U.S. patent application number 11/183831 was filed with the patent office on 2005-11-10 for crypthecodinium cohnii biomass cultured to synthesize docosahexaenoic acid.
This patent application is currently assigned to The University of Hull. Invention is credited to Anderson, Alistair James, de Swaaf, Martin E., Grantham, David John, Kanagachandran, Kanagasooriyam, Ratledge, Colin, Sijtsma, Lolke, Stephenson, Janet Christine.
Application Number | 20050250190 11/183831 |
Document ID | / |
Family ID | 10857245 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250190 |
Kind Code |
A1 |
Ratledge, Colin ; et
al. |
November 10, 2005 |
Crypthecodinium cohnii biomass cultured to synthesize
docosahexaenoic acid
Abstract
C. cohnii is cultured in a suitable growth medium with acetic
acid/acetate as the main carbon source. The acetate is provided,
and replenished, by adding acetic acid to the growth medium in
response to an increase in pH resulting from the utilisation of
acetic acid/acetate by C. cohnii. The C. cohnii produces relatively
high levels of docosahexaenoic acid (DHA). A stationary phase is
not essential for satisfactory DHA production.
Inventors: |
Ratledge, Colin; (Beverly,
GB) ; Anderson, Alistair James; (Hull, GB) ;
Kanagachandran, Kanagasooriyam; (London, GB) ;
Grantham, David John; (Hull, GB) ; Stephenson, Janet
Christine; (Hull, GB) ; de Swaaf, Martin E.;
(Utrecht, NL) ; Sijtsma, Lolke; (Renkum,
NL) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
700 THIRTEENTH ST. NW
SUITE 300
WASHINGTON
DC
20005-3960
US
|
Assignee: |
The University of Hull
North Humberside
GB
HU6 7RH
ATO BV
Wageningen
NL
6708 PD
|
Family ID: |
10857245 |
Appl. No.: |
11/183831 |
Filed: |
July 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11183831 |
Jul 19, 2005 |
|
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|
10030700 |
Jan 14, 2002 |
|
|
|
10030700 |
Jan 14, 2002 |
|
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PCT/GB00/02695 |
Jul 13, 2000 |
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Current U.S.
Class: |
435/134 ;
435/252.3 |
Current CPC
Class: |
A23D 9/00 20130101; A23L
33/12 20160801; A23K 10/16 20160501; C12N 1/125 20210501; C12N 1/12
20130101; C12P 7/6472 20130101; C12R 2001/89 20210501; C12P 7/6427
20130101 |
Class at
Publication: |
435/134 ;
435/252.3 |
International
Class: |
C12P 007/64; C12P
007/54; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 1999 |
GB |
9916537.5 |
Claims
1-39. (canceled)
40. A Crypthecodinium cohnii biomass cultured in an aqueous
nutrient medium containing a compound selected from the group
consisting of acetic acid and acetate ions, the Crypthecodinium
cohnii biomass consuming the acetic acid or acetate ions as the
primary carbon source to synthesize docosahexaenoic acid, wherein
the nutrient medium has an initial concentration of acetic acid or
acetate ions of between 4 and 16 g/l.
41. The Crypthecodinium cohnii biomass according to claim 40,
wherein the initial concentration of acetic acid or acetate ions in
the nutrient medium is about 8 g/l.
42. The Crypthecodinium cohnii biomass according to claim 40,
wherein the culturing process parameters are controlled in a manner
that results in the absence of a stationary phase.
43. The Crypthecodinium cohnii biomass according to claim 40,
wherein the total concentration of docosahexaenoic acid synthesized
by the microorganism is at least 0.9 g/l of cultured medium.
44. The Crypthecodinium cohnii biomass according to claim 40,
wherein at least 29.3 percent docosahexaenoic acid is recovered
from an oil produced by the Crypthecodinium cohnii biomass.
Description
[0001] The invention relates to a method of culturing a
microorganism for the synthesis of a polyunsaturated fatty acid,
particularly docosahexaenoic acid, by the microorganism. The
invention also relates to oils and preparations of fatty acids,
particularly docosahexaenoic acid, prepared from microorganisms
cultured in accordance with the method.
