WO2004074423A2

WO2004074423A2 - Reactor for industrial culture of photosynthetic micro-organisms

Info

Publication number: WO2004074423A2
Application number: PCT/EP2004/001797
Authority: WO
Inventors: Mario Tredici; Liliana Rodolfi
Original assignee: Universita'degli Studi Di Firenze
Priority date: 2003-02-24
Filing date: 2004-02-24
Publication date: 2004-09-02
Also published as: ITFI20030047A1; WO2004074423A3; EP1599570A2; IL170429A

Abstract

The present invention refers to a reactor for the culture of photosynthetic micro- organisms or plant cells suspended in a suitable culture medium, comprising a culture chamber made of a material which is transparent to photosynthetically active radiation, a large-meshed grid structure (2) suitable to contain the culture chamber, and an external support framework (1,3).

Description

"Reactor for industrial culture of photosynthetic micro-organisms"

Field of the invention

The present invention concerns a low-cost reactor for the culture of photosynthetic micro-organisms or plant cells, easy to scale up at industrial level and able to achieve high volumetric productivity and high cell concentration. State of the art

The industrial exploitation of photosynthetic micro-organisms, microalgae and cyanobacteria in particular, is limited by the difficulties encountered in scaling up the culture system or in other words in increasing the size of the reactors, which is necessary to make them commercially profitable. These difficulties, as will be explained later, derive mainly from the need of conciliating the large size modules required in industrial plants with an efficient culture system. With the exclusion of few special cases (production of high value products, such as labelled molecules), the industrial production of microalgae and derived products requires plants able to produce tens or hundreds of tons of biomass per year. Considering that the volumetric productivity of culture systems for phototrophic micro-organisms, or "photobioreactors" (hereinafter referred to as "PBR") rarely exceeds 2 grams per litre per day, industrial plants must make use of culture systems of tens or hundreds of cubic meters. The minimum size of the culture unit (module) in commercial plants is therefore in the range 0.2-0:5 m³.

In particular, the difficulties in scaling up PBR derive from the following technical and biological constraints:

- the PBR must have a high illuminated surface to volume ratio (hereinafter referred to as "Si/V") in order to achieve high volumetric productivities and maintain high cell concentrations. The need for high cell concentrations and volumetric productivities is related to the cost for harvesting and for preparing and moving the culture medium; the higher the cell concentration and the productivity per unit of volume, the lower are these costs. Similarly, the capacity of the culture to withstand contamination from invading micro-organisms will be higher, the higher are these two parameters;

- inside the reactor, oxygen is generated as a result of oxygenic photosynthesis; if this oxygen is not removed from the culture medium, it can reach levels that are toxic to the organism cultivated. The rate of oxygen production and of oxygen accumulation in the culture medium are related to the Si/V of the reactor;

- inside a closed PBR, the temperature tends to reach values which do not permit the growth of the cultivated organism; this is due to the fact that a transparent wall reactor kept outdoors behaves as a solar collector. An adequate control of temperature is therefore required;

- the culture has to be continuously mixed in order to prevent thermal stratification, sedimentation and/or aggregation of the cells, which may cause nutritional deficiencies, and in order to provide the cells with adequate light/dark cycles; - the reactor walls must be made from materials having high transparency to the photosynthetically active radiation (hereinafter referred to as "PAR") of wavelength ranging between 400 and 700 nm for oxygenic phototrophs and between 400 and more than 900 nm for anoxygenic phototrophs. Materials have moreover to be resistant to weathering and to mechanical stress, and preferably have low cost; - biofouling, i.e. the adhesion of cells or particulate or pigmented matter to the reactor walls, which may reduce transmission of the radiation useful to the growth of the cultivated micro-organisms, must be avoided;

- the carbon source for the culture, typically CO₂, must be supplied in gaseous form. Up to today, several types of bioreactors for growing phototrophic micro-organisms have been developed; most of these reactors however do not solve efficiently the problems mentioned above. Besides, even when the above described difficulties are overcome, as in some of the industrial plants currently in operation, this is done thanks to the adoption of very expensive designs and devices, as for example described in Tredici M.R. (1999), Photobioreactors. In: Enciclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Vol. 1. Flickinger M.C. and Drew S.W. (eds). John Wiley Sons, Inc. New York, pp. 395- 419. The need of a reactor that complies with the above mentioned requirements and is easily scalable, is therefore deeply felt. Summary of the invention The Applicant has devised a low-cost system for the culture of photosynthetic micro-organisms, in particular microalgae and cyanobacteria, or plant cells, suspended in a suitable culture medium, which presents good scalability and efficiently solves the above reported problems.

