Journal of Applied Microbiology 2002, 92, 737±744
Yarrowia lipolytica as a potential producer of citric acid from raw glycerol S. Papanikolaou1*, L. Muniglia1, I. Chevalot1, G. Aggelis2 and I. Marc1 1
Laboratoire des Sciences du GeÂnie Chimique, Vandúuvre-leÁs-Nancy France, and 2Laboratory of General and Agricultural Microbiology, Agricultural University of Athens, Greece
374/4/00: received 30 April 2000, revised 4 October 2001 and accepted 4 November 2001
S . P A P A N I K O L A O U , L . M U N I G L I A , I . C H E V A L O T , G . A G G E L I S A N D I . M A R C . 2002.
Aims: To study the biochemical response of Yarrowia lipolytica LGAM S(7)1 during growth on raw glycerol (the main by-product of bio-diesel production units) in order to produce metabolic products of industrial signi®cance. Methods and Results: Yarrowia lipolytica was cultivated on raw glycerol or glucose in ¯asks. Although nitrogen-limited media were employed, growth was not followed by production of reserve lipid. Nitrogen limitation led to citric acid excretion. Growth and citric acid production parameters on glycerol were similar to those obtained on glucose. When high initial glycerol media were used, citric acid up to 35 g l)1 (yield 0á42±0á44 g acid g)1 glycerol consumed) was produced. Conclusions: Raw glycerol was an adequate substrate for Y. lipolytica. Growth was not followed by reserve lipid accumulation, but amounts of citric acid were produced. Signi®cance and Impact of the Study: Raw glycerol is an industrial feedstock appearing in increasing quantities as the main by-product of bio-diesel production facilities. The present study describes an alternative way of glycerol valorization, with the production of remarkable amounts of citric acid, in addition to its main valorization way (production of 1,3-propanediol by bacteria). INTRODUCTION Yarrowia lipolytica, as well as the fungus Aspergillus niger, are considered as potential producers of citric acid (Hamissa and Radwan 1977; Aiba and Matsuoka 1979; Klasson et al. 1989; Wojtatowicz et al. 1991; Roukas and Kotzekidou 1997). The growth of these micro-organisms on sugars is followed by a signi®cant citric acid accumulation in the culture medium. Only a few Y. lipolytica strains have been reported to accumulate signi®cant amounts of storage lipid, growing on osidic nitrogen-limited media (Ratledge 1994). However, Y. lipolytica strain LGAM S(7)1 grown on an industrial derivative of animal fat presented high cellular
Correspondence to: Ivan Marc, Laboratoire des Sciences du GeÂnie Chimique, 13, rue du Bois de la Champelle, 54500, Vandúuvre-leÁs-Nancy, France (e-mail:
[email protected]). *Present address: Laboratory of General and Agricultural Microbiology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos, 11855, Athens, Greece. ã 2002 The Society for Applied Microbiology
lipid accumulation, while negligible amounts of organic acids were produced (Papanikolaou 1998). Numerous carbon sources, such as molasses, n-paraf®ns, hexadecanes, edible oils, glucose, starch hydrolysates etc., have been used to produce citric acid from Y. lipolytica (Wojtatowicz et al. 1991; Rane and Sims 1993; Barth and Gaillardin 1997). However, few investigations have been carried out with glycerol as the sole substrate (Barth and Gaillardin 1997) although glycerol has been widely used as a carbon source in the production of 1,3-propanediol (Biebl et al. 1992; Menzel et al. 1997). Whatever the fermentation mentioned, pure glycerol was used as the carbon substrate, as the salts presented in the raw glycerol exert important inhibitory effects on many micro-organisms. As far as is known, few strains of Clostridium butyricum have been reported to grow on raw glycerol (Petitdemange et al. 1995). Raw glycerol can become an important feedstock when bio-diesel is applied on a large commercial scale. With the production of 10 kg of bio-diesel from rapeseed oil, 1 kg of glycerol becomes available (Meesters et al. 1996).
