U.S. patent application number 15/405559 was filed with the patent office on 2017-05-11 for production of docosahexaenoic acid and/or eicosapentaenoic acid and/or carotenoids in mixotrophic mode by nitzschia.
The applicant listed for this patent is FERMENTALG. Invention is credited to Pierre CALLEJA, Francois GODART, Khadidja ROMARI.
Application Number | 20170130190 15/405559 |
Document ID | / |
Family ID | 46754530 |
Filed Date | 2017-05-11 |
United States Patent
Application |
20170130190 |
Kind Code |
A1 |
ROMARI; Khadidja ; et
al. |
May 11, 2017 |
PRODUCTION OF DOCOSAHEXAENOIC ACID AND/OR EICOSAPENTAENOIC ACID
AND/OR CAROTENOIDS IN MIXOTROPHIC MODE BY NITZSCHIA
Abstract
New strains of microalgae belonging to the Nitzschia genus allow
high-yield production of lipids such as docosahexaenoic acid (DHA)
and eicosapentaenoic acid (EPA), and/or carotenoids such as
fucoxanthin. Biomass containing the microalgae can be charged with
fucoxanthin representing more than 0.2% by weight of the dry
matter.
Inventors: |
ROMARI; Khadidja;
(Clermont-ferrand, FR) ; GODART; Francois;
(Genissac, FR) ; CALLEJA; Pierre; (Bordeaux,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FERMENTALG |
Libourne |
|
FR |
|
|
Family ID: |
46754530 |
Appl. No.: |
15/405559 |
Filed: |
January 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14385502 |
Sep 16, 2014 |
9580730 |
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PCT/FR2013/050542 |
Mar 15, 2013 |
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15405559 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 13/00 20130101;
C12P 7/6427 20130101; C12P 7/6472 20130101; C12P 23/00 20130101;
C12N 1/12 20130101; C12R 1/89 20130101 |
International
Class: |
C12N 1/12 20060101
C12N001/12; C12N 13/00 20060101 C12N013/00; C12P 23/00 20060101
C12P023/00; C12R 1/89 20060101 C12R001/89; C12P 7/64 20060101
C12P007/64 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2012 |
FR |
1252378 |
Aug 16, 2012 |
FR |
1257843 |
Claims
1. Biomass consisting essentially of microalgae of the genus
Nitzschia and water, wherein said microalgae of the genus Nitzschia
are charged with fucoxanthin, said fucoxanthin representing more
than 0.2% by weight of the biomass dry matter.
2. The biomass of claim 1, wherein said fucoxanthin represents more
than 0.25% by weight of the biomass dry matter.
3. The biomass of claim 1, wherein the biomass comprises at least
30 g/L of dry matter.
4. The biomass of claim 1, wherein the biomass is product of
fermentation.
5. The biomass of claim 4, wherein the biomass is obtained from a
culture that has been cultured in mixotrophic mode over several
generations in a culture medium comprising a carbon-containing
substrate.
6. The biomass of claim 1, wherein said microalgae of the genus
Nitzschia is the strain FCC 810, deposited with the CCAP (Culture
Collection of Algae and Protozoa) under the accession number CCAP
1052/21.
Description
[0001] The invention relates to a method of culture in mixotrophic
mode, in particular in the presence of discontinuous and/or
variable illumination with light, of a microalga of the genus
Nitzschia, in particular of the species Nitzschia brevirostris. The
method makes it possible to obtain a high yield of biomass and
enrichment of the thus cultured microalgae in lipids and more
particularly in docosahexanoic acid (DHA) and/or eicosapentaenoic
acid (EPA). The method also makes it possible to obtain an
enrichment in carotenoids of the thus cultured microalgae, and more
particularly, in fucoxanthin. The method thus makes it possible to
select strains of Nitzschia, in particular of Nitzschia
brevirostris, with mixotrophic character, and having a high yield
of lipids and more particularly of polyunsaturated fatty acids,
and/or a high yield of carotenoids, more particularly fucoxanthin.
The invention also relates to a novel strain of microalga belonging
to the species Nitzschia brevirostris, particularly suitable for
the production of carotenoids. This novel strain of Nitzschia
brevirostris is useful for producing docosahexaenoic acid (DHA)
and/or eicosapentaenoic acid (EPA) and/or fucoxanthin in
mixotrophic mode.
PREAMBLE
[0002] Microalgae are autotrophic photosynthetic microorganisms,
i.e. they have the ability to grow autonomously by
photosynthesis.
[0003] Microalgae develop both in the marine aquatic media and in
fresh or brackish waters, as well as in various land habitats.
[0004] Most species of the microalgae found in fresh water or in
the oceans are generally autotrophic, i.e. they can only grow by
photosynthesis. For these species, the presence in their
environment of organic carbon-containing substrates or organic
matter is not favourable to them, and does not improve their
growth. However, a certain number of species of microalgae, of very
varied families and origins, are found to be not strictly
autotrophic. Thus, some of them, said to be heterotrophic, are
capable of developing in the total absence of light, by
fermentation, i.e. by using organic matter.
