WO2001004263A2

WO2001004263A2 - Microorganism culture method and apparatus

Info

Publication number: WO2001004263A2
Application number: PCT/GB2000/002628
Authority: WO
Inventors: Jonathan Mortimer
Original assignee: Biofence Limited
Priority date: 1999-07-08
Filing date: 2000-07-10
Publication date: 2001-01-18
Also published as: AU5995100A; WO2001004263A3; GB9915874D0

Abstract

A method of culturing microorganisms in a bioreaction system comprising providing at least one substantially liquid impermeable flexible elongate tubular envelope having an inlet and an outlet. The outlet is spaced downstream from the inlet. The envelope comprises a longitudinally extending flowbed and a longitudinally extending roof connected to the flowbed. A gaseous atmosphere is provided between the flowbed and the roof of the envelope. Culture medium is introduced to the flowbed and allowed to flow along the flowbed from upstream to downstream.

Description

Microorganism Culture Method and Apparatus

The present invention relates to microorganism culture, and more particularly to culture of photosynthetic microorganisms .

Microorganism culture systems for cultunng photosynthetic microorganisms (photobioreactors) are well known. For example, FR-A-2596412 discloses a photobioreactor comprising an inclined longitudinal conduit through which flows a nutrient solution charged with microorganisms .

The ob ect of the present invention is to provide an improved bioreaction system and method of cultunng microorganisms, suitable for use as a photobioreactor.

Accordingly, the present invention provides a method of cultunng microorganisms m a bioreaction system, which method comprises providing at least one substantially liquid impermeable flexible elongate tubular envelope having an inlet and an outlet, the outlet being spaced downstream from the inlet, the envelope comprising a longitudinally extending flowbed and a longitudinally extending roof connected to the flowbed; providing a gaseous atmosphere between the flowbed and the roof; introducing culture medium to the flowbed; and allowing the culture medium to flow along the flowbed from upstream to downstream.

The present invention further provides a bioreaction system for cultunng microorganisms, the system including at least one substantially liquid impermeable flexible elongate tubular envelope having an inlet and an outlet, the outlet being spaced downstream from the inlet, the envelope comprising a longitudinally extending flowbed and a longitudinally extending roof connected to the flowbed; wherein the envelope is arranged to provide a gaseous atmosphere between the flowbed and the roof, and the flowbed is arranged such that culture medium introduced to the flowbed can flow along the flowbed from upstream to downstream.

A flexible elongate tubular envelope is generally relatively cheap to manufacture, such that the bioreaction system according to the present invention is advantageously more cost effective than known culture systems.

As the tubular envelope is generally cheap and easy to manufacture, the envelope may be disposable. It is generally possible, therefore, to culture hazardous microorganisms and/or use hazardous reagents for the culture of microorganisms m the bioreaction system of the present invention.

The flowbed of the envelope is preferably inclined such that the culture medium flows under gravity from upstream to downstream. The culture medium is preferably introduced together with nutrients into the tubular envelope, such that the culture medium with the nutrients therein can flow from upstream to downstream of the envelope.

The culture medium is generally arranged to flow m a relatively shallow liquid stream (preferably a thin film) along the flowbed of the envelope. This has benefits m that photo-synthetic microorganism culture along the flowbed is highly efficient auto-trophically (photo- trophically) . A relatively large culture medium surface area further maximises contact of the culture medium with oxygen for highly efficient fermentation reactions when the system is used to cultivated fermentative microorganisms.

The tubular envelope used m the present invention is effectively closed from the atmosphere (closed system) . It is preferred that the tubular envelope is substantially gas impermeable. This desirably reduces external contamination of culture inside the envelope.

In one embodiment, air or other suitable gas (preferably substantially non-contaminating and/or filtered air/gas) may be introduced into the tubular envelope so as to provide internal positive pressure within the envelope. This beneficially aids m supporting the tubular envelope m a non-collapsed condition and may be used to inflate the envelope. Means are preferably provided for introducing gas into the tubular envelope. The introduced gas may De intentionally supplied as relatively warmer or cooler than ambient air temperature m order to cool or warm the culture environment according to optimum process conditions .

In preferred embodiment of the present invention, the tubular envelope should be susceptible to sterilisation, for example, by irradiation, ultraviolet techniques or, by use of an autoclave .

The tubular envelope may therefore be sterilised prior to use for cultunng microorganisms. A sterilised envelope may thus be used when it is important to maintain a sterile culture environment.

The internal walls of photobioreactors and other bioreactors for cultunng microorganisms will generally, after a period of time, become coated with microorganisms present m the culture medium. Several patent specifications relate to cleaning mechanisms which prevent the accumulation of microorganisms on the internal walls of these bioreactors, for example, WO 98/15362.