738 S . P A P A N I K O L A O U ET AL.
The aim of the present investigation was to study the biochemical response of Y. lipolytica LGAM S(7)1 on raw glycerol in high C/N media. Growth parameters were compared with those obtained from glucose as glucose, broadly employed in the literature, can be considered as a reference substrate. M A T E R I A LS A N D M E T H O D S Micro-organism and culture conditions Yarrowia lipolytica strain LGAM S(7)1 came from the culture collection of the Laboratory of General and Agricultural Microbiology (Agricultural University of Athens). The strain had been kept on potato dextrose agar at 4°C. The culture medium used contained (g l)1): KH2PO4 7, Na2HPO4 2á5, MgSO4.7H2O 1á5, CaCl2 0á15, FeCl3.6H2O 0á15, ZnSO4.7H2O 0á02, MnSO4.H2O 0á06. Buffered media contained KH2PO4 and Na2HPO4 each at 12 g l)1. In some experiments, a volume (500±600 ll) of KOH at 5 mol l)1 was periodically added in order to maintain a medium pH value greater than 5. Ammonium sulphate and yeast extract (Fluka, nitrogen content: 7% (w/w)), 0á5 g l)1 of each, were used as nitrogen sources. The carbon sources used were glucose monohydrate (Fluka), pure glycerol (Prolabo) and technical raw glycerol (French Agro-Chemical Industry Novance, CompieÁgne, purity 60%). Culture conditions and measurement of cell growth The experiments were performed in 250 ml Erlenmeyer ¯asks containing 50 ml growth medium inoculated with 108 cells and incubated at 28°C in a rotary shaker. The agitation rate was 185 rev min)1. Cells were harvested by centrifugation, washed once with distilled water and dried at 80°C until constant weight was reached. Determination of glucose, glycerol, organic acids and nitrogen Filtered aliquots of the culture medium were analysed by a Waters Association HPLC (Column Polypore H 25 cm ´ 7 mm Brownlee, Perkin Elmer, Les Ulis, France). The eluant was H2SO4 0á02 mol l)1. The organic acids were detected by a u.v. detector (kmax481; Waters, Saint-Quentinen-Yvelines, France), whereas glycerol and glucose were detected by an R.I. detector (differential refractometer 410Waters). In order to proceed with a more judicious determination of iso-citric acid, an enzymatic method was used. This was based on the measurement of the NADPH produced during conversion of iso-citric to a-ketoglutaric acid, a
reaction catalysed by isocitrate dehydrogenase. Nitrogen in the culture medium was measured by the Kjeldahl method. Cellular lipid extraction and analysis Cellular lipids were extracted according to Folch et al. (1957), as modi®ed by Aggelis et al. (1995). In order to determine the lipid fractions, thin layer chromatography (TLC) was carried out on 60G silica gel plates (Merck) using petroleum ether± diethyl ether±acetic acid (70:30:1 v/v/v) as the solvent system. Lipid fractions were visualized by iodine vapour. Cellular lipids were esteri®ed according to the AFNOR method (AFNOR 1984) and analysed in a Delsi DI200 gas chromatograph (Quad Service, Poissy, France) equipped with a silica capillary column Carbowax (30 m ´ 0á32 mm, ®lm thickness 0á25 lm; Alltech, Templemars, France) and a Flame Ionization Detector (F.I.D.). Conditions of analysis were as follows: oven temperature 120±240°C with a 10°C min)1 gradient; injector temperature 250°C; detector temperature 300°C; carrier gas helium; ¯ow 1á5 ml min)1. Identi®cation of methyl-esters was based on the comparison of retention times with known standards. Abbreviations and units X ± biomass, g l)1; Glc ± glucose, g l)1; Glol ± glycerol, g l)1; Cit ± citrate, g l)1; N ± total extracellular nitrogen, g l)1; YX/ Glc (YX/Glol) ± biomass yield, grams of biomass against grams of glucose (glycerol) consumed; YCit/Glc (YCit/Glol) ± citric acid yield, grams of citric acid produced against grams of glucose (glycerol) consumed; l ± speci®c growth rate h)1; pCit ± speci®c citric acid production rate evaluated in the stationary growth phase, grams of citric acid per gram of biomass corresponding per h; mGlc (mGlol) ± speci®c rate of glucose (glycerol) consumption, grams of glucose (glycerol) per gram of biomass per hour. Indices max, 0 and f correspond to the maximum, initial and ®nal quantity of the elements, respectively. RESULTS Growth on glucose Growth on glucose can be considered as a basis for comparison as this substrate has been broadly used in the literature. Growth of Y. lipolytica on this substrate was accompanied by the production of organic acids, resulting in a decrease in the pH of the culture medium (Table 1). Cellular lipid accumulation did not occur. Only 5±9% w/w of lipid was accumulated in dry cellular mass. Citrate was the principal organic acid produced. Its maximum concentration was signi®cantly in¯uenced by the pH value. Therefore, buffered culture media resulted in increased citric acid concentrations and in higher carbon
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
CITRIC ACID BY YARROWIA LIPOLYTICA
739
Table 1 Growth and citric acid production by Yarrowia lipolytica on glucose
U.B.M.* U.B.M. B.M.à B.M.§
Glc consumed (g l)1)
C/N molar
X (g l)1)
YX/Glc (g g)1)
pH0
pHf
Citmax (g l)1)
Carbon recovery (% w/w)
30 30 28 29
97 97 97 115
5á8 6á5 5á2 6á1
0á19 0á22 0á19 0á21
6á0 5á0 6á5 6á5
3á1 2á7 4á3 5á4
5á9 < 0á5 10á9 9á7
47 37 66 61
All calculations were made at maximum citric acid concentration. U.B.M. unbuffered medium; B.M. buffered medium. *Incubation time 69 h, initial glucose 30 g l)1. Incubation time 110 h, initial glucose 30 g l)1. à Incubation time 110 h, initial glucose 28 g l)1. § Incubation time 118 h, initial glucose 34á5 g l)1.
