AU736564B2

AU736564B2 - Culture of micro-organisms

Info

Publication number: AU736564B2
Application number: AU52317/98A
Authority: AU
Inventors: Stephen Skill
Original assignee: Photosynthesis Jersey Ltd
Current assignee: Photosynthesis Jersey Ltd
Priority date: 1996-12-06
Filing date: 1997-12-03
Publication date: 2001-08-02
Anticipated expiration: 2017-12-03
Also published as: WO1998024879A1; GB2320031A; AU5231798A; GB2320031B; EP0946708A1; IL130319A0; WO1998024879A9; GB9625400D0

Description

-1- CULTURE OF MICRO-ORGANISMS The invention relates to the culture of plant or animal micro-organisms to provide an economic harvest.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

GB-A-1547373 (1979) discloses a method of growing plant cells by passing a liquid suspension through a passageway defined by two sheets of light transparent plastics. The document teaches that by adjusting the flow rate it is possible to maintain a steady S 10 growth of biomass and a continuous harvesting operation. There is no disclosure of the S. means of circulating the suspension. US-A-4473970 (1984) discloses a similar method and teaches the inclusion of at least one pump for continuously agitating the biomass during the growth cycle of the biomass. The structure of the various pumps in the apparatus is not identified, but a header tank is present, which means high pressure.

15 US-A-5179012 (1993) discloses a closed photobioreactor including a pump of undefined o00* o structure to circulate the culture medium. WO-A-95/06111 discloses a photobioreactor which avoids the use of pumps because these cause damage to cultivated cells.

GB-A-2118572 (1983) discloses a photobioreactor comprising parallel passageways which define a continuous flow path, the wall of the passageway being transparent to light. The liquid medium is circulated in the passageways under high pressure, and in turbulent manner.

In one or more embodiments, the present invention provides an apparatus for the culture of plant or animal micro-organisms to provide an economic harvest in which a pump is -2present and selected to circulate the culture medium at a low flow rate which will encourage growth without generating harmful turbulence.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

According to one aspect, the present invention provides apparatus for use in growing a culture of micro-organisms, the apparatus comprising an arrangement of elongate passageways disposed in generally parallel relation and connected together to define a substantially continuous flow path, at least one side of the passageways being at least partially transparent to light, pumping means being present within at least one or more of the passageways and arranged to circulate a liquid culture of micro organisms through the apparatus, the pumping means comprising a vaned rotor which is elongate in the direction of its axis of rotation and which is disposed adjacent mouths of said elongate passageways, said mouths being distriubed along the length of the rotor.

According to another aspect, the present invention provides apparatus for the culture of photosynthetic micro-organisms, the apparatus comprising a deck having a floor and a roof, partion walls being present between the floor and the roof to form generally parallel passageways, the passageways opening into a main plenum conduit at each end, an inlet being present at one conduit and an outlet present at the other, the roof being formed of a light transmission material, and a pump device extending across each passageway, the pump device comprising an axially elongate vaned rotor extending across the passageways.

The invention also relates to apparatus for use in growing a culture of micro-organisms, the apparatus comprising an arrangmeent of elongate passageways disposed in generally -3parallel relation and connected together to define a substantially continuous flow path, at least one side of the passageways being at least partially transparent to light, pumping means being present within at least one or more of the passageways arranged to circulate a liquid through the apparatus, each pump comprising a flexible impeller housed within the respective passageway which is dimensioned so as to compress the vanes of the impeller, thereby to circulate the culture liquid at a low flow rate and to generate a low level of turbulence.

It is a preferred feature of the invention that each passageway be provided with independent or dedicated pump means. The pump means is preferably located adjacent 10 the inlet end of the passageway so as to circulate the liquid culture through the length of S•the passageway. Where the passageway is very long extra pump means may be provided, e.g. at intervals of about 10 to about 100 metres.

The pump means may take a wide variety of forms. Preferably the pump means comprises a rotor in a stator therefor, the stator forming part, e.g. a wall, of the 15 passageway. The rotor may be made of metal, plastics, timber, composites, rubber or the oooo like. Preferably each pump has a flexible impeller which is housed within the passageway dimensioned so as to compress vanes of the impeller, thereby to accelerate the movement of the culture liquid. Preferably the vanes are formed of a natural or synthetic flexible material, e.g. neoprene, food grade rubber, plastics, VITON, and the like.