[0002] Such a method is known from EP 0515460 which describes the
culture of the dinoflagellate Crypthecodiniwn cohnii (C. cohnii)
with glucose. The C. cohnii uses the glucose as the carbon source
and synthesises docosahexaenoic acid.
[0003] According to a first aspect of the invention there is
provided a method of culturing a microorganism for the synthesis of
docosahexaenoic acid by the microorganism, comprising culturing the
microorganism with an organic species comprising an acidic group or
an ionised form of an acidic group, the microorganism using the
species as a carbon source and synthesising docosahexaenoic
acid.
[0004] The use of such a species as a carbon source may allow the
production of comparable or greater amounts of docosahexaenoic
acid, for given culture conditions, compared to culture methods
that do not use such a species. Additionally, by choosing suitable,
cheap sources of such species, it may be possible to produce
docosahexaenoic acid more cheaply. Further, in the culture method
described in EP 0515460, it is necessary to impose a stationary
phase in order to achieve satisfactory production of
docosahexaenoic acid. Such a phase may not be necessary when such a
species is provided as a carbon source.
[0005] According to a second aspect of the invention there is
provided a method of culturing a microorganism for the synthesis of
docosahexaenoic acid by the microorganism, comprising culturing the
microorganism with an organic species comprising an acidic group or
an ionised form of an acidic group, the microorganism synthesising
docosahexaenoic acid containing carbon from the species.
[0006] According to the third aspect of the invention there is
provided a method of culturing a microorganism for the synthesis of
a polyunsaturated fatty acid by the microorganism, comprising
culturing C. cohnii with an organic species comprising an acidic
group or an ionised form of an acidic group, the C. cohnii using
the species as a carbon source and synthesising a polyunsaturated
fatty acid.
[0007] The following is a more detailed description of embodiments
of the invention, by way of example, reference being made to the
appended drawings in which:
[0008] FIG. 1 is a schematic representation of part of a
fermentation apparatus;
[0009] FIG. 2 is a graph showing certain parameters over time
during culture of a first microorganism;
[0010] FIG. 3 is a similar graph to FIG. 2 showing the parameters
during culture of a second microorganism;
[0011] FIG. 4 is a similar graph to FIG. 2 showing the parameters
during culture of a third microorganism;
[0012] FIG. 5 is a graph showing dry cell weight over time during
culture of the first microorganism after different
pretreatments;
[0013] FIG. 6 is a graph showing lipid produced over time during
culture of the first microorganism after the pretreatments of FIG.
5;
[0014] FIG. 7 is a graph showing dry cell weight over time during
culture of the first microorganism under different conditions;
[0015] FIG. 8 is a graph showing lipid production over time during
culture of the first microorganism under the different conditions
of FIG. 7;
[0016] FIG. 9 is a similar graph to FIG. 8 in which lipid
production is expressed in a different manner;
[0017] FIG. 10 is a graph showing dry cell levels achieved using
different initial concentrations of sodium acetate;
[0018] FIG. 11 is a graph showing lipid levels achieved using
different initial concentrations of sodium acetate;
[0019] FIG. 12 is a graph showing DHA levels achieved using
different initial concentrations of sodium acetate; and
[0020] FIG. 13 includes two graphs showing various values at
different time intervals during a culture of C. cohnii.
EXAMPLE 1
[0021] In Example 1, six microorganisms are cultured, separately,
in a growth medium to which an organic acid is added. The
microorganisms synthesise docosahexaenoic acid containing carbon
from the organic acid.
[0022] The culture method will be described in detail for one of
the six microorganisms--the strain of C. cohnii available from the
American Type Culture Collection and identified by the number 30772
(C. cohnii ATCC 30772). The remaining five microorganisms are also
strains of C. cohnii available from the American Type Culture
Collection. They are identified, respectively, by the numbers:
30541; 50298; 40750; 30555; and 30557. These five strains of C.
cohnii are cultured in an identical manner to C. cohnii 30772 and
their culture will not be described in detail. Other strains which
are also available from the ATCC or from other sources would be
expected to behave similarly to those described here.
