papanikolaou et al. 2004b). Specifically, in C. echinulata percentage, lipid yield, and biomass yield. N-excess media cultivated in multiple limited media, when the C:N ratio with different levels of nitrogen showed more biomass was increased from 83.5 to 133.5, lipid content in C. yield than N-limited media. However, the lipid yield in N- echinulata subsequently increased from 36 to 47%. excess media with different levels of nitrogen was less than Papanikolaou et al. (2004b) also found that, at a high C:N that observed in N-limited media. Despite the effects of ratio varying from 150 to 340, lipid productivity increased excess nitrogen on microorganisms, the lipid yield when from 0.05 to 0.07 g/h. the nitrogen level was held between 0.5–1 g/L was around The role of the C:N ratio in lipid accumulation has been 15%. This finding disagrees with the old hypothesis shown to depend on the microorganism and the fermenta- suggesting that microorganisms can only accumulate lipids tion liquid medium (Vani et al. 1988). During growth-phase in N-limited media. However, to date, few studies have batch fermentation with an initial C:N ratio of 17, the lipid focused on N-concentration. Certik et al. (1999) used yield of the cells increased from 0.25 to 0.48 at the end of different concentrations of nitrogen (NaNO ) at the begin- fed-batch mode, when the initial C:N was 30. When the C: ning of culture in modified Czapek–Dox media. On the N was further increased to 40 in fed-batch mode, the lipid second day of cultivation, the percent lipid accumulation yield decreased to 0.33. Another study of yeast reported was between 5 and 10% when using 1.2 g/L NaNO and that the optimum C:N ratio was 420, but at this ratio, biomass and lipid production was very low (Yong-Hong et al. 1.2 2006). 31 31 1.0 Growth and accumulation of reserve lipids in N-excess media 24 24 0.8 Growth and lipid accumulation were studied by feeding 0.6 17 17 cultures with ammonium tartrate and by controlling the ammonium tartrate concentration (Fig. 2); cultures were fed 0.4 thrice daily with ammonium tartrate (1 g/L). 7 7 With three times daily feeding, biomass was (13 g/L) 0.2 Nitrogen concentration (g/l) and lipid accumulation was (16%). Nitrogen concentration Glucose concentration (g/l) 0.0 0 was between 0.4 and 0.6 g/L in the first days, and glucose 0 7 17 24 24 31 31 41 41 48 48 55 55 65 65 72 72 79 79 89 89 96 96 103 103 113 113120 120 gradually decreased (Fig. 3). Time h Based on the above findings, we focused more on Fig. 3 Evaluation of reduced glucose (g/L ♦) and nitrogen concen- nitrogen level by controlling the nitrogen concentration tration before and after feeding with ammonium tartrate (g/L) during (0.2–0.8, 0.5–1 and 0.8–1 g/L) for 2 days of culture C. bainieri 2A1 growth in N-excess media with thrice daily feeding with ammonium tartrate (1 g/L) (Table 2). There were significant differences among the GLA % (wt/wt lipid) Biomass (g/l) Nitrogen concentration (g/l) Lipid % (wt/wt ) Glucose concentration (g/l) Specific activity ICDH (nmol/min.mg) 620 Ann Microbiol (2010) 60:615–622 Table 2 Growth of C. bainieri 2A1 in N-excess media of increasing nitrogen concentration resulting in increased biomass production but decreased oil accumulation, the Nitrogen X (g/L) Y Y L/X Pro X Pro L x/Glc L/Glc only difference being that, even when we controlled the level (g/L) (% wt/wt) (mg/ml h) (mg/ml h) level of nitrogen, the microbes still accumulate lipid but not 0.2-0.8 8.3 0.55 0.12 21 0.17 0.039 the same as in limited media. The glucose