[0005] Other species of microalgae, for which photosynthesis
remains essential for their development, are capable of benefiting
both from photosynthesis and from organic matter present in their
environment. These intermediate species, said to be mixotrophic,
can be cultured in the presence of both light and organic
matter.
[0006] This particularity of so-called "mixotrophic" algae seems to
be related with their metabolism, which allows them to carry out
photosynthesis and fermentation simultaneously. Both types of
metabolism co-exist with a positive overall effect on the growth of
the algae [Yang, C. et al. (2000); Biochemical Engineering Journal,
6: 87-102].
[0007] Microalgae are currently the subject of numerous industrial
projects since some species are capable of accumulating or
secreting major quantities of lipids, in particular polyunsaturated
fatty acids.
[0008] Among these polyunsaturated fatty acids, certain highly
unsaturated fatty acids (HUFA) of the omega-3 series
(PUFA-.omega.3), in particular eicosapentaenoic acid (EPA or C20:5
107 3) and docosahexaenoic acid (DHA or C22:6 107 3), and of the
omega-6 series (PUFA-.omega.6), in particular arachidonic acid (ARA
or AA or also eicosatetraenoic acid C20:3 107 6) have a recognized
nutritional importance, and have strong potential in terms of
therapeutic applications.
[0009] Regarded as an essential nutrient, DHA is necessary to the
normal functional development of cells, and plays a crucial role in
various biochemical processes and functions. Its polyunsaturated
character confers on it a crucial importance in relation to the
properties of the cell membrane, both in plants and in animals:
fluidity, flexibility and selective permeability allowing for
example efficient adaptation, and even survival, at low
temperatures in particular in fish.
[0010] DHA is a major structural constituent of the human brain.
DHA represents 15-20% of the cerebral cortex (an adult's brain
contains at least 20 g of DHA) and 30-60% of the retina. It is
essential for the development of the nervous system and for retinal
function, by incorporation into the cell membranes, and plays a
major role in the acquisition and the satisfactory maintenance of
the mechanisms of vision and memory.
[0011] Fish oils, from the fishing industry, are currently the main
commercial source of these types of fatty acids. However, while
these oils find new applications (food supplement in aquaculture,
incorporation into margarines), marine halieutic resources are
becoming scarce because of intensive fishing activity.
[0012] Therefore, new sources of these fatty acids such as EPA, DHA
and ARA, have to be sought in order to meet, in the future, the
increasing demand for these types of polyunsaturated fatty
acids.
[0013] In addition to their capability of synthesizing fatty acids
de novo, microalgae provide several advantages compared to fish
oils: they may be cultured in vitro under controlled conditions,
which allows production of a biomass of a relatively constant
biochemical composition, and, in addition, unlike fish oils, they
do not have an unpleasant odour and their lipids contain little or
no cholesterol.
[0014] Finally, the lipids produced by microalgae have a simpler
fatty acid profile than that of fish oils, which limits the steps
for separating the fatty acids of interest.
[0015] At present, the classification of algae is still based
widely on morphological criteria and on the character of the
photosynthetic pigments that their cells contain. Consequently, it
is not very indicative of the autotrophic, heterotrophic or
mixotrophic character of the algal species, whereas the latter
cover a very great diversity of species and forms [Dubinsky et al.
(2010); Hydrobiologia, 639:153-171].
[0016] The taxonomic classification of eukaryotic algae contains 14
phyla. Large variations exist among the species of the different
classes making up these phyla that produce fatty acids, as regards
the polyunsaturated fatty acid content of the microalgae. Moreover,
the relative proportions of lipids, in particular of EPA, DHA and
of ARA in the lipid profiles, vary according to the species and the
culture conditions.
[0017] On the other hand, carotenoids are also molecules of
interest. They are generally used as pigments, but they also have
an important role for human health as antioxidant agents. Finally,
they have the ability to stimulate the immune system. Fucoxanthin
is an example of a carotenoid, and is in particular contained in
wakame, an alga used in Japanese cuisine.
[0018] To implement the production of fatty acids and/or
carotenoids by microalgae on an industrial scale, several factors
must be taken into account. For example, cultures may be carried
out under autotrophic, mixotrophic or heterotrophic conditions
depending on the strain, the temperature, the lighting conditions
and the size of the fermenters. For example, cultures may also be
carried out in 1 L containers, in a laboratory, in
photo-bioreactors, and in 100,000 L containers or in open ponds
(several hectares). However, the costs of energy and other
resources such as manpower and the ease of monitoring the culture
must be taken into account for developing ideal culture
conditions.
[0019] In any case, it is desirable that the microalgae are
cultured under optimum conditions for increasing the yield of the
fatty acid(s) and/or carotenoid(s) to be produced. Thus, it is
preferable to have a yield that is as high as possible (for example
biomass above 30 g/l of dry matter, and more than 20% of fatty
acids by weight relative to the dry matter). For carotenoids, a
yield above 0.2% of microalgal dry matter is desirable.
[0020] The microalgae of the genus Nitzschia are marine diatoms
that are generally found in cold seas such as those of the Arctic
or the Antarctic. These microalgae are known mainly for the
production of eicosapentaenoic acid (EPA) in heterotrophic
mode.