[0023] The culture of C. cohnii 30772 is performed in a 5 L
fermenter 10 of known type (see FIG. 1). The fermenter 10 is
provided with a pH electrode 11 that is positioned in the fermenter
10 for monitoring the pH of the growth medium in the fermenter 10.
The pH electrode 11 is connected via a control device 12 to a pump
13. The control device 12 is programmed to cause the pump 13 to
pump a liquid from a reservoir 14 into the fermenter 10 when the pH
electrode 11 detects a pH greater than a predetermined value. This
process is described in greater detail below.
[0024] The fermenter 10 is also provided with a thermometer (not
shown) that is positioned for monitoring the temperature of the
growth medium (and also the C. cohnii 30772) in the fermenter 10.
The thermometer is connected via a control device (not shown) to a
heater (not shown). The control device is programmed to control the
heater on the basis of the temperature determined by the
thermometer so as to maintain the growth medium (and the C. cohnii
30772) at a desired temperature within the fermenter 10.
[0025] The fermenter 10 is also provided with an aerator (not
shown) that is connected to a source of air (not shown) for
aerating the growth medium within the fermenter 10. An oxygen
sensor (not shown) is positioned for measuring dissolved oxygen
concentration in the growth medium within the fermenter 10. A
stirrer (not shown) is provided for stirring the growth medium (and
the C. cohnii 30772). The oxygen sensor is connected to the stirrer
by a control device (not shown) for varying the speed of the
stirrer in response to the oxygen concentration detected by the
oxygen sensor so as to maintain the oxygen concentration at a
desired level.
[0026] Before initiating the culture, the C. cohnii 30772 used to
inoculate the fermenter 10 (the "inoculum") is prepared from a
standing culture of this organism using a pre-inoculation medium.
The pre-inoculation medium contains (initially) glucose (27 g/L),
yeast extract (3.8 g/L ) and sea salts (25 g/L--commercial
preparation purchased from SIGMA, Dorset, UK). Before use, the pH
of the pre-inoculation medium is corrected to 6.5 by the addition
of 1M KOH and the medium is autoclaved at 121.degree. C. for 30
minutes (the glucose is sterilised separately to avoid
caramelisation).
[0027] The inoculum is prepared by adding 10 ml of the standing
culture (4 days old) to a 250 ml flask containing 100 ml of the
pre-inoculation medium. The flask is incubated at 27.degree. C.
while shaking at 200 rpm for 3 days. 70 ml of the incubated mixture
is then added to a 2 L flask containing 700 ml of pre-inoculation
medium. The 2 L flask is then incubated at 27.degree. C. while
shaking at 200 rpm for 3 days after which the full content of the 2
L flask forms the inoculum.
[0028] The growth medium used for culture in the fermenter 10
contains (initially) sodium acetate (1 g/L), yeast extract (7.5
g/L) and sea salts (25 g/L). Before use, the pH of the growth
medium is corrected to 6.5 by the addition of 1M KOH and the medium
is autoclaved at 121.degree. C. for 1 hour.
[0029] Glacial acetic acid mixed with water to give a 50% (v/v)
solution is added to the reservoir 14.
[0030] The culture is initiated by adding the inoculum into the
fermenter 10. The total volume in the fermenter 10 is made up to
3.5 L with growth medium. The fermenter 10 is then closed.
[0031] The control device connected to the thermometer and the
heater is set so that the heater maintains the growth medium and
the C. cohnii 30772 at 27.degree. C.
[0032] The aerator is set to aerate the growth medium at a rate of
1 volume of air per volume of medium per minute. The control device
connected to the oxygen sensor and the stirrer is set so that the
dissolved oxygen concentration is maintained above 20% of air
saturation. In order to achieve this, the speed of the stirrer
varies between 300 to 1000 rpm.
[0033] Under these conditions, the C. cohnii 30772 grows readily.
