World Journal
of Microbiology
& Biotechnology
10. 417422
/lAwor---a source of cocoa butter and gamma-linolenic acid M.P. Roux, J.L.F. Kock,* A. Botha, J.C. du Preez, G.V. Wells and P.J. Botes Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:O) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGS) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SW-TAGS and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% l&O in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGS. Key words: Acetic acid, cocoa butter, gamma-linolenic
acid, lipid, Mucor, oil, triacylglycerol.
Although the production of oils and fats by fungi is well established, no fungal process, with the exception of two processes, has ever reached commercial realization. This can be attributed to the high costs involved in biotechnological routes, which cannot compete against the low costs of agricultural seed oil production (Ratledge 1992). According to Ratledge (1992), there are two main markets on which oil from fungi or single-cell oil (SCO) may impact, namely the markets for cocoa butter and gamma-linolenic acid. Cocoa butter is used extensively in the manufacture of chocolate (Beavan et al. 1992). The chemical and physical properties of cocoa butter are responsible for the melting behaviour, texture and mouth-feel of chocolate. These characteristics of cocoa butter are dependent on the fatty acid ratios of approximately 30% stearic acid (18:0), 30% palmitic acid (l&O) and 30% oleic acid (l&l). Consequently, the major symmetrical triacylglycerol (SUS-TAG) is POS-TAG (sn-lpalmitoyl-2-oleoyl-3-stearoyl-glycerol) and this is, to a large extent, responsible for the melting characteristics of cocoa butter. Attempts to select fungi capable of producing cocoa butter equivalents (CBEs), with a high l&O content in their triacyglycerols, have been unsuccessful. Mutation techniques have therefore been applied and the l&O content of the yeast M.P. Roux, J.L.F. Kock, A. Botha, J.C. du Preez and P.J. Botes are with the Department of Microbiology and Biochemisby, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa; fax: 27 51 482 004. G.V. Wells is with Sasol Waxes, P.O. Box 1, Sasolburg, South Africa. *Corresponding author.
0 1994 Rapid Communications
Cyptococcus curvatus (formerly Candida curvata and Apiotrichum curWum) was eventually increased by partial deletion of the A9 desaturase which is responsible for the transformation of 18:0 to l&l (Ykema et al. 1990). Unfortunately, the economics of the process with the mutant C. curvatus is such that it cannot compete with cocoa butter itself which, at present, is sold at the low price of US $3OOO/ton (Ratledge 1992). Another target for commercial SC0 production is an oil containing gamma-linolenic acid (GLA) which has been obtained, for many years, from the seeds of the evening primrose and is now prescribed for the treatment of eczema (Graham 1984; Ratledge 1992). GLA is also the precursor for the physiologically active human hormones, the prostaglandins. At present, the UK, European and Japanese markets amount to approximately 500 to 800 tons/year, with a price ranging from US $30 to US $60/kg (Ratledge 1992). Currently, only Japan is producing GLA commercially, using the fungus Mortierella (Ratledge 1993). Recently, Kock & Botha (1993) reported on a novel biotechnological route which effectively produced the two main targets for commercial SC0 production: namely a GLArich lipid as well as a CBE, from a low-cost substrate (acetic acid) that originates from a petrochemical process. Both GLA and CBE are produced by the fungus Mucor circinelloides f. circinelloides CBS 108.16 when fed acetic acid at sub-toxic levels as sole carbon source. Since this is, to our knowledge, the first report on a species of Mucor capable of producing both
of Oxford Lfd World journal
ofMicrobiology
b Biotechnology, Vol IO, 1994
417
M.P.Roux etal. these
high-value
lipids,
screening
of fungi
GLA-rich
oils
acid
as sole
Materials
the aim
of the genus
and carbon
CBEs
when
of the present
Mucor grown
study
for the ability with
glucose
was
the
to produce or acetic
source.
column temperature and, following a 10 to 240°C at the same were 170 and 25O”C, at 5 ml/mm. Peaks standards.
of 145°C was increased by J”C/min to 225°C min isothermal period, was then increased rate. The inlet and detector temperatures respectively. N, was used as a carrier gas were identified by reference to authentic
AnaZyses of
and Methods
CBE and GLA Production with Glucose as Carbon
source
Microorganisms and Cultivation. The Mucor strains
used in this study (Table 1) were obtained from the Centraal Bureau for Schimmelcultures (CBS) in Delft, The Netherlands, and from the University of the Orange Free State (UOFS), Bloemfontein, South Africa. Each strain was transferred from YM agar slants into l-l conical shake flasks and grown at 3O”C, 160 rev/min, for approximately 5 days. Each flask contained 200 ml medium, pH 5.5, containing (g/B: glucose, 50; (NH,l,SO, 1.7; KHJQ,, 2.0; Mg!SOJI-I~O, 0.5; CaC1,.2H,O, 0.2; yeast extract, 1.0; FeSOJH,O, 0.01; ZnSOJH~O, 0.01; MnSO,.4H,O, 0.001; and CuSO~5H,O, 0.0005. The medium was sterilized by autoclaving at 121°C for 20 mins. Fungal cultures were harvested by filtration through Whatman No. 1 filter paper and washed with 800 ml distilled water. The cells were immediately frozen with liquid N, and then freeze-dried.
Lipid Extraction. Lipids
were extracted from the freeze-dried material as described by Kendrick & Ratledge (1992a). This included extraction with chloroform/methanol (21 v/v) according to Folch et al. (19571, followed by two washes with distilled water and final evaporation of the organic phase under vacuum. Subsequently, the lipid material was dissolved in diethyl ether and transferred to preweighed vials. Prior to determination of lipid weights, samples were dried to constant weight in a vacuum oven over P20s at 55°C.
Fractionation
of Extracted Lipid. Extracted lipid was dissolved in chloroform and applied to a column (140 mm x 20 mm) of silicic acid, activated by heating overnight. Neutral, sphingoand glycolipids (as a combined fraction), as well as polar lipids, were eluted by successive applications of organic solvents as described by Kendrick & Ratledge (1992a). Final solvent removal and storage was as for whole lipid extracts. Each fraction was further separated by TLC to evaluate the fractionation ability of the silicic acid column.
Thin-layer Chromatography. TLC of the neutral, sphingoand glyco-lipid fractions was performed on silica gel thin-layer plates backed with ahnninium (Merck). The polar fraction, containing phospholipids, was separated using chloroform/methanol/ water/acetic acid (65 : 43 : 3 : 1 by vol.). The neutral lipid fraction was separated using petroleum ether fb.p. 60 to 80’C)/diethyl ether/acetic acid (85: 15 : 1, by vol.). Plates were developed onedimensionally by the ascending technique and visualized by exposure to I, vapbur. Further identification was achieved by running authentic standards along with experimental samples.