[0021] However, it is to be noted that the mixotrophic mode has
been studied on the species Nitzschia laevis in order to assess its
impact on the synthesis of EPA.
[0022] Thus, it was after numerous experiments under unusual
lighting conditions and with the addition of various substrates
that the applicant succeeded in isolating microalgal strains of the
species Nitzschia brevirostris that can be cultured in mixotrophic
mode, allowing, under the conditions of the present invention, a
high-yield production of polyunsaturated fatty acids, in particular
DHA and/or EPA and/or fucoxanthin.
[0023] One strain (FCC 810) representing novel strains of Nitzschia
brevirostris thus isolated and selected, was deposited at the CCAP
(Culture Collection of Algae and Protozoa, Scottish Association for
Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37
1QA, Scotland, United Kingdom) according to the provisions of the
Treaty of Budapest, under the accession number CCAP 1052/21.
[0024] The method of culture and selection consisted more
particularly of culturing the microalgae under mixotrophic
conditions, in the presence of variable and/or discontinuous
illumination, notably in the form of flashes, with a range of
specific variations of light intensity and frequency.
[0025] The frequent alternation of illuminated phases and phases of
darkness or of lower light intensity, generally perceived as
stressful for microalgae, surprisingly, made it possible to obtain
a high production of biomass, of lipids and more particularly of
polyunsaturated fatty acids, and/or one or more carotenoid(s), in
particular fucoxanthin, from the strains of Nitzschia brevirostris.
The application of such strains according to the invention opens
the perspective of industrial production of polyunsaturated fatty
acids, in particular of DHA and/or of EPA and/or of fucoxanthin, in
fermenters benefiting from a reduced light supply, and should
therefore make possible energy savings compared to autotrophic
modes of culture.
[0026] The different aspects and advantages of the invention are
detailed below.
DETAILED DESCRIPTION
[0027] The present invention therefore relates to a method of
culture of microalgae of the genus Nitzschia, in particular of the
species Nitzschia brevirostris, in mixotrophic mode, under
conditions of illumination that is discontinuous and/or variable
over time, the illumination having variations in intensity, the
amplitude of which is generally comprised between 5
.mu.molm.sup.-2s.sup.-1 and 1,000 .mu.molm.sup.-2s.sup.-1,
preferably between 30 and 400 .mu.molm.sup.-2s.sup.-1. These
variations may generally take place between 2 and 3,600 times per
hour, preferably between 2 and 200 times per hour. The method
according to the invention allows an enrichment of the microalgae
of the genus Nitzschia in polyunsaturated fatty acids, more
particularly in DHA and/or EPA, and/or carotenoid(s), more
particularly in fucoxanthin.
[0028] These culture conditions make it possible to supply a
defined quantity of light. This light supply may comprise phases of
discontinuous and/or variable illumination, with variations in
intensity that may have identical or different amplitudes. The
illumination may in particular be in the form of flashes.
[0029] The advantage of this method is to increase the yield of
biomass obtained from the culture. The other is to enrich the thus
cultured microalgae in polyunsaturated fatty acids, more
particularly in docosahexaenoic acid (DHA) and/or eicosapentaenoic
acid (EPA), and/or carotenoid(s), more particularly in fucoxanthin.
This method can also be used for selecting strains of the genus
Nitzschia, in particular of the species Nitzschia brevirostris,
with a mixotrophic character, and having a high yield of
polyunsaturated fatty acids, in particular of DHA and/or EPA,
and/or of carotenoid(s), more particularly of fucoxanthin.
[0030] Culture of this microalga in mixotrophic mode is preferably
carried out in the presence of 5 mM to 1 M, preferably from 50 mM
to 800 mM, more preferentially from 70 mM to 600 mM, and even more
preferentially from 100 mM to 500 mM of an organic
carbon-containing substrate. The substrate is supplied continually
during the culture, so as to allow the cells to accumulate a
significant concentration of lipids. Additional substrate is added
to the culture medium during the culture process so as to maintain
a constant concentration. This organic carbon-containing substrate
comprises preferentially, in pure form or as a mixture: glucose,
derivatives of cellulose, lactate, lactose, saccharose, acetate
and/or glycerol.
[0031] The organic carbon-containing substrate contained in the
culture medium may consist in complex molecules or in a mixture of
substrates. The products resulting from the biotransformation of
starch, for example starting from maize, wheat or potato, notably,
starch hydrolysates, which constituted of small sized molecules,
for example, organic carbon-containing substrates which may be used
for mixotrophic culture of the microalgae according to the
invention.
[0032] This method is more particularly intended for the use of
novel strains of microalgae of the genus Nitzschia (Division:
Bacillariophyta, Order: Bacillariaceae, Family: Bacillariaceae)
[ITIS Catalogue of Life, 2010] selected for their mixotrophic
character, notably for their capability to be cultured with a light
supply greater than 10 .mu.E, in a mineral medium, for example the
f medium plus silica enriched in nitrogen [Guillard, R. R. L.