As the C. cohnii 30772 grows it uses the acetate from the growth
medium as a carbon source. In fact, it is believed that the C.
cohnii 30772 takes up acetic acid formed from the acetate-acetic
acid being formed in accordance with the equilibrium reaction:
acetate +H.sup.+=acetic acid. Acetate is, of course, a carboxylate
ion--it comprises the ionised form of the carboxylic acid group. As
the cells use the acetate/acetic acid, the pH of the growth medium
increases. As indicated above, the pH of the growth medium is
monitored by the pH electrode 11 and when the pH exceeds a pre-set
value (slightly greater than 6.5), the control device 12 causes the
pump 13 to pump acetic acid solution from the reservoir 14 to the
fermenter 10 where it mixes with (and is incorporated into) the
growth medium. The addition of the acetic acid solution reduces the
pH of the growth medium. The arrangement is such that the pH of the
growth medium remains at about 6.5 during the culture.
[0034] Acetic acid added in this way dissociates to form, in
equilibrium with the acid as indicated above, acetate and protons.
The acetic acid derived from the added. acetic acid solution, and
the acetate formed from this acetic acid, are used as a carbon
source by the C. cohnii 30772. As indicated above, it is believed
that it is acetic acid that is actually take up by the C. cohnii
30772. However acetate may also be taken up (if it is not, it is
used as a carbon source by first being converted to acetic acid).
The use of the acetic acid/acetate (derived from the added acetic
acid) by the C. cohnii similarly leads to an increase in the pH of
the growth medium and this is counteracted by the addition of more
acetic acid--so as to maintain the pH at about 6.5--as described
above.
[0035] By adding acetic acid over a period of time, a considerable
amount of acetic acid/acetate can be provided as a carbon source
for the C. cohnii 30772 while, at any particular moment in time,
the concentrations of the acetate and the acetic acid are
sufficiently low so as not to be detrimental to growth of the C.
cohnii 30772. An advantage of this method of cultivation is that it
maintains the acetate/acetic acid concentration approximately
constant so that it is optimal for the process. The system of
adding acetic acid in response to an increase in pH caused by use
of acetic acid/acetate is a convenient way of providing acetic
acid/acetate as it is needed to replenish acetic acid/acetate used
by the microorganisms. The addition of acetic acid in this way
allows optimum growth of the C. cohnii 30772, a high final biomass
(consisting of cells of C. cohnii 30772), a high content of oil in
the cells and a high proportion of docosahexaenoic acid within the
oil.
[0036] The acetic acid/acetate is the sole or main carbon source
for the C. cohnii 30772--small amounts of other carbon sources may
be provided in the yeast extract.
[0037] The yeast extract present in the growth medium is provided,
primarily, as a source of nitrogen, essential vitamins, amino acids
and growth factors. The sea salts are needed for osmo-protection of
the C. cohnii 30772, which are marine cells in origin.
[0038] If, during the culture, the pH falls below 6.5, the pH can
be brought back to 6.5 by adding NaOH (2M). This can be done
automatically.
[0039] During the culture method, samples of the C. cohnii 30772
together with growth medium are taken at time intervals. Each
sample is analysed to determine dry cell weight, lipid content,
docosahexaenoic acid content, and polysaccharide content. The
results of these analyses are shown in FIG. 2 which shows that dry
cell weight, lipid content, docosahexaenoic acid content and
polysaccharide content increase with time over the course of the
culture method. Significantly, FIG. 2 shows that docosahexaenoic
acid forms a substantial part of the lipid synthesised by the C.
cohnii 30772.
[0040] As the acetic acid/acetate is a carbon source, the
docosahexaenoic acid will contain carbon derived from the acetic
acid/acetate.
[0041] Growth of C. cohnii 30772 proceeds until the cells reach
their maximum biomass yield (after about 140 hours of culture in
this Example). This is marked by the fact that the pump 13 ceases
to operate, indicating that the C. cohnii 30772 is no longer
consuming acetic acid/acetate. At this stage, no further production
of lipid (including docosahexaenoic acid) will occur.