recoveries (biomass structure was considered as C5H7NO2). In contrast, maximum biomass value and yield YX/Glc were not affected by the pH value. The production of iso-citrate, acetate and a-ketoglutarate was low (< 20% w/w of the amount of citric acid) and unaffected by the pH value. The kinetics of pH evolution and citric acid production are presented in Fig. 1. A rapid decrease in the pH value resulted in a lower maximum citric acid concentration. However, citric acid productivity was relatively higher in unbuffered medium than in the buffered medium (0á4 against 0á25 g l)1 h)1, respectively). The kinetics of one characteristic case (buffered medium) is presented in Fig. 2. The maximum speci®c growth rate lmax was 0á25 h)1 and the overall yield YX/Glc 0á19 g g)1. Relatively high quantities of citric acid were produced 7
12
6
(maximum concentration 11 g l)1, YCit/Glc 0á39 g g)1). The speci®c consumption rate of glucose (mGlc) was 0á08 g g)1 h)1, while the speci®c citrate production rate (pCit) was 0á04 g g)1 h)1. Two well-de®ned phases were observed: the growth phase, in which the substrate was mainly used for biomass production, and the stationary phase, in which glucose was principally transformed into citric acid (yield YCit/Glc about 0á80 g g)1). After the sugar has been exhausted, some consumption of the citric acid produced was observed. Growth on raw glycerol High cell growth was observed on raw glycerol, followed by a signi®cant acidi®cation of the culture medium (Table 2). Cellular lipid was found to be 5±9% w/w in dry matter. Citrate was the principal organic acid produced. In buffered media, increased concentrations of citric acid and higher carbon recoveries were obtained. Maximum biomass
10 40
16
35
14
30
12
25
10
20
8
15
6
10
4
5
2
pH
6 3 4 2 1
2
0
0 0
20
40
60 80 Time (h)
100
120
140
0
Fig. 1 Growth of Yarrowia lipolytica on glucose. Change of pH (m, n) and citric acid (n, h) in non-buffered media (open symbols) and in buffered media (®lled symbols). Culture conditions for buffered medium: initial glucose concentration 28 g l±1, molar ratio C/N 97. For unbuffered medium: initial glucose concentration 30 g l±1, molar ratio C/N 97
–1 X, Cit (g L )
4
Glc (g L–1)
8
–1 Citric acid (g L )
5
0 0
20
40
60
80
100
120
140
Time (h)
Fig. 2 Biomass (s), glucose (n) and citric acid (d) changes of Yarrowia lipolytica growing on glucose (buffered medium). Culture conditions: initial glucose concentration 28 g l±1, molar ratio C/N 97
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
740 S . P A P A N I K O L A O U ET AL.
Table 2 Growth and citric acid production by Yarrowia lipolytica on raw glycerol
U.B.M.* U.B.M. B.M.à B.M.§
Glol consumed (g l)1)
C/N molar
X (g l)1)
YX/Glc (g g)1)
pH0
pHf
Citmax (g l)1)
Carbon recovery (% w/w)
30 29 32 39
98 98 98 147
6á5 6á0 7á7 7á8
0á22 0á21 0á24 0á20
6á0 5á0 6á5 6á5
3á5 2á4 4á5 4á5
1á2 < 0á5 11á9 13á7
46 42 73 64
All calculations were made at maximum citric acid concentration. U.B.M. unbuffered medium; B.M. buffered medium. *Incubation time 88 h, initial glucose 30 g l)1. Incubation time 88 h, initial glucose 30 g l)1. à Incubation time 106 h, initial glucose 32 g l)1. § Incubation time 118 h, initial glucose 46 g l)1.