It is therefore subject of the present invention a reactor for the culture of photosynthetic micro-organisms and plant cells comprising:

- a culture chamber delimited by walls of a material transparent to PAR and suitable for holding the micro-organisms or the cells to be cultivated, suspended in a suitable culture medium;

- a grid structure suitable for containing the said culture chamber; and/or - a rigid framework comprising a base and a series of vertical uprights, suitable for containing the said culture chamber and/or the said grid structure. A further subject of the invention is the use of the above said reactor for the industrial culture of photosynthetic micro-organisms or cells, and the plant for the culture of phototrophic micro-organisms or cells comprising one or more of the above said reactors.

Brief description of the figures

As non-limiting examples:

Figure 1 shows two views, 1a and 1b, of the framework and of the grid structure for the containment of the culture chamber according to the invention; Figure 2 shows a particular of the grid and of the bulges of the culture chamber; Figure 3 shows a plant made of several reactors according to the invention, placed parallel and vertically on the ground;

Figure 4 shows a plant made of several reactors according to the invention, placed on the ground so as to create a tunnel having triangular section; Figure 5a shows a frontal view of a circular reactor according to the invention; and Figure 5b shows a top view of the same reactor. Detailed description of the invention

With reference to the figures briefly illustrated above, the reactor -of the invention, and its applications and advantages are described in detail herein below. Figure 1 shows two different views of the preferred embodiment of the present reactor, wherein both the grid structure and the containment external framework are present. In particular, Figure 1a shows the containment framework (1 ,3) and the grid structure (2), which consists of large-meshed grids, preferably made of a metallic material. Figure 1 b better shows the vertical cross section of the reactor, substantially rectangular in shape, which becomes slightly elliptic as a consequence of the hydrostatic pressure exerted by the culture. The vertical cross section of the culture chamber, typically rectangular or slightly elliptical, can be varied by a different and non-parallel arrangement of the grid structure and/or of the containment framework and may assume trapezoidal, or buckled, or triangular or other suitably chosen shape. Furthermore, Figure 1 b shows the grid structure (2) the base (3) onto which the reactor rests, and the uprights (1) comprised in the external metallic framework that gives stability to the whole structure.

The present reactor is suitable for building culture units or modules, in any number according to the production needs and to the available area, wherein the single module may have the following size: a) length ranging from 1 to 50 m, preferably from 10 to 25 m; b) height corresponding to the height of the reactor and ranging between 0.5 and 3 m, preferably between 1 and 1.5 m; c) width corresponding to the width of the culture chamber, and ranging between 0.01 and 0.2 m, preferably between 0.02 and 0.08 m. A module of 10 x 1 x 0.04 m (length, height, width) will contain a culture volume of 0.4 m³, which may increase up to 0.5-0.6 m³ as a consequence of the width increase caused by the internal hydrostatic pressure. According to a preferred embodiment of the invention, the present reactor , comprises the culture chamber, the grid structure containing the culture chamber, and the containment framework containing the grid structure and the culture chamber. According to another embodiment of the invention, the present reactor comprises the culture chamber, and a structure containing the culture chamber selected from the grid structure and the containment framework.

The grid structure and the containment framework are designed to contain the culture chamber. In the preferred embodiment of the invention, the grid structure and the culture chamber are both contained in a rigid external framework that gives stability to the whole structure. According to a preferred embodiment of the invention, the culture chamber is placed within the grid structure and/or the external containment framework without any connection, such as by welding or by any other type of connection; when both the grid structure and the external framework are present, they are not connected together nor to the culture chamber. The walls of the culture chamber must necessarily be transparent so as to allow transmittance of PAR to the cells, which are kept inside the chamber; for example, the walls can be made of transparent plastic sheets, films or tubes, preferably having a thickness lower than 1 mm. Preferably, the walls of the culture chamber are made of flexible plastic film. Alternatively, the walls of the chamber can be made from sheets of rigid transparent material as. for example, PVC (polyvinyl chloride), poiymethacrylate, polycarbonate, glass, fibreglass and similar.