[0042] At this stage, the C. cohnii 30772 cells and the growth
medium are removed from the fermenter 10 and the biomass (cells of
C. cohnii 30772) is separated. The oil content of the biomass is
extracted using known methods. This oil contains docosahexaenoic
acid and is useful as a food or food supplement. The
docosahexaenoic acid may be partially or totally purified from the
oil and again the purified preparations are useful as food or food
supplements. Docosahexaenoic acid in its free acid form, and also
esters of docosahexaenoic acid with simple alcohols (e.g. methanol,
ethanol, etc) may be prepared from the oil.
[0043] Table 1 shows dry cell content, lipid content and the
content of docosahexaenoic acid expressed as a percentage of the
total fatty acid content of the lipid, as measured after C. cohnii
30772 has ceased to grow. Dry cell content and lipid content are
expressed as g per L of growth medium and biomass. Table 1 also
shows the maximum lipid content in the cells. This is the highest
value of lipid content per dry cell weight over the course of the
culture. Additionally, Table 1 shows the same parameters measured
after culturing the other five strains of C. cohnii (strain Nos.
30541, 50298, 40750, 30555, and 30557) under identical
conditions.
1TABLE 1 Growth of six different strains of Crypthecodinium cohnii
obtained from the American Type Culture Collection (ATCC) on
acetate/acetic acid as described in Example 1. Values after 140 h
of growth DHA in total Maximum lipid in Strain Dry cells Lipid
fatty acid from cell (g lipid/100 g (ATCC no) (g/L) (g/L) lipid (%)
cells) 30772 17 7.48 59 44 30541 45.5 8.32 33 24 50298 45.5 12.3 31
30 40750 17 3.31 46 22 30555 20.7 2.8 30 15 30557 25.6 4.7 40 18
DHA - Docosahexaenoic acid
[0044] Table 2 shows further measured parameters and derived values
relating to the end points (140 hours of growth) of cultures of the
six strains of C. cohnii. Specifically, Table 2 shows the amounts
of acetic acid utilised and the final volumes of the cultures.
Table 2 also shows the yields of biomass, docosahexaenoic acid and
lipid.
2TABLE 2 Yields of biomass, lipid and DHA of six different strains
of C. cohnii obtained from the American Type Culture Collection
(ATCC) on acetate/acetic acid as described in Example 1. Acetic
acid Yields Yields Yields Strain utilised/ of Biomass of DHA of
Lipid ATCC no Final volume (g/g acetic acid utilised) 30772 607
g/4.2 L 0.118 0.03 0.05 30541 1039 g/4.6 L 0.2 0.012 0.037 50298
1107 g/5.2 L 0.21 0.018 0.06 40750 340 g/4.5 L 0.22 0.026 0.044
30555 498 g/4.2 L 0.18 0.007 0.024 30557 754 g/4.7 L 0.16 0.01
0.029
[0045] As will be seen from Tables 1 and 2, there is a considerable
variation in the production and yields of dry cells/biomass, lipid
and docosahexaenoic acid between the different strains of C.
cohnii. Each of these strains, however, produces potentially useful
amounts of docosahexaenoic acid.
[0046] FIGS. 3 and 4 show the same parameters as those shown in
FIG. 2--during the culture of C. cohnii 30541 and 50298,
respectively. The results are similar to those with C. cohnii
30772.
[0047] It will be appreciated that the culture method of Example 1
constitutes a convenient method for the production of
docosahexaenoic acid containing biomass. Moreover, this method may
produce comparable or greater amounts of docosahexaenoic acid
compared to methods in which glucose is used as the sole carbon
source.
[0048] It will be appreciated that the method of Example I may be
varied.
[0049] For example, additional nutrients could be added to the
acetic acid solution in reservoir 14 so that the nutrients are
added to the fermenter 10 together with the acetic acid. Suitable
additional nutrients comprise, for example, sources of nitrogen,
sources of phosphorus, vitamins and additional salts.
Alternatively, instead of adding the nutrients directly to the
acetic acid solution in the reservoir 14, the nutrients could be
provided in the form of a separate solution in a separate
reservoir. Nutrients from the separate reservoir would be pumped
into the fermenter 10 by a separate pump. The separate pump would
be controlled by the control device 12 and may, for example,
operate at the same time as the pump 13 operates. If additional
nutrients are added using a separate pump, the separate pump may be
operated to add the nutrients during part, but not all, of the
culture period.
