METHOD FOR CULTIVATING A MICROORGANISM OF INTEREST AND ASSOCIATED FACILITY

Abstract

A method for cultivating at least one microorganism of interest, by heterotrophism or mixotrophism, in an aqueous culture medium, contaminating microorganisms developing naturally in the culture medium. A portion of the culture medium with the microorganism of interest and the contaminating microorganisms is sampled. The microorganism of interest and the contaminating microorganisms in the portion of culture medium is physically separated. The contaminating microorganisms thus separated is lysed to produce a lysate. The lysate is reintroduced into the culture medium.

Claims

1-12. (canceled)

13. A method for cultivating at least one microorganism of interest, by heterotrophy or mixotrophy, in an aqueous culture medium, contaminating microorganisms developing naturally in the aqueous culture medium, the method comprising: (a) sampling a portion of the aqueous culture medium comprising the microorganism of interest and the contaminating microorganisms; (b) physically separating the microorganism of interest and the contaminating microorganisms in the portion of aqueous culture medium; (c) lysis of the contaminating microorganisms thus separated to produce a lysate; and (d) reintroducing the lysate into the aqueous culture medium.

14. The cultivation method of claim 13, wherein the microorganism of interest separated is reintroduced into the aqueous culture medium, alone or in a mixture with the lysate.

15. The cultivation method of claim 13, further comprises supplying an organic carbon into the aqueous culture medium.

16. The cultivation method of claim 15, by mixotrophy, wherein the aqueous culture medium is initially devoid of the organic carbon and the microorganism of interest is left in autotrophic culture for a predetermined time before supplying the organic carbon and before sampling the portion of the aqueous culture medium.

17. The cultivation method of claim 13, further comprises concentrating the contaminating microorganisms separated in the portion of aqueous culture medium.

18. The cultivation method of claim 13, wherein the aqueous culture medium comprises several different microorganisms of interest, the method comprises upstream from the physically separating step, a prior step of isolating a single type of microorganisms of interest selected or a set of different microorganisms of interest selected within the sampled portion, such that when the sample portion undergoes the physical separation, the sample portion only comprises said single type of microorganisms of interest selected or said set of different microorganisms of interest selected.

19. The cultivation method of claim 13, comprising a repeated cycle of the steps (a)-(d).

20. The cultivation method of claim 13, wherein the microorganism of interest is the cyanobacterium Aphanizomenon flos-aquae (AFA).

21. A facility to cultivate at least one microorganism of interest, by heterotrophy or mixotrophy, comprising: at least one open or closed culture compartment configured to receive an aqueous culture medium, said at least one microorganism of interest and contaminating microorganisms; at least one separating apparatus configured to perform at least one physical separation of said at least one microorganism of interest and the contaminating microorganisms within a portion of the aqueous culture medium; a lysis device configured to perform a lysis of the contaminating microorganisms separated by said at least one physical separation to produce a lysate; and a transport device configured to at least sample a portion of the culture medium comprising said at least one microorganism of interest and the contaminating microorganisms, and configured to reintroduce the lysate into said at least one open or closed culture compartment.

22. The cultivation facility of claim 21, wherein the transport device is further configured to: unify the lysate with said at least one microorganism of interest, separated by said at least one physical separation, to form a mixture and reintroduce the mixture into said at least one open or closed culture compartment; and supply an organic carbon to the aqueous culture medium directly into said at least one open or closed culture compartment, into the lysate, into the mixture or to said at least one microorganism of interest separated by said at least one physical separation.

23. The cultivation facility of claim 21, further comprising at least one concentration system configured to concentrate the contaminating microorganisms separated by said at least one physical separation.

24. The cultivation facility of claim 21, further comprising a first drainer to drain the contaminating microorganisms, a second drainer to drain the aqueous culture medium devoid of the contaminating microorganisms and of said at least one microorganism of interest and a collection device to collect said at least one microorganism of interest.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0055] The invention will be better understood on reading the following description, given by way of non-limiting example, and with reference to the figures which represent:

[0056] FIG. 1: illustrates the steps comprised by the cultivation method according to the present invention according to implementations;

[0057] FIG. 2: illustrates a cultivation facility according to the present invention according to an embodiment; and

[0058] FIG. 3: graph of the progression of the mass concentration of Aphanizomenon flos-aquae (AFA) over time in a culture medium according to the implementation of the method according to the present invention by autotrophy or by mixotrophy with reintroduction of different lysate concentrations.