The passageways may be made of a wide variety of materials which are transparent (or at least translucent) to light radiation. The light may be natural or artificial, e.g. incandescent, fluorescent, discharge, light emitting diodes, or the like. The light 3Asource may be external to the apparatus or it may be internal. In the latter case, the heat generated may be used to maintain the temperature of the culture being treated.

The passageways may be of rigid or flexible tubing. The passageways may be of any suitable cross-sectional shape, such as circular, oval, box section, triangular, or the like.

Most preferably the passageways are rigid and formed of polycarbonate or the like.

In one specific embodiment, the invention provides apparatus for the culture of micro-organisms, the apparatus comprising a deck having a floor and a roof, partition 9 9 S 0 9*9*

S

90 0 S S 0 00*00* 0 WO 98/24879 WO 9824879PCU1GB97/03331 4 walls being present between the floor and the roof to form generally parallel passageways, the passageways opening into a main plenum conduit at each end, an inlet being present at one conduit and an outlet present at the other, the roof being formed of a light transmission material, a pump device extending across each passageway, the device comprising a rotor having radially spaced apart flexible vanes, the wall of the passageway in the region of the rotor being dimensioned and shaped to flex vanes as the rotor rotates, thus to pump a suspension of micro organisms along the respective passageway at a low flow rate.

Apparatus of the invention may take a variety of forms. For example, it may be a purpose built wall in the open, or it may form the lining to an existing building, e.g.

bam roof. The apparatus may be generally arched or curved according to the circumstances of the installation. Extra layers may be present, e.g. to prevent or reduce the risk of overheating in the day and overcooling at night. The layers may be arranged to provide a flowing cooling or heating medium, e.g. gas or liquid. If one uses triple wall polycarbonate sheet, outer surface passageways may be plugged at pump and non-pump end. This enables good winter and summer operation. In winter, the extra layer is full of air or preferably less heat conducting media. This extra layer will essentially act as double glazing, reducing conductive heat loss. In summer this layer is filled with a conducting liquid, such that excess heat from culture is conducted through extra layer liquid phase to the exterior.

Circulation of extra layer liquid may not be necessary. Further enhanced cooling may be effected by sprinkling water onto external surface-evaporative cooling. At night the extra layer liquid would be drained from the reactor to leave an air filled insulating channel. During the day, extra layer may be filled with liquid for cooling.

The extra layer liquid (not culture) may incorporate wavelength adjusting molecules to convert the non photosynthetically active radiation (PAR) wavelength present in sunlight into PAR wavelengths. This will increase the efficiency of sunlight usage and also reduce heat accumulation by the culture, fluorescence). Wavelength modifying chemicals are not added to the culture containing passageways. The extra layer liquid may be an organic solvent enabling a wider range of dye shifting agents to be used.

Wavelength shift dyes or absorbents or other agents may be added to the medium to affect the culture, e.g. to induce a specific secondary metabolite accumulation or diminution in the biomass. Photochemicals may be added to absorb UV from sunlight.

10 According to a further aspect, the present invention provides a method of growing micro-organisms, the method comprising pumping a suspension of the micro-organisms along generally parallel passageways arranged in a continuous flow path through apparatus having at least one wall at least partially transparent to natural or artificial sunlight, the pumping being done by means of at least one pump comprising an axially 15 elongate vaned rotor extending across the passageways.

Preferably the pump includes a flexible impeller located adjacent the inlet end of the off 0 •passageway.

A wide variety of micro organisms may be treated in the apparatus of the invention.

Examples include microalgae, macroalgae tissue, photosynthetic bacteria, photosynthetic archaebacteria, heterotrophic bacteria fungi, plant tissue culture, water fleas; and the like.

Apparatus of the invention may be used in the culture of micro-organisms for the production of colourings and anti-oxidants, fishfeeds, pharmaceuticals and the like.

WO 98/24879 PCT/GB97/03331 6 Apparatus of the invention is able to maintain continuously growing cultures of Haematococcus for several months to provide commercial harvests of Astaxanthin.

Apparatus of the invention can be used for the culture of strains of Dunaliella, a unicellular, flagellated green algae without a cell wall which contain high concentration (up to 10%) of beta-carotene when growing under high light intensities and high salinities (100g/I). Beta-carotene has traditionally been used as a food colouring agent and may have medical uses. One major benefit of the apparatus of the invention is purity of the products. Although several companies grow Dunaliella in open ponds, product contamination regularly occurs. Apparatus of the invention is able to maintain continuously growing contaminant- free cultures of Dunaliella resulting in high purity beta-carotene.