Triucylglycerols. Triacylglycerol analyses were performed according to the method of Deffense as reported by Christie (1992). HPLC of the symmetrical triacylglycerols was carried out on an ODS column (Spherisorb 5 ODS 2; 240 mm x 4.6 mm; Phasesep, UK), using a mixture of acetone/ acetonitrile (62.5 : 37: 5 v/v) as the mobile phase at a flow-rate of 1 .l ml/min, and with refractive index detection. Peaks were identified by reference to authentic standards.
CBE and GLA Production with Acetic Acid as Carbon Source Inoculum. A spore
suspension of the Mucor strain was washed from a 3-day-old culture on Sabouraud dextrose agar slants with 0.05 M sterile KH,PO, containing 0.1% Tween 80. This suspension, of about lo7 spores/ml, was diluted to give a count of about 10s spores/ml after inoculation into the fermenter.
Cultivation and Lipid Analysis. Each of the five Mucor strains, representing three Mucor species (Table 21, was grown at 30°C in a 12-1 glass air-lift fermenter (Chemap, CF 3000), containing 11 1 medium and fitted with a marine propeller which was operated at 150 rev/min to improve mixing. The dissolved Oz tension was monitored with an Ingold polarographic probe and maintained above 20% by adjusting the rate of air sparging from 2 to 13 l/ min. The medium contained (g/l): citric acid, 0.16; NH&l, 0.20; MgSOJH~O, 0.5; KH,PO, 1.88; CaC1,.2H,O, 0.03; yeast extract, 0.5; glucose, 0.5; Na acetate, 31; and trace elements as before. The fermenter and medium were sterilized in situ, with the exception of the acetate solution which was autoclaved separately and aseptically added to the fermenter. The medium was subsequently adjusted to pH 7 with 10 M NaOH. After inoculation, a 6-h period was allowed for spore germination, after which the culture was adjusted to pH 6 with 5 M H,SO,. The acetic acid concentration in the culture during the 100-h cultivation period was maintained at about 2 g/l by automatic titration with a 50% (v/v) solution of acetic acid, to maintain a constant pH. Lipid extraction and the analytical procedures were as above.
Analyses
of Triacylglycerols. Fractionation of the cocoa butter was effected by addition of acetone to the neutral lipid fraction [seven times the volume, according to the method of Beaven et al. (199211 which was then shaken at 30°C for 2 h, followed by cooling to 20°C for about 2 h. The latter step was accompanied by the seeding of the mixture with a grain of cocoa butter. The liquid upper phase was separated from the precipitate (fraction 1) and left overnight at lO”C, allowing another precipitate (fraction 2) to form. The top layer was recovered and cooled further at 4°C overnight to enhance precipitation (fraction 31. On evaporation of the remaining solvent, the remnant was designated fraction 4.
DSC Annlyses. Differential Fatty Acid Analyses. Lipid
was dissolved in chloroform and methylated by the addition of trimethyl sulphonium hydroxide (TMSH) as described by Butte (1983). The fatty acid methyl esters were analysed by GC, with a flame ionization detector and a Supelcowax 10 capillary column (30 mx0.75mm). The initial
418
World @mad
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& Biotechnology, Vol lo,1994
scanning calorimetry @SC) analyses were performed on a Perkin Elmer DSC-4 instrument fitted with an Intracoler II sub-ambient accessory and controlled by a Perkin Elmer 3700 data station with TADS software. The instrument purge was N, at 2 atm. Standard aluminium sample pans and lids were used and sample sizes were approx. 0.5 mg. Instrument
source of CBE and GLA
Mucor-a calibration was performed using indium obtained from Perkin Elmer. The analytical procedure was according to ASTM D4419, using a preparatory cooling rate of 5”C/min and a scanning heating rate of lO”C/min over a temperature range of -20 to 70°C. Chemic&. All organic chemicals and solvents used were of analytical reagent grade and obtained from major retailers. Silicic acid (100 mesh) was from Aldrich. Neutral and phospholipid standards, as well as the triacylglycerols and fatty acid standards, were from Sigma.
Results and Discussion CBE and GLA Production with Glucoseas Carbon Source The results of the lipid analyses of the 28 strains, representing 22 Mucor species, grown on glucose as carbon source, are summarized in Table 1. Only four strains, M. circinelZoides f. circinelloides UOFS 100, M. recurvus var. indicus, M. rouxii and M. tuberculisporus, accumulated more than 20% (mol/mol) crude oil and can be considered oleaginous under these growth conditions, according to the definition of Ratledge Table
1. Lipid composition
of strains
of various
hfucor
Culture no.
species
Crude
Neutral lipid in biomass (% w/w)
Oil
(% w/w)
M M hf. M. M. M. M. M. M. M. M.
amphibiomm ardhlaengiktus azygosporus bainieri circinelloides f. griseocyanus circine//oides f. circinelloides circinelloides f. janssenii circinelloides f. circinelloides circinelloides f. circinelloides circinelloides f circinelloides flaws
M. fragifis
grown
(1988). These fungi produced crude oil which consisted mainly (> 80%) of neutral lipids. The individual strains differed extensively in their percentage of neutral lipids in the dry biomass, which varied from 0.2% in M. ardhluengiktus to 42% in M. circinelloides f. circinelloides UOFS 100. The percentage of 18 : 0 ranged from 1.3% in M. oblongiellipticus to as high as 27% in M. flaws. This was an interesting finding because attempts to select fungi capable of naturally producing CBEs, and therefore with a high 18:O content in their triacylglycerols, have so far been unsuccessful according to the published literature. Four strains had a particularly high content of SLJS-TAGS, namely M. flavus (65%), M. fuscus (68%), M. oblongisporus (63%) and M. subtilissimus (61%) (Table 1). These fungi, therefore, have potential as a source of CBEs if the content of SOSTAGS (1,3-distearoyl-2-oleoyl-glycerol) could be increased to the content required in CBEs. CBEs contain approximately 20% SOS-TAGS, necessary for the formation of the nucleate centres of the cocoa butter crystals and responsible for the physical characteristics of this lipid (Schlichter-Aronhime & Garti 1988). Although M. fuscus had a higher percentage of total SUS-TAGS than M. flavus, the former did not produce any on glucose GLA (mg/g biomass)
CBS 763.74 CBS 210.8
2.7 0.5
1.4 0.2
0.1 0.1
CBS 292.63 CBS 293.63
16.7
12.1 6.9
12.3 10.6
4.4 9.8
5.8 14.6
11.7 15.5 47.7
8.7 13.5 41.6
2.9 32.3
CBS 116.08 CBS 119.08 CBS 232.29 CBS 106.16 UOFS 100 CBS 203.26
6.9 7.4 14.2
38.3 14.5
17.0
12.8
CBS 126.70 CBS 236.35
11.2 11.4
9.0 8.6
1.4 9.6
M.