[0050] Additional carbon source compounds could be added together
with the acetic acid. Suitable compounds comprise, for example,
oils and lipids containing various precursors of docosahexaenoic
acid. Glycerol and glycerophosphate may also contribute to good
lipid production.
[0051] The acetic acid used could be industrial grade or may be
produced by other chemical processes either as a primary or a
secondary product. The presence of other aliphatic acids within the
acetic acid solution would not be deleterious and may provide
additional carbon sources. Waste solutions from industrial
processes comprising acetic acid together with other compounds may
be suitable for use in place of the acetic acid solution in
reservoir 14.
[0052] Instead of acetic acid/acetate, other organic species
comprising an acidic group-or an ionised form of an acidic group,
or mixtures of such species, can be used as a carbon source.
Preferably, the carbon source is a carboxylic acid or a carboxylate
ion, or mixtures of these species. If the carbon source is provided
by adding an organic acid to the growth medium, then, preferably
the utilisation of the organic acid (and/or its ionised form) by
the microorganism results in an increase in the pH of the growth
medium.
[0053] The volume of the inoculum may vary. Preferably, this volume
will be between 1% and 20% of the initial volume of the culture
mixture. More preferably, the volume will be between 5 and 10% of
the initial volume of the culture mixture.
[0054] Whereas the method of Example 1 is a fed-batch process, it
will be appreciated that the culture method may be adapted to be
performed as a continuous or semi-continuous process. In this case,
the acetic acid and other compounds needed for cell growth, cell
maintenance and for oil production would be added into the
fermenter throughout the course of the culture. Cells and spent
medium would be removed from the fermenter either continuously
during the course of the culture or from time to time depending on
the volume of the culture or density of the cells. Unlike the
culture method described in EP 0515460, which uses glucose as an
apparently sole carbon source, the use of acetic acid/acetate as a
carbon source does not require the imposition of a stationary phase
in order to cause satisfactory production of docosahexaenoic acid.
This is particularly advantageous as it will facilitate adaptation
of the method to be a continuous or semi-continuous process.
[0055] Although the method of Example 1 is performed in a 5 L
fermenter 10, it will be appreciated that the method can be
performed in larger, industrial scale fermenters. When culture is
performed in larger fermenters, it may be advantageous to add the
acetic acid at a plurality of different points into the fermenter.
This would aid dispersion of the acid and avoid build-up of any
local high concentration of acid.
[0056] Whereas, in the method of Example 1, the oxygen
concentration is controlled by controlling the stirring speed, it
will be appreciated that oxygen concentration may be controlled by
controlling the aeration rate or by controlling both aeration rate
and stirring speed.
[0057] The configuration of the fermenter 10 may be changed and an
air-lift fermenter, or a fermenter having appropriate aeration and
mixing without mechanical stirring, may be used.
[0058] Microorganisms other than C. cohnii may be used. When C.
cohnii is used, it may be used to synthesise other polyunsaturated
fatty acids instead of, or in addition to docosahexaenoic acid.
[0059] The initial concentration of sodium acetate in the growth
medium may be varied. It may be between 0.5 g/L and 20 g/L.
Preferably, it will be between 1 g/L and 10 g/L.
[0060] The pH of the growth medium may be maintained at values
other than 6.5 during the culture. The pH may be maintained at
values between 5 and 8. Preferably, the pH is maintained between 6
and 7.5. The acceptable and preferred pH values will depend on the
microorganism used.
EXAMPLE 2
[0061] In the method of Example 2, C. cohnii 30772 is grown, prior
to culture in the fermenter 10 as described above in Example 1,
with acetic acid/acetate. It is hypothesised that this will allow
the cells to adapt to acetic acid/acetate so that they will grow
more quickly during culture in the fermenter 10.