DETAILED DESCRIPTION OF THE INVENTION

[0059] It is noted as of now that the figures are not to scale.

[0060] More generally, the scope of the present invention is not limited to the implementations and embodiments described above by way of non-limiting examples, but extends on the contrary to any modifications to the scope by a person skilled in the art. Each feature of an embodiment can be advantageously implemented separately or combined with any other feature of any other embodiment.

[0061] FIG. 1 illustrates a method 20 for cultivating at least one microorganism of interest, by heterotrophy or mixotrophy, in an aqueous culture medium, contaminating microorganisms developing naturally in said culture medium, according to a particular implementation. The method includes a step 21 of sampling a portion of the culture medium comprising the microorganism of interest and contaminating microorganisms. Preferably, the method 20 is carried out by mixotrophy, the aqueous culture medium being initially devoid of organic carbon and the microorganism of interest is left in autotrophic culture for a time t before the step 21.

[0062] The method 20 includes a step 22 of physically separating the microorganism of interest and the contaminating microorganisms in the portion of the culture medium.

[0063] The embodiment of the physical separation step 22 can be based on a morphological or phenotypic difference between the microorganisms of interest and the contaminating microorganisms. For example, it can consist of a difference in size, shape, surface properties, density, aggregation or flocculation tendency.

[0064] According to an implementation example, the physical separation step 22 includes a step of gravitational filtration or separation so as to separately obtain the concentrated contaminating microorganisms on one hand and the nutrient medium on the other. An example of filtration used in a preferred implementation is tangential filtration or frontal filtration. The filtration can be performed using a membrane.

[0065] The method 20 further includes an optional step 23 of concentrating the contaminating microorganisms isolated in step 22.

[0066] The optional step 23 of concentrating the contaminating microorganisms can be based on a morphological or phenotypic feature of the contaminating microorganisms. For example, it can consist of their size, their shape, their surface properties, their density, their tendency to aggregate or flocculate.

[0067] According to a preferred implementation, the concentration step 23 includes a step of gravitational filtration or separation so as to separately obtain the concentrated contaminating microorganisms on one hand and culture medium devoid of contaminating microorganisms on the other. As for the physical separation, in a particular implementation, an example of filtration used is tangential filtration or frontal filtration, and the filtration can particularly be performed using a membrane.

[0068] The method 20 includes a step 24 of lysis of the contaminating microorganisms separated by the physical separation so as to produce a lysate. This lysate advantageously represents nutrients digestible by the microorganism of interest, particularly organic carbon. In a particular implementation, the lysis step 24 can comprise a chemical lysis and/or a thermal lysis and/or a mechanical lysis, of the contaminating microorganisms.

[0069] The method 20 also includes a step 25 reintroducing said lysate into the culture medium.

[0070] According to a preferred embodiment example, the step 25 is carried out such that the lysate is reintroduced into the culture medium in a mixture with the microorganism of interest isolated by the physical separation step 22.

[0071] Finally, the method 20 includes an optional step 26 of supplying organic carbon to the culture medium. This organic carbon originates from a source other than the lysate of contaminating microorganisms. Indeed, this supply of organic carbon in step 26 is an additional supply to the supply of nutrients (of which organic carbon) present in the lysate of contaminating microorganisms.

[0072] Preferably, the step 26 of supplying organic carbon in the culture medium is performed by introducing said organic carbon into the lysate and microorganism of interest mixture, into the lysate alone, into the microorganism of interest isolated by the physical separation step 22 or into the aqueous culture medium directly.

[0073] According to a preferred implementation, the method includes a repeated cycle of steps 21 to 26, steps 23 and 26 remaining optional.

[0074] According to an implementation example wherein the cultivation method is by heterotrophy, the step 26 of supplying organic carbon is introduced in a cycle C1 of steps 21 to 25, after the implementation of at least one cycle C1, thus creating a cycle C2 of steps 21 to 26 which is hereinafter repeated as many times as is necessary.

