WO1999001537A1

WO1999001537A1 - A bioreactor for the growth of photosynthetic microorganisms

Info

Publication number: WO1999001537A1
Application number: PCT/EP1998/004118
Authority: WO
Inventors: Davide Di Giuseppe Stefanori
Original assignee: Ingredient Technology Corporation International
Priority date: 1997-07-04
Filing date: 1998-07-03
Publication date: 1999-01-14
Also published as: AU8731498A

Abstract

Photosynthetic microorganisms are cultivated in an enclosure, and harmonic surface waves are generated, which increase the production yield of the microorganism culture.

Description

A BIOREACTOR FOR THE GROWTH OF PHOTOSYNTHETIC

MICROORGANISMS

FIELD OF THE INVENTION

The present invention relates to bioreactors and methods for the growth of photosynthetic microorganisms.

BACKGROUND OF THE INVENTION

Photosynthetic organisms represent a renewable source of economically important compounds such as carotenoids; phytosterols, or phycobiliproteins, phenols, flavonoids. thiols, isoprenoids and lipoic acids. Harvesting these compounds from higher plants, however, is costly and the yield is low. Photosynthetic microorganisms such as algae, on the other hand, may contain up to 20% of their dry weight as valuable components, and are thus up to 20 times more productive than higher plants as sources of these compounds.

Large scale cultivation of photosynthetic microorganisms is limited mainly by two factors. One is the inability of light to penetrate deep into dense cultures of microorganisms, thus restricting growth to a small volume just below the surface of the liquid growth medium. Another limitation in closed systems bioreactors is the accumulation of oxygen. In closed systems if the produced oxygen is not evacuated it accumulates within the culture. This accumulated oxygen imposes a photolimitation in the cultural growth. Photosynthetic microorganisms exposed to intense light produce oxygen during the light dependent reactions of photosynthesis which -competes with CO₂ for binding to the enzyme ribulose bisphosphate carboxylase/oxygenase. and thus inhibits photosynthesis.

SUMMARY OF THE INVENTION

The present invention is based upon the novel finding that generating surface waves in a liquid culture of photosynthetic microorganisms increases the obtainable cell biomass per unit of light receiving surface of the culture. While not wishing to be bound by any particular theory, it is believed that as waves pass over the surface of the culture, flows of culture medium are generated below the surface such that an individual organism moves in a circular or an elliptical path defining a plane perpendicular to the surface.

Each cell is, thus exposed to a flash of intense light when at the top of its elliptical path (near the surface) followed by exposure to dim light at the other parts of the path (deeper below the surface). It is believed that intermittent exposure to intense light optimizes the photosynthetic efficiency of the cells and further permits the oxygen produced during the light flashes to diffuse away from the cells during the episodes of dim light. Furthermore, by having a large number of cells cycling out of phase with one another, a larger fraction of the culture volume can be utilized for growth.

The increase in production yield in accordance with the invention is thus a result of a flash effect. Rather than being subjected to random turbulent flows, the photosynthetic cells undergo an ordered rotational motion. The individual cells do not undergo medium translation, but rather move in circles, or in the case of sinusoidal harmonic wave of the shallow-water type, in ellipse, with a radius determined by the amplitude of the wave and a period essentially equal to that of the wave. As will be appreciated, in the case of sinusoidal harmonic waves of the shallow-water type, the radius of the ellipse decreases exponentially with increase in depth. Where. as in accordance with a preferred embodiment of the invention, the generated waves are harmonic, the energy requirement is minimal.

In addition, the generation of surface waves in accordance with the invention, improves exchange of gas and nutrients between the cells and the liquid culture, and exchange of gas between the culture and the environment.

Accordingly, it is the object of the present invention to provide a bioreactor for the growth of photosynthetic microorganisms comprising (a) an enclosure for containing a liquid culture of photosynthetic microorganisms;

(b) a wave generator for generating surface waves in the culture.

Light for the photosynthetic activity of the microorganisms typically is made to come from above but may also be additionally made to come from other sides or even from within as will be explained further below.

The enclosure in which the microorganisms are grown, may have a variety of shapes and forms. For example, the enclosure may have the form of a trough, it may be an open pond, etc.

The waves generated by the wave generator are preferably harmonic waves, typically of the shallow-water sinusoidal harmonic wave type. This type of wave propagates without change in its shape, as its velocity depends on the wave frequency (or wave length).

The wave generator may have a variety of different designs. For example, in a bioreactor of the invention having the form of a trough or pond, the wave generator may be a paddle reciprocating in a forward-rearward direction, to generate waves along an axis, preferably a longitudinal axis, of the trough or pond. Typically, the paddle is pivotly fixed at its bottom end and oscillates in a rearward-forward direction about its pivot. In the case of an enclosure which has the form of a trough or a rectangular pond, the end of the enclosure opposite the wave generator is typically provided with an inclined plane extending downward from the surface to the bottom surface of the enclosure. Such an inclined surface maintains the wave in a state of equilibrium, avoids fall of the wave and also prevents excessive interference due to the phenomena of multiple reflection which may occur in long waves. The slope depends on the length of the resulting wave as well as on the length of the enclosure.