(1975). Culture of phytoplankton for feeding marine invertebrates,
in Culture of Marine Invertebrate Animals, pp 26-60. Smith W. L.
and Chanley M. H (Eds.) Plenum Press, New York], to which an
organic carbon-containing substrate is added. Preferably, the
organic carbon-containing substrate comprises glucose, or lactate,
at a concentration that is equivalent to or greater than 5 mM.
[0033] These novel strains of Nitzschia, more particularly
Nitzschia brevirostris, may be isolated and selected by the method
of selection and culture according to the invention described
hereafter.
[0034] A representative strain of the Nitzschia brevirostris
strains according to the invention is the strain FCC 810 isolated
by the applicant and deposited with the CCAP, under the accession
number CCAP 1052/21. These strains are capable of producing
significant quantities of biomass as well as lipids, and more
particularly of DHA and/or EPA, when they are cultured in
mixotrophic mode with a variable or discontinuous light supply,
according to the invention. They are also capable of producing
significant quantities of one or more carotenoid(s), more
particularly fucoxanthin.
[0035] According to the taxonomic analyses carried out, the strain
CCAP 1052/21 belongs to the species Nitzschia brevirostris. The
invention relates to any strain of the species Nitzschia
brevirostris, capable of growing under mixotrophic culture
conditions such as described in the present application, and
capable of producing fatty acids, such as DHA. The invention also
relates to any strain of microalga of the genus Nitzschia, capable
of growing under mixotrophic culture conditions such as described
in the present application, and capable of producing fatty acids,
such as DHA and/or EPA and/or carotenoids, more particularly
fucoxanthin.
[0036] The isolated strains of Nitzschia brevirostris according to
the invention make it possible to produce, under mixotrophic
conditions, significant quantities of biomass as well as lipids
rich in DHA and/or EPA, said DHA and/or EPA being capable of
representing more than 20%, more than 25%, or more than 30% of the
total lipids contained in the microalgae. Also the isolated strains
of Nitzschia brevirostris according to the invention make it
possible to produce, under mixotrophic conditions, significant
quantities of carotenoid(s), more particularly fucoxanthin, capable
of representing more than 0.2% of dry matter, more preferentially
more than 0.25% of dry matter, of the total microalgal dry matter
(which includes the carotenoids content contained in said
microalga).
[0037] In the present invention, the biomass obtained with the
strain FCC 810, isolated by the applicant, from a culture under
mixotrophic conditions in the presence of variable and/or
discontinuous illumination, notably in the form of flashes, is from
10 to 60%, more generally from 20 to 50%, greater than that of a
culture with the same strain carried out in heterotrophic mode. By
heterotrophic mode, it is meant culture conditions with an
identical culture medium, but without a light supply.
[0038] The invention thus relates to a method for culturing
microalgae of the genus Nitzschia, in particular of the species
Nitzschia brevirostris in mixotrophic mode, in the presence of an
illumination that is variable and/or discontinuous over time, for
example, in the form of flashes, notably with a view to producing
polyunsaturated fatty acids, such as DHA and/or EPA, and/or one or
more carotenoid(s), such as fucoxanthin.
[0039] The invention thus relates to a method for selecting strains
of the genus Nitzschia, in particular of the species Nitzschia
brevirostris, with a mixotrophic character, and having a high yield
of polyunsaturated fatty acids such as DHA and/or EPA, and/or a
high yield of one or more carotenoid(s), more particularly of
fucoxanthin, in the presence of an illumination that is variable
and/or discontinuous over time.
[0040] It appeared that variable and/or discontinuous illumination
of the cultures, in particular when used in a culture in
mixotrophic mode, had a favourable impact on the development of the
algae and made it possible to increase the productivity of the
latter, in particular with regard to their production of lipids
and/or carotenoids. Without being bound to theory, the inventor
believes that a discontinuous and/or variable light supply to the
microalgae has the effect of causing a "stress" favourable to the
growth and to the synthesis of lipids and/or carotenoids. This
phenomenon may be partly explained by the fact that, in nature,
microalgae tend to accumulate lipids and/or carotenoids reserves,
to withstand the constraints of their environment.
[0041] By "discontinuous illumination", it is meant an illumination
punctuated by periods of darkness. The periods of darkness may be
more than a quarter of the time, preferably, half of the time or
more, during which the algae are cultured.
[0042] According to a preferred aspect of the invention, the
illumination is discontinuous and, more preferentially, in the form
of flashes. A flash, within the meaning of the invention, is an
illumination with light of short duration, i.e. of less than 30
minutes. The duration of the flash may be less than 15 minutes,
preferably less than 5 minutes or yet more preferentially less than
1 minute. According to certain embodiments of the invention, the
duration of the flash may be less than a second. For example, the
duration of the flash may be 1/10 of a second, or 2/10 of a second,
or 3/10 of a second, or 4/10 of a second, or 5/10 of a second, or
6/10 of a second, or 7/10 of a second, or 8/10 of a second, or 9/10
of a second. The illumination with light, or the flash, generally
lasts longer than 15 seconds. The duration of the flash is
generally comprised between 5 seconds and 10 minutes, preferably
between 10 seconds and 2 minutes, more preferentially between 20
seconds and 1 minute.