[0075] According to an implementation example wherein the cultivation method 20 is by mixotrophy, with the aqueous culture medium initially devoid of organic carbon and the microorganism of interest left in autotrophic culture for a time t before step 21, step 26 of supplying organic carbon is introduced from the implementation of the first cycle of steps 21 to 25, before the sampling step 21, thus commencing the method 20 with the cycle C2, said step 26 being carried out by supplying organic carbon directly into the aqueous culture medium at least in the first cycle C2.

[0076] In a particular implementation, the cultivation method 20 comprises a step (not shown in the figures) of clarifying the lysate obtained by means of the lysis step 24, a concentration and/or an adjustment of pH of the lysate, before the reintroduction step 25.

[0077] FIG. 2 illustrates a facility 27 for cultivating at least one microorganism of interest 28 by heterotrophy or mixotrophy. The facility does not advantageously need to be under aseptic conditions, either beforehand, or during the use therefore for cultivation.

[0078] The facility 27 includes an open or closed culture compartment 29 designed to receive an aqueous culture medium 30, said at least one microorganism of interest 28 and contaminating microorganisms 32.

[0079] The facility 27 further includes at least one separating apparatus 33 designed to perform at least one physical separation of the microorganism of interest 28 and the contaminating microorganisms 32 within a portion of the culture medium 30.

[0080] The facility 27 includes a lysis device 34 designed to perform a lysis of the contaminating microorganisms 32 separated so as to produce a lysate 35.

[0081] According to a particular embodiment, the facility further includes at least one concentration system 36 designed to concentrate the contaminating microorganisms 32 separated before the lysis thereof.

[0082] The facility 27 includes transport means designed for at least: [0083] sampling a portion of the culture medium 30 comprising the microorganism of interest 28 and contaminating microorganisms 32; [0084] reintroducing the lysate 35 into the culture compartment 29.

[0085] Preferably, the transport means are furthermore designed to unify the lysate 35 with the separated microorganism of interest 28, so as to form a mixture and reintroduce said mixture into the culture compartment 29.

[0086] Preferably, the transport means are designed to supply organic carbon to the culture medium 30 directly in the culture compartment 29 or to the lysate 35 or to the microorganism of interest 28 separated by the physical separation or to the mixture of lysate 35 and microorganism of interest 28 or in a combination of at least two thereof.

[0087] According to a preferred embodiment, the transport means include means: [0088] for generating a first flow 37 of culture medium 30 comprising the microorganism of interest 28 and contaminating microorganisms 32, from the culture compartment 29 to the separating apparatus 33; [0089] for generating a second flow 38 of the microorganism of interest 28 outflowing from the separating apparatus 33; [0090] for generating a third flow 39 of contaminating microorganisms 32 from the separating apparatus 33 to the concentration system 36; [0091] for generating a fourth flow 40 of concentrated contaminating microorganisms 32 from the concentration system 36 to the lysis device 34; [0092] for generating a fifth flow 41 of culture medium 30 devoid of contaminating microorganisms, from the concentration system 36 to the culture compartment 29; [0093] for generating a sixth flow 42 of lysate 35 from the lysis device 34 to the culture compartment 29; [0094] for generating a seventh flow 43 of organic carbon; [0095] for unifying the second flow 38, the sixth flow 42 and the seventh flow 43 so as to form an eighth single current flow 44 to the culture compartment 29.

[0096] According to a particular embodiment example, the facility 27 comprises an organic carbon tank 45. The seventh flow 43 is preferably generated such that it comes out of the tank 45.

[0097] The fifth flow 41 is preferably devoid of microorganisms of interest.

[0098] According to a particular embodiment, the means for generating the different flows include at least a motor and/or a pump (not illustrated in the figures).

[0099] In a particular embodiment, the facility 27 includes a first device 46 for draining contaminating microorganisms 32, a second device 47 for draining culture medium 30 devoid of microorganisms of interest and contaminating microorganisms and a device 48 for collecting the microorganism of interest 28.

[0100] The method 20 for cultivating at least one microorganism of interest 28, by heterotrophy or mixotrophy, is detailed hereinafter according to an implementation wherein a facility 27 according to the present invention is particularly used according to one of the embodiments thereof:

[0101] An aqueous culture medium 30 and microorganism of interest 28 is previously introduced into the culture compartment 29. The microorganism of interest 28 is preferably left in autotrophic culture for a time t.

