Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source by daicavn


									 Production of Docosahexaenoic Acid by Crypthecodinium cohnii
        Grown in a pH-Auxostat Culture with Acetic Acid
                  as Principal Carbon Source
        Colin Ratledge*, K. Kanagachandran, Alistair J. Anderson, David J. Grantham, and Janet C. Stephenson
                    Lipid Research Centre, Department of Biological Sciences, University of Hull HU6 7RX, United Kingdom

ABSTRACT: Crypthecodinium cohnii, a marine alga used for                   also produce DHA at high concentrations within its intracel-
the commercial production of docosahexaenoic acid (DHA),                   lular oil, when it is grown on acetic acid. Preliminary work
was cultivated in medium containing sodium acetate as princi-              (de Swaaf, M.E., personal communication) indicated that C.
pal carbon source; the pH was maintained at a constant value               cohnii could utilize sodium acetate, but the rise in pH that ac-
by addition of acetic acid, which also provided an additional              companied growth resulted in poor cell yield (<3 g/L). Also,
carbon source in a controlled manner. The accumulation of
                                                                           published work (18) indicated that C. cohnii, as well as other
lipid by C. cohnii in this pH-auxostat culture was significantly
greater than previously reported for batch cultures using glu-
                                                                           microalgae, might be able to grow on acetate, but only if the
cose as principal carbon source. Of six strains tested in pH-              concentration was kept low (1 g/L or less).
auxostat cultures, C. cohnii ATCC 30772 was the best, with the                 Growth of microorganisms on sodium acetate results in a
cells reaching 20 to 30 g dry weight per liter after 98 to 144 h           rise in pH. This is because Na+ remains in the medium and
and containing in excess of 40% (w/w) total lipid, with DHA                CH3COO− is gradually replaced with OH− and other anions.
representing approximately half of the total fatty acids in the tri-       Sodium hydroxide is a much stronger base than sodium acetate,
acylglycerol fraction. A productivity of 36 mg DHA L−1 h−1 was             and hence the pH rises. An increase in pH is characteristic of
achieved during cultivation for 98 h using a 5% (vol/vol) inocu-           growth of microorganisms on salts of organic acids, or organic
lum, and DHA production was in excess of 3 g per liter of cul-             acids that have been neutralized by the addition of alkali. In the
ture. Most of the DHA was present in neutral lipids.                       present work, we have used a pH-auxostat culture system in
    Paper no. L8870 in Lipids 36, 1241–1246 (November 2001).
                                                                           which a low initial concentration of sodium acetate is included
                                                                           in the initial growth medium and acetic acid is used to main-
With the current increasing demand for oils rich in docosa-                tain a constant pH value and supply a further carbon source for
hexaenoic acid, DHA (22:6 n-3), not only for improved in-                  growth. The concept of the pH-auxostat was first described in
fant nutrition (1–3) but also as a dietary supplement for adults           1976 (19), and a pH-auxostat fed with acetic acid has been used
(4–7), attention has increasingly focused on the potential of              to cultivate Mucor circinelloides for the production of biomass
microorganisms to meet these requirements (8,9). Nonmicro-                 and an oil rich in γ-linolenic acid (20). In this paper we report
bial supplies of pure DHA are restricted to such sources as                the cultivation of C. cohnii in a pH-auxostat and DHA produc-
the eye-socket of the tuna or to expensive fractionation of fish            tion in excess of 3 g per liter of culture.
oils (4), but in the latter case the DHA is nearly always ac-
companied by notable amounts of eicosapentaenoic acid
                                                                           MATERIALS AND METHODS
(20:5n-3) (10), which may detract from the efficacy of DHA
as a nutritional supplement (1–7). However, it has long been               Growth of C. cohnii. Crypthecodinium cohnii strain ATCC
known that the marine dinoflagellate, Crypthecodinium                      30772 was used except where stated otherwise. Strains were
cohnii, produces DHA as its sole polyunsaturated fatty acid                grown at 27°C for 4–5 d in screw-capped tubes (25 mL) con-
(11). Development of this organism as a commercial source                  taining 5 mL of medium (ATCC Culture Medium 460 A2E6)
of oil rich in DHA has taken place over the past 6 to 8 yr,                and used to inoculate starter cultures. Starter cultures were
mainly through the efforts of Martek Co. Inc. (Columbia,                   grown at 27°C for 4 d in static flasks (250 mL) containing 100
MD) (12–14), and numerous patents have been taken out to                   mL medium containing (per liter): glucose (9 g), yeast extract
protect the process and the product (e.g., Refs. 15–17).                   (2 g), and sea salts (25 g). Shake-flask cultures were grown in
   In an attempt to improve upon the existing process, which               flasks (250 mL) containing 100 mL medium composed of
uses glucose as the principal carbon source, we have discov-               (per liter): glucose (27 g), yeast extract (3.8 g), and sea salts
ered that the organism will grow to high cell densities, and               (25 g). These cultures were inoculated (10% vol/vol) with sta-
                                                                           tic culture and grown at 27°C for 3 d with shaking at 180 rpm.
*To whom correspondence should be addressed.                                   Small pH-auxostat cultures were grown in fermenters (1
                                                                           L) containing 800 mL of medium composed of (per liter):
Abbreviations: ATCC, American Type Culture Collection; DHA, docosa-
hexaenoic acid; EPA, eicosapentaenoic acid; GC, gas chromatography; TLC,   yeast extract (7.5 g), sea salts (25 g), and sodium acetate
thin-layer chromatography; vvm, volume of air/volume of medium/min.        (1–16 g, as required). The pH of the medium was adjusted to