12
7 6
biomass formation with a YX/Glol of 0á48 g g)1. Finally, after total exhaustion of glycerol from the culture medium, some consumption of the accumulated citrate was observed. A similar experiment using pure glycerol (purity 98%) as the sole carbon source was also conducted, giving equivalent kinetic parameters (lmax 0á2 h)1, YX/Glol 0á23 g g)1, maximum citric acid concentration 11 g l)1, YCit/Glol 0á36 g g)1 and pCit 0á04 g g)1 h)1). Growth on mixtures of raw glycerol and glucose Glucose and raw glycerol were used simultaneously as carbon sources in order to compare their uptake rates and microbial speci®city. In all cases, higher assimilation rates of glycerol were observed compared with those of glucose. Glycerol was exhausted from the culture medium whereas signi®cant quantities of glucose remained unconsumed,
10
pH
4 6 3 4 2
Glol (g L–1)
8
–1 Citric acid (g L )
5
40
16
35
14
30
12
25
10
20
8
15
6
10
4
5
2
–1 X, Cit (g L )
value and yield YX/Glol were not affected by the pH value, whereas the production of other organic acids was low. Curves for the evolution of pH and citric acid concentration in the culture medium are presented in Fig. 3. As in the case of glucose, a rapid decrease in the medium pH caused a lower maximum citric acid concentration. Characteristic kinetics (in the case of buffered medium) are presented in Fig. 4. The speci®c growth rate, estimated in the early exponential growth phase, was 0á2 h)1 and the yield YX/Glol was 0á24 g g)1. Relatively high amounts of citric acid were produced (12 g l)1, yield YCit/Glol 0á38 g g)1 and pCit 0á04 g g)1 h)1. Glycerol was completely consumed (mGlol 0á05 g g)1 h)1). Citric acid was produced principally during the stationary growth phase (YCit/Glol 0á5 g g)1), while in the ®rst fermentation step, glycerol was used for
2
1
0
0 0
20
40
60
80
100
120
140 0
Time (h)
0 0
Fig. 3 Growth of Yarrowia lipolytica on raw glycerol. Change of pH (m, n) and citric acid (n, h) in unbuffered media (open symbols) and in buffered media (®lled symbols). Culture conditions for buffered medium: initial glycerol concentration 31 g l±1, molar ratio C/N 98. For unbuffered medium: initial glycerol concentration 30 g l±1, molar ratio C/N 98
20
40
60
80
100
120
140
T ime (h)
Fig. 4 Biomass (s), glycerol (n) and citric acid (d) changes of Yarrowia lipolytica growing on raw glycerol (buffered medium). Culture conditions: initial glycerol concentration 31 g l±1, molar ratio C/N 98
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
CITRIC ACID BY YARROWIA LIPOLYTICA
741
Table 3 Growth and citric acid production by Yarrowia lipolytica on mixtures of raw glycerol and glucose in buffered media Glc consumed (g l)1)
Glol consumed (g l)1)
C/N molar
X (g l)1)
pH0
pHf
Citmax (g l)1)
Carbon recovery (% w/w)
6* 11 10à 4§
19 15 21 32
90 120 120 160
6á1 6á4 8á0 6á0
6á5 6á5 6á5 6á5
5á1 4á5 5á1 4á3
9á1 9á8 10á1 14á9
75 76 73 68
All calculations were made at maximum citric acid concentration. *Incubation time 98 h, initial glucose 9 g l)1, initial glycerol 19 g l)1. Incubation time 115 h, initial glucose 21 g l)1, initial glycerol 15 g l)1. à Incubation time 135 h, initial glucose 16 g l)1, initial glycerol 21 g l)1. § Incubation time 150 h, initial glucose 16 g l)1, initial glycerol 32 g l)1.