[0102] Organic carbon is then introduced directly into the culture compartment 29 according to step 26, via the seventh flow 43 of organic carbon outflowing from the tank 45. The culture then switches to mixotrophy and contaminating microorganisms 32 develop in the culture medium 30 with the microorganism of interest 28.

[0103] In step 21, the sampling of a portion of the culture medium 30 comprising the microorganism of interest 28 and the contaminating microorganisms 32 is carried out by generating the first flow 37 of culture medium 30 from the compartment 29 to the separating apparatus 33.

[0104] In the separating apparatus 33, the step 22 of physically separating the microorganism of interest 28 and the contaminating microorganisms 32 in the portion of the culture medium 30 sampled is performed. Preferably, this step 22 is carried out by tangential membrane filtration.

[0105] After the physical separation step 22, the second flow 38 of microorganism of interest 28 outflowing from the separating apparatus 33 is generated, as well as the third flow 39 of contaminating microorganisms 32 from the separating apparatus 33 to the concentration system 36.

[0106] In the concentration system 36, the optional step 23 of concentrating the contaminating microorganisms 32 separated by the step 22 takes place. Preferably, this step 23 is carried out by tangential membrane filtration. This step 23 can be considered as a second physical separation for separating, on one hand, the contaminating microorganisms 32 in a concentrated manner and, on the other, culture medium 30 devoid of contaminating microorganisms 32 and microorganism of interest 28.

[0107] After this step 23, the fourth flow 40 of concentrated contaminating microorganisms 32 is generated from the concentration system 36 to the lysis device 34, and the fifth flow 41 of culture medium 30 devoid of contaminating microorganisms 32 and of microorganism of interest 28, is generated from the concentration system 36 to the culture compartment 29.

[0108] In the lysis device 34, the step 24 of lysis of the separated and concentrated contaminating microorganisms 32 then takes place, so as to produce the lysate 35.

[0109] The third flow 39 is directly targeted toward the lysis device 34 in an implementation where the concentration step 23 does not take place. In such a particular implementation, the lysis step 24 is carried out on the separated contaminating microorganisms 32 originating directly from the separating apparatus 33 via the third flow 39.

[0110] Then, in the reintroduction step 25, the lysate 35 is reintroduced into the culture medium 30 in the culture compartment 29 by generating the sixth flow 42 of lysate 35. Preferably, the second flow 38 of microorganisms of interest 28 and the sixth flow 42 of lysate 35 are unified so as to form a current mixture to the compartment 29.

[0111] This cycle C2 of the cultivation method can be repeated several times.

[0112] After the first cycle C2, in the steps 26 of each following cycle C2, the seventh flow 43 of organic carbon outflowing from the tank 45 which is generated is preferably unified with the second flow 38 and the sixth flow 42 so as to form the single eighth flow 44 of current mixture to the compartment 29 wherein it is reintroduced.

Example Applied to the Cultivation of Aphanizomenon flos-aquae (AFA) by Mixotrophy in Open Medium

[0113] Microorganisms Used

[0114] The microorganism of interest 28 used in this study is an isolated Aphanizomenon flos-aquae (AFA) strain. AFA is a cyanobacterium of industrial and commercial interest.

[0115] Model contaminating microorganisms 32 are formed by a mixture of 5 strains of bacilli having different morphological and phenotypic characteristics. This mixture is presented in the form of a live bacterial solution, concentrated and presented in liquid formulation. The solution marketed under the reference “Bactilit™” by the company CG PACKAGING can for example be used.

[0116] In order to simplify the method, the contaminating microorganisms 32 are initially introduced into the cyanobacteria culture at a rate of one cell of the microorganism of interest 28 for one cell of contaminating microorganism 32. Moreover, it should be noted that a filament is considered as a cyanobacteria cell, regardless of its size.

[0117] Culture Media

[0118] The culture medium used is a BG11 medium (detailed composition provided in tables A to D), native for cultivation by autotrophy, or, supplemented with whey powder at a final concentration of 4.5 g.L.sup.−1 for cultivation by mixotrophy.