According to a particularly preferred embodiment of the invention the material of the walls, either rigid or flexible, has anti-adhesive properties, so as to limit biofouling.

According to a particular embodiment of the invention, shown for example in Figure 1 , the said culture chamber, said grid structure and/or the said rigid framework have a substantially parallelepipedal shape and a width much smaller than length. According to another embodiment of the invention, the said culture chamber, said grid structure and/or the said rigid framework have a curvilinear shape, forming for example a semicircle; the curvature may be increased until the two extremities of the said culture chamber, said grid structure and/or the said rigid framework, meet, and the reactor assumes a closed shape, for example triangular, square, rectangular, trapezoidal, hexagonal, more or less elliptical, or circular. If the reactor assumes a closed shape, the culture chamber may not necessarily be interrupted, but may be continuous. For example, if the shape is circular, as in the embodiment illustrated in Figure 5, the culture chamber can have an annular section. These modifications of the reactor shape can be applied to reactors having the culture chamber walls made of both flexible and rigid materials.

Figure 5 shows a circular reactor in which the culture chamber is made of flexible plastic, a metal grid structure contains and gives support to the culture chamber from the outside (Figure 5a), and both a metal grid structure and a rigid framework support the chamber from inside (Figure 5b).

The culture chamber of the reactor shown in Figure 5, and of other reactors of similar closed shape, may be built using rigid plastic material of limited thickness, for example using rolls of fibreglass 1-3 m high and 1-2 mm thick. In this case, the culture chamber may be supported from outside by a grid structure or by a net or by flat rings placed at different heights as hoops in a barrel. The reactor according to the invention is provided with one or several perforated tubes placed for example at the bottom of the culture chamber; typically the tubes, made of either plastic or metal, have a diameter ranging between 0.5 and 1 cm, and are provided with holes or injectors of diameter ranging between 0.5 and 1 mm, which are placed at a distance ranging between 4 and 10 cm from each other and allow the tubes to be in communication with the culture chamber. In said tubes compressed air, or compressed air mixed with CO₂ of with other gasses suitably chosen, is introduced; the air exits from the holes into the culture achieving mixing of the culture and removal of the dissolved oxygen. In the typical case in which air is injected, this will provide the required oxygen for cell respiration during the dark period, as well. Air bubbling achieves turbulent mixing of the culture and thus provides a suitable light-dark cycle to the cells and, at least partially, cleans the internal surface of the reactor walls, thus reducing the risk of biofouling.

In the reactor according to the invention, the control of the temperature is achieved by two different systems, which can be operated alternatively or in combination. The first system consists of one or more tubes or serpentines made of metal or any other material having high thermal conductivity. The serpentine may cross the reactor longitudinally at different heights, typically near the bottom. Inside the tubes or serpentines a thermoregulated liquid is circulated. A temperature probe is connected to an actuator that opens a valve or activates a pump, which circulates the thermoregulated fluid in the serpentine according to the thermal needs of the culture. The second system for controlling the culture temperature, more suitable to cool the culture, consists of a plastic tube provided with sprinklers placed outside the reactor so as to sprinkle or nebulise water on the reactor walls and achieves evaporative cooling. The opening of the sprinklers is regulated, as in the previous example, by a temperature probe and an actuator. According to a preferred embodiment of the invention, the liquid sprayed onto the walls is collected by a suitable drain and recycled. Carbon dioxide is provided as a mixture with air or other gasses, otherwise it is supplied separately as pure gas using the aeration tubes described above or a different tube placed for this purpose in the reactor or in some zones of the reactor. According to a particular embodiment of the present reactor, the culture chamber is divided in bubbled and non-bubbled zones, so as to obtain circulation of the culture as in air lift reactors.

