GB2118572A - Culture growth and apparatus therefor - Google Patents

Abstract

The photobioreaction of a liquid culture is carried out in a reactor system having a light admitting flow part 5, 6 and a return part 7. The flow part includes a header reservoir 1 for the ingress of said culture and an egress part 3 for the egress of said culture and is so arranged and configured that culture flowing therethrough is illuminated whilst flowing in a turbulent manner. In the return part 7, at least one passageway for return flow of the medium to the header reservoir 1 has pumping means 8 for inducing the return flow by propulsive action of a kind such that the flow path of the part of the culture being propelled is generally independent of the propulsive action. A peristaltic pump or a gas injector 9 is preferably used for the pumping. <IMAGE>

Description

SPECIFICATION Culture growth and apparatus therefor The present invention relates to culture growth and apparatus therefor. Objects of the present invention are the provision of a reactor system and a method by which the photobioreaction of cultures can be achieved in a convenient manner.

In accordance with the present invention, there is provided a reactor system for use in the photobioreaction of a liquid culture (usually a culture of a microorganism), said reactor system comprising a flow part constructed at least in part of a transparent material and a return part, said flow part including a header reservoir for the ingress of said culture, an egress part for the egress of said culture and being so arranged and configured that the culture when flowing therethough is illuminated via the transparent material whilst flowing in a turbulent manner, said return part providing at least one combination of a passageway for return flow of said medium from the egress part to the header reservoir, and pumping means for inducing said return flow by propulsive action on the culture when in said passageway, of a kind such that the flow path of the part of the culture to which the propulsive action is applied is generally independent of said propulsive action.

Further in accordance with the present invention, there is provided a method of cultivation by photobioreaction in a liquid culture medium characterised by the use of a reactor system as aforesaid.

The primary application of the invention is to the cultivation of microorganisms, unicellular algae or bacteria. It may however be applied also to the cultivation of other vegetable matter such as plant tissue cells and rotifers.

By having the culture flowing in a turbulent manner during its illumination, the long germ shading of significant proportion of the individual unicellular or other organisms by overlying parts of a dense culture is reduced. It is found that the intermittent illumination of the individual organism resulting from the turbulence makes efficient use of the incident radiation whilst enabling a dense culture to be employed.

A major application of the invention is to the capture of solar energy by photosynthesis and it is found that such capture per unit area can readily be made 10 or more times that typically achieved in conventional agriculture and several times that achieved by algal culture in an open pond. Photosynthesis in a culture of such density that the effect of the turbulence is substantial can lead to economy (including economy in the energy requirements) in subsequent processing of the culture. Examples of final products which may be produced are starch, fats, proteins, and glycerol. The residues can be employed for various purposes, eg. fish foods or fertilisers and, in some cases, as sources of vegetable fibre or the production of methane by fermentation.

Nitrogen fixation may be achieved by the application of the invention to the culture of blue-green algae. Another application of the invention is to the treatment of waste matter to produce a biomass and hydrogen.

In one preferred form of the apparatus, the pumping means is a peristaltic pump arranged to form at least a part of said passageway. In a more preferred alternative, the passageway is constituted, at least in part, by a riser section and the pumping means is a gas injector for injecting gas into the riser section.

The operation of both of these arrangements inevitably produces some directional changes within the flow of the culture. These changes are however small, even insignificant, compared with those produced by pumping systems involving reciprocating components and valves or involving centrifugal impellers. Although turbulence is required at the illumination stage, the shear forces usually involved in pumping other than by the above-indicated preferred arrangements, are found to be harmful to the cultivated matter, even in the case of unicellular organisms.

Various forms of the flow part rnay be employed in accordance with the invention including forms in which the header reservoir is horizontally extensive and is arranged to provide illumination of the culture whilst therein. For most purposes however, it is preferred to employ a header reservoir of tanklike configuration.

In a preferred construction the flow part has, leading from the header reservoir to the return part, an irradiation section constructed to provide for the culture at least one radiation-admitting flow channel having a closed cross section and arranged to produce a flow pattern of the culture such that substantially the whole thereof subjected intermittently to radiation admitted by the transparent material.