Apparatus of the invention can be used to produce live feeds, on site, or dried and processed algae based feeds that are shipped to users.

Micro algae cultivated in the apparatus of the invention provide the most effective biological means known to remove carbon dioxide from the atmosphere. At the same time, the biomass they produce can be used as fuel.

Micro algae can remove carbon dioxide from the atmosphere 25 times faster than the fastest growing trees. Carbon dioxide is removed from stack gases before it even enters the atmosphere. Trees, on the other hand, remove carbon dioxide after it enters the earth's atmosphere. Biomass from micro algae can be used as a fuel.

Micro algae produced at the site of a fossil fuel burning plant could be directly fed into the fuel combustion process.

WO 98/24879 PCT/GB97/03331 7 Micro algae cultivated in accordance with the invention can supply quantities adequate for all purposes ranging from screening to production.

Apparatus of the invention may be incorporated into a multistage waste water treatment system. For example, a high strength organic waste or substrate may be used as the starting material which is passed through successive stage of treatment with one or more of high rate anaerobes photosynthetic anaerobes photosynthetic aerobes algae aerobes algae microfauna In one specific method, the apparatus is first filled with the water. Raw effluent is then introduced into the reactor at low rate. Samples of algal and/or bacterial innocula are added to the influent, innocula obtained from a farm yard when treating livestock rearing effluents). The pump system is set at a low circulation rate to generate low turbulence. Slime layers form on passageway inner walls. Within the slime layer consortia of bacteria, algae, microflora and fauna colonise. The pump rate is gradually increased to operating velocity, not sufficient to dislodge slime layer). This colonisation may take 2 to 8 weeks dependent on effluent. The effluent input rate is increased gradually according to the quality of discharge.

-8- One will observe that different types of biomass occupy different zones in a linear sequence within the reactor. The zone adjacent to the effluent input will be dominated by an anaerobic (oxygen free) consortium of organisms, this is succeeded by oxygen producing microalgae which are in turn replaced by a complex community of algae and bacteria and the organisms which feed upon them (protozoa, rotifers and nematodes). As effluent enters the reactor, the anaerobes assimilate the complex organic molecules present in the effluent. They in turn release metabolites utilisable by bacteria and/or algae in the neighbouring zone, which in turn provide feed for the rotifers, etc. The organic strength and nutrient content of the effluent is gradually decreased as it passes 10 along the length of the reactor. Excess biomass will shear from the inner walls and ,.oOeither re-adhere to the passageway walls further down the reactor or be washed out of the system in the final discharge where they will be readily sedimented and/or collected for 0 **9 other uses.

The end product (and intermediate products) may be used for a variety purposes, e.g. as 9 9 *9 15 a feed for fish; or the like. The apparatus may also be useful in connection with conventional tissue or microbial fermentations e.g. brewing. The multistage operation may be a continuious cultivation or a batch process.

9 Apparatus of the invention may also be used in the treatment of gases.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

8A- In order that the invention may be well understood it will now be described, by way of example only, with reference to the accompanying diagrammatic drawings, in which ooooo o*o ooooo WO 98/24879 PCT/GB97/03331 Figure 1 is a plan view of one embodiment of apparatus of the invention; Figure 2 is a cross-sectional view taken of one pump in the apparatus Figure 1; Figure 3 shows end views of different embodiments of apparatus; Figure 4 shows one mounting of apparatus of Figure 1; Figure 5 is a perspective view of apparatus of the invention mounted on the side of a tank containing a culture to be treated; Figure 6 is an exploded view of one pump in apparatus of the invention; Figure 7 is a perspective view of an elongate arch incorporating apparatus of the invention, and Figure 8 is a front elevation and side elevation of two forms of apparatus mounted on the roof of existing buildings.

The apparatus shown in Figures 1 to 4 comprises a deck or platform 1 formed of floor 2 and a roof 3 having vertical partitions 4 to define parallel passageways As shown in Figure 3 the passageways 5 are rectangular in cross-section. The ends of the adjacent partitions are linked together by end couplers 6, which extend longitudinally of the apparatus so that the passageways together define a WO 98/24879 PCT/GB97/03331 continuous serpentine or zig zag flow path. An inlet 7 is present at one side, and an outlet 8 at the other of one of the couplers. The platform is made of a light transmissive plastics material such as polycarbonate. Such items are commercially available at reasonable prices.