[0043] In general, the number of flashes is comprised between about
2 and 3,600 per hour. It may be, for example, comprised between 100
and 3,600 flashes per hour. It may also be comprised between 120
and 3,000, or between 400 and 2,500, or between 600 and 2,000, or
between 800 and 1,500 flashes per hour. It may also be comprised
between 2 and 200, preferentially between 10 and 150, more
preferentially between 15 and 100, and yet more preferentially
between 20 and 50 per hour. The flashes may be emitted at regular
or irregular time intervals. In the case of emission at regular
intervals, the number of flashes per hour then corresponds to a
frequency (F) having a time period (T), it being considered that
F=1/T. This time period may be comprised between 1 second and 30
minutes, or between 1 second and 36 seconds, or between 1.2 second
and 30 seconds, or between 1.44 second and 9 seconds, or between
1.8 second and 6 seconds, or between 2.4 seconds and 4.5 seconds.
This frequency may also be comprised between 18 seconds and 30
minutes, preferentially between 24 seconds and 6 minutes, more
preferentially between 36 seconds and 4 minutes, and yet more
preferentially between 72 seconds and 3 minutes. The number of
flashes per hour is selected as a function of the intensity and
duration of the flashes (see below). In general, the intensity of
the light supplied in the form of flashes is comprised between 5
and 1,000 .mu.molm.sup.-2s.sup.-1, preferably between 5 and 500
.mu.molm.sup.-2s.sup.-1, or 50 and 400 .mu.molm.sup.-2s.sup.-1, and
more preferentially between 150 and 300 .mu.molm.sup.-2s.sup.-1. By
definition, 1 .mu.molm.sup.-2s.sup.-1 corresponds to 1 .mu.E
m-.sup.2s.sup.-1 (Einstein), a unit often used in the
literature.
[0044] According to a particular embodiment of the invention, the
intensity of the light is comprised between 50 and 200
.mu.molm.sup.-2s.sup.-1, the frequency of the flashes is comprised
between 10 seconds and 60 minutes for a flash duration comprised
between 1 second and 1 minute.
[0045] According to another embodiment of the invention, the
illumination may be variable, which means that the illumination is
not interrupted by phases of darkness, but instead the light
intensity varies over time. This variation of the light intensity
is regular and may be periodic or cyclic. According to the
invention, light may also be supplied combining phases of
continuous and discontinuous illumination.
[0046] According to the invention, regardless of the illumination
conditions, the light intensity supplied to the algae in culture,
expressed in micromoles of photons per second per square metre
(.mu.molm.sup.-2s.sup.-1), varies at least once in any one hour.
The amplitude of this variation of light intensity is generally
between 5 and 1,000, or between 50 and 800, or between 100 and 600
.mu.molm.sup.-2s.sup.-1. The intensity of the light may also vary
between 5 and 400 .mu.molm.sup.-2s.sup.-1. Preferably, the
amplitude of the variation of light intensity is between 70 and 300
.mu.molm.sup.-2s.sup.-1 and more preferentially between 100 and 200
.mu.molm.sup.-2s.sup.-1.
[0047] Said light intensity may attain successively, under
conditions of variable illumination, for example, the values 50
.mu.molm.sup.-2s.sup.-1 and 100 .mu.molm.sup.-2-1, or 5 and 400
.mu.molm.sup.-2-1, or 50 and 800 .mu.molm.sup.-2s.sup.-1 several
times every hour. Said light intensity may attain successively,
preferably, the values 50 and 200 .mu.molm.sup.-2s.sup.-1.
Alternatively, under conditions of discontinuous illumination, said
light intensity may attain successively, several times per hour,
for example, the values 0 and 50 .mu.molm.sup.-2s.sup.-1, the
values 0 and 100 .mu.molm.sup.-2s.sup.-1 or still more
preferentially the values 0 and 200 .mu.molm.sup.-2s.sup.-1. It may
also attain successively, several times per hour, for example, the
values 0 and 300 .mu.molm.sup.-2s.sup.-1, the values 0 and 600
.mu.molm.sup.-2s.sup.-1, the values 0 and 800
.mu.molm.sup.-2s.sup.-1 or also the values 0 and 1,000
.mu.molm.sup.-2s.sup.-1.
[0048] According to an embodiment of the invention, regardless of
the illumination conditions, the intensity of the light supplied to
the culture varies as a function of the cell density. The denser
the culture becomes, the more intense the light may be. The cell
density is the number of cells per ml and it is measured by the
techniques known to one skilled in the art.
[0049] At the initial stage of the culture, when the cell density
is between about 10.sup.5 and 5.times.10.sup.5 cells per ml, the
light intensity may be between 5 and 15 .mu.molm.sup.-2s.sup.-1,
preferably between 5 and 10 .mu.molm.sup.-2s.sup.-1. When the
culture reaches a density between 10.sup.6 and 10.sup.7 cells per
ml, the light intensity may be increased to between 15 and 200
.mu.molm.sup.-2s.sup.-1, for example, preferably, between 20 and 50
.mu.molm.sup.-2s.sup.-1. When the culture, at the final stage,
reaches a density between 10.sup.7 and 10.sup.8 cells per ml, the
light intensity may be increased to between 50 and 400
.mu.molm.sup.-2s.sup.-1 for example, preferably, between 50 and 150
.mu.molm.sup.-2s.sup.-1.