fuscus
CBS 132.22
M. M. M. M M. M. M.
hiemaks f. hiemalis minutus mousenensis oblongiellipticus
CBS 110.19 CBS 586.67
12.2 7.9 12.4
9.1 5.5 7.9
7.0 2.8
CBS 999.7
3.4
2.0
0.5
CBS 566.7 CBS 569.7 CBS 111.07
11.4 14.0
5.3 9.7 3.4
1.2 2.0 4.1
7.3 9.7
8.2 4.2
OblOngiSpONS
plumbeus prayagensis
5.5 9.6
in shake
flasks
at 30°C
Neutral
GLA
POP
ND
ND
ND
ND
9.8 2.6
19.8 25.7
21.5
3.2
44.5
22.0 3.1
2.2 4.8
11.5 5.9
14.2 3.7
6.4
3.3 23.9 9.2
1.2
24.5
0.4
18.3
6.4 27.1
13.64 20.0
8.0 32.5
11.3 1.5
10.8 26.8
11.2
2.3
32.9
6.7
4.4 12.8
50.6 7.5 14.7
17.3
3.5 2.6
9.5 5.5 7.8 12.1
5.9
2.3 2.1
1.3 15.6
33.3 25.4
12.0 11.2
8.9 3.8
ND 1.7 10.7
CBS 786.7
18.6 21.9
17.4
12.5
4.3 7.2
5.9 8.0
CBS 416.77 CBS 204.74
22.6 3.3
18.9 1.5
17.0 3.2
9.0 21.3
2.9 5.2
5.5 5.2
27.9
33.0
4.5
ND
5.6
4.8
0.6
1.2
21.9
22.0
16.8 10.2 9.6
0.5 9.4 15.0
0.3 9.2 15.6
9.9 7.1 15.2
7.5 22.4 14.4
1.0 12.9 10.2
fatty acid on sn 2 position
33.3 62.9 ND 1.7
0.7 0.9
15.4 39.1
3.3
21.7 14.9 13.0
IJ = unsaturated
23.3 5.9
ND
4.0 10.3
CBS 562.66 CBS 564.66 CBS 837.7
fatty acid on sn 1,3 positions,
64.7 39.6 67.9 12.8
0.1
CBS 735.7
); GLA-gamma-linolenic stearoyl-2-oleoyl-3-stearoyl
21.6 12.2 -
5.3 8.5
2.5 31 .o
analyses SOS-sn-1
31.3 2.2 4.8
9.1
var. indicus
(S = saturated
5.6
2.3 8.8
var. recurvus
ND-Not determined (oil content too low for triacyiglycerol PCS+%-1 -palmitoyl-2-oieoyl-3-stearoyl triacylglycerol;
SUS-TAGS
1.9
M. recurvus
sinensis subtilissimus tuberculispzws variabilis variosporus
SOS
13.7
M. rscutvus
M. M. M. M M.
POS
6.0 10.2
CBS 616.7 CBS 317.52
M rouxii
(% w/w)
0.4
15.3 13.1 14.9
4.0
18:O
lipid composltion
acid; POP-sn-1 -palmitoyl-2-oleoyl-3-palmitoyl triacylglycerol; SUS-TAGs-symmetrical
8.8 7.7 7.7
60.7
0.1
8.6 35.3 27.3
2.7
triacylglycerol; triacylglycerois
and TAG = triacylglyceroi).
World Journal ofMicrobiology 6’ Biotechnology, Vol 10, 1994
419
M. P. Roux et al. Table 2. Lipid accumulation fermenter.
by five strains,
representing
three
Mucor
species,
grown
at 30°C with acetic
Parameter
Mucor
Total lipid content of mould (% w/w) Relative 16:0 content (% of neutral lipid) Relative gamma-linolenic GLA content of biomass Biomass
acid (GLA) content (mg/g)
(% of neutral
lipid)
Lipid concentration GLA concentration
acid assimilated)
(@) (ms/l)
‘I-M. circinelloides f. circinelloides M. recurvus var indiws CBS 766.7;
CBS 106.16; 2-M. circinelloides 6-M. rouxii CBS 416.77.
a
1. The
production
d
c
b
of
symmetrical
e
4
40.3 27.3
26.3 12.9
31 .I 19.0
58.1 11.7
40.0 13.4
5.0 19.6
11.3 26.9
6.2 22.5
4.0 20.6
6.3 30.2
circinelloides UOFS 100; CBS
420
CBS d--M.
416.77;
203.28; recurvus
STD-standard
World Journal ofMicrobiology
4.62
3.64
4.16
5.2
3.67
0.26 0.005
0.24 0.006
0.26 0.006
0.30 0.006
0.20 0.006
100 0.10
100 0.06
100 0.06
100 0.17
100 0.06
1.66 90
0.96 110
1.34 110
3.03 120
1.47 120
circinelloides
f. circinelloides
CBS 203.26;
3-M.
UOFS
100; 4-
whereas M. circinelloides f. circinelloides UOFS 100 was characterized by the highest GLA content of 38.3 mg GLA/g dry cells (Table 1). CBE and GLA Production with Acetic Acid as a Carbon Source Table 2 summarizes the results obtained when five strains, representing three Mucor species, were grown at 30°C in an air-lift fermenter in a pH-stat mode with acetic acid as titrant
(SUS-
SOS-TAGS (m) grown at 30°C in source. a-M. circinelloides f.
c-M. circinelloides f. var. indicus CBS 786.7; (cocoa
5
ST0
triacylglycerols
TAGS), consisting of POP- ( ), POS- (0) and (see Table 1 for explanation), by strains of Mucor an air-lift fermenter with acetic acid as carbon circinelloides f. circinelloides CBS 106.16; b-M.
strain’ 3
f. ckcinelloides
SOSTAGs. As is also evident from the literature (Ratledge 1992), there appears to be a correlation between a high percentage of 18 : 0 and a high total percentage of SE-TAGS, with the exception of M. circinelloides f. griseocyunus CBS 116.08, which produced only 2.2% SUS-TAGS from 22% 18 : 0 (Table 1). In this case, most of the 18: 0 was probably associated with non-symmetrical triacylglycerols. Mucor circinelloides f. circinelloides CBS 108.16 produced the highest percentage of GLA (24%) in the neutral lipid fraction,
In an air-lift
2
(g0)
Production time (h) Lipid yield (g lipid/g acetic
source
1
Biomass yield (g/g acetic acid assimilated) GLA yield (g GLA/g acetic acid assimilated)
Figure
acid as carbon
circinelloides e-M. rouxii
butter).