A pump 10 is present and extends across each passageway. As shown in Figures 2 and 5, each pump comprises a rotor shaft 11 having radially spaced apart flexible blades or vanes 12, which have a reinforcement 13 at their free ends. The rotor is formed of metal, plastics, wood or the like; the vanes are of rubber, neoprene or the like. As shown in Figure 2, the rotor 11 is mounted in a stator block 14 having through holes 15 to receive ends of passageway pipe 5. The block has a lid 16 defining a flat wall to flex the vanes 12, thereby to provide a pumping impulse to the flow. In this way, a culture suspension is given a gentle urge forward each time the rotor rotates a full circle so adding turbulence to the culture. A motor 17 (Fig. 4) is provided to rotate the rotors 11.

As shown in Figure 3, the deck 1 may take a variety of forms, and may be mono or multi layer, and the passageways may be of different internal shapes.

As shown in Figure 4, the platform 1 is pivotally mounted on a support 20 so that it may be inclined towards a natural light source, i.e. the sun, or an artificial one, not shown.

In contrast to other bioreactors where a single pump or agitation mechanism moves a large volume of liquid, it will be noted that apparatus of this invention incorporates WO 98/24879 PCT/GB97/03331 11 one pump for each passageway. (There may be circumstances in which two or more passageways share a pump).

In the embodiment shown in Figure 6, the impeller 11 is mounted on a rotary shaft 18 powered by a motor 17 (Figure 4) at one end. Bearings 21 and seals 22 are present, together with end plates 23 as required. The rotor extends across to the opposite side of the apparatus where the rotor portion 24 has no impeller. Different lengths of rotor may be joined together to rotate in synchronism.

As shown in Figures 6 to 8 apparatus of the invention may form part of building structure-area under sunlight contact surface may be used for industrial, agricultural, domestic purposes. Retrofits of existing building structures, e.g. installation of wastewater treatment facilities where lack of land area prohibits new buildings, are also possible.

Apparatus of the invention may include specially designed polycarbonate or plastic sheet extrusions where the channels are substantially circular for biomass passage.

Circular section is preferred because flow characteristics and turbulence distribution are more uniform in a substantially circular channel.

Where the apparatus is a continuous bioreactor one can utilise wavelength enhancing dyes at different stages along the reactor, e.g. for the induction of beta carotene synthesis and accumulation in Dunaliella species, beta carotene inducing wavelengths may be minimised in the early growth stages while the later stages towards the end of the reactor beta carotene inducing wavelengths are enhanced.

Partitions may be present along the length of the apparatus so that different WO 98/24879 PCT/GB97/03331 12 conditions may apply to different passageways, e.g. temperature, illumination and pH and the like. The method may be applied to Haematococcus or Prophyridium cruentum.

In the case of waste water treatment (fixed film growth) the following may apply: Anaerobes Photosynthetic Photosynthetic aerobes Crustacae etc anaerobes Secondary treatment Tertiary Quaternary The following is an example of Spirulina growth media:- Dissolve the following weight of salt in the appropriate volume of water from the hot tap.

Volume 1 litre 10 litres 20 litres Salt (grams) NaHCO 3 18 180 360

K

2

HPO

4 0.5 5 NaNO 3 2.5 25 K2 S04 1 10 NaCI 1 10 MgSO 4 0.2 2 4 CaCl2 0.04 0.4 0.8 When the basal salts are completely dissolved add (using the recipes below): Solution A 1 ml 10ml Solution B 1ml 10ml Fe-EDTA solution 1ml 10ml WO 98/24879 WO 9824879PCT/GB97/03331 13 For lOX micronutnient Solution A ml 0. 1 1 2 SolutionB Ml 0.1 1 2 Fe-EDTA solution 1 10 ml Trace element solution A (to make 1 litre) 1lOxsoI

H

3 B0 3 g 2.86 28.60 MnCI 2 g 1.81 18.10 ZnSO 4 .7H- 2 0 g 0.22 2.20

H

2 0 g 0.079 0.79 M00 3 g 0.015 0.15 Trace element solution B (to make 1 litre) 1lOxsoI

NH

4

VO

3 mg 22.96 229.60

K

2 Ca 2 (S04 )4.24H mg 96 960.00 NiSO 4 .7 H 2 0 mg 47.85 478.50 Na 2 W0 4 .2 H 2 0 mg 17.94 179.40 Te 2 (S0 4 )3 mg 40 400.00 Co(N0 3 2 .6 H 2 0 mg 43.98 439.80 The pH of the resulting media should be around 9.5; pH 9-10 is acceptable for Spirulina growth. CO 2 Iair mixture 1:20 to 100% 002 bubbled into culture.