Conveniently the flow channel is formed of transparent tubing, one advantage thereof being that it will admit direct or diffuse radiation from anywhere within a wide angular range and another advantage being the simplicity with which lengths of the tubing may be joined together. An alternative arrangement is to provide the flow channel in the form of a transparent sheet material secured to a transparent or non-transparent backing. For example the channel may be constructed of transparent facing sheeting, eg. formed of glass or polyvinyl chloride or other resinous material adhesively secureed to an opaque or transparent backing, the facing sheeting, the backing, or both, being corrugated. This con struction can provide a set of parallel channel sections which may be connected in parallel or in series as preferred.

Advantageously the irradiation section is constructed to provide for the culture a plurality of said flow channels and said return part provides a plurality of combinations as aforesaid, one for each flow channel. This arrangement is convenient in the commerical application of the invention. Each flow channel can be provided by a simple continuous structure for example a transparent tube provided with its own return flow passageway and pumping means. A series of the channels can be mounted together in proximity, to intercept incident radiation over the required effective area. The provision of gas injectors for the pumping gives a very economic construction. Each need involve no more than a simple inlet for admitting the gas all the inlets or a group of the inlets, may be supplied from a common gas supply line fed by a single gas pump.

Where necessary, the design of nozzles to eject the gas at a suitable rate and bubble-size and the selection of the pump may be achieved by simple routine experiment. Inlets each in the form of one or more hypodermic needles are satisfactory in most cases.

When the irradiation section is constructed of tubing, the length of the tubing should normally be, at the very least, 20 times the average cross-sectional dimension. This average must be low enough to promote adequate turbulence and the length determines the area exposed to the irradiation.

The reservoir, whether of tank-like construction or not is, for most applications, enclosed and has a gas exit passageway in communication therewith. This exit passageway may be provided with gas pumping means for supplying gas from such gas exit passageway to said gas injector. In some important applications of the invention a product of the growing culture is a gas (eg. oxygn). This gas may be circulated as the pumping gas or part thereof and withdrawn from the system at an appropriate rate.

A convenient arrangement is to provide the header reservoir with an output passageway for the withdrawal of culture as harvest and a supply passageway of nutrient medium to the culture.

For simplicity of control, the system of the invention may have at least one sensor responsive to a composition parameter of the culture to produce an output signal representing said parameter. Additionally the system may be provided with supply means responsive to said output signal for maintaining said parameter within a determined range. By providing a number of sensor responsive to different parameters and supply, or withdrawal means, responsive thereto. the system may be arranged to operate automatically at least as regards parameters which vary rapidly.

Automatic operation is most conveniently achieved by logic circuitry, preferably of the electronic type. The design and construction thereof is preferably based on computer technology. Response of the system to fluctuations of the incident solar radiation is readily achieved, thus enabling the system to be operated efficiently in regions of pronounced fluctation such as the United Kingdom. For regions such as desert regions where conditions change relatively slowly routine manual adjustment, especially when made with reference to signals from appropriate sensors, can yield reasonably optimum results.

A simple and convenient form of temperature sensor is a resistance thermometer. An analyser of the infra-red type may be provided for sensing the concentration of carbon dioxide gas taken from and/or supplied to the culture. Using a gas injector supplied with gas from the reservoir to provide the pumping action greatly simplifies the monitoring and control of the carbon dioxide.

The following description in which reference is made to the accompanying diagrammatic drawings is given in order to illustrate the invention. In the drawings: Figure 1 shows a simple form of the apparatus, Figure 2 shows a form of the apparatus provided for use on a larger scale than the apparatus of Fig. 1.

Figure 3 shows an apparatus incorporating a computer controller automatic control system, and Figure 4 is a flow sheet for the computer system.

The apparatus of Fig. 1, designed primarily for experimental use, has a flow part in the form of a tank 1 which serves as a header reservoir. Liquid culture 2 (total volume 4.6 litres) flows from a bottom outlet 3 via a vertical conduit 4 to and through an irradiation section 5 formed of 52 tubes of colourless Pyrex glass--only 1 2 are shown for simplicity of illustration--each having an internal diameter of 1 cm gad a length 1 m.

These tubes are connected together in series by silicone rubber U-bends 6 to provide a continuous flow path in which the culture may be irradiated. Vertical conduit 4 may be transparent or opaque.

Culture leaving section 5 enters a vertical riser section 7 of internal diameter 7mm. By injecting a stream of air or other gas through injector tube 8, communicating if necessary with riser section 7 by a nozzle at 9, the culture is induced to return to tank 1 for recirculation. Gas which separates from the culture passes out from the top of tank 1 via outlet 10 for discharge or recycling. Recycling with partial draw-off of the gas for use is appropriate in cases where a gaseous product of the photobioreaction is required.