b Biotechnology, Vof IO, 1994
1
3
2
4
STD
Fraction
Flgure
2. Symmetrical ), POS- (0)
triacylglycerols and SOS-TAGS
(SUS-TAGS), consisting of (m), in the lipid fractions
produced by M. circinelloides f. circinelloides CBS 108.16 when grown at 30°C in an air-lift fermenter with acetic acid as carbon source. STD-standard (cocoa butter).
source of CBE and GLA
Mucor-a
a
oca
-wm
ta
iom
am
lam
am
Temperature
(“C)
Temperature
(“C)
am
d
iom
Temperature
(“C)
rm f
C
Figure 3. Differential scanning CBS 108.18 and cocoa butter, cooling and (f) mixture remelt.
calorimetry plots of cocoa butter and a mixture of 20% of lipid fraction 3 from M. circinelloidesf. circinelloides showing (a) cocoa butter melt, (b) cocoa butter cooling, (c) cocoa butter remelt, (d) mixture melt, (e) mixture
and carbon source. The total lipid content of M. recurvus var. indicus was the highest at 58% crude oil. Mucor circinelloides f. circinelloides CBS 108.16 produced the highest percentage of 18:0 (27%) in the NL fraction, whereas M. circinelloides f. circinelloides CBS 203.28 produced the highest percentage of GLA (11.3%) in the NL fraction. The SUS-TAG composition of the five organisms, as obtained by HPLC analyses, is presented in Figure 1. Although M. circinelloides f. circinelloides CBS 203.28, M. recurvus var indicus and M. rouxii accumulated
approximately the same percentage of total SUS-TAGS (approx. 40%), the ratios of the individual SUS-TAGS (SOS, POP and POS; Figure 1) differed considerably. Whereas M. circinelloides f. circinelloides UOFS 100 produced only 22% total SUS-TAGS, M. circinelloides f. circinelloides CBS 108.16 produced more than 70% total SUSTAGs. Consequently, the latter strain was selected for the fractionation of its NL fraction. The results are depicted in Figure 2. Fractions 1 and 4 differed from the cocoa butter standard, whereas the compo-
World @md
of Microbiology
& Biotechnology, Vol 10.1994
421
M. P. Roux et al. sition of the other two fractions was similar to that of the standard. Preliminary DSC analyses of these two fractions, using a melt/cool/remelt regime (results not shown) and a mixture containing 20% of fraction 3 with cocoa butter (Figure 3) revealed profiles similar to those of cocoa butter. A comparison of lipid production by the five strains grown on glucose in shake flasks and on acetic acid in an air-lift fermenter revealed a much higher accumulation of total lipids (crude oil) by M. circinelloides f. circinelloides strains CBS 108.16 and CBS 203.28, M. recurvus var. indicus and M. rouxii when grown on acetate. On glucose, these fungi accumulated only 16%, 17.0%, 22% and 23%, respectively, of total lipids, whereas on acetic acid these values increased to 40%, 26%, 58% and 40%, respectively. Mucor circinelloides f. circinelloides UOFS 100, by contrast, produced less lipids (32%) when grown on acetic acid than on glucose (48%). At present these different patterns of lipid accumulation cannot be explained. A lower GLA content of the NL fraction (expressed as mg GLA/g biomass) was found when M. circinelloides f. circinelloides strains CBS 108.16 and UOFS 100 were grown on acetic acid in an air-lift fermenter than when they were grown as shake-flask cultures on glucose. On glucose, these strains produced 32 mg GLA/g biomass and 38 mg GLA/g biomass, respectively, compared with only 20 mg GLA/g biomass and 23 mg GLA/g biomass, respectively, on acetic acid. In contrast, M. circinelloides f. circinelloides CBS 203.28, M. recurvus var. indicus and M. rouxii accumulated more GLA in the biomass when grown on acetic acid (Table 2) than on glucose (Table 1). These results cannot be explained and it is evident that these differences are strain specific. The five strains had a higher content of 18:O in the NL fraction when grown on acetic acid than when grown on glucose (Tables 1 and 2). On glucose, M. circinelloides f. circinelloides CBS 108.16 produced 6% 18:O and 6.4% SUS TAGS, whereas on acetic acid 27% 18:O and 72% SUS-TAGS were produced. This dramatic increase may be due to an inhibitory effect of acetic acid on the production of NADPH associated with the membranes of this fungus, because NADPH supplies the reducing power necessary for fatty acid desaturation (Kendrick & Ratledge 199213). In conclusion, we have demonstrated that, contrary to general belief, certain Mucor strains accumulate a high percentage of 18:0 and therefore a high percentage of SUS-TAGS, when grown on glucose and acetic acid as carbon sources. Furthermore, a higher content of 18 : 0 and of SUSTAGs was found in the lipids of Mucor grown on acetic acid, than when the fungus was grown on glucose. Preliminary DSC analyses showed that the CBEs obtained from M. circinelloides f. circinelloides CBS 108.16 were compatible with cocoa butter. Because acetic acid is a relatively low-cost substrate, the fungal biomass can be considered a potential source of CBEs. This study showed that it is possible to produce a lipid which contains both CBEs and GLA from Mucor grown on acetic acid.
422
World Journal ofMicrobiology
& Biotechnology, Vol lo,1994
Acknowledgements We would like to thank Prof. C. Ratledge of the Department of Applied Biology, University of Hull, UK, for his valuable advice and encouragement as well as M. Immelman of the University of the Grange Free State for the fermenter cultivations. This work was supported by grants from the Foundation for Research and Development and from Sasol.