TABLE-US-00001 TABLE A composition of stock solution 1 Product Concentration g/L Na.sub.2MgEDTA 0.1 Ferric Ammonium Citrate 0.6 Citric acid, H.sub.2O 0.6 CaCl.sub.2, 2H.sub.2O 3.6 Osmosed water, sterilization 20 minutes at 121° C. or filtration (storage at 4° C.)

TABLE-US-00002 TABLE B composition of stock solution 2 Product Concentration g/L MgSO.sub.4, 7H2O 7.5 Osmosed water, sterilization 20 minutes at 121° C. or filtration (storage at 4° C.)

TABLE-US-00003 TABLE C composition of stock solution 3 Product Concentration g/L K2HPO4 0.6 Osmosed water, sterilization 20 minutes at 121° C. or filtration (storage at 4° C.)

TABLE-US-00004 TABLE D composition of BG11 nutrient medium solution Product Concentration g/L or mL/L Stock solution 1 (mL) 10 Stock solution 2 (mL) 10 Stock solution 3 (mL) 10 Na.sub.2CO.sub.3 (g) 0.02 NaNO.sub.3 (g) 1.5 Osmosed water, pH adjusted to 9, sterilization 20 minutes at 121° C.

[0119] Culture Operating Conditions

[0120] The AFA culture is produced in an air-conditioned room in which the temperature is regulated at 25±1° C. The borosilicate glass culture compartment 29 (total volume =250 mL, culture volume =200 mL, effective height =70 mm, effective diameter =60 mm) is equipped with blade stirring (water propeller type mobile stirring device, mobile device diameter =30 mm, stirring speed =60 rpm).

[0121] Light is supplied by a light-emitting diode panel (irradiance of photosynthetically active radiation measured on the outer wall of the reaction vessel =200 μmol.s.sup.−1.m.sup.−2).

[0122] The culture inoculation rate was set at 5.10.sup.5 and 1.10.sup.6 cells.mL.sup.−1.

[0123] Unitary Operations and Operating Conditions for Physically Separating the Microorganism of Interest from the Contaminating Microorganisms and for Concentrating the Contaminating Microorganisms

[0124] As regards the physical separation between Aphanizomenon Flos-Aquae and the model contaminating microorganisms (various strains of bacilli in a mixture), the method used consists of a first tangential membrane filtration.

[0125] The concentration of the model contaminating microorganisms makes use of a second physical separation between said contaminating microorganisms and the BG11 culture medium carried out by a second tangential membrane filtration.

[0126] The first filtration makes use of a membrane having a porosity of 5 μm. It is possible, for the first tangential filtration, to use a membrane having a porosity between 0.65 μm and 20 μm, and preferably between 1.2 μm and 20 μm, preferably between 3 μm and 20 μm, and more preferably between 5 μm and 20 μm.

[0127] The second filtration makes use of a membrane having a porosity of 0.2 μm. It is possible, for the second tangential filtration, to define a porosity threshold less than 5 μm, and preferably less than 3 μm, and preferably less than 1.2 μm, and preferably less than 0.65 μm, and preferably less than 0.2 μm.

[0128] Concerning the permeation rate to be applied for the first tangential filtration (i.e., the sampling rate of a portion of the culture medium), a daily rate greater than or equal to one time the volume of the cultivation facility 27, and preferably greater than or equal to three times the volume of the cultivation facility 27, is preferably selected.

[0129] Concerning the tangential rates to be applied in the first tangential filtration, it is preferable to apply rates less than or equal to 2.1 m.s.sup.−1, and preferably less than or equal to 0.7 m.s.sup.−1.

[0130] Unitary Operations and Operating Conditions for the Lysis of the Contaminating Microorganisms

[0131] Concerning the lysis of the contaminating microorganisms, a hot alkaline lysis is used. A volume of 5N soda was added to the concentrated contaminating microorganism solution in order to reach a final concentration of 0.1N. This solution was then kept at 70° C. for 10 minutes before being cooled.

[0132] Results Obtained

[0133] Before the sampling step 21 of the cultivation method, the microorganism of interest is left in autotrophic culture in the culture medium 30 for a time t in order to validate the correct physiological state of the strain and establish reference data in respect of the growth performances. After the time t, the method is launched in a repeated cyclical fashion according to the cycle C2 with the step 26 of supplying organic carbon and the concentration step 23.