The production yield of the culture may at times be increased by adding artificial light sources, e.g. light transmitted through optical fibers with their ends embedded within the liquid culture.

The cells in the liquid culture do not undergo median translation but rather move in circles or more precisely, in the case of a sinusoidal harmonic wave of the shallow-water type, in ellipses. The circular motion of the cells has a radius and a period equal to that of the surface wave. As will be appreciated, the radius of the ellipse decreased exponentially with an increase in me depth of the enclosure, with the circular motion approaching closely that of a circle.

The circular motion of each of the photosynthetic microorganisms improves the photosynthetic process, both on a single microorganism level as well as on the culture as a whole, and thus significantly increases the culture yield. The circular motion induces a flash effect which increases photosynthetic induced production in each of the photosynthetic microorganisms.This flash effect is a result of periodic movement of the cells from the upper levels of the culture to deeper levels of the culture. When the cells are at the crest of their path, they accumulate photochemical energy, which is then utilized for biosynthesis when the cells are in deeper levels of the culture where they are protected from excessive radiation by upper culture volume. In addition, this circular motion increases exposure of a larger mass of microorganisms in the culture to photosynthetically effective solar light to allow a much higher amount of the solar energy to be converted into chemical energy. This circular motion also prevents cells from reaching light saturation levels, which for most photosynthetic microorganisms, including algae, is about 4500 Lux.

The wave period in the case of an outdoor enclosure, which may depend on exact design particulars of the enclosure and may be within the range of about 0.4 to about 3 Hz, preferably within the range of from about 0.65 Hz to about 1.5 Hz, most preferably within the range of from about

0.8 to about 1.2 Hz, typically about 1 Hz.

Typically, in conventional bioreactors of photosynthetic microorganisms, the effective culture volume, for growth and cultivation of the photosynthetic microorganisms is 120 L/m^", seeing that the thickness of the effective culture basin up to about 12 cm. In the case of the bioreactor of the invention, the effective volume is increased by about 5-6 times. In addition, from reasons which have not yet been elucidated, in a case of the algae Dicnaliella bardawill, it was found that after culturing in a bioreactor of the invention, the β-carotene content in each cell is considerably increased. The enclosure may be open, or alternatively, the enclosure may have a transparent cover. A transparent cover is useful both in order to induce a greenhouse effect, giving rise to heating of the liquid culture within the enclosure by the sun, which may be important, particularly in outdoor cultivation in cold regions. Furthermore, a closed bioreactor has the advantage of preventing contamination of the culture by other types of photosynthetic microorganisms or predators which feed on the photosynthetic microorganisms.

The enclosure having the form of a trough may have transparent side walls, which in fact may further increase the production yield. In addition, at times the bottom wall may also be made transparent and the enclosure will then be placed above ground, optionally combined with providing some light irradiation from below, e.g. by diverting some sunlight, e.g. by means of mirrors, to illuminate the enclosure from below. Nutritive substances may be added to the liquid culture continuously so as to support the growing biomass of the culture. Inorganic carbon may be supplied in a gaseous form by bubbling CO₂ into the liquid culture medium. In the case of Dunaliella it was found that in order to obtain a productivity of 20 g/m"/day, 37 g CO₂/m^"/day is required. The bubbling of the C0₂ into the culture liquid may be regulated by a pH control apparatus ma taining the inorganic carbon of an approximately constant level.

Harvesting of the photosynthetic microorganisms may be performed as known per se. Typically, in the case of many microalgae, the harvesting may take place after about 4-5 days. The present invention also provides a method for growth and cultivation of photosynthetic microorganisms, comprising:

(a) inoculating the photosynthetic microorganisms into a liquid culture medium contained in an enclosure;

(b) generating a surface wave propagating along an axis of said enclosure.

Photosynthetic microorganisms which may be grown and cultivated in accordance with the invention mav be selected from a wide variety of such microorganisms. These may include numerous genera of unicellular and multicellular algae and other photosynthetic phytoplankton. Examples are: "Spirulina platensis (Cyanophyceae), Haemetococcus pluvialis (Chlorophyceae), Chlorella sorokiniensis (Chlorophyceae), Dunaliella salina, Dunaliella barawill (Chlorophyceae), Isochrysis galbana (Haptophyceae). A preferred photosynthetic microorganism in accordance with the invention are the algae Dunaliella bardawill. cultivated for the purpose of obtaining a natural, β-carotene enriched preparation.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be demonstrated by way of non-limiting examples with reference to the accompanying drawings in which:

Fig. 1 shows a cutaway view of a bioreactor according to an embodiment of the invention, comprising a trough having a rectangular cross-sectional shape.