regardless of the initial amounts of each substrate used (Table 3). Relatively high quantities of citric acid (10±15 g l)1) were produced. One of the cases of glycerol/glucose co-utilization, showing the speci®city of the micro-organism on these substrates, is presented in Fig. 5. Signi®cant cell growth took place and total consumption of glycerol was realised. Glucose uptake was clearly lower than that of glycerol. Citric acid production from raw glycerol Industrial glycerol seems to be a suitable substrate for citric acid production from Y. lipolytica. Growth and the parameters of citric acid production have been found to be similar to those obtained from glucose, whereas glycerol uptake was higher than that of glucose. 25
8
Citrate excretion and extracellular nitrogen
7 20 6 5
15
4 10
3
–1 X (g L )
Glc, Glol (g L–1)
In order to increase citric acid production, growth of Y. lipolytica was carried out in media with high concentrations of industrial glycerol (80 and 120 g l)1) and increased initial C/N molar ratios. Buffered media were employed, and 500±600 ll 5 mol l)1 KOH were periodically added to the ¯ask in order to maintain a pH value of between 5 and 6. Yarrowia lipolytica proceeded to grow with high glycerol assimilation and increased citric acid production (33±35 gl)1), while YCit/Glol was slightly higher (0á42±0á44 g g)1) than that obtained from the previous experiments (Table 4). Other organic acids, produced in lower quantities, were iso-citric, acetic and a-ketoglutaric acids. Citric acid production occurred in the stationary growth phase (®nal concentration 34 g l)1, YCit/Glol 0á51 g g)1, pCit 0á04 g g)1 h)1, productivity 0á15 g l)1 h)1 (Fig. 6). During the ®rst growth steps, glycerol was mainly used for biomass formation with YX/Glol 0á45 g g)1.
2 5 1 0
0 0
20
40 60 Time (h)
80
100
Fig. 5 Biomass (s), glycerol (n) and glucose (n) changes of Yarrowia lipolytica growing on a mixture of glucose and raw glycerol (buffered medium). Culture conditions: initial glycerol concentration 18 g l±1, initial glucose concentration 9 g l±1, molar ratio C/N 90
In the ®rst growth phase, growth of Y. lipolytica proceeded with rapid nitrogen assimilation. The stationary growth phase, followed by citrate biosynthesis, began when the concentration of the nitrogen reached a critical value of 0á02 g l)1 (Fig. 7). Morphology and cellular lipid of Y. lipolytica Yeast morphology, and cellular lipid quantity and composition, were studied during all of the experimental runs. In all cases, Y. lipolytica developed its yeast form, whereas the presence of true mycelium was negligible. Lipids in dry cellular mass were, in all cases, between 5 and 9% (w/w). They were mainly composed of polar fractions and sterols. Although high molar C/N ratios were employed in the culture medium, triglycerides were not detected. In experiments with glucose as the substrate, microbial growth was accompanied by a change in the cellular fatty acid composition as a function of the fermentation time (Table 5).
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
742 S . P A P A N I K O L A O U ET AL.
Table 4 Citric acid production by Yarrowia lipolytica growing on raw glycerol Glol consumed (g l)1)
C/N molar
X (g l)1)
YX/Glol (g g)1)
Citmax (g l)1)
YCit/Glol (g g)1)
77* 83
261 388
6á8 5á8
0á09 0á07
33á6 35á1
0á44 0á42
Fermentation stopped 236 h after inoculation. All calculations were made at maximum citric acid concentration. *Initial glycerol 80 g l)1. Initial glycerol 120 g l)1. Table 5 Cellular fatty acid composition (% w/w) of Yarrowia lipolytica growing on glucose (mean of three experiments)
40
100
35 80
25
60
20 40
15
–1 X, Cit (g L )
Glol (g L–1)
30
10
Growth phase
C16 : 0
C18 : 0
C18 : 1
C18 : 2
Exponential (17 h) Early stationary (25á5 h) Stationary (51 h) Stationary (90 h) Stationary (120 h)
18 13 15 17 10
25 8 12 13 9
23 51 40 42 45
14 19 19 16 17
Culture conditions: buffered medium, glucose 28 g l±1, molar ratio C/ N 97.
20 5
20
In the exponential growth phase, the percentage of stearic acid was 25% w/w, but it decreased during the stationary growth phase to 8±13% w/w. In contrast, the percentage of oleic acid was 23% w/w in the exponential growth phase, increasing signi®cantly at the early stationary phase to 51% w/w. Throughout the stationary phase it was 45%. Cell growth on raw glycerol was not accompanied by signi®cant changes in cellular fatty acid composition. Cellular lipid composition was oleic 45%, linoleic 20%, stearic 10% and palmitic 15% acids. Similar fatty acid composition was observed when cells of Y. lipolytica were grown on mixtures of glucose and raw glycerol used as cosubstrates (data not presented).