The apparatus of Fig. 1, designed primarily for experimental use, has a flow part in the form of a tank 1 which serves as a header reservoir. Liquid culture 2 (total volume 4.6 litres) flows from a bottom outlet 3 via a vertical conduit 4 to and through an irradiation section 5 formed of 52 tubes of colourless Pyrex glassonly 1 2 are shown for simplicity of illustration-each having an internal diameter of 1 cm and a length of 1 m.

These tubes are connected together in series by silicone rubber U-bends 6 to provide a continuous flow path in which the culture may be irradiated. Vertical conduit 4 may be transparent or opaque.

Culture leaving section 5 enters a vertical riser section 7 of internal diameter 7mm. By injecting a stream of air or other gas through injector tube 8, communicating if necessary with riser section 7 by a nozzle at 9, the culture is induced to return to tank 1 for recirculation. Gas which separates from the culture passes out from the top of tank 1 via outlet 10 for discharge or recycling. Recycling with partial draw-off of the gas for use is appropriate in cases where a gaseous product of the photobioreaction is required.

In the apparatus of Fig. 2, culture flows from the tank 1 via a downwardly descending conduit 4' which extends horizontally to connect with the higher ends of a set of transparent inclined tubes 12 each 50m in length.

Tubes 1 2 are supported by a framework indicated diagrammatically at 1 3 with a fall of from 1 to 2 metres over their lengths. At its lowermost end, each tube communicates with its own riser 7' which leads upwardly to the horizontal part 1 4 of a common return conduit which connects with tank 1 by an ascending part 1 5.

At or near its bottom end, each riser is connected with a gas supply tube 8' fed with gas recycled from the upper part of tank 1 by a gas recycle pump 16. Supply tube 8' is connected with the risers 7' by control valves, not shown, set to give a uniform distribution of gas to the risers.

Fig 2 shows the transparent tubes 1 2 spaced widely apart for simplicity of illustration. In practice they are arranged close together to intercept a high proportion of the solar radiation received over the occupied area. Reflecting material, not necessarily of the specular type, may be positioned under the tubes, eg. simply on the ground, if desired. Simple access to the tubes along their length may be provided by positioning the tubes in groups of convenient width with gangways therebetween.

As shown in Fig. 2, the risers are supplied with gas direct from a single tube 8'. An alternative arrangement which facilitates uniformity of the gas supply to the tubes is to supply groups of the risers each from its own branch of a main gas tube. A number of main gas tubes, each with its own pump may be provided where preferred.

Culture is withdrawn from tank 1 as required by a harvest outlet 1 7 and the medium is replenished with nutrient solution (including trace elements which, in some cases, are recovered from the harvested culture) via nutrient input 1 8. Branch 1 9 of the recirculating gas system is provided for the withdrawal of gaseous products and branch 20 for replenishment of the circulating gas.

Line 21 indicates means for withdrawing gas or culture from tank 1 for testing purposes. The testing is preferably by automatic equipment coupled with an automatic control system.

Fig. 3 shows an automatic system in diagrammatic form. This system, which is of general application, is shown applied to an apparatus having an irradiation part 5 an a riser section 7 similar in construction and dimensions to those of Fig. 1. The particular apparatus is designed for evaluation purposes and is provided with an artificial source of radiation in the form of six mercury metal halide lamps 23 (Wotan HQI-R25QW) behind a radiation control in the form of a remotely actuated venetian blind. These lamps provide a wavelength distribution over the photosynthetic range (400 to 700 nm) which approximates to that of sunlight. The blind serves to vary the intensity of illumination as required, eg. to simulate the diurnal variation.

Automatic control is provided by an electronic computer system in the form of a micro-computer 25 connected in operational relationship with an interface 26 and provided with a video display and keyboard unit 27 and a printer 28. Some of the connections between the interface and parameter sensors and controls are indicated by broken lines.

Fig. 4 is a flow sheet for the computer systerm.

Gas withdrawn from the tank 1 via line 29 is passed through Brooks Thermal Mass Flowmeter 30 and a BOC Sirius 1 infra-red gas analyser 31 which measures the percentage of carbon dioxide. The supply of gas to the apparatus is controlled by a valve 32 and measured by a thermal mass flow meter 32a.