The tubular envelope of the present invention preferably comprises plastic sheeting material. It has been discovered by the present inventors that there is minimal accumulation of microorganisms on the internal surfaces of tubular envelopes comprising plastic sheeting material.

It is a preferred feature of the present invention, that the tubular envelope is seamless m at least one of a longitudinally extending direction and a transverse direction. More preferably, the element is seamless both in a longitudinally extending direction and also m a transverse direction. This may be achieved by using an envelope comprising blown plastic sheeting, the envelope preferably having longitudinally spaced first and second ends .

In a seamless tubular envelope, accumulation of microorganisms, including contaminating microorganisms, may advantageously be minimised as there are essentially no crevices or internal structures for attachment of the microorganisms thereto. This further alleviates the need to provide a cleaning mechanism for the bioreaction system according to the present invention. The seamless tubular envelope may be easily cleaned with a high pressure hose or the like.

The flowbed of the tubular envelope is preferably a substantially planar bed surface. It is a further preferred feature that the flowbed rests on a support surface (which may be inclined) . The flowbed preferably conforms to and lies flat when resting on the support surface .

In an alternative embodiment, the roof of the tubular envelope may be supported by support members .

In a still further embodiment, the flowbed of the tubular element may be substantially non-rigid and the roof of the envelope may be more rigid than the flowbed. It is a preferred feature of the present invention that the inlet of the tubular envelope communicates with an upstream part of a flowpath, and the outlet of the envelope communicates with a downstream part of the flowpath. Such communication may be with an upstream/downstream flowpath defining structure for the inlet/outlet respectively.

The bioreaction system preferably includes a trough connecting with, and upstream of, the flowbed of the tubular envelope. The trough is preferably formed integrally with the flowbed, the integrally formed trough preferably comprising a flexible walled enclosure portion of greater depth dimension than the flowbed.

Culture medium may be continuously provided to the trough, the overflow of culture medium beneficially forming a thin uniform film along the flowbed connected to the trough.

It is preferred that the bioreaction system includes a fluid circuit for recycling at least some of the culture medium from downstream of the envelope to upstream of the envelope. The fluid circuit therefore facilitates recirculating flow of the culture medium. The recycled culture medium may be pumped around the fluid circuit preferably by a pump. Pumping the culture medium around the fluid circuit desirably induces recirculating flow.

Tne fluid circuit is preferably substantially entirely enclosed from an external atmosphere, such that the external atmosphere is desirably substantially barred from the fluid circuit. This may be achieved by the upstream and downstream flowpath defining structures comprising liquid tight (preferably substantially airtight) structures .

Desirably the wetted perimeter of the upstream (and also preferably downstream) portion of the flowpath is substantially less than the wetted perimeter of the flowbed of the tubular envelope.

It is preferred that the bioreaction system includes a vessel (which may be a tank) connecting with the flowbed of the tubular envelope, the vessel holding a volume of the culture medium. Culture medium is preferably drawn from the vessel, passed along the flowbed and subsequently returned to the vessel .

Sand and/or gravel or the like, may be contained within the vessel, beneficially filtering culture medium passing through the vessel. This filtering action may be used as a means of cleaning water or other liquids, or as a means of concentrating microorganisms m the culture medium, such as algae.

In one embodiment, the vessel may be formed integrally with, and downstream of, the flowbed of the tubular envelope. The integrally formed vessel preferably comprising a flexible walled enclosure portion of greater depth dimension than the flowbed. Desirably the bioreaction system includes means permitting harvesting of culture from the system. The harvesting means is desirably provided to permit harvest from a culture medium holding vessel . Such harvesting means may be arranged to permit batch and/or continuous harvesting of culture.

The bioreaction system preferably includes means permitting entry of microorganism culture medium into the system.

The bioreaction system preferably includes means permitting entry of nutrients into the system.

Encry of the microorganism culture medium and/or the nutrients may be batch or continuous.

In one embodiment of the invention, the system flowpath may include a plurality of tubular envelopes. The envelopes may be arranged hydraulically m parallel

(preferably connected to one another by respective inlet and outlet manifold arrangements) .

In an augmented or alternative embodiment, the system flowpath may include a plurality of tubular envelopes arranged m a cascade or series formation. In this embodiment, the adjacent envelopes extend end to end m a stacked configuration. The adjacent envelopes may be inclined m opposed directions. This has space saving benefits for the bioreaction system. The tubular envelope may comprise a translucent and/or transparent plastic material, to permit light to penetrate the culture medium to beneficially be utilised by photosynthetic microorganisms.

The bioreaction system according to the present invention, may be used to culture photo-synthetic microorganisms such as algae. A light stage of the culture (where algal photo-synthetic reactions can ensue) is typically provided by a transparent and/or translucent tubular envelope and a dark stage of the culture (where algal respiration can ensue) is typically provided by the culture medium containing vessel .