[0134] In autotrophy, a quick observation makes it possible to demonstrate that the strain is growing with a first phase, up to 100 h, corresponding to exponential growth then, a second phase of linear growth, between 100 h and 600 h. Finally, a deceleration phase is observed after 600 h. These trends are conventionally observed in the case of non-balanced growth, i.e., in the case where an environmental factor becomes limiting. This may consist of a nutritional factor (substrate, metabolism product) or physicochemical factors. In this case, it is reasonable to hypothesize that the light energy can become limiting beyond a certain cell density (shading phenomenon).

[0135] By applying a linear regression, it is possible to determine a representative equation of the growth rate, namely: y=0.002x+0.4453.

[0136] It is also possible to evaluate the volume productivity of the strain thanks to the mass concentration measurements of the microorganism of interest.

[0137] The cellular concentration in the cultures is estimated by measuring the absorbance of a liquid sample. The absorbance measurement of the medium is carried out at 600 nm by means of a spectrophotometer. This absorbance (or optical density) is considered as having a linear relationship with the concentration (Beer-Lambert law, Abs=ϵ.I.C) for values between 0.1 and 0.4 units. Dilutions are made so as to be situated in this validity range.

[0138] The mass cellular concentration is also measured by measuring the suspended solids (SS). The concentration of suspended solids ([SS]) is estimated by weighing the dry mass of a known volume of cell suspension. The latter is filtered on a membrane of pore size 0.45 μm (Φ=4.7 cm) previously dried at 60° C. and 200 mm Hg for 24 hours and weighing. After filtration, the membrane and the cell deposit are dried under the same conditions and then weighed. The difference in mass with respect to the volume makes it possible to obtain the concentration of suspended solids in the filtered suspension.

[0139] The microbial growth is evaluated in terms of doubling time (or generation time, t.sub.g) and volume productivity (r.sub.x).

[0140] The autotrophic growth parameters of the AFA strain are a maximum suspended solid concentration ([SS]max) of 0.75 g.L.sup.−1 and a maximum volume productivity (r.sub.x max) of 26.10.sup.−3 g.L.sup.−1.j.sup.−1.

[0141] When the method according to the present invention is implemented by mixotrophy, i.e., when the first cycle C2 is initiated starting with the step 26 of supplying organic carbon to the culture medium, a gain in productivity is observed despite the consumption of some of the organic carbon supplied by the contaminating microorganisms. Indeed, adding lysate of contaminating microorganisms to the culture makes it possible to supply highly digestible organic carbon and therefore, to recycle some of the initial organic carbon consumed by the contaminating microorganisms.

[0142] Calculation of the Productivity Yield Gain for Cultivating AFA with the Cultivation Method by Mixotrophy

[0143] The mass concentration of AFA was recorded over time (every hour for 150 h) in a native BG11 culture medium (autotrophy: Control). In parallel, the mass concentration of AFA was recorded over time in BG11 culture media during implementation tests of the cultivation method according to the present invention carried out by mixotrophy, the step of reintroducing the lysate into the culture medium being carried out with different lysate concentrations (0.1 g/L or 1 g/L or 5 g/L) according to the tests.

[0144] The progression of the mass concentration of AFA over time can be seen in the graph in FIG. 3.

[0145] These results indicate that adding lysate makes it possible to increase the productivity and the maximum AFA concentration in the cultures. This effect is all the more pronounced as the lysate concentrations are high for values between 0.1 and 5 g/L.

[0146] A stoichiometric analysis shows that adding 5 g/L of lysate (corresponding to 165 mg of organic carbon per liter) makes it possible to increase the maximum AFA titer by 111 mg of AFA per liter. This being equivalent to a heterotrophic production yield of microorganism of interest of 0.6 grams of AFA per gram of organic carbon supplied by the lysate.

[0147] A kinetic analysis shows that adding 5 g/L of lysate makes it possible to increase, for up to 29 h of culture, the AFA productivity from 26.4 mg of AFA per liter per hour to 91.2 mg of AFA per liter per hour, i.e., +340%.