concentration 0.5-1 9.3 0.6 0.09 15 0.19 0.03 may divert the carbon source to either lipid-free cellular 0.8-1 10 0.7 0.08 11 0.2 0.02 mass production or storage lipid synthesis. However, when the nitrogen source has no effect on glucose uptake, the C: X total biomass,Y total biomass yield on glucose consumed, Y x/Glc L/Glc N is an important factor affecting lipogenesis, which means reserve lipid yield on glucose consumed, L/X maximum ratio of lipid that when carbon uptake rate was high, lipid accumulation accumulated, Pro X productivity of biomass, Pro L productivity of reserve lipid during 48 h of culture occurred, even in the presence of relative high amounts of nitrogen in the growth medium (Fakas et al. 2008c). From around 15% when using 0.4 g/L NaNO . On the fourth day this explanation, we can conclude that, when C. bainieri of cultivation, the percent lipid accumulation was around 2A1 is cultured in N-excess media, glucose uptake was 10% when using 1.2 g/L NaNO and around 20% when high, so lipid accumulation occurred, even in the presence using 0.4 g/L. NaNO levels and NAD ICDH activity of nitrogen. increased concomitantly with NaNO concentration. This There have been insufficient previous studies of N- study thus indicated that lipid accumulation in C. echinulata excess media to explain the phenomena observed in the is indirectly correlated with nitrogen concentration. present study since all previous studies have used N-limited In contrast, in C. echinulata cultivated on hydrolyzed media. The hypotheses for N-limited media were that tomato waste (Fakas et al. 2007), the lipid content increased oleaginous species could accumulate more than 10% lipids to up to 25% of the biomass during the growth and and that they would then degrade AMP in order to release lipogenic phases. Although approximately 0.4 g/L of additional nitrogen from ammonium. This would result in organic nitrogen were measured in the growth environment, an increased energy charge ratio inside the cell, resulting in lipogenesis still commenced, indicating that this nitrogen inhibition of ICDH and intra-cellular citric acid accumula- could not be assimilated. Acid treatment for hydrolyzed tion. Acetyl-CoA would then be produced by citric acid tomato waste (TWH) oil yield reached around 40% at the cleavage via the ATP:citrate lyase reaction (Holdsworth and highest nitrogen concentration utilized (Fakas et al. 2008b), Ratledge 1988; Holdsworth et al. 1988). However, in the the main trend observation being that increasing nitrogen present study, the microorganisms accumulated lipids to concentration increased biomass production but decreased around 15% despite the presence of excess nitrogen (0.5– oil accumulation, except for growth on TWH. Our 1 g/L). Thus, further studies are required to explain lipid observation agreed with (Fakas et al. (2008b) in the case production in N-excess media. Table 3 Fatty acid composition C:N Culture time (h) Fatty acid concentration (wt/wt) in total mycelium lipids of total lipid produced by C. bainieri 2A1 in N-limited media Δ9 Δ9,12 Δ6,9,12 C16:0 C18:0 C18:1 C18:2 C18:3 C24:0 42 24 18.8 10.0 40.2 14.6 8.6 2.5 48 19.3 10.5 40.11 13.3 9.2 3.1 72 17.8 10.4 39.6 15.0 9.6 3.2 96 16.6 8.4 41.11 15.4 11.0 3.1 120 16.3 8.3 40.4 16.1 11.2 3.2 69 24 18 27.1 31.2 9.4 4.5 4.3 48 19.3 23.6 33.3 9.7 5 4.1 72 18.1 23.6 33.8 9.7 5.5 4.3 69 17.4 22.4 34.3 9.9 6.4 4.5 120 18.9 18.7 36.4 10.4 6.5 4.0 97 24 19.2 24.6 33.4 8.8 5.1 3.7 48 18.3 18.9 37.6 8.8 6.0 5.3 72 19.2 16.2 38.6 10.9 6.9 3.4 96 18.6 16.4 39.0 