[0062] The method of Example 2 is similar to the method of Example
1 with the exception of the preparation of the inoculum. The
inoculum is prepared by adding 10 ml of the standing culture of C.
cohnii 30772 to a 250 ml flask containing 100 ml of the
pre-inoculation medium described above in Example 1. The flask is
incubated at 27.degree. C. while shaking at 200 rpm for 3 days.
After 3 days, 70 ml of the incubated mixture is added to a 1 L
fermenter and the volume in the 1 L fermenter is made up to 700 ml
with the growth medium described above in Example 1. The 1 L
fermenter is then closed and the fermenter is heated and aerated in
the same manner as the 5 L fermenter 10 of Example 1. Additionally,
acetic acid is added in response to increases in pH in the same
manner as described in Example 1. The C. cohnii 30772 is allowed to
grow in the 1 L fermenter for 72 hours after which the content of
the 1 L fermenter forms the inoculum.
[0063] After this period, 700 ml of inoculum is taken from the 1 L
fermenter and cultured with the growth medium of Example 1 in the 5
L fermenter 10, exactly as described above in Example 1. During the
culture in the 5 L fermenter, samples are taken at time intervals
for analysis of dry cell weight and lipid content. The results of
these analyses are shown in FIGS. 5 and 6, respectively. For
comparison, FIGS. 5 and 6 also show comparable analyses on samples
taken during the culture of C. cohnii 30772 during Example 1. As
will be seen from FIGS. 5 and 6, both dry cell weight and lipid
content increase more rapidly during culture in the 5 L fermenter
10 when the C. cohnii 30772 has been pre-grown with acetic
acid/acetate (in Example 2), as opposed to being pre-grown with
glucose as a carbon source (in Example 1).
EXAMPLE 3
[0064] In Example 3, C. cohnii 30772 is grown with acetic
acid/acetate in the same manner as in Example 1, and with different
concentrations of sea salts.
[0065] A first inoculum of C. cohnii 30772 is prepared as described
above in Example 2. The first inoculum is then cultured in the 5 L
fermenter 10, exactly as described in Example 1.
[0066] A second inoculum is prepared in the same way as the first
inoculum. The second inoculum is then cultured in the 5 L fermenter
10, as described in Example 1, but with an initial concentration of
sea salts in the growth medium of 16 g/L (rather than 25 g/L in
Example 1).
[0067] During the culture of the first and second inocula, samples
are taken at time intervals for the analysis of dry cell weight and
lipid content. The results of these analyses are shown in FIGS. 7,
8 and 9. As will be seen from these Figures, culture of the C.
cohnii 30772 with 16 g/L sea salts results in poorer lipid
production compared to culture with 25 g/L sea salts.
EXAMPLE 4
[0068] In Example 4, C. cohnii 30772 was cultured in seven separate
batch cultures. Each culture was performed as described above in
Example 1 with the exception that in six of the cultures, the
initial sodium acetate concentrations in the growth media were,
respectively, 4, 6, 8, 10, 12 and 16 g/L. In the seventh culture
the initial sodium acetate concentration in the growth medium was 1
g/L (i.e. this culture was performed identically to the culture of
C. cohnii 30772 in Example 1).
[0069] The results are shown in FIGS. 10 to 12. FIG. 10 shows the
dry cell levels (in g/L) at various time intervals over the 140
hour cultures. FIG. 11 shows the lipid production (in g/L) at the
same time intervals and FIG. 12 shows the DHA production (in g/L)
at the same time intervals. The highest levels of dry cells, lipid
and DHA were obtained in the culture that used 8 g/L (initial
concentration) sodium acetate in the growth medium. The lowest
levels of dry cells, lipid and DHA were seen in the culture that
used 16 g/L (initial concentration) in the growth medium. The
levels of dry cells, lipid and DHA were broadly similar in the
cultures that used 1, 4, 6 and 12 g/L sodium acetate.
[0070] The results may indicate that initial concentrations of
sodium acetate of 16 g/L or above are inhibitory for growth of C.
cohnii 30772.
EXAMPLE 5
[0071] C. cohnii was cultured generally as described in Example 1,
but using a growth medium containing 10 g/L yeast extract (compared
to 7.5 g/L in Example 1) and 8.0 g/L sodium acetate (compared to I
g/L in Example 1). The growth medium also contained 25 g/L sea salt
as per the growth medium of Example 1.