[0050] According to certain embodiments, for example, when the
duration of the flashes is for example less than a minute, or less
than a second, the intensity of the light may be higher than the
values stated above. At the initial stage of the culture, when the
cell density is between about 10.sup.5 and 5.times.10.sup.5 cells
per ml, the light intensity may be between 5 and 200
.mu.molm.sup.-2s.sup.-1, preferably between 5 and 100
.mu.molm.sup.-2s.sup.-1. When the culture reaches a density between
10.sup.6 and 10.sup.7 cells per ml, the light intensity may be
increased to between 30 and 500 .mu.molm.sup.-2s.sup.-1, for
example, preferably, between 50 and 400 .mu.molm.sup.-2s.sup.-1.
When the culture, at the final stage, reaches a density between
10.sup.7 and 10.sup.8 cells per ml, the light intensity may be
increased to between 100 and 1,000 .mu.molm.sup.-2s.sup.-1 for
example, preferably between 200 and 500
.mu.molm.sup.-2s.sup.-1.
[0051] According to an embodiment of the invention, the quantity of
light supplied to the culture per hour remains between certain
values. It is comprised between about 2,000 and 600,000, preferably
between 2,000 and 300,000 .mu.molm.sup.-2. It may be comprised
between 4,000 and 200,000 .mu.molm.sup.-2, per hour.
[0052] According to an embodiment of the invention, the culture is
illuminated with 30 flashes per hour, each flash having a duration
of 30 seconds and an intensity of 10 .mu.molm.sup.-2s.sup.-1. The
latter gives a total supply of light per hour of 9,000
.mu.molm.sup.-2. According to another embodiment of the invention,
the culture is illuminated with 20 flashes per hour, each flash
having a duration of 30 seconds and an intensity of 20
.mu.molm.sup.-2s.sup.-1. The latter gives a total supply of light
per hour of 12,000 .mu.molm.sup.-2. According to another embodiment
of the invention, the culture is illuminated with 45 flashes per
hour, each flash having a duration of 15 seconds and an intensity
of 5 .mu.molm.sup.-2s.sup.-1, which gives a total supply of light
per hour of 3,375 .mu.molm.sup.-2.
[0053] According to another embodiment of the invention, the
culture is illuminated with 120 flashes per hour, each flash having
a duration of 10 seconds and an intensity of 200
.mu.molm.sup.-2s.sup.-1, which gives a total supply of light per
hour of 240,000 .mu.molm.sup.-2.
[0054] As described for the light intensity above, and according to
an embodiment of the invention, the quantity of light supplied to
the culture per hour may vary as a function of the cell density. At
the initial stage of the culture, when the cell density is 10.sup.5
and 5.times.10.sup.5 cells per ml, the total supply of light per
hour is generally comprised between about 1,500 and 8,000,
preferably 1,500 and 6,000 .mu.molm.sup.-2, yet more preferably
between 2,000 and 5,000 .mu.molm.sup.-2. When the culture reaches a
density between 10.sup.6 and 10.sup.7 cells per ml, the total
supply of light per hour may be increased until between 6,000 and
67,000 .mu.molm.sup.-2, preferably between 6,000 and 50,000, and
yet more preferably between 12,000 and 45,000 .mu.molm.sup.-2, for
example. At the final stage of the culture, at a cell density
between 10.sup.7 and 10.sup.8 cells per ml, the total supply of
light per hour may be increased to between 45,000 and 300,000, for
example preferably, between 45,000 and 200,000 .mu.molm.sup.-2, and
for example, yet more preferably, between 50,000 and 150,000
.mu.molm.sup.-2.
[0055] According to an embodiment of the invention, at the initial
stage of the culture (at a cell density between 10.sup.5 and
5.times.10.sup.5 cells per ml), the culture is illuminated with 30
flashes per hour, each flash having a duration of 30 seconds and an
intensity between 5 and 10 .mu.molm.sup.-2s.sup.-1, which gives a
total supply of light per hour of 2,250 .mu.molm.sup.-2 to 4,500
.mu.molm.sup.-2. Then, at the intermediate stage (at a cell density
between 10.sup.6 and 10.sup.7 cells per ml), the culture is
illuminated with 30 flashes per hour, each flash having a duration
of 30 seconds and an intensity between 15 and 50
.mu.molm.sup.-2s.sup.-1, which gives a total supply of light per
hour of 13,500 to 45,000 .mu.molm.sup.-2. Then, at the final stage
of the culture (at a cell density between 10.sup.7 and 10.sup.8
cells per ml), the culture is illuminated with 30 flashes per hour,
each flash having a duration of 30 seconds and an intensity between
50 and 150 .mu.molm.sup.-2s.sup.-1, which gives a total supply of
light per hour of 45,000 to 135,000 .mu.molm.sup.-2.