10.2 6.7 4.1 Culture conditions: initial pH 6, temperature 30°C and C:N ratios 120 18.4 15.2 39.6 10.2 7.0 4.3 42, 69 and 97 Ann Microbiol (2010) 60:615–622 621 Table 4 Fatty acid composition of total lipid produced by C. bainieri 2A1 in N-excess media Culture Fatty acid concentration (wt/wt) in total mycelium lipid time (h) Δ9 Δ9,12 Δ6,9,12 C16:0 C18:0 C18:1 C18:2 C18:3 C24:0 24 15.5 16.0 41.3 9.2 6.4 4.1 48 15.4 18.4 37.8 11.3 6.5 4.9 72 15.9 15.5 37.4 12.6 6.3 6.4 96 15.8 14.9 37.1 15.6 6.9 4.9 120 14.7 12.6 37.1 15.2 7.8 6.8 Fig. 4 GLA (g/L) in the reserve lipid versus lipid-free materials determine (g/L) in N-limited media (■) and N-excess media (♦) Culture conditions: initial pH 6, temperature 30°C, glucose, 30 g/L, and thrice daily feeding with ammonium tartrate (1 g/L) Conclusion Fatty acid composition of total lipid produced by C. bainieri 2A1 Lipid biosynthesis in C. bainieri 2A1 occurred in N- limited media at C:N ratios of 42, 69, and 93, and activity In C. bainieri 2A1 cultivated on N-limited media at C:N 42, of NAD - ICDH at C:N 42 appeared in high activity Δ9 the principal cellular fatty acid was oleic acid ( C18), the during the period of growth; this activity may be due to concentration of which remained constant during growth. In the build-up of lipid-free material which is produced until contrast, the concentrations of palmitic (C16:0) and stearic the stationary phase,. The lipid percentage was around (C18:0) acids slightly decreased and that of linolenic 35% and GLA was around 11%. However, there were no Δ9,12 ( C18:3) acid slightly increased (Table 3). In the first significant differences among the three types of C:N ratio phase of growth, γ-linolenic acid GLA content was elevated in total reserve lipids, whereas there were significant during lipid accumulation. Our results demonstrate the effect differences in GLA content. Statistical analysis of the lipid of the C:N ratio on fatty acid concentration. γ-linolenic acid profile demonstrated a significant difference between the GLA content reached 11.2% during lipid accumulation when first day of culture and the third day, whereas there were using C:N 42, but only reached 6.5% and 7.0% when using no significant differences from the second to the fifth C:N ratios of 69 and 97, respectively (Table 3). These findings days. In N-excess media, lipid biosynthesis still occurred indicate that the C:N ratio increases have effects contrary to even when N was still detected in the media at levels of those of GLA concentration. The fatty acid composition of N- 0.2–0.8, 0.5–1, and 0.8–1 g/L, where the lipid percentages excess media differed from that of N-limited media; palmitic were 21, 15, and 11, respectively. NAD -ICDHactivity acid (C16:0) remained almost constant during growth, and the was strongly detected on all the 5 days of culture. γ-linolenic acid GLA content remained constant (Table 4 ). A Microbial growth and production of reserve lipids were negative relationship was observed between ammonium not negatively affected by N-excess media and this may be tartrate level and γ-linolenic acid concentration (Table 5). due to the high uptake of glucose. Fatty acid composition A correlation between the quantity of GLA produced and was thus affected by the use of N-excess and N-limited the amount of lipid-free material was established in both media media, by different C:N ratios, and by different levels of types. Y wasdeterminedto be 0.22inN-limited media nitrogen. This result proved that the strain C. bainieri 2A1 GLA/Xf and 0.1 