Besides, to increase the contact time between the liquid and the gas phase of the culture thus enhancing mass transfer, the culture chamber is provided with sections or channels, for example made by welding the opposite reactor's alls, suitable to force the ascending gas bubbles to follow predetermined routes. Preferably, at the air inlets and outlets there may be filters and devices to keep sterile the culture chamber and carry out the harvesting, the addition of the culture medium and similar operations under axenic conditions.

Through suitable openings provided at the top of the culture chamber, electrodes, probes and other sensors for measurement and regulation of the main chemical- physical culture parameters (temperature, pH and pO₂) may be introduced.

If the culture chamber is made from a plastic sheet, the chamber will be open at the top and might be closed, hermetically or not, by a suitable cover sheet provided with outlets for air and gasses and inlets for electrodes and probes. If a large flexible plastic tube is used to make the culture chamber, suitable holes for gas exit and probes will be provided in the upper part of the tube.

Similarly, if devices for cleaning the walls of the culture chamber are provided, they may consist of suitable brushes introduced from the top opening if the culture chamber is open at the top; if the culture chamber is closed, they may -consist -of devices dragged by ropes or by magnets longitudinally along the reactor. At the bottom of the culture chamber suitable valves may be provided for harvesting the culture and/or emptying the reactor. According to a preferred embodiment of the present reactor, a module of 10 m length x 1 m height x 0.04 m width can be built from a 10 x 2.2 m rectangular film (or from a flexible plastic tube 10 m long and 1.1 m high) made of transparent and flexible plastic, having a thickness lower than 0.4 mm. The film is introduced inside a parallelepipedal cage made by metal grids, 2.5 m long and 1 m high, placed vertically and in two parallel rows, at a distance of 0.04 m from each other. The lateral edges of the film (or of the tube) are welded or glued or hermetically sealed. The grids are placed inside a suitable metal framework as that indicated as (1 , 3) in Figure 1.

The grids have large meshes, for example comprised between 10 x 50 cm and 5 x 10 cm and typically 5 x 20 cm, so as not to intercept a significant part of the impinging radiation.

When the culture chamber is filled with the culture, its vertical cross section becomes slightly elliptic because of the hydrostatic pressure that pushes the chamber walls against the grids. Besides, the hydrostatic pressure causes bulging of the culture chamber walls if these are made of a flexible plastic. The bulges protrude from the grid meshes outwards, thus increasing the illuminated surface area of the reactor and achieving a "light dilution effect" (Tredici & Chini Zittelli, Biotechnol. Bioeng., 1998, 57: 187-197). This embodiment of the present invention is illustrated in Figure 2, where it is shown a particular of a reactor whose chamber walls are made from a plastic film, which because of its flexibility, forms bulges (4) that protrude from the grid meshes (2).

The reactors according to the invention, may be placed vertically on the ground as shown in Figure 3, preferably in parallel east-west oriented rows. Alternatively, the reactors of the invention may be placed on the ground with an inclination different from the vertical; besides also the orientation and the distance between the reactors may vary depending from the climatic and topographic conditions and from the photochemical requirements of the culture. An example of arrangement of the reactors with an inclination different from the vertical is shown in -Figure 4, where the reactors have alternatively an opposite inclination so as that they converge at the top and form a sort of tunnel having triangular section. This arrangement has a further advantage as the space inside the tunnel may be used, for example, to house devices for artificial illumination; therefore this arrangement makes it possible the combined use of natural light (intercepted by the walls facing upwards) and artificial light impinging from below.

The reactors according to the invention offer the possibility to be connected so as to have a continuity of the culture medium and build modules of bigger size. Typically, the connection consists of a tube of suitable diameter inserted into the reactors at the bottom near the close extremities of the two reactors to be connected. In order to obtain a continuous flow of the culture from a reactor to the next, an internal zone of the reactor will be partially isolated, for example by welding the walls, at the level of the connecting tube. This zone is not bubbled. The culture in this non-bubbled zone has a higher specific weight and moves down and then along the connection tube to a second reactor. This latter is provided with a second tube at the opposite side, again in a non-bubbled zone, which returns the culture to the first reactor or to a series of reactors similarly connected to each other. The reactor according to the invention shows the following advantages that make it suitable to overcome the limitations typical of the reactors currently in use and described above, and thus it will be able to be scaled up to industrial level maintaining its efficiency:

- the reactor of the invention has a high Si/V typically between 25 and 200 m^"1. Thus it allows to achieve high volumetric productivities and maintain high cell concentrations with consequent lower cost for preparation and movement of the culture medium and an easier control of contamination by unwanted microorganisms;

- thanks to the special structure of the reactor, an efficient degassing and circulation of the culture medium is attained without using pumps or other mechanical devices for mixing, which may damage the cells. A good efficiency of utilisation of solar radiation is achieved thanks to the light dilution effect and to the fact that a large amount of diffuse and/or reflected radiation is also intercepted;

- temperature and pH are efficiently controlled; - the materials of the reactor can be easily found and have low cost; the construction is easy. Thanks to above mentioned advantages, it can be concluded that the reactor of the invention is suitable to realise a system for the culture of photosynthetic oxygenic, and also anoxygenic, micro-organisms, suspended in a suitable culture medium, and is particularly suited to be scaled up to commercial level. Any culture medium commonly used in the culture of photosynthetic micro- organisms and plant cells, can be used in the reactor according to the invention. Preferably, the culture medium is an aqueous solution comprising salts and nutrients that are required for the metabolism and/or the growth of the cultivated organism. Examples of micro-organisms that can be cultured with the present reactor are: Chlorella and other green microalgae, Nannochloropsis, Tetraselmis, Isochrysis, diatoms, dinoflagellates, cyanobacteria, red and green photosynthetic bacteria, and similar. These micro-organisms can be used to produce biomass and products which are useful as labelled molecules, natural pigments, biopesticides, chemicals, pharmaceuticals, neutraceuticals, aquaculture feed, probiotics, food and feed ingredients, and in bio-processes such as bio-remediation or solar energy conversion into fuels.

Besides the reactor is characterised by its ease of operation, flexibility and low cost in comparison with the photobioreactors currently used at industrial level. To give an idea of the efficacy of the reactors of the invention, it suffices to say that a plant made of 400 reactors, 25 m long, 1 m high and placed at a distance of 1 m from each other, will contain 400 m³ of culture suspension and display 20.000 m² of illuminated surface area (thus achieving a significant light dilution effect), and has the potential for producing 60 tons of dry biomass per year.

Claims

1. A reactor for the culture of photosynthetic micro-organisms and plant cells comprising:

- a culture chamber delimited by walls of a material transparent to 5 photosynthetically active radiation and suitable for holding the micro-organisms or the cells to be cultivated, suspended in a suitable culture medium;

- a grid structure suitable for containing the said culture chamber; and/or

- a rigid framework comprising a base and a series of vertical uprights, suitable for containing the said culture chamber and/or the said grid structure. lό

2. The reactor according to claim 1 , wherein the said material transparent to the photosynthetically active radiation is flexible, and gives rise to bulges outwards under the hydrostatic pressure caused by the culture medium.

3. The reactor according to claim 1 , wherein the said material transparent to photosynthetically active radiation is a rigid material selected from the group

15 consisting of PVC (polyvinyl chloride), polycarbonate, polymethacrylate, glass, and fibreglass, in the form of sheets or foils or tubes.

4. The reactor according to claim 2, wherein the said material transparent to photosynthetically active radiation is a flexible plastic material in the form of sheets, films, or tubes, having a thickness lower than 1 mm. 0

5. The reactor according to claim 1 , wherein the said material transparent to photosynthetically active radiation has anti-adhesive properties.

6. The reactor according to claim 1 , wherein the said culture chamber, the said grid structure and/or the said rigid framework have a substantially parallelepipedal shape and width much smaller than length, and wherein the said culture chamber 5 takes an elliptic shape under the hydrostatic pressure caused by the culture medium.

7. The reactor according to claim 1 , wherein the said grid structure consists of a metal grid with mesh size ranging between 10 x 50 cm and 5 x 10 cm.

8. The reactor according to claim 1 , further comprising one or more perforated 0 tubes for injecting inside the culture chamber compressed air, compressed air mixed with CO₂, pure CO₂ or other suitable gas.