System 33 measures the readiation received at part 5 from the lamps 23 by suitably positioned photodiodes (not shown) and is arranged to actuate the venetian blind 24 when it is required to simulate changing solar radiation.

Culture withdrawn from and returned to tank 1 is passed through a temperature sensor and controller 34.

Ammonium hydroxide solution is supplied as a nutrient, periodically in response to computer signals, to the culture from a vessel 35 carried by a digital balance 36 and enclosed within a housing 37 to avoid evaporation losses. Trace elements are supplied from a reservoir 38.

A glass electrode pH meter and controller is shown at 39.

An antifoam agent eg. polypropylene glycol (molecular weight 2000) is supplied to the tank 1 from a reservoir 39 by a manually set timer 40.

Harvested culture is collected in a vessel 41 and liquid collected therefrom by centrifugation is return as a nutrient from a vessel 42, after purification in appropriate cases.

A pump at 43 operates to withdraw culture as harvest and to return nutrient to tank 1 as required. The pump is actuated to maintain the volume of the culture substantially constant. There is a finite possibility of failure of the computer system causing the pump to drain the system. To prevent this the pump is powered via a relay connected to a timer which operates to open the relay after an hour unless reset by a signal from the computer.

The computer is programmed to generate a re-set signal 30 minutes after ammonia has been supplied from vessel 35.

The apparatus of Fig. 3 was used in photosynthetic culture of consortium MA003 (Lee and Pirt, J.Chem.Tech.Biotechnol 31(1981), 295)) which consists of a Chlorella-like green alga and three heterotroplic bacterial species and shows optimum growth at 37"C and pH 6.5. A deposit of the consortium has been made with the Culture Centre of Algae and Protozoa, 36, Storey's Way, Cambridge, England. The date of deposit is July 1981 and the accession number is PAC 455.

A non-sterilised culture medium, having the following composition was employed (amounts in g L-'). (Part A) KH2PO4, 2.63; NH4CI was initially supplied at a concentration of 0.053 g L-1 and maintained at this level automatically by pH control; (part B) MgSO4.7H20, 1.50: CaCl2.2H20, 0.16; (part C) FeCl3.6H20, 0.096; MnsO4.4H20, 0.016; ZnS04.7H20, 0.018; CuSO4.5H20, 0.008; Na2B407.10 H20, 0.035; NaVO3, 0.0005; NiSO4.7H20, 0.0008; NaMoO4.2H20, 0.01; CoCI2.6H20, 0.005.

The medium, which allowed the culture to grow up to a maximum concentration of 259 dry weight per litre, was made up from three separate aqueous stock solutions. Solution (A) was made at 2X strength and adjusted to pH 6.7 with NaOH. Solution (B) was made at 2X strength and adjusted to pH 6.5 with NaOH, Solution (C) was made at 23X strength and adjusted to pH 2.0 with HCI. Each stock solution was prepared separately and solutions (A) and (B) were mixed just before use.

Solutions (A) and (B) constituted the bulk of the culture medium. Solution (C) was fed intermittently into the culture a- the same rate as a feed of aqueous ammonia, provided as a source of nitrogen, i.e. both stock solution (C) and ammonia feed pumps were activated simultaneously by the pH control system. Supplying trace elements in this manner was found satisfactory up to a biomass output rate of at least 1.1 X 10-4 Kg m-3 s-1 (0.4 9 L-1h-'). Stock solution (C) contributed less than 1 % of the total culture volume.

After growth of the culture had become established, the pH was maintained at 6.5 and the temperature at 37"C. Carbon dioxide was injected into the air supply at a rate sufficient to maintain the culture entering the illuminated section saturated. The air supply was regulated to give a Reynolds' number of 1 200 in the riser 1. The light intensity was adjusted to provide excess light.

Growth inhibition by the accumulation of oxygen was avoided by removal of oxygen by the ascending gas in the riser.

Turbulence in the irradiation section adequate to ensure that all the cells of the culture are frequently exposed to the radiation was obtained at Reynolds numbers of 2000 and above. The Reynolds number is a convenient indication of the turbulence. Its useful lower limit can be ascertained by experiment in particular cases. For most cases it should be at least 1000 and normally at least 1500.

Reynolds' numbers substantially greater than those required are wasteful of energy and can, in extreme experiments with apparatus in which gas is injected into the photostage itself result in the formation of spaceconsuming gas pockets and inadequate turbulence.