in N-excess media by linear regression analysis of has an alternative behavior in lipid biosynthesis that GLA produced and units of lipid free biomass (Fig. 4). differs from yeast. Table 5 Fatty acid composition Ammonium tartrate Culture time Fatty acid concentration (wt/wt) in total mycelium lipid of total lipid produced by C. (g/L) (h) bainieri 2A1 in N-excess media Δ9 Δ9,12 Δ6,9,12 C16:0 C18:0 C18:1 C18:2 C18:3 C24:0 0.2 -0.8 24 22.2 14.4 38.1 10.4 6.4 3 48 20.2 17.3 36.4 9.7 6.4 4 0.5-1 24 22.3 15.6 34.9 10.4 5.5 3.4 48 20.7 18.5 35 9.2 5.2 5.2 Culture conditions: initial pH 6, 0.8-1 24 20.6 20.9 31.5 11.3 4.5 5.3 temperature 30°C, glucose 30 g/L, and different levels of ammonium 48 20.3 19.5 33.6 11.4 4.7 5.0 tartrate 622 Ann Microbiol (2010) 60:615–622 Holdsworth JE, Veenhuis M, Ratledge C (1988) Enzyme activates in References oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids. J Gen Microbiol 134:2907–2915 Botham AP, Ratledge C (1979) A biochemical explanation for lipid Igamberdiev UA, Gardestrom P (2003) Regulation of NAD and NAD- accumulation in candida 107 and other oleaginous micro dependent isocitrate dehydrogenases by reduction levels of organism. J Gen Microbiol 114:361–375 pyridine nucleotides in mitochondria and cytosol of pea leaves. Bradford MM (1976) Arapid and sensitive method for the quantitation Biochim Biophys Acta: Bioenergetics 1606:117–125 of microgram quantities of protein utilizing the principle of Kendrich A, Ratiedge C (1992) Desaturation of polyunsaturated fatty protein dye-binding. Anal Biochem 72:248–254 acid in mucorcircinelloides and the inevolvement of a novel Certik M, Sajbidor J, Stredanska S (1993) Effect of carbon and membrane-bound malic enzyme. Eur J Microbiol 209:667–673 nitrogen sources on growth, lipid production and fatty acid Kornberg A (1955) Isocitrate dehydrogenase of yeast. Meth Enzymol composition of Mucormucedo F1384. Microbiol 74:7–15 1:705–7010 Certik M, Megova J, Horenitzky R (1999) Effect of nitrogen source Makri A, Fakas S, Aggelis G (2010) Metabolic activities of biotechno- on the activities of lipogenic enzymes in oleaginous fungus logical interest in Yarrowia lipolytica grown on glycerol in repated Cunninghamella echinulata. J Gen Appl Microbiol 45:289–293 bach cultures. Bioresour Technol 101:2351–2358 Chaney AL, Marbach EP (1962) Modified reagents for determination Moreton RS (1988) Physiology of lipid accumulation yeasts. In: of urea and ammonium. Clin Chem 8:130–132 Moreton RS (ed) Single cell oil. Longman, Essex, UK, pp 1–32 Chen H-C, Chang C-C (1996) Production of gamma-linoleic acid by Papanikolaou S, Aggelis G (2003) Modeling lipid accumulation and the fungus Cunninghamella echinulata CCRC 31840. Biotechnol degradation in Yarrowia lipolytic cultivated on industrial fats. Prog 12:338–341 Curr Microbiol 46:398–402 Evans TC, Ratledge C (1984) Effect of nitrogen source on lipid Papanikolaou S, Komaitis M, Aggelis G (2004a) Single cell oil (SCO) accumulation in oleaginous yeasts. J Gen Microbiol 130:1693–1704 production by Mortierella isabellina grown on high-sugar Evans TC, Ratledge C (1985) The role of mitochondrial NAD :isocitrate content media. Bioresour Technol 95:287–291 dehydrogenase in lipid accumulation by the oleaginous yeast Papanikolaou S, Sarantou S, Komaitis M, Aggelis G (2004b) Repression Rhodosporidium toruloides CBS14. Can J Microbiol 31:845–850 of reserve lipid turnover in Cunninghamella echinulata and Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, Mortierella isabellina cultivated in multiple-limited