Copyright © 2001 by AOCS Press                                         1241                                      Lipids, Vol. 36, no. 11 (2001)
1242                                                    C. RATLEDGE ET AL.

approximately 6.5 with NaOH, prior to autoclaving for 60               Determination of polysaccharide. Samples of culture (100
min at 121°C. pH-auxostat cultures were inoculated (10%            mL) were centrifuged (4000 × g at 5°C for 10 min). The su-
vol/vol) with culture grown in shake-flasks as described           pernatant (25 mL) was mixed with 2-propanol (75 mL) and
above. The pH was maintained at 6.5 by the automatic addi-         allowed to stand at room temperature for 30 min. The precip-
tion of acetic acid (50% vol/vol) and, if necessary, KOH (2        itated material was transferred to a preweighed filter (What-
M). The temperature was maintained at 27°C. The cultures           man GF/A) and dried to constant weight at 65°C.
were stirred at 300–1000 rpm and aerated at 0.2 to 1.0 vol air/
vol medium/min (vvm), increasing the stirring speed and
aeration as required to maintain a dissolved oxygen concen-
tration above 30% of air saturation. Foaming was controlled        Optimization of the initial nutrient concentrations for growth
by addition of polypropylene glycol (2000 grade). For growth       of C. cohnii in a pH-auxostat. When C. cohnii ATCC 30772
in a fermenter with glucose as principal carbon source, the        was grown in a series of pH-auxostat cultures with the initial
shake-flask culture medium described above was used and in-         concentration of sodium acetate in the medium ranging from
oculated (10% vol/vol) with culture grown in the same              1 to 16 g/L, the growth rate and cell density attained after
medium. The growth conditions were otherwise as described          growth for 140 h were greatest with sodium acetate at 8 g/L
for pH-auxostat culture except that the pH was maintained at       (Fig. 1A). This concentration also produced the highest lipid
6.5 by the automatic addition of KOH (2 M) and, if necessary,      content in the cells (Fig. 1B). The concentration of sodium
HCl (2 M).                                                         acetate in the medium had no significant effect on the propor-
    Larger pH-auxostat cultures were grown in the same             tion of DHA, which represented approximately 40 to 50% of
medium (3.5 L) in fermenters (5 L) and inoculated (5%              the total fatty acids (Fig. 1C).
vol/vol) with pH-auxostat culture grown for 3 d in a 1-L pH-           The effect of independently varying the concentrations of
auxostat culture. The culture was aerated at 1 vvm and the         sea salts and yeast extract in the growth medium was tested.
dissolved oxygen concentration was maintained above 30%            No significant improvement in lipid accumulation or DHA
of air saturation by increasing the stirrer speed as required,     production was achieved by modest increases or decreases in
from an initial speed of 300 rpm to a maximum of 1000 rpm.         concentration (data not shown).
The growth temperature was 27°C and the pH was maintained              Performance of other strains in pH-auxostat culture. The
at 6.5 as described above. Foaming was controlled as before.       performance of six strains of C. cohnii, including ATCC
    Determination of dry weight. Samples of culture (50 to 100     30772, all grown in pH-auxostat cultures with 8 g sodium
mL) were centrifuged (4000 × g at 5°C for 10 min) and              acetate/L as the initial concentration, is summarized in Table
washed twice with water. The resulting cells were freeze-          1. All the strains tested produced DHA, and the highest pro-
dried until constant weight was achieved.                          ductivity was achieved using strain 30772. On the basis of the
    Lipid extraction. Lipid was extracted from freeze-dried        amount of acetic acid consumed, strain 30772 produced the
cells (usually 250 to 500 mg) by standing overnight in ap-         lowest yield of cells and the highest yield of DHA.
proximately 100 mL chloroform/methanol (2:1, vol/vol) and              All cultures of C. cohnii became viscous toward the end of
then following established procedures (21), with the final         the growth period, and material, presumably exopolysaccha-
lipid amount being determined gravimetrically.                     ride, could be precipitated from the supernatant fluid by treat-
    Lipid fractionation. The total lipid extract (approximately    ment with 2-propanol (Table 1). The highest concentration of
120 mg) was fractionated following the procedure of Christie       precipitable material was found for strain ATCC 30772.
(22) using a 70 mL/10 g SPE Isolute NH2 column (Interna-               Effect of inoculum substrate on pH-auxostat growth. It is
tional Sorbent Technology, Mid-Glamorgan, United King-             well known that the utilization of acetate requires the induc-
dom) which was washed initially with hexane, and then the          tion of several enzymes that are not active in glucose-grown
lipid extract, in chloroform, was added. Fractions were eluted     cells. The performance of C. cohnii in the pH-auxostat was,
progressively with diethyl ether (to elute neutral triacylglyc-    however, similar for cultures inoculated with glucose or ac-
erols), acetone/pyridine (1:1, vol/vol) (to elute sterol esters,   etate (pH-auxostat) cultures. No significant differences were
sterol glycosides, and any digalactosyldiacyl glycerols),          seen with respect to growth performance (Fig. 2A), lipid pro-
methanol (to elute choline-containing lipids), and finally         duction (Fig. 2B), or DHA production (Fig. 2C), and both
chloroform/methanol/28% (wt/vol) aqueous ammonia/0.05              lipid and DHA production were substantially greater than
M ammonium acetate (4:1:0.08:0.02, by vol), to remove polar        achieved during batch fermentation with glucose as carbon
phospholipids. The solvent from each lipid fraction was evap-      source (Figs. 2B, 2C). Presumably the inducible enzymes
orated and the residue weighed. The identity of each lipid         needed for the utilization of acetate were synthesized soon
component was confirmed by standard thin-layer chromatog-           after exposure of the cells to acetate, and this occurred during
raphy (TLC) analysis (23).                                         the lag period, prior to rapid growth of the culture.
    Fatty acid analysis. Fatty acyl groups of the total lipid or       Lipid fractionation. The lipid produced by cells grown
the lipid fractions were transesterified using trimethylsulfo-     with glucose as principal carbon source was fractionated into
nium hydroxide (24) and analyzed by gas chromatography             its major components (Table 2). Comparable data for cells
(25).                                                              grown on acetate in a pH-auxostat are shown in the lipid from