15
DISCUSSION
0
0 0
50
100 150 Time (h)
200
250
Fig. 6 Biomass (s), glycerol (n) and citric acid (h) changes of Yarrowia lipolytica growing on raw glycerol (buffered medium). Culture conditions: periodical addition of KOH 5 mol l±1 (pH 5±6), initial glycerol concentration 80 g l±1, molar ratio C/N 261 25
0.20
0.10 10
X, Cit (g L–1)
–1
N (g L )
0.15
0.05 5
0.00
0 0
50
100
150
Time (h)
Fig. 7 Biomass (s), citric acid (d) and total nitrogen (m) in the medium of Yarrowia lipolytica growing on raw glycerol (buffered medium). Culture conditions: periodical addition of KOH 5 mol l±1 (pH 5±6), initial glycerol concentration 80 g l±1, molar ratio C/N 261
Yarrowia lipolytica strain LGAM S(7)1 showed signi®cant cell growth on glucose or raw industrial glycerol. Cell growth was accompanied by relatively high citric acid production, while cellular lipid accumulation was not observed. The biomass yield on glycerol was slightly higher than that obtained on glucose, whereas citric acid production was similar for both substrates used. When the culture medium was buffered, maximum concentrations of citric acid, as well as citrate yields (YCit/Glol, YCit/Glc), were relatively high (10±15 g l)1 and 0á35±0á4 g g)1, respectively). In contrast, unbuffered media presented lower maximum citrate concentrations and decreased carbon recoveries. An increased energy of maintenance requirement, as well as excessive CO2 production during culture in unbuffered media, could explain this ®nding. After the
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
CITRIC ACID BY YARROWIA LIPOLYTICA
exhaustion of sugars from the culture medium, some consumption of the citric acid produced was observed. This is in agreement with Gutierrez et al. (1993) who investigated citric acid production from glucose and galactose by a Candida guillermondii strain. The use of glucose and raw glycerol as co-substrates showed that the amounts of glycerol assimilated by Y. lipolytica were much higher than those of glucose. Since carriers of glucose and glycerol are different, it can be assumed that the enzymatic activity of the catabolic C6 pathway of glycolysis is decreased due to the energy excess in the C3 pathway (glycerol catabolism). This assumption is in agreement with the ®ndings of Biebl and Marten (1995), who reported that a Clostridium butyricum strain simultaneously consumed glucose and glycerol used as co-substrates, with a glucose uptake signi®cantly lower than that of glycerol. Technical raw glycerol therefore seems to be an ef®cient substrate for citric acid production. The use of this substrate in high concentrations (80 and 120 g l)1) resulted in a signi®cant increase in citric acid production (®nal concentration 33±35 g l)1). High citric acid production (overall yields YCit/Glc 0á5±0á8 g g)1 with ®nal concentrations 50±80 g l)1) has already been reported for strains of Candida sp. (mainly Y. lipolytica) growing on glucose (150±200 g l)1) under nitrogen limitation (C/N molar ratios higher than 150) by several investigators in batch, fed-batch and continuous cultures (Briffaud and Engasser 1979a, b; Klasson et al. 1989; Wojtatowicz et al. 1991; Rane and Sims 1993, 1994, 1995). During batch fermentations, citric acid formation occurs during the stationary growth phase when the extracellular nitrogen is the limiting factor for cell growth (Briffaud and Engasser 1979a; Rane and Sims 1993). In general, during the ®rst bioconversion steps, biomass formation is observed (partial yields YX/Glc 0á30±0á47; Briffaud and Engasser 1979a; Klasson et al. 1989; Wojtatowicz et al. 1991). After the nitrogen source has been depleted, Y. lipolytica uses most of the remaining sugar for citrate production, with some further increase in biomass (Briffaud and Engasser 1979a; Wentworth and Cooper 1996). In addition, citric acid biosynthesis is favoured under ef®cient aeration conditions of the culture medium and for this reason, growth has been observed in highly aerated and agitated stirred tank reactors (Briffaud and Engasser 1979a; Klasson et al. 1989; Wojtatowicz et al. 1991; Rane and Sims 1993, 1994, 1995). Yarrowia lipolytica LGAM S(7)1 growing on glucose or raw glycerol develops its yeast form. The presence of true mycelium is negligible. However, growth of Y. lipolytica on industrial fats composed of saturated free fatty acids used as the sole carbon substrate was followed by a signi®cant production of true mycelia (Papanikolaou 1998). In general, the morphology of this fungus depends on a variety of