WO 98/24879 PCT/GB97/03331 14 The following is an example of culture of anaerobes in apparatus of the invention.

Anaerobic photosynthetic bacteria such as Rhodopsuedomonas sp. require complex carbon substrates such as lactic acid.

In use, a culture of micro-organisms capable of photosynthesis is added to an aqueous nutrient medium to form a suspension. The culture is introduced to the apparatus via the inlet. The apparatus is then presented to the light, and the pumps energised by the motors so as to circulate the suspension through the apparatus with a degree of turbulence. The agitation of exposure to the light enhances the rate of growth of the micro-organisms. Nutrients are added via the inlet as required, e.g. mineral salts and CO 2 The inclination of the platform is adjusted for maximum exposure, either manually or automatically.

The invention provides apparatus useful in the large scale culture of microorganisms and having a combination of useful properties, in particular good light utilisation, efficiency and control of turbulence, temperature and gas transfer. The apparatus may readily be sterilised and may be used to cultivate a wide range of micro organisms.

Claims

1. Apparatus for use in growing a culture of micro-organisms, the apparatus comprising an arrangement of elongate passageways disposed in generally parallel relation and connected together to define a substantially continuous flow path, at least one side of the passageways being at least partially transparent to light, pumping means being present within at least one or more of the passageways and arranged to circulate a liquid culture of micro organisms through the apparatus, the pumping means comprising a vaned rotor which is 10 elongate in the direction of its axis of rotation and which is disposed adjacent mouths of said elongate passageways, said mouths being distributed along the length of the rotor.

2. Apparatus according to Claim 1, wherein the pump means is present adjacent 15 the inlet ends of the passageways. Apparatus according to Claim 1 or 2, wherein the rotor has flexible vanes and is housed within passageways dimensioned so as to compress vanes of the impeller, thereby to accelerate the movement of the culture liquid.

4. Apparatus according to any preceding Claim, wherein the passageways are generally box section passageways of performed plastics body.

16- Apparatus according to Claim 4, wherein the body is formed of polycarbonate or the like. 6. Apparatus according to Claim 4 or 5, wherein the body has a plurality of layers of partitions. 7. Apparatus for the culture of photosynthetic micro-organisms, the apparatus comprising a deck having a floor and roof, partition walls being present 10 between the floor and the roof to form generally parallel passageways, the passageways opening into a main plenum conduit at each end, an inlet being present at one conduit and an outlet present at the other, the roof being formed of a light transmission material, and a pump device extending across each passageway, the pump device comprising an axially elongate vaned rotor 15 extending across the passageways. S: 8. Apparatus as claimed in Claim 7, wherein the rotor has radially spaced apart S"flexible vanes, the walls of the passageways in the region of the rotor being dimensioned and shaped to flex the vanes as the rotor rotates, thus to pump a suspension of micro organisms along the passageways. -17- 9. A method of growing micro-organisms, the method comprising pumping a suspension of the micro-organisms along generally parallel passageways arranged in a continuous flow path through apparatus having at least one wall at least partially transparent to natural or artificial sunlight, the pumping being done by means of at least one pump comprising an axially elongate vaned rotor extending across the passageways. A method according to Claim 9, wherein the pumping is done using a pump 0 including a flexible impeller located adjacent the mouths of the passageways. ooo 1 9 9 9 11. A method according to Claim 9 or 10, wherein raw effluent is added to water in the apparatus, the pump is run to cause the liquid culture to circulate at a low circulation low turbulence rate to cause or allow slime layers to form on the inner walls of the passageways. 12. A method according to Claim 11, wherein different types of biomass occupy different zones in a linear sequence within the apparatus. 13. A method according to Claim 12, wherein the apparatus contains successively from the inlet: anaerobic organisms; oxygen producing algae; and protozoa, rotifers and nematodes.

18- 14. A method according to any preceding Claim, including adding gas to the apparatus. 15. Apparatus for use in growing a culture of micro-organisms, substantially as described hereinabove with reference to any one of the Examples and/or Figures 1 to 8 of the accompanying drawings, but excluding comparative examples. 16. A method of growing micro organisms, substantially as herein described with reference to any one of the Examples but excluding comparative examples. DATED this 15 th day of June 2001 PHOTOSYNTHESIS (JERSEY) LIMITED eo by istered Atto of The Insti e of Patent and rk Attorneys of Australia of BALDWIN SHELSTON WATEPC