Lipids, Vol. 36, no. 11 (2001)
                                                   DHA PRODUCTION BY CRYPTHECODINIUM COHNII                                                              1243

                                                                                    FIG. 1. Growth (A), lipid content of cells (B), and docosahexaenoic acid
                                                                                    (DHA) content of total fatty acids (C) for Crypthecodinium cohnii grown
                                                                                    in pH-auxostat cultures in media containing various concentrations of
                                                                                    sodium acetate: ● 4, ● 6, ■ 8, ▲ 10, ▼ 12, ▲ 16 g/L. The data pre-
                                                                                    sented are typical of numerous fermentations carried out under the con-
                                                                                    ditions described.

                                                                                    dinoflagellate would therefore seem beneficial not only for
                                                                                    the improved growth of the organism but also to promote both
                                                                                    lipid accumulation and DHA production beyond that seen
                                                                                    with glucose as the feedstock.

                                                                                    The large-scale production of DHA-rich oil derived from C.
Table 3. In both cases the major fraction, constituting 75% of                      cohnii has been carried out since the mid-1990s with the oil
the total lipids, was the neutral lipids. These were shown by                       being offered as an over-the-counter dietary supplement for
TLC to be wholly triacylglycerols. The proportion of the                            adults and as a beneficial and important polyunsaturated fatty
other lipid components was also similar for cells produced in                       acid for inclusion in infant formulae. Although technical as-
glucose and acetate pH-auxostat cultures. Of possible signifi-                       pects of the commercial process are obviously restricted, it is
cance, though, was the increased content of DHA not only in                         nevertheless understood that the organism is cultivated in
the total extracted lipid but also in the triacylglycerol fraction                  large stirred fermenters with glucose as principal carbon
obtained from the acetate-grown cells compared to the glu-                          source (12,13). Yields of biomass, lipid, and DHA are, of
cose-grown cells. The triacylglycerol fraction, which is the                        course, closely guarded company secrets.
commercially most valuable part of the oil, contained over                             Nevertheless the results being reported here would indi-
50% more DHA when isolated from the acetate-grown cells                             cate that an alternative process based on acetic acid as the
than was in the same fraction obtained from the glucose-                            principal carbon feedstock might seek to rival the traditional
grown cells. Acetate as a feedstock for the cultivation of this                     glucose feedstock process. The performance of C. cohnii