The Reynolds' number NR is given by the equation NR = vrp/rl where v is the liquid velocity, r is the radius of the tube, p is the liquid density and 17 is the vicosity.

With a given apparatus the rate of growth of a biomass at a constant level of illumination is a function of the rate of flow of the culture through the irradiation section. The optimum rate may be found experimentally when required. The foregoing reference to Reynolds numbers may be used as guide to the range over which the experiments are to be conducted.

Using the apparatus described, the maximum rate of carbon dioxide uptake was directly proportional to the partial pressure over the range of from 0.05 to 0.3 atmospheres under C02-limited conditions at liquid flow rate of 3.79 X 10-5, and a gas flow rate of 4.17 X 10-6, m3s-1.

Calculation from results obtained using electrical illumination indicated a photosynthetic efficiency corresponding with 16.6% storage of incident solar energy. Allowance was made for solar energy received indirectly by reflection to the transparent material of the egress part. However, this allowance was deliberately made small and it is quite probable that an efficiency of 20% could in fact be obtained using natural solar illumination under com mercial conditions. In typical commercial practice the expected annual yield is about 70 tonnes dry weight per square hectare. This is of the order of ten times the yield obtained by grass production in conventional agriculture.

Control apparatus operating in the manner described with reference to Figs. 3 and 4 can be applied to the form of apparatus shown in Fig. 2. Having the tubes 1 2 associated with a single tank 1, a single pumping system and a single control apparatus yields an economic construction. In cases where apparatus must be duplicated to cover an enlarged area, a single computer system may be arranged to control the individual installations, eg. on a time-sharing basis.

Claims (17)

1. A reactor system for use in the photobioreaction of a liquid culture, said reactor system comprising a flow part constructed at least in part of a transparent material and a return part, said flow part including a header reservoir for the ingress of said culture, an egress part for the egress of said culture and being so arranged and configured that the culture when flowing therethrough is illuminated via the transparent material whilst flowing in a turbulent manner, said return part providing at least one combination of a passageway for return flow of said medium from the egress part to the header reservoir, and pumping means for inducing said return flow by propulsive action on the culture when in said passageway, of a kind such that the flow path of the part of the culture to which the propulsive action is applied is generally independent of said propulsive action.

2. A system according to claim 1 in which the pumping means is a peristaltic pump arranged to form at least a part of said passageway.

3. A system according to claim 1 in which said passageway is constituted, at least in part, by a riser section and the pumping means is a gas injector for injecting gas into the riser section.

4. A system according to any one of claims 1 to 3 in which the flow part has, leading from the header reservoir to the return part, an irradiation section constructed to provide for the culture at least one radiationadmitting flow channel having a closed cross section and arranged to produce a flow pattern of the culture such that substantially the whole thereof is subjected intermittently to radiation admitted by the transparent material.

5. A system according to claim 4 in which the flow channel is formed of transparent tubing.

6. A system according to either of claims 4 or 5 in which the irradiation section is constructed to provide for the culture a plurality of such flow channels and said return part provides a plurality of combinations as aforesaid, one for each flow channel.

7. A system according to any one of claims 1 to 6 in which said header reservoir is of tank-like configuration.

8. A system according to any one of claims 1 to 7 in which said header reservoir is an enclosed reservoir and has a gas exit passageway in communication therewith.

9. A system according to claim 8 as appendent to claim 3 having pumping means for supplying gas from said gas exit passageway to said gas injector.

10. A system according to any one of claims 1 to 9 in which said header reservoir is provided with an output passageway for the withdrawal of culture as harvest and a supply passageway for the supply of nutrient medium to the culture.

11. A system according to any one of claims 1 to 10 having at least one sensor responsive to a composition parameter of the culture to produce an output signal representing said parameter.

1 2. A system according to any one of claims 1 to 10 having supply means responsive to said output signal for maintaining said parameter within a determined range.

1 3. A reactor system for use in the photobioreaction of a liquid culture of a microorganism, substantially as hereinbefore described and illustrated by reference to the accompanying drawings.

1 4. A method of cultivation by photobioreaction in a liquid culture medium, characterised by the use of a reactor system in accordance with any one of the preceding claims.

15. A method according to claim 14 when applied to the cultivation of a microorganism.

1 6. A method of cultivation by photobioreaction, substantially as hereinbefore described and illustrated by reference to the accompanying drawings.

17. A product of photobioreaction when produced by a method in accordance with any one of claims 14 to 16.