[0056] According to an embodiment of the invention, for example,
when the duration of the flashes is for example of less than a
minute, or less than a second, at the initial stage of the culture
(at a cell density between 10.sup.5 and 5.times.10.sup.5 cells per
ml), the culture is illuminated with 30 flashes per hour, each
flash having a duration of 10 seconds and an intensity between 50
and 100 .mu.molm.sup.-2s.sup.-1, which gives a total supply of
light per hour of 15,000 .mu.molm.sup.-2 to 30,000 .mu.molm.sup.-2.
Then at the intermediate stage (at a cell density between 10.sup.6
and 10.sup.7 cells per ml), the culture is illuminated with 50
flashes per hour, each flash having a duration of 10 seconds and an
intensity between 200 and 300 .mu.molm.sup.-2s.sup.-1, which gives
a total supply of light per hour of 100,000 to 150,000
.mu.molm.sup.-2. Then, at the final stage of the culture (at a cell
density between 10.sup.7 and 10.sup.8 cells per ml), the culture is
illuminated with 120 flashes per hour, each flash having a duration
of 10 seconds and an intensity between 350 and 450
.mu.molm.sup.-2s.sup.-1, which gives a total supply of light per
hour of 420,000 to 540,000 .mu.molm.sup.-2.
[0057] The light supply to the cultures may be obtained by lamps
distributed around the external wall of the fermenters. A clock
triggers these lamps for defined illumination times. The fermenters
are preferentially located in an enclosure, shielded from daylight,
whose ambient temperature may be controlled.
[0058] As the applicant could ascertain, the fact that the thus
selected strains have good growth capabilities in mixotrophic mode,
in the presence of discontinuous and/or variable light, predisposes
said strains to a higher production of polyunsaturated fatty acids,
notably DHA and/or EPA and/or a higher production of one or more
carotenoid(s), more particularly fucoxanthin.
[0059] The method of culture according to the invention thus allows
selection of strains of the genus Nitzschia, in particular of the
species Nitzschia brevirostris, with mixotrophic character, similar
to that isolated by the applicant and deposited at the CCAP under
the accession number CCAP 1052/21, and having a high yield of
polyunsaturated fatty acids and/or carotenoid(s). This method of
culture is characterized in that it comprises the following
steps:
[0060] a) culture, in mixotrophic mode, of one or more strains of
the genus Nitzschia, under conditions of illumination that is
discontinuous and/or variable over time, the illumination having
variations in intensity, the amplitude of which is comprised
between 5 .mu.molm.sup.-2s.sup.-1 and 1,000, preferably between 5
and 400 .mu.molm.sup.-2s.sup.-1, these variations taking place
between 2 and 3,600, preferably 5-400 times per hour,
[0061] b) a step of maintaining said culture over several
generations, in the presence of an organic carbon-containing
substrate in the culture medium, and optionally
[0062] c) a step of recovery of the thus cultured microalgae.
[0063] By "step of recovery", it is meant more particularly the
isolation of the strain or strains for which the number of cells
increased the most during said generations.
[0064] Advantageously, culture in mixotrophic mode is carried out
under conditions of illumination that is discontinuous and/or
variable over time, the illumination having variations in
intensity, the amplitude of which is comprised between 5
.mu.molm.sup.-2s.sup.-1 and 400 .mu.molm.sup.-2s.sup.-1, these
variations taking place between 2 and 200 times per hour.
[0065] For carrying out selection of the strains, different strains
of the genus Nitzschia, in particular of the species Nitzschia
brevirostris, may be cultured, in parallel, on microplates in one
and the same enclosure, with precise monitoring of the conditions
and evolution of the different cultures. It is, thus, easy to
determine the response of the different strains to discontinuous
and/or variable illumination and, if applicable, to the addition of
one or more organic carbon-containing substrates to the culture
medium. The strains that respond favourably to the discontinuous
and/or variable illumination and to the organic carbon-containing
substrates, generally provide a better yield for the production of
carotenoids and lipids in terms of quality (polyunsaturated fatty
acids more abundant in the lipid profile and fucoxanthin more
abundant among the carotenoids) and in terms of quantity (the
lipids contain a higher proportion of DHA and/or EPA).
[0066] The microalgae may be selected in a fermenter from a
heterogeneous population, and from which one aims to select the
variants favoured by the manner of selection according to the
invention, combining discontinuous and/or variable light, having a
specific range of light intensity and a specific frequency, with
mixotrophic culture conditions. In this case, culture is carried
out by maintaining the microalgae in cultures over many
generations, and then an isolation of the components that have
become predominant in the culture medium, is performed at the end
of culture.
[0067] The method of culture according to the invention also makes
it possible to produce lipids.
[0068] In this case, the method according to the invention further
comprises the following steps:
d) a step of recovery of the lipids from the microalgae, and
optionally e) the extraction of the DHA and/or the EPA from the
recovered lipids.
[0069] The method of culture according to the invention also makes
it possible to produce carotenoids.
[0070] In this case, the method according to the invention further
comprises the following steps:
d) a step of recovery of the hydrophobic matter from the
microalgae, and optionally e) the extraction of the DHA and/or the
EPA and/or the fucoxanthin from the hydrophobic matter
recovered.