743
nutritional and environmental conditions (Novotny et al. 1994; Bhushan and Hoondal 1997; Zinjarde et al. 1998). It is well known that the immediate precursor of cellular lipid accumulation in oleaginous micro-organisms is cellular citric acid (Botham and Ratledge 1979; Boulton and Ratledge 1980; Ratledge 1994). In the case of the strain used in the present study, the percentage of lipids in dry matter was only 5±9% (w/w), whereas the cellular lipids were composed mainly of polar fractions and sterols. In spite of the high initial C/N molar ratios used in the culture medium, cell growth was followed by signi®cant citric acid production, without cellular lipid accumulation. It can be assumed, therefore, that the ATP-citrate lyase (key enzyme for lipid biogenesis) is not active under these experimental conditions. However, it was found recently that this strain of Y. lipolytica produced signi®cant quantities of lipid when grown on glucose in continuous culture (Aggelis and Komaitis 1999). Cellular fatty acid composition of the yeast growing on glucose showed a decrease in stearic acid and an increase in oleic acid during the late cell growth steps. In contrast, the amount of stearic acid in the exponential growth phase was noticeable (25% w/w). The change in the cellular lipid pro®le as a function of the fermentation time has already been observed by Aggelis et al. (1996) in oleaginous yeasts growing on glucose. In contrast to the results obtained on glucose, cell growth on raw glycerol, or on mixtures of glucose and glycerol, was not accompanied by signi®cant modi®cations of the cellular fatty acid composition. In all growth phases, the percentages (w/w) of oleic and linoleic acids were high (about 45 and 15%, respectively), whereas those of stearic and palmitic acids were lower. The signi®cant presence of unsaturated fatty acids in cellular lipids of Y. lipolytica, even in the early cell growth phase, could be related to increased biological activity, explaining the high uptake rate of glycerol. It is known that cellular unsaturated fatty acids are required as components of yeast mitochondrial lipids to permit the coupling of electron transport to phosphorylation and active cation transport. In addition, the physical state of the lipid component has been deduced to be a factor in¯uencing the membrane capacity for oxygen uptake. Arrhenius plots of membrane-bound enzymes show that unsaturated fatty acids induced a lowering of the energy of activation, due to presumed conformational changes in the membrane structure in the immediate vicinity of the enzymes (Rattray et al. 1975). In conclusion, raw glycerol appears to be a suitable substrate for citric acid production by Y.a lipolytica LGAM S(7)1. Relatively large amounts of this acid have been produced (33±35 g l)1), although it is plausible to wait for improved productivities with an optimization of the operating conditions (use of bioreactors, growth on a constant medium pH, increase of agitation and aeration rate).
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
744 S . P A P A N I K O L A O U ET AL.
ACKNOWLEDGEMENTS The authors wish to express their appreciation to the State Scholarship Foundation, Athens, Greece, for the fellowship of S.P. and the partial ®nancial support of the project. REFERENCES AFNOR (1984) Recueil Des Normes FrancËaises Des Corps Gras/Grains OleÂagineux et Produits DeÂriveÂs 3rd edn, pp. 60±233. ed. Paris: AFNOR. Aggelis, G. and Komaitis, M. (1999) Enhancement of single-cell oil production by Yarrowia lipolytica growing in the presence of Teuchrium polium L. aqueous extract. Biotechnology Letters 21, 747± 749. Aggelis, G., Komaitis, M., Papanikolaou, S. and Papadopoulos, G. (1995) A mathematical model for the study of lipid accumulation in oleaginous microorganisms. I. Lipid accumulation during growth of Mucor circinelloides CBS 172±27 on a vegetable oil. Gracas y Aceites 46, 169±173. Aggelis, G., Stathas, D., Tavoularis, N. and Komaitis, M. (1996) Composition of lipids produced by some strains of Candida species. Production of single cell oil in chemostat culture. Folia Microbiologica 41, 299±302. Aiba, S. and Matsuoka, A. (1979) Identi®cation of a metabolic model: citrate production from glucose by Candida lipolytica. Biotechnology and Bioengineering 21, 1373±1386. Barth, G. and Gaillardin, C. (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiology Reviews 19, 219±237. Bhushan, B. and Hoondal, G.S. (1997) Short communication: Relationship between lipase production and pseudomycelia formation in a dimorphic Candida sp. World Journal of Microbiology and Biotechnology 13, 129±130. Biebl, H. and Marten, S. (1995) Fermentation of glycerol to 1,3-propanediol: Use of co-substrates. Applied Microbiology and Biotechnology 44, 15±19. Biebl, H., Marten, S., Hippe, H. and Decker, W.D. (1992) Glycerol conversion to 1,3-propanediol by newly isolated Clostridia. Applied Microbiology and Biotechnology 36, 592±597. Botham, P. and Ratledge, C. (1979) A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous microorganisms. Journal of General Microbiology 114, 361±375. Boulton, C. and Ratledge, C. (1980) Regulatory studies on citrate synthase in Candida 107, an oleaginous yeast. Journal of General Microbiology 121, 441±447. Briffaud, J. and Engasser, J.M. (1979a) Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor. Biotechnology and Bioengineering 21, 2083±2092. Briffaud, J. and Engasser, J.M. (1979b) Citric acid production from glucose. II. Growth and excretion kinetics in a trickle-¯ow fermentor. Biotechnology and Bioengineering 21, 2093±2111. Folch, J., Lees, M. and Slane-Stanley, J. (1957) A simple method for the isolation and puri®cation of total lipids from animal tissues. Journal of Biological Chemistry 226, 497±509. Gutierrez, N., McKay, I., French, C., Brooks, J. and Maddox, I. (1993) Repression of galactose utilization by glucose in a citrate-producing
yeast Candida guilliermondii. Journal of Industrial Microbiology 11, 143±146. Hamissa, F.A. and Radwan, A. (1977) Production of citric acid from cane molasses on a semi-pilot scale. Journal of General and Applied Microbiology 23, 325±329. Klasson, T.K., Clausen, E.C. and Gaddy, J.L. (1989) Continuous fermentation for the production of citric acid from glucose. Applied Biochemistry and Biotechnology 21, 491±509. Meesters, P.A.E.P., Huijberts, G.N.M. and Eggink, G. (1996) High cell density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Applied Microbiology and Biotechnology 45, 575±579. Menzel, K., Zeng, A.P. and Deckwer, W.D. (1997) Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. Journal of Biotechnology 56, 135±142. Novotny, C., Dolezalova, L. and Lieblova, J. (1994) Dimorphic growth and lipase production in lipolytic yeasts. Folia Microbiologica 39, 71±73. Papanikolaou, S. (1998) Etude du comportement physiologique d'une souche de Yarrowia lipolytica en croissance sur des co-produits industriels: production orienteÂe des lipides cellulaires. TheÁse de Doctorat I.N.P.L.-C.N.R.S., Nancy, France. Petitdemange, E., DuÈrr, C., Abbad-Andaloussi, S. and Raval, G. (1995) Fermentation of raw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. Journal of Industrial Microbiology 15, 498±502. Rane, K. and Sims, K. (1993) Production of citric acid by Candida lipolytica Y 1095: Effect of glucose concentration on yield and productivity. Enzyme and Microbial Technology 15, 646±651. Rane, K. and Sims, K. (1994) Oxygen uptake and citric acid production by Candida lipolytica Y 1095. Biotechnology and Bioengineering 43, 131±137. Rane, K. and Sims, K. (1995) Citric acid production by Candida lipolytica Y 1095 in cell recycle and fed-batch fermentors. Biotechnology and Bioengineering 46, 325±332. Ratledge, C. (1994) Yeasts, moulds, algae and bacteria as sources of lipids. In Technological Advances in Improved and Alternative Sources of Lipids ed. Kamel, B.S. and Kakuda, Y. pp. 235±291. London: Blackie Academic and Professional. Rattray, J., Schibeci, A. and Kidby, D. (1975) Lipids of yeasts. Bacteriological Reviews 39, 197±231. Roukas, T. and Kotzekidou, P. (1997) Pretreatment of date syrup to increase citric acid production. Enzyme and Microbial Technology 21, 273±276. Wentworth, S. and Cooper, D. (1996) Self-cycling fermentation of a citric acid producing strain of Candida lipolytica. Journal of Fermentation and Bioengineering 81, 400±405. Wojtatowicz, M., Rymowicz, W. and Kautola, H. (1991) Comparison of different strains of the yeast Yarrowia lipolytica for citric acid production from glucose hydrol. Applied Biochemistry and Biotechnology 31, 165±174. Zinjarde, S., Pant, A. and Desphande, M. (1998) Dimorphic transition in Yarrowia lipolytica isolated from oil-polluted sea water. Mycological Research 102, 553±558.
ã 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 737±744