[0072] As shown in FIG. 13, it was found that increases in dry cell
weight, total lipid and DHA levels continued up until 209 hours of
culture (as opposed to 140 hours in Example 1), with higher overall
levels of dry cell weight, total lipid and DHA being achieved after
209 hours than those achieved after 140 hours in Example 1. The dry
cell weight, total lipid and total DHA content increased linearly
between 72 and 209 hours of cultivation. A stationary phase was not
necessary in order to obtain satisfactory production of DHA. The
maximum overall volumetric DHA productivity was achieved after 209
hours of cultivation. The dry cell weight, total lipid and total
DHA were then, 51.1, 27.6 and 8.0 g/L, respectively.
[0073] It is believed that the increased productive culture time
and the increased levels of dry cell weight, lipid and DHA may be
caused by the additional yeast extract.
[0074] FIG. 13 also shows that the percentage content of lipid in
the cells increased over time, to a maximum of 54.0% at 209 hours.
The DHA content of the extracted lipid varied between 30% and 40%,
and was stable at about 30% between 67 and 239 hours.
[0075] Values of dry cell weight, total lipid content and DHA
content are given at various time intervals in Table 3 which also
gives various other values. It is noteable that the overall
volumetric production of DHA was 38.5 mg/l/h.
3TABLE 3 time H Ac DCW lipid DHA tlipid tDHA QDHA h g/l g/l % % g/l
g/l mg/l/h 0 0.0 0.7 8.2 33.9 0.1 0.0 0.0 19 3.3 1.8 5.7 37.3 0.1
0.0 2.1 24 6.3 2.7 5.8 36.4 0.2 0.1 2.4 43 30.4 7.0 9.1 36.4 0.6
0.2 5.5 47 39.7 9.0 10.3 35.5 0.9 0.3 7.0 67 76.9 12.0 20.0 31.0
2.4 0.7 11.2 72 86.5 14.0 19.5 31.5 2.7 0.9 12.0 91 123.6 19.3 28.3
29.3 5.4 1.6 17.5 119 178.4 26.7 40.4 29.6 10.8 3.2 26.9 141 220.5
32.6 45.8 28.9 14.9 4.3 30.7 163 262.3 37.6 49.4 29.6 18.6 5.5 33.7
186 297.0 42.3 52.3 29.6 22.1 6.5 35.2 209 327.1 51.1 54.0 29.1
27.6 8.0 38.5 239 361.2 51.1 54.2 30.0 27.7 8.3 34.8 Abbreviations
Explanation H Ac Total amount of acetic acid added (in g) DCW Dry
cell weight (in g/l) lipid Lipid content of the dry cells (in %)
DHA DHA content of the lipid (in %) tlipid Total lipid
concentration (in g/l) tDHA Total DHA concentration (in g/l) QDHA
Overall volumetric productivity of DHA (in mg/l/h)
EXAMPLE 6
[0076] In a system where both acetic acid and glucose were fed it
was demonstrated that acetic acid was used by C. cohnii and not
glucose as the glucose added to the culture was still present in
the medium. Apparently, C. cohnii prefers acetic acid above glucose
and consumes only acetic acid when both acetic acid and glucose are
offered.
[0077] The medium (800 ml) consisted of 10 g yeast extract, 25 g
sea salt, 4.5 g glucose and 6.3 g sodium acetate. The inoculum (100
ml) was grown on 25 g/l sea salt, 6.2 g/l sodium acetate, 25 g/l
glucose and 5.5 g/l yeast extract.
[0078] The feed was added via the pH control system described in
Example 1 in order to maintain the pH at 6.5. The feed contained
273 g/l glucose and 280 g/l acetic acid. Microorganisms cultured by
the methods described above can be used as a food or food
supplement. Before such use the microorganisms may be separated
from the culture medium and, if desired, subjected to processes
such as drying, freeze-drying, etc. The cultured microorganisms may
be particularly suitable as a food or food supplement for fish.
* * * * *