DESCRIPTION OF A SPECIFIC EMBODIMENT

Fig. 1 is shows a cross-sectional view through bioreactor 10 according to the invention in which one wall of a rectangular trough 12 has been removed to reveal the liquid culture of photosynthetic microorganisms 14 contained within the trough 12. The walls of the trough are preferably transparent or translucent. Means for generating surface waves in the culture medium 14 comprises a paddle 16 near one end of the trough which is hinged through a hinge 18 at its bottom edge to the bottom of the trough. A rod 20 is pivotally attached at the top end to the paddle 16 while the other end of rod 20 is pivotally attached off-center to a pulley 22. Pulley 22 can be made to revolve by means of motor 24.

Pulley 22 revolves in a direction indicated by arrow 26 and causes back and forth oscillations of paddle 16 about its binge 18 as indicated by the two-directional arrow 28, which in turn generates surface waves in the culture 14. At the end of the trough distal to the paddle 16 there is an inclined panel 30 which bring to maximization of the constructive superimposition of reflected and incident waves. The angle of the inclined panel 30 above bottom surface 32 of the trough depends on the frequency of the generated surface wave and on the length of the container.

An individual microorganism in the culture will undergo an elliptical movement in a vertical plane as indicated for one particular individual organism by ellipse 34. The elliptical paths of other individual microorganisms will in general be displaced or out of phase with the elliptical path 34 shown. In addition, during their elliptical movement the microorganisms also rotate about their horizontal axis. Illumination 36 is from above and may be natural or artificial light, natural (sunlight) being preferred. The microorganisms thus experience a flash of intense light when at the top 38 of their elliptical paths 34. otherwise they are exposed to dimmer light.

The bioreactor shown in Fig. 1 may have a depth of from about

40 to about 100 cm, typically from about 50 to about 90 cm, and preferably from about 60 to about 80 cm; and the oscillation of paddle 4 may be at a frequency within the range from about 0.65 Hz to about 1.5 Hz, preferably within the range of from about 0.8 to about 1.2 Hz and typically about 1 Hz.

As will be appreciated, a similar design principle to that of the bioreactor shown in Fig. 1, may also be applied to a larger enclosure, e.g. in the form of an outdoor pond. In case of a large enclosure, rather than a single wave generator, e.g. a paddle such as paddle 4 in Fig. 1, a plurality of such generators may at times be employed.

The trough shown in Fig. 1, is typically provided with a transparent cover 40 attached to the side walls of the trough in a fluid-tight manner. Cover 40 may be provided with an exhaust vent 42. Typically, the bioreactor will be filled to about 20-80%, preferably 20-50%. of its volume to leave some free spare (80-20%, preferably 50-20%. respectively) for accumulation of oxygen. Nutrients may be added batch-wise or continuously through appropriate ports (not shown) and CO₂ may be continuously bubbled through liquid culture by a suitable arrangement, as known per se (also not shown). Excess gas, e.g. undesired oxygen produced during photosynthesis (which accumulation may inhibit further photosynthesis and thus decrease production yield )may be moved by a suitable exhaust system also known er se (equally not shown).

Claims

CLA╬ôMS:

1. A bioreactor for the growth of photosynthetic microorganisms comprising (a) an enclosure for containing a liquid culture of photosynthetic microorganisms;

(b) a wave generator for generating surface waves in the culture.

2. A bioreactor according to Claim 1. wherein the generated waves are harmonic waves.

3. A bioreactor according to Claim 2. wherein the harmonic waves are shallow-water sinusoidal harmonic waves.

4. A bioreactor according to any one of Claims 1-3, wherein said enclosure is a trough or pond.

5. A bioreactor according to Claim 4, wherein the wave generator is a paddle reciprocating in a rearward-backward direction to generate waves along an axis of the enclosure.

6. A bioreactor according to Claim 5, wherein the paddle is pivotally fixed at its bottom and oscillates about the pivot in a forward-rearward direction to generate said waves.

7. A bioreactor according to any one of Claims 2-5, wherein the waves are generated along a longitudinal axis of the enclosure.

8. A bioreactor according to any one of Claims 2-5, wherein the wall of the enclosure opposite the wave generator is inclined.

9. The bioreactor according to Claim 8, wherein the inclination is such so as to maintain the wave in a state of equilibrium so as to prevent fall of the wave as it approaches the opposite wall.

10. A bioreactor according to any one of the previous claims, comprising a cover.

11. A bioreactor according to Claim 10. wherein the culture fills from about 20% to about 80% of the volume of the reactor.

12. A bioreactor according to any one of Claims 1-11, where the photosynthetic microorganisms are Dunaliella bardawill, cultivated for the purpose of obtaining a natural, ╬▓-carotene enriched preparation therefrom.

13. A method for growth and cultivation of photosynthetic microorganisms, comprising:

(a) inoculating the photosynthetic microorganisms into a liquid culture medium contained in an enclosure; (b) generating a surface wave propagating along an axis of said enclosure.

14. A method according to Claim 13, wherein the generated waves are shallow-water sinusoidal harmonic waves.

₁5. A method according to Claim 13 or 14, wherein the photosynthetic microorganisms are Dimaliella bardawill, which are cultivated for obtaining a natural, ╬▓-carotene enriched preparation therefrom.

RECTIFIED SHEET (RULE 91) ISA EP