[0071] The method of culture according to the invention may also be
applied to any species of the genus Nitzschia, capable of growing
under the mixotrophic conditions according to the invention, and
capable of producing DHA and/or EPA and/or fucoxanthin.
[0072] The method of culture according to the invention makes it
possible to optimize the production of the biomass obtained from
the culture. It also makes it possible to enrich the thus cultured
microalgae in polyunsaturated fatty acids, more particularly in DHA
and/or EPA, and/or enrich the microalgae thus cultured in
carotenoid(s), more particularly in fucoxanthin.
[0073] Therefore, the invention is also directed to optimizing the
production of biomass, as well as the production of lipids, notably
of fatty acids, through the culture of microalgae of the
genusNitzschia with mixotrophic character, preferably cultured or
selected according to the methods mentioned above, then the
recovery of the thus cultured microalgae in order to extract the
lipids therefrom, in particular DHA and/or EPA. The invention is
also directed to optimizing the production of carotenoid(s), more
particularly of fucoxanthin. The strains of the species Nitzschia
brevirostris are especially concerned. The methods for selectively
extracting the lipids, including EPA and ARA, are known to one
skilled in the art and are, for example, described by [Bligh, E. G.
and Dyer, O. K. (1959); A rapid method of total lipid extraction
and purification, Can. J. Biochem. Physiol., 37:911-917] and by
[McCreary D K, Kossa W C, Ramachandran S, Kurtz R R. (1978), "A
novel and rapid method for the preparation of methyl esters for gas
chromatography: application to the determination of the fatty acids
of edible fats and oils", J Chromatogr Sci. 16(8):329-31].
[0074] The methods of extraction and analysis of the carotenoids,
including fucoxanthin, are known to one skilled in the art and are,
for example, described by Wright et al. (1991) (S. W. Wright, S. W.
Jeffrey, R. F. C. Mantoura, C. A. Llewellyn, T. Bjornland, D.
Repeta, N. Welschmeyer: Improved HPLC method for the analysis of
chlorophylls and carotenoids from marine phytoplankton. Marine
ecology progress series: Vol. 77: 183-196, 1991).
[0075] The invention also relates to the microalgae of the genus
Nitzschia, which can be obtained according to the method of the
invention as previously described. These microalgae are enriched in
polyunsaturated fatty acids. The total lipids of such microalgae
generally comprise more than 20%, often more than 25% and sometimes
even more than 30% of DHA and/or EPA with respect to the total
percentage of lipids. The microalgae can also comprise a
fucoxanthin content of more than 0.2% of dry matter, preferably
more than 0.25% of dry matter of the microalgal dry matter.
EXAMPLE 1
[0076] The cultures of Nitzschia brevirostris FCC 810 were carried
out in 2 L usable capacity fermenters (bioreactors) with dedicated
automatic controllers with computerized supervision. The pH of the
system is adjusted by adding base (a 1N sodium hydroxide solution)
and/or acid (a 1N sulphuric acid solution). The culture temperature
is set to 25.degree. C. Stirring is achieved using 2 stirring
rotors mounted on the shaft according to the Rushton configuration:
three-blade impellers with downward pumping. The stirring speed and
the air flow rate are regulated to a minimum=100 rpm and to a
maximum=250 rpm and Qmin=0.5 vvm/Qmax=2 vvm respectively. The
bioreactor is equipped with an external lighting system surrounding
the transparent tank.
[0077] The reactors are inoculated with a preculture prepared on a
stirring table (140 rpm) in a controlled-temperature enclosure
(25.degree. C.) and illuminated between 80 and 100 .mu.E.
Pre-cultures and cultures in bioreactors are carried out in the F
medium plus silica enriched with nitrogen. The organic
carbon-containing substrate used for the mixotrophic culture in a
bioreactor is glucose at concentrations between 100 mM and 150
mM.
Monitoring of the Cultures:
[0078] The total biomass concentration is monitored by measuring
the dry mass (filtration on a Whatman GFB filter, then oven drying,
at 100.degree. C. for a minimum of 24 h before weighing).
[0079] Regarding the quantification of the total lipids, 7.10.sup.8
cells/mL were extracted. Methods for extracting lipids are known to
one skilled in the art.
Illumination:
[0080] The culture is illuminated with 180 flashes per hour, each
flash having a duration of 5 seconds and an intensity of 300
.mu.molm.sup.-2-1.
[0081] The light supply to the cultures in the bioreactor was
obtained by LED (light-emitting diode) lamps distributed around the
external wall of the fermenter. The power supply to the LEDs is
triggered by automated control for the illumination time or the
flashes.
Results:
TABLE-US-00001 [0082] Dry mass Total lipids Fucoxanthin (g/L) (% of
dry mass) % DHA % EPA (mg/g) Mixotrophy with flashes 35.1 +/- 0.7
23.6 +/- 0.4 0.6 +/- 0.5 26 +/- 1 2.2 +/- 0.1 Heterotrophy 25.4 +/-
0.2 24 +/- 0.3 0.8 +/- 0.4 22 +/- 1 0.8 +/- 0.1
* * * * *