References Beavan, M., Kligerman, A., Droniuk, R., Drouin, C., Goldenberg, B., Effio, A., Yu, I’., Giulliany, B. & Fein, J. 1992 Production of microbial cocoa butter equivalents. In Industrial Applications of Single Cell Oils, eds Kyle, D.J. & Ratledge, C. pp. 156-184. Champaign, IL: American Oil Chemists’ Society. Butte, W. 1983 Rapid method for the determination of fatty acid profiles from fats and oils using trimethylsulphonium hydroxide for transesterification. Journal of Chromatography 261,142-145. Christie, W.W. fed) 1992 Gas Chromatography and Lipids. Ayr, UK: Oily Press. Folch, J., Lees, M. & Sloane-Stanley, G.H. 1957 A simple method for the isolation and purification of total lipid from animal tissues. Journal of Biological Chemistry X%,497-509. Graham, J. (ed) 1984 Evening Primrose Oil. Wellingborough, UK: Thorsons. Kendrick, A.J. & Ratledge, C. 1992a Lipids of selected molds grown for production of n-3 and n-6 polyunsaturated fatty acids. Lipids 27,15-20. Kendrick, A.J. & Ratledge, C. 1992b Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. European’ journal of Biochemistry 209,667-673. Kock, J.L.F. & Botha, A. 1993 Acetic acid-a novel source of cocoa butter equivalents and gamma-linolenic acid. South African Journal of Science 89,465. Ratledge, C. 1988 Yeasts for lipid production. Biochemical Society Transactions 16,1088-1090. Ratledge, C. 1992 Microbial lipids: commercial realities or academic curiosities. In Industrial Applications of Single Cell Oil, eds Kyle, D.J. & Ratledge, C. pp. l-15. Champaign, IL: American Oil Chemists’ Society. Ratledge, C. 1993 Single cell oils-have they a biotechnological future? Trends in Biotechnology 11,278-284. Schlichter-Aronhime, J & Garti, N. 1988 Solidification and polimorphism in cocoa butter and the blooming problems in Crystallization and Polimorphism of Fats and Fatty Acids, eds Garti, N. & Sato, K. pp. 363-391. New York and Basel: Marcel Dekker. Ykema, A., Verbree, E.C., Verwoert, I.I.G.S., Van Der Linden, K.H., Nijkamp, H.J.J. & Smith, H. 1990 Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiofrichum curvafum. Applied Microbiology and Biotechnology 33,176-182. (Received 10 January 1994; accepted 31 January 1994)
of Microbiology
& Biotechnology
10. 417422
/lAwor---a source of cocoa butter and gamma-linolenic acid M.P. Roux, J.L.F. Kock,* A. Botha, J.C. du Preez, G.V. Wells and P.J. Botes Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:O) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGS) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SW-TAGS and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% l&O in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGS. Key words: Acetic acid, cocoa butter, gamma-linolenic
acid, lipid, Mucor, oil, triacylglycerol.
Although the production of oils and fats by fungi is well established, no fungal process, with the exception of two processes, has ever reached commercial realization. This can be attributed to the high costs involved in biotechnological routes, which cannot compete against the low costs of agricultural seed oil production (Ratledge 1992). According to Ratledge (1992), there are two main markets on which oil from fungi or single-cell oil (SCO) may impact, namely the markets for cocoa butter and gamma-linolenic acid. Cocoa butter is used extensively in the manufacture of chocolate (Beavan et al. 1992). The chemical and physical properties of cocoa butter are responsible for the melting behaviour, texture and mouth-feel of chocolate. These characteristics of cocoa butter are dependent on the fatty acid ratios of approximately 30% stearic acid (18:0), 30% palmitic acid (l&O) and 30% oleic acid (l&l). Consequently, the major symmetrical triacylglycerol (SUS-TAG) is POS-TAG (sn-lpalmitoyl-2-oleoyl-3-stearoyl-glycerol) and this is, to a large extent, responsible for the melting characteristics of cocoa butter. Attempts to select fungi capable of producing cocoa butter equivalents (CBEs), with a high l&O content in their triacyglycerols, have been unsuccessful. Mutation techniques have therefore been applied and the l&O content of the yeast M.P. Roux, J.L.F. Kock, A. Botha, J.C. du Preez and P.J. Botes are with the Department of Microbiology and Biochemisby, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa; fax: 27 51 482 004. G.V. Wells is with Sasol Waxes, P.O. Box 1, Sasolburg, South Africa. *Corresponding author.
0 1994 Rapid Communications
Cyptococcus curvatus (formerly Candida curvata and Apiotrichum curWum) was eventually increased by partial deletion of the A9 desaturase which is responsible for the transformation of 18:0 to l&l (Ykema et al. 1990). Unfortunately, the economics of the process with the mutant C. curvatus is such that it cannot compete with cocoa butter itself which, at present, is sold at the low price of US $3OOO/ton (Ratledge 1992). Another target for commercial SC0 production is an oil containing gamma-linolenic acid (GLA) which has been obtained, for many years, from the seeds of the evening primrose and is now prescribed for the treatment of eczema (Graham 1984; Ratledge 1992). GLA is also the precursor for the physiologically active human hormones, the prostaglandins. At present, the UK, European and Japanese markets amount to approximately 500 to 800 tons/year, with a price ranging from US $30 to US $60/kg (Ratledge 1992). Currently, only Japan is producing GLA commercially, using the fungus Mortierella (Ratledge 1993). Recently, Kock & Botha (1993) reported on a novel biotechnological route which effectively produced the two main targets for commercial SC0 production: namely a GLArich lipid as well as a CBE, from a low-cost substrate (acetic acid) that originates from a petrochemical process. Both GLA and CBE are produced by the fungus Mucor circinelloides f. circinelloides CBS 108.16 when fed acetic acid at sub-toxic levels as sole carbon source. Since this is, to our knowledge, the first report on a species of Mucor capable of producing both
of Oxford Lfd World journal
ofMicrobiology
b Biotechnology, Vol IO, 1994
417
M.P.Roux etal. these
high-value
lipids,
screening
of fungi
GLA-rich
oils
acid
as sole
Materials
the aim
of the genus
and carbon
CBEs
when
of the present
Mucor grown
study
for the ability with
glucose
was
the
to produce or acetic
source.
column temperature and, following a 10 to 240°C at the same were 170 and 25O”C, at 5 ml/mm. Peaks standards.
of 145°C was increased by J”C/min to 225°C min isothermal period, was then increased rate. The inlet and detector temperatures respectively. N, was used as a carrier gas were identified by reference to authentic
AnaZyses of
and Methods
CBE and GLA Production with Glucose as Carbon
source
Microorganisms and Cultivation. The Mucor strains
used in this study (Table 1) were obtained from the Centraal Bureau for Schimmelcultures (CBS) in Delft, The Netherlands, and from the University of the Orange Free State (UOFS), Bloemfontein, South Africa. Each strain was transferred from YM agar slants into l-l conical shake flasks and grown at 3O”C, 160 rev/min, for approximately 5 days. Each flask contained 200 ml medium, pH 5.5, containing (g/B: glucose, 50; (NH,l,SO, 1.7; KHJQ,, 2.0; Mg!SOJI-I~O, 0.5; CaC1,.2H,O, 0.2; yeast extract, 1.0; FeSOJH,O, 0.01; ZnSOJH~O, 0.01; MnSO,.4H,O, 0.001; and CuSO~5H,O, 0.0005. The medium was sterilized by autoclaving at 121°C for 20 mins. Fungal cultures were harvested by filtration through Whatman No. 1 filter paper and washed with 800 ml distilled water. The cells were immediately frozen with liquid N, and then freeze-dried.