media. J Appl Aggelis G (2006) Lipid of Cunninghamella echinulata with Microbiol 97:867–875 emphasis of γ-linolenic acid distribution among lipid classes. Ratledge C (1982) Microbial oils and fats: an assessment of their Appl Microbiol Biotechnol 73:676–683 commercial potential. In: Ball JM (ed)Progress in industrial Fakas S, Galiotoupanayotou M, Papanikolaou S, Komaitis M, Aggelis microbiology 16. Elsevier, Oxford, pp 119–206 G (2007) Compositional shifts in lipid fraction during lipid Ratledge C (1994) Yeasts, moulds, algae and bacteria as sources of lipid. turnover in Cunninghamella echinulata. Enzyme Microb Technol In: Kamel BS, Kakuda Y (eds) Technological advances in improved 1321–1327 and alternative sources of lipids. Blackie, London, pp 235–291 Fakas S, Papapostolou I, Papanikolaous S, Georgiou DC, Aggelis G Ratledge C (2002) Regulation of lipid accumulation in oleaginous (2008a) Susceptibilit to peroxidation of the major mycelia lipids microorganisms. Biochem Soc Trans 30:1047–1050 of Cunninghamella echinulata. Eur J Lipid Sci Technol Ratledge C (2004) Fatty acid biosynthesis in microorganism being 110:1062–1067 used for single cell oil production. Biochimie 86:807–815 Fakas S, Certik M, Papanikolaou S, Aggelis G, Komaitis M, Galiotou- Ratledge C, Boulton AC (1985) Fat and oil. In: Murray Moo–Young (ed) Panayotou M (2008b) γ-Linolenic acid production by Cunning- Comprehensive biotechnology. Peramon, New York, pp 983–1003 hamella echinulata growing on complex organic nitrogen sources. Szczesha-Antczak M, Antczak T, Piotrowicz-Wasiak M, Rzyska M, Bioresour Technol 99:5986–5990 Binkowska N, Bielecki S (2006) Relationships between lipases Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, and lipids mycelia of two mucor strains. Enzyme Microb Technol Aggelis G (2008c) Organic nitrogen of tomato waste hydrolysate 39:1214–1222 enhances glucose uptake and lipid accumulation in Cunning- Tao J-YL, Zhang X-Y (2007) Growth low of gamma-inoleic acid hamella echinulata. J Appl Microbiol 105:1364–5072 production by Cunninghamella echinulata. J US-China Med Sci Fakas S, Makri A, Mavromati M, Tselepi M, Aggelis G (2009) Fatty 1:55–60 acid composition in lipid fraction lengthwise the mycelium of Vani S, Sharma CD, Bhagat SD, Saini VS, Adhikari DK (1988) Lipid Mortierella isabellina and lipid production by solid state and fatty acid biosynthesis by Rhodotorula minuta. JAOCS fermentation. Bioresour Technol 100:6118–6120 75:501–505 Folch J, Lees M, Sloane–Stanley GH (1957) Simple method for the Wynn PJ (1998) Microorganisms as sources of nutritionally important isolation and purification of total lipids from animal tissues. J polyunsaturated fatty acid. Forum Appl Biotechnol 1:1215–1222 Biol Chem 226:497–509 Wynn PJ, Hamid AA, Li Y, Ratledge C (2001) Biochemical events Gema H, Kavadia A, Dimou D, Tsagou V, Komaitis M, Aggelis G leading to the diversion of carbon into storage lipids in the (2002) Production of γ- linolenic acid by Cunninghamella oleaginous fungi Mucor circinelloides and Mortierella alpine. echinulata cultivated on glucose and orange peel. Appl Microbiol Microbiology 147:2857–2864 Biotechnol 58:303–307 Yong–Hong Li, BO Liu, ZHAO Zong–Bao, AB Feng Wa (2006) Holdsworth EJ, Ratledge C (1988) Lipid turnover in oleaginous Optimization of culture condition for lipid production by yeasts. J Gen Microbiol 134:339–346 Rhodosporidium toruloides. Chin J Biotechnol 22:650–656

Journal

Annals of MicrobiologySpringer Journals

Published: Jul 28, 2010

There are no references for this article.