9. The reactor according to claim 8, wherein the said perforated tubes are arranged so that inside the reactor both bubbled and non-bubbled zones are formed.

10. The reactor according to claim 8, further comprising interspaces or channels inside the culture chamber that force the ascending gas bubbles to follow predetermined routes.

11. The reactor according to claim 8, wherein inlets and outlets of the said tubes are provided with filters or other devices able to maintain axenicity of the environment.

12. The reactor according to claim 1 , further comprising one or more systems for controlling temperature inside the culture chamber.

13. The reactor according to claim 12, wherein the said system for controlling temperature consists of one or more tubes made of a material having high thermal conductivity, and running longitudinally through the reactor, inside which tubes a liquid for thermoregulation circulates according to the thermal requirements of the culture thanks to an electrically operated valve or an injection pump guided by a regulator connected to a temperature probe.

14. The reactor according to claim 12, wherein the said system for controlling temperature consists of a plastic tube placed externally to the reactor and provided with nozzles, whose opening is regulated by a temperature probe, in a way that water or other liquid is sprinkled on the wall of the reactor for evaporative cooling according to the thermal requirements of. the culture.

15. The reactor according to claim 1 , further comprising electrodes, probes and other sensors introduced in the said culture chamber for the measurement and regulation of pH, temperature and pO₂.

16. The reactor according to claim 1 , further comprising valves for harvesting the product and for emptying the reactor, placed in the lower part of the culture chamber.

17. The reactor according to claims 1-16, having length ranging from 1 to 50 m, height ranging from 0.5 to 3 m, and width ranging from 0.01 to 0.2 m.

18. The reactor according to claim 17, having length ranging from 10 to 25 m, height ranging from 1 to 1.5 m, and width ranging from 0.02 to 0.08 m.

19. The reactor according to claim 1 , wherein the said grid structure and/or the said rigid framework have a parallel arrangement and the vertical cross section of the said culture chamber is substantially rectangular or slightly elliptical.

20. The reactor according to claim 1 , wherein the said grid structure and/or the said rigid framework have a non-parallel arrangement and the vertical cross section of the said culture chamber is trapezoidal, buckled, triangular or of other suitably chosen non-rectangular shape.

21. The reactor according to claim 1 , wherein the said culture chamber is placed within the grid structure and/or the external containment framework without any connection, such as by welding or by any other type of connection; when both the grid structure and the external framework are present, they are also not connected together nor to the culture chamber.

22. The reactor according to claim 1 , further comprising a device for cleaning the walls of the culture chamber consisting of suitable brushes introduced from the top opening if the culture chamber is open at the top; or consisting of devices dragged by ropes or by magnets longitudinally along the reactor, if the culture chamber is closed.

23. The reactor according to claim 1 , further comprising a suitable cover sheet provided with outlets for air and gasses and inlets for electrodes and probes.

24. The reactor according to claim 1 , wherein the said culture chamber, the grid structure and/or the rigid framework have a curvilinear shape.

25. The reactor according to claim 24, wherein the said culture, chamber, the grid structure and/or the rigid framework have a circular closed shape.

26. The reactor according to claim 24, wherein the culture chamber is continuous.

27. The reactor according to claim 25, wherein the said culture chamber is delimited by walls of rigid plastic material.

28. The reactor according to claim 27, further comprising flat rings supporting from the outside the said culture chamber made of plastic rigid material and placed at different heights as hoops in a barrel.

29. A plant for the culture of phototrophic micro-organisms or plant cells, comprising one or more reactors as described in claims 1-28, connected with each other by a tube of suitable diameter inserted at the extremities of contiguous reactors in such a way to create a continuous flow of culture from a reactor to the next.

30. The plant according to claim 29, wherein the said reactors are disposed in parallel rows and vertical with respect to the ground.

31. The plant according to claim 29, wherein the said reactors have alternatively opposite inclination and converge at the top so as to form a tunnel having triangular section for the possible housing of artificial illumination devices.

32. Use of the reactor as described in claims 1-28 for the industrial culture of photosynthetic micro-organisms and plant cells.

33. Use of the reactor according to claim 32, in which the said photosynthetic micro-organisms are selected among oxygenic and anoxygenic micro-organisms.