Lipid Extraction. Lipids
were extracted from the freeze-dried material as described by Kendrick & Ratledge (1992a). This included extraction with chloroform/methanol (21 v/v) according to Folch et al. (19571, followed by two washes with distilled water and final evaporation of the organic phase under vacuum. Subsequently, the lipid material was dissolved in diethyl ether and transferred to preweighed vials. Prior to determination of lipid weights, samples were dried to constant weight in a vacuum oven over P20s at 55°C.
Fractionation
of Extracted Lipid. Extracted lipid was dissolved in chloroform and applied to a column (140 mm x 20 mm) of silicic acid, activated by heating overnight. Neutral, sphingoand glycolipids (as a combined fraction), as well as polar lipids, were eluted by successive applications of organic solvents as described by Kendrick & Ratledge (1992a). Final solvent removal and storage was as for whole lipid extracts. Each fraction was further separated by TLC to evaluate the fractionation ability of the silicic acid column.
Thin-layer Chromatography. TLC of the neutral, sphingoand glyco-lipid fractions was performed on silica gel thin-layer plates backed with ahnninium (Merck). The polar fraction, containing phospholipids, was separated using chloroform/methanol/ water/acetic acid (65 : 43 : 3 : 1 by vol.). The neutral lipid fraction was separated using petroleum ether fb.p. 60 to 80’C)/diethyl ether/acetic acid (85: 15 : 1, by vol.). Plates were developed onedimensionally by the ascending technique and visualized by exposure to I, vapbur. Further identification was achieved by running authentic standards along with experimental samples.
Triucylglycerols. Triacylglycerol analyses were performed according to the method of Deffense as reported by Christie (1992). HPLC of the symmetrical triacylglycerols was carried out on an ODS column (Spherisorb 5 ODS 2; 240 mm x 4.6 mm; Phasesep, UK), using a mixture of acetone/ acetonitrile (62.5 : 37: 5 v/v) as the mobile phase at a flow-rate of 1 .l ml/min, and with refractive index detection. Peaks were identified by reference to authentic standards.
CBE and GLA Production with Acetic Acid as Carbon Source Inoculum. A spore
suspension of the Mucor strain was washed from a 3-day-old culture on Sabouraud dextrose agar slants with 0.05 M sterile KH,PO, containing 0.1% Tween 80. This suspension, of about lo7 spores/ml, was diluted to give a count of about 10s spores/ml after inoculation into the fermenter.
Cultivation and Lipid Analysis. Each of the five Mucor strains, representing three Mucor species (Table 21, was grown at 30°C in a 12-1 glass air-lift fermenter (Chemap, CF 3000), containing 11 1 medium and fitted with a marine propeller which was operated at 150 rev/min to improve mixing. The dissolved Oz tension was monitored with an Ingold polarographic probe and maintained above 20% by adjusting the rate of air sparging from 2 to 13 l/ min. The medium contained (g/l): citric acid, 0.16; NH&l, 0.20; MgSOJH~O, 0.5; KH,PO, 1.88; CaC1,.2H,O, 0.03; yeast extract, 0.5; glucose, 0.5; Na acetate, 31; and trace elements as before. The fermenter and medium were sterilized in situ, with the exception of the acetate solution which was autoclaved separately and aseptically added to the fermenter. The medium was subsequently adjusted to pH 7 with 10 M NaOH. After inoculation, a 6-h period was allowed for spore germination, after which the culture was adjusted to pH 6 with 5 M H,SO,. The acetic acid concentration in the culture during the 100-h cultivation period was maintained at about 2 g/l by automatic titration with a 50% (v/v) solution of acetic acid, to maintain a constant pH. Lipid extraction and the analytical procedures were as above.
Analyses
of Triacylglycerols. Fractionation of the cocoa butter was effected by addition of acetone to the neutral lipid fraction [seven times the volume, according to the method of Beaven et al. (199211 which was then shaken at 30°C for 2 h, followed by cooling to 20°C for about 2 h. The latter step was accompanied by the seeding of the mixture with a grain of cocoa butter. The liquid upper phase was separated from the precipitate (fraction 1) and left overnight at lO”C, allowing another precipitate (fraction 2) to form. The top layer was recovered and cooled further at 4°C overnight to enhance precipitation (fraction 31. On evaporation of the remaining solvent, the remnant was designated fraction 4.
DSC Annlyses. Differential Fatty Acid Analyses. Lipid
was dissolved in chloroform and methylated by the addition of trimethyl sulphonium hydroxide (TMSH) as described by Butte (1983). The fatty acid methyl esters were analysed by GC, with a flame ionization detector and a Supelcowax 10 capillary column (30 mx0.75mm). The initial
418
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& Biotechnology, Vol lo,1994
scanning calorimetry @SC) analyses were performed on a Perkin Elmer DSC-4 instrument fitted with an Intracoler II sub-ambient accessory and controlled by a Perkin Elmer 3700 data station with TADS software. The instrument purge was N, at 2 atm. Standard aluminium sample pans and lids were used and sample sizes were approx. 0.5 mg. Instrument
source of CBE and GLA
Mucor-a calibration was performed using indium obtained from Perkin Elmer. The analytical procedure was according to ASTM D4419, using a preparatory cooling rate of 5”C/min and a scanning heating rate of lO”C/min over a temperature range of -20 to 70°C. Chemic&. All organic chemicals and solvents used were of analytical reagent grade and obtained from major retailers. Silicic acid (100 mesh) was from Aldrich. Neutral and phospholipid standards, as well as the triacylglycerols and fatty acid standards, were from Sigma.
Results and Discussion CBE and GLA Production with Glucoseas Carbon Source The results of the lipid analyses of the 28 strains, representing 22 Mucor species, grown on glucose as carbon source, are summarized in Table 1. Only four strains, M. circinelZoides f. circinelloides UOFS 100, M. recurvus var. indicus, M. rouxii and M. tuberculisporus, accumulated more than 20% (mol/mol) crude oil and can be considered oleaginous under these growth conditions, according to the definition of Ratledge Table
1. Lipid composition
of strains
of various
hfucor
Culture no.
species
Crude
Neutral lipid in biomass (% w/w)
Oil
(% w/w)
M M hf. M. M. M. M. M. M. M. M.