Comparison of Crypthecodinium cohnii Strains After 140 h in pH-Auxostat Cultures
ATCC                                                                                                                                              Precipitable
strain             Dry weight        Yield of cellsa          Lipid      Lipid               DHA               DHA          Yield of DHAb          materialc
number               (g L−1)      [mg (g acetic acid)−1]     (g L−1)   (% dry wt)     (% total fatty acids)   (g L−1)    [mg (g acetic acid)−1]      (g L−1)
30772                 17.0                  120               7.5           44                 59               4.4                30                 8.0
30541                 45.5                  200               8.3           24                 33               2.7                12                 2.8
50298                 45.5                  210              12.3           30                 31               3.8                18                 3.8
40750                 17.0                  220               3.3           22                 46               1.5                26                 3.3
30555                 20.7                  180               2.8           15                 30               0.8                 7                 5.0
30557                 25.6                  160               4.7           18                 40               1.9                10                 3.9
     Yield calculated on basis of acetic acid utilized.
    Material precipitated by treatment of culture supernatant with 2-propanol and inferred to be polysaccharide (see Ref. 28).

                                                                                                                                 Lipids, Vol. 36, no. 11 (2001)
1244                                                        C. RATLEDGE ET AL.

                                                                       FIG. 2. Growth (A), lipid production (B), and DHA production (C) by
                                                                       Crypthecodinium cohnii in glucose culture (■), and in pH-auxostat cul-
                                                                       tures with 8 g sodium acetate/L, inoculated with cells grown on glucose
                                                                       (● ) or acetate (●) medium. The data presented are typical of numerous
                                                                       fermentations carried out under the conditions described.

                                                                       the medium on the growth and lipid accumulation by C. cohnii
                                                                       ATCC30772 has been studied in shake-flask culture (26). In
                                                                       pH-auxostat culture, no improvement in growth or production
                                                                       of lipid and DHA was achieved by changes in the concentra-
                                                                       tions of either sea salts or yeast extract.
                                                                           All of the six strains of C. cohnii that we have examined
                                                                       produced DHA in acetate pH-auxostat culture. It is possible
                                                                       that other strains of this organism (the American Type Cul-
                                                                       ture currently lists some 45 strains) might have even better
ATCC 30772 in acetate pH-auxostat culture is certainly supe-           DHA productivity than strain ATCC 30772. The pH-auxostat
rior to that achieved with this strain using medium containing         is clearly a useful method of cultivation of C. cohnii on ac-
glucose as principal carbon source (26). Overall productivities        etate, since the carbon source is delivered in a controlled
for total lipid and DHA were calculated as 78 and 36 mg L−1h−1,        manner so that its concentration never becomes inhibitory to
respectively, for 98-h cultivation in a pH-auxostat culture using      growth. The pH-auxostat might also be of value for the culti-
a 5% inoculum taken from a similar culture (Figs. 2B, 2C). The         vation of a wide range of oleaginous organisms on acetic acid,
influence of the concentrations of sea salts and yeast extract in       which is an inexpensive carbon source.

               TABLE 2
               Fractionation, by Column Chromatography, of Lipid Extracted from Crypthecodinium cohnii ATCC 30772
               Grown with Glucose as Principal Carbon Source
                                                                    Relative % (w/w) of fatty acyl groups
                                                                         Sterolglycosides and
               Fatty                                Total    Neutral      digalactosyldiacyl       Choline        Acidic
               acid                                 lipid     lipids           glycerols            lipids     phospholipids
               10:0                                    7        7                Tracea             Trace           Trace
               12:0                                   24       19                  19                  3               6
               14:0                                   26       22                  16                11              16
               16:0                                    8        9                  18                19              27
               16:1                                    1        2                   1                  1               2
               18:0                                    1        1                   4                  3               8
               18:1                                    7        9                  12                11              16
               22:6                                   26       31                  30                52              25
               Weight applied (mg)                   121       —                   —                 —               —
               Weight recovered (mg)                 111       82                   9                18              1.3
               Recovery (% original lipid)            92       68                   7                15                1
               Recovery (% total recovered lipid)   (100)      75                   8                16                1
                Trace (<0.5%).