amphibiomm ardhlaengiktus azygosporus bainieri circinelloides f. griseocyanus circine//oides f. circinelloides circinelloides f. janssenii circinelloides f. circinelloides circinelloides f. circinelloides circinelloides f circinelloides flaws
M. fragifis
grown
(1988). These fungi produced crude oil which consisted mainly (> 80%) of neutral lipids. The individual strains differed extensively in their percentage of neutral lipids in the dry biomass, which varied from 0.2% in M. ardhluengiktus to 42% in M. circinelloides f. circinelloides UOFS 100. The percentage of 18 : 0 ranged from 1.3% in M. oblongiellipticus to as high as 27% in M. flaws. This was an interesting finding because attempts to select fungi capable of naturally producing CBEs, and therefore with a high 18:O content in their triacylglycerols, have so far been unsuccessful according to the published literature. Four strains had a particularly high content of SLJS-TAGS, namely M. flavus (65%), M. fuscus (68%), M. oblongisporus (63%) and M. subtilissimus (61%) (Table 1). These fungi, therefore, have potential as a source of CBEs if the content of SOSTAGS (1,3-distearoyl-2-oleoyl-glycerol) could be increased to the content required in CBEs. CBEs contain approximately 20% SOS-TAGS, necessary for the formation of the nucleate centres of the cocoa butter crystals and responsible for the physical characteristics of this lipid (Schlichter-Aronhime & Garti 1988). Although M. fuscus had a higher percentage of total SUS-TAGS than M. flavus, the former did not produce any on glucose GLA (mg/g biomass)
CBS 763.74 CBS 210.8
2.7 0.5
1.4 0.2
0.1 0.1
CBS 292.63 CBS 293.63
16.7
12.1 6.9
12.3 10.6
4.4 9.8
5.8 14.6
11.7 15.5 47.7
8.7 13.5 41.6
2.9 32.3
CBS 116.08 CBS 119.08 CBS 232.29 CBS 106.16 UOFS 100 CBS 203.26
6.9 7.4 14.2
38.3 14.5
17.0
12.8
CBS 126.70 CBS 236.35
11.2 11.4
9.0 8.6
1.4 9.6
M.
fuscus
CBS 132.22
M. M. M. M M. M. M.
hiemaks f. hiemalis minutus mousenensis oblongiellipticus
CBS 110.19 CBS 586.67
12.2 7.9 12.4
9.1 5.5 7.9
7.0 2.8
CBS 999.7
3.4
2.0
0.5
CBS 566.7 CBS 569.7 CBS 111.07
11.4 14.0
5.3 9.7 3.4
1.2 2.0 4.1
7.3 9.7
8.2 4.2
OblOngiSpONS
plumbeus prayagensis
5.5 9.6
in shake
flasks
at 30°C
Neutral
GLA
POP
ND
ND
ND
ND
9.8 2.6
19.8 25.7
21.5
3.2
44.5
22.0 3.1
2.2 4.8
11.5 5.9
14.2 3.7
6.4
3.3 23.9 9.2
1.2
24.5
0.4
18.3
6.4 27.1
13.64 20.0
8.0 32.5
11.3 1.5
10.8 26.8
11.2
2.3
32.9
6.7
4.4 12.8
50.6 7.5 14.7
17.3
3.5 2.6
9.5 5.5 7.8 12.1
5.9
2.3 2.1
1.3 15.6
33.3 25.4
12.0 11.2
8.9 3.8
ND 1.7 10.7
CBS 786.7
18.6 21.9
17.4
12.5
4.3 7.2
5.9 8.0
CBS 416.77 CBS 204.74
22.6 3.3
18.9 1.5
17.0 3.2
9.0 21.3
2.9 5.2
5.5 5.2
27.9
33.0
4.5
ND
5.6
4.8
0.6
1.2
21.9
22.0
16.8 10.2 9.6
0.5 9.4 15.0
0.3 9.2 15.6
9.9 7.1 15.2
7.5 22.4 14.4
1.0 12.9 10.2
fatty acid on sn 2 position
33.3 62.9 ND 1.7
0.7 0.9
15.4 39.1
3.3
21.7 14.9 13.0
IJ = unsaturated
23.3 5.9
ND
4.0 10.3
CBS 562.66 CBS 564.66 CBS 837.7
fatty acid on sn 1,3 positions,
64.7 39.6 67.9 12.8
0.1
CBS 735.7
); GLA-gamma-linolenic stearoyl-2-oleoyl-3-stearoyl
21.6 12.2 -
5.3 8.5
2.5 31 .o
analyses SOS-sn-1
31.3 2.2 4.8
9.1
var. indicus
(S = saturated
5.6
2.3 8.8
var. recurvus
ND-Not determined (oil content too low for triacyiglycerol PCS+%-1 -palmitoyl-2-oieoyl-3-stearoyl triacylglycerol;
SUS-TAGS
1.9
M. recurvus
sinensis subtilissimus tuberculispzws variabilis variosporus
SOS
13.7
M. rscutvus
M. M. M. M M.
POS
6.0 10.2
CBS 616.7 CBS 317.52
M rouxii
(% w/w)
0.4
15.3 13.1 14.9
4.0
18:O
lipid composltion
acid; POP-sn-1 -palmitoyl-2-oleoyl-3-palmitoyl triacylglycerol; SUS-TAGs-symmetrical
8.8 7.7 7.7
60.7
0.1
8.6 35.3 27.3
2.7
triacylglycerol; triacylglycerois
and TAG = triacylglyceroi).
World Journal ofMicrobiology 6’ Biotechnology, Vol 10, 1994
419
M. P. Roux et al. Table 2. Lipid accumulation fermenter.
by five strains,
representing
three
Mucor
species,
grown
at 30°C with acetic
Parameter
Mucor
Total lipid content of mould (% w/w) Relative 16:0 content (% of neutral lipid) Relative gamma-linolenic GLA content of biomass Biomass
acid (GLA) content (mg/g)
(% of neutral
lipid)
Lipid concentration GLA concentration
acid assimilated)
(@) (ms/l)
‘I-M. circinelloides f. circinelloides M. recurvus var indiws CBS 766.7;
CBS 106.16; 2-M. circinelloides 6-M. rouxii CBS 416.77.
a
1. The
production
d
c
b
of
symmetrical
e
4
40.3 27.3
26.3 12.9
31 .I 19.0
58.1 11.7
40.0 13.4
5.0 19.6
11.3 26.9
6.2 22.5
4.0 20.6
6.3 30.2
circinelloides UOFS 100; CBS
420
CBS d--M.
416.77;
203.28; recurvus
STD-standard
World Journal ofMicrobiology
4.62
3.64
4.16
5.2
3.67
0.26 0.005
0.24 0.006
0.26 0.006
0.30 0.006
0.20 0.006
100 0.10
100 0.06
100 0.06
100 0.17
100 0.06
1.66 90
0.96 110
1.34 110
3.03 120
1.47 120
circinelloides
f. circinelloides
CBS 203.26;
3-M.
UOFS
100; 4-
whereas M. circinelloides f. circinelloides UOFS 100 was characterized by the highest GLA content of 38.3 mg GLA/g dry cells (Table 1). CBE and GLA Production with Acetic Acid as a Carbon Source Table 2 summarizes the results obtained when five strains, representing three Mucor species, were grown at 30°C in an air-lift fermenter in a pH-stat mode with acetic acid as titrant
(SUS-
SOS-TAGS (m) grown at 30°C in source. a-M. circinelloides f.
c-M. circinelloides f. var. indicus CBS 786.7; (cocoa
5
ST0
triacylglycerols
TAGS), consisting of POP- ( ), POS- (0) and (see Table 1 for explanation), by strains of Mucor an air-lift fermenter with acetic acid as carbon circinelloides f. circinelloides CBS 106.16; b-M.
strain’ 3
f. ckcinelloides
SOSTAGs. As is also evident from the literature (Ratledge 1992), there appears to be a correlation between a high percentage of 18 : 0 and a high total percentage of SE-TAGS, with the exception of M. circinelloides f. griseocyunus CBS 116.08, which produced only 2.2% SUS-TAGS from 22% 18 : 0 (Table 1). In this case, most of the 18: 0 was probably associated with non-symmetrical triacylglycerols. Mucor circinelloides f. circinelloides CBS 108.16 produced the highest percentage of GLA (24%) in the neutral lipid fraction,
In an air-lift
2
(g0)
Production time (h) Lipid yield (g lipid/g acetic
source
1
Biomass yield (g/g acetic acid assimilated) GLA yield (g GLA/g acetic acid assimilated)
Figure
acid as carbon
circinelloides e-M. rouxii
butter).
b Biotechnology, Vof IO, 1994
1
3
2
4
STD
Fraction
Flgure
2. Symmetrical ), POS- (0)
triacylglycerols and SOS-TAGS
(SUS-TAGS), consisting of (m), in the lipid fractions
produced by M. circinelloides f. circinelloides CBS 108.16 when grown at 30°C in an air-lift fermenter with acetic acid as carbon source. STD-standard (cocoa butter).
source of CBE and GLA
Mucor-a
a
oca
-wm
ta
iom
am
lam
am
Temperature
(“C)
Temperature
(“C)
am
d
iom
Temperature
(“C)
rm f
C
Figure 3. Differential scanning CBS 108.18 and cocoa butter, cooling and (f) mixture remelt.
calorimetry plots of cocoa butter and a mixture of 20% of lipid fraction 3 from M. circinelloidesf. circinelloides showing (a) cocoa butter melt, (b) cocoa butter cooling, (c) cocoa butter remelt, (d) mixture melt, (e) mixture
and carbon source. The total lipid content of M. recurvus var. indicus was the highest at 58% crude oil. Mucor circinelloides f. circinelloides CBS 108.16 produced the highest percentage of 18:0 (27%) in the NL fraction, whereas M. circinelloides f. circinelloides CBS 203.28 produced the highest percentage of GLA (11.3%) in the NL fraction. The SUS-TAG composition of the five organisms, as obtained by HPLC analyses, is presented in Figure 1. Although M. circinelloides f. circinelloides CBS 203.28, M. recurvus var indicus and M. rouxii accumulated
approximately the same percentage of total SUS-TAGS (approx. 40%), the ratios of the individual SUS-TAGS (SOS, POP and POS; Figure 1) differed considerably. Whereas M. circinelloides f. circinelloides UOFS 100 produced only 22% total SUS-TAGS, M. circinelloides f. circinelloides CBS 108.16 produced more than 70% total SUSTAGs. Consequently, the latter strain was selected for the fractionation of its NL fraction. The results are depicted in Figure 2. Fractions 1 and 4 differed from the cocoa butter standard, whereas the compo-
World @md
of Microbiology
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M. P. Roux et al. sition of the other two fractions was similar to that of the standard. Preliminary DSC analyses of these two fractions, using a melt/cool/remelt regime (results not shown) and a mixture containing 20% of fraction 3 with cocoa butter (Figure 3) revealed profiles similar to those of cocoa butter. A comparison of lipid production by the five strains grown on glucose in shake flasks and on acetic acid in an air-lift fermenter revealed a much higher accumulation of total lipids (crude oil) by M. circinelloides f. circinelloides strains CBS 108.16 and CBS 203.28, M. recurvus var. indicus and M. rouxii when grown on acetate. On glucose, these fungi accumulated only 16%, 17.0%, 22% and 23%, respectively, of total lipids, whereas on acetic acid these values increased to 40%, 26%, 58% and 40%, respectively. Mucor circinelloides f. circinelloides UOFS 100, by contrast, produced less lipids (32%) when grown on acetic acid than on glucose (48%). At present these different patterns of lipid accumulation cannot be explained. A lower GLA content of the NL fraction (expressed as mg GLA/g biomass) was found when M. circinelloides f. circinelloides strains CBS 108.16 and UOFS 100 were grown on acetic acid in an air-lift fermenter than when they were grown as shake-flask cultures on glucose. On glucose, these strains produced 32 mg GLA/g biomass and 38 mg GLA/g biomass, respectively, compared with only 20 mg GLA/g biomass and 23 mg GLA/g biomass, respectively, on acetic acid. In contrast, M. circinelloides f. circinelloides CBS 203.28, M. recurvus var. indicus and M. rouxii accumulated more GLA in the biomass when grown on acetic acid (Table 2) than on glucose (Table 1). These results cannot be explained and it is evident that these differences are strain specific. The five strains had a higher content of 18:O in the NL fraction when grown on acetic acid than when grown on glucose (Tables 1 and 2). On glucose, M. circinelloides f. circinelloides CBS 108.16 produced 6% 18:O and 6.4% SUS TAGS, whereas on acetic acid 27% 18:O and 72% SUS-TAGS were produced. This dramatic increase may be due to an inhibitory effect of acetic acid on the production of NADPH associated with the membranes of this fungus, because NADPH supplies the reducing power necessary for fatty acid desaturation (Kendrick & Ratledge 199213). In conclusion, we have demonstrated that, contrary to general belief, certain Mucor strains accumulate a high percentage of 18:0 and therefore a high percentage of SUS-TAGS, when grown on glucose and acetic acid as carbon sources. Furthermore, a higher content of 18 : 0 and of SUSTAGs was found in the lipids of Mucor grown on acetic acid, than when the fungus was grown on glucose. Preliminary DSC analyses showed that the CBEs obtained from M. circinelloides f. circinelloides CBS 108.16 were compatible with cocoa butter. Because acetic acid is a relatively low-cost substrate, the fungal biomass can be considered a potential source of CBEs. This study showed that it is possible to produce a lipid which contains both CBEs and GLA from Mucor grown on acetic acid.
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Acknowledgements We would like to thank Prof. C. Ratledge of the Department of Applied Biology, University of Hull, UK, for his valuable advice and encouragement as well as M. Immelman of the University of the Grange Free State for the fermenter cultivations. This work was supported by grants from the Foundation for Research and Development and from Sasol.
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