Lipids, Vol. 36, no. 11 (2001)
                                            DHA PRODUCTION BY CRYPTHECODINIUM COHNII                                                      1245

               TABLE 3
               Fractionation, by Column Chromatography, of Lipid Extracted from Crypthecodinium cohnii ATCC 30772
               Grown in pH-Auxostat Culture with Acetic Acid as Principal Carbon Source
                         Relative % (w/w) of fatty acyl groups
                                                                               Sterolglycosides and
               Fatty                                    Total     Neutral       digalactosyldiacyl     Choline       Acidic
               acid                                     lipid      lipids            glycerols          lipids    phospholipids
               10:0                                     Tracea     Trace              Trace             Trace         Trace
               12:0                                         7         8                13                  5          Trace
               14:0                                       21        16                 18                13             16
               16:0                                       18        16                 19                15             28
               16:1                                         2         2                  2              Trace            2
               18:0                                         2         1                  3                4              5
               18:1                                       11        10                 11                11             14
               22:6                                       39        47                 34                52             35
               Weight applied (mg)                       132        —                  —                 —              —
               Weight recovered (mg)                     125        93                 12                19            1.4
               Recovery (% original lipid)                95        70                   9               14              1
               Recovery (% total recovered lipid)       (100)       74                 10                15              1
                Trace (<0.5%).

    Fractionation of the lipid produced from glucose (Table 2)              7. Ykema, A., Verbree, E.C., van Verseveld, H.W., and Smit, H.
and acetate (Table 3) cultures confirmed the observations (27)                  (1986) Mathematical Modelling of Lipid Production by Oleagi-
                                                                               nous Yeasts in Continuous Cultures, Ant. van Leeuwenhoek 52,
that DHA is present in both neutral and polar lipids of C.
cohnii and that choline lipids contain the highest proportion               8. Ratledge, C. (2001) Microorganisms as Sources of Polyunsatu-
of DHA. In the present study, however, most of the DHA is                      rated Fatty Acids, in Structured and Modified Lipids (Gunstone,
present in neutral lipids, simply because of the much greater                  F., ed.) pp. 351–399, Marcel Dekker, New York.
lipid accumulation achieved.                                                9. Singh, A., and Ward, O.P. (1997) Microbial Production of Do-
                                                                               cosahexaenoic Acid (DHA, C22:6), Adv. Appl. Microbiol. 45,
    The material that could be precipitated from supernatant
fluid with 2-propanol has recently been identified as polysac-               10. Shahidi, F., and Wanasundara, U.N. (1998) Omega-3 Fatty Acid
charide (28). The production of exopolysaccharide is likely to                 Concentrates: Nutritional Aspects and Production Technologies,
affect yield and is therefore significant for large-scale cultiva-              Trends Food. Sci. Technol. 9, 230–240.
tion of C. cohnii for DHA production. Thus, carbon and energy              11. Harrington, G.W., and Holz, G.G. (1968) The Monoenoic and
                                                                               Docosahexanenoic Fatty Acids of a Heterotrophic Dinoflagel-
source will be wasted in production of polysaccharide, and it
                                                                               late, Biochim. Biophys. Acta 164, 137–139.
becomes increasingly difficult to supply cultures with sufficient          12. Kyle, D.J. (1992) Production and Use of Lipids from Microal-
dissolved oxygen as their viscosity increases. The presence of                 gae, Lipid Technol. 4, 59–64.
polysaccharide may also interfere with the recovery of the                 13. Kyle, D.J. (1996) Production and Use of a Single Cell Oil Which
DHA-containing oil.                                                            Is Highly Enriched in Docosahexaenoic Acid, Lipid Technol. 8,
    A patent application (GB 00/02695) covering the main as-
                                                                           14. Kyle, D.J., Behrens, P., Bingham, K., Arnett, K., and Lieber-
pects of this work has been filed.                                              man, D. (1998) Microalgae as a Source of EPA-Containing Oils,
                                                                               in Biotechnology for the Fats and Oils Industry (Applewhite,
                                                                               T.H., ed.) pp. 117–122, AOCS Press, Champaign.
ACKNOWLEDGMENT                                                             15. Kyle, D.J. (1994) Microbial Oil Mixtures and Uses Thereof,
This work was funded by the European Community (FAIR CT97-                     Martek Corporation, U.S. Patent 5,374,657.
3146).                                                                     16. Kyle, D.J., Reeb, S.E., and Sicotte, V.J. (1995) Infant Formula
                                                                               and Baby Food Containing Docosahexaenoic Acid Obtained
                                                                               from Dinoflagellates, Martek Biosciences Corporation, U.S.
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Lipids, Vol. 36, no. 11 (2001)

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