The invention will now be further described m specific embodiments, by way of example only, and with reference to the accompanying drawings, m which:

Figure 1 is a schematic representation of an exemplary embodiment of a bioreaction system according to the invention (for use m carrying out a method according to the invention) ;

Figure 2 is an enlarged schematic sectional view along line A-A of Figure 1 ;

Figure 3 is a schematic plan view of a modification of a bioreaction system according to the invention;

Figure 4 is a schematic side view of an alternative embodiment of a bioreaction system according to the mvention, which includes cascaded tubular envelopes; and

Figure 5 is a schematic side view of an alternative embodiment of a bioreaction system according to the invention m which a culture medium holding vessel and trough are integrally formed with the flowbed of the tubular envelope.

Referring to the drawings, and initially to Figures 1 and 2 m particular, there is shown a bioreaction system for cultunng microorganism (generally designated 1) . System 1 comprises a "closed loop" fluid circuit arrangement for continuously recirculating culture medium about a flowpath including a tubular envelope 2 and a culture medium holding vessel 3. The closed loop arrangement includes a fluid pump 4 arranged to pump the culture medium along a fluid conduit 5 to the upstream end of tubular envelope 2, and a fluid conduit 6 connecting the downstream end of tubular envelope 2 to holding vessel 3.

Water and nutrients are introduced into the closed loop

(sealed) system from store 7 via inlet line 8. Initial microorganism culture medium is introduced into the system via inlet line 9 from unit 10. C0₂ (or other suitable gas) is introduced into the system via inlet 21. The addition of microorganism culture medium and water/nutrients may be operated continuously, or as a batch introduction process or as an initial inoculation.

Culture medium holding vessel 3 is provided with a culture harvest takeoff 11, permitting either batch or continuous harvestmg of culture from the system.

Air (or other suitable gas) is pumped into the tubular envelope via a gas inlet duct 12. The air is pumped along ducts 12 by pump 13, the air being drawn via an air puπfymg/fliter unit 14.

The bioreaction system illustrated m Figures 1 and 2 is particularly suitable for photo-trophic reactions for culture of photo-synthetic microorganisms (such as algae) .

Initial algae culture may be introduced into the photobioreactor system 1 via inlet line 9 from unit 10

(which may be a continuous algae culture reactor) . Algal photo-bio reactions may occur m a transparent and/or translucent tubular envelope 2 and algal respiration typically takes place m holding vessel 3.

Tubular envelope 2 comprises a flexible plastics liquid- tight and airtight tubular elongate envelope 15 (shown most clearly m section m Figure 2) . The envelope 15 has a flowbed 16 and a spaced, integrally connected roof 17. The flowbed 16 m use may be substantially planar and may be supported to be inclined along its length such that the culture medium flows into the tubular envelope 2 and then flows downwardly under gravity as a relatively thin stream (or film) passing over the flowbed 16. In some embodiments, the flowbed 16 may be substantially non-rigid and the roof 17 may be more rigid relative to the flowbed 16. The flowbed 16 may be supported upon a support; the support may be banked or inclined. Alternatively, the flowbed 16 may be supported proximate its upper and lower extents and be self supporting over an intermediate length .

Because culture medium is arranged to flow as a relatively thin stream/film along the flowbed 16 through the tubular envelope 2, the bioreaction system 1 is highly efficient photo- trophically m view of the relatively large surface area for maximised light absorption. The bioreaction system 1 is also highly efficient for fermentation reactions, as the relatively large culture medium surface area maximises contact with oxygen. Where there is gravitational flow down an inclined flowbed 16, this induces enhanced C0₂ absorption from the pumped air. Because the tubular envelope 2 is substantially closed from the atmosphere contamination of the culture medium is substantially alleviated.

The air (or other suitable gas) pumped via pump 13 and ducted into envelope 15 via duct 12 ensures that the air/ gas within envelope 15 is at positive pressure with respect to ambient pressure. This aids m inflating the roof 17 to be spaced from the flowbed 16 of envelope 15, and also aids in C0₂ penetration into the flowing culture medium. Furthermore, the air/gas may be conditioned m unit 14 (by cooling or heating for example) to benefit culture process conditions as required. A vent 20 is arranged to permit the air/gas internally of the .envelope 15 to vent to an external atmosphere at a predetermined pressure internally of envelope 15

As shown m Figure 3, the envelopes 15a, 15b may be mcluded m banks of two or more connected together m parallel by respective inlet manifolds 18 and outlet manifolds 19.

In the embodiment shown m Figure 4, two tubular envelopes 2a, 2b are arranged m a 'series' cascade configuration m which respective flowbeds 16a, 16b may be inclined m complementary senses. Such a cascaded arrangement can permit minimisation of the overall area taken up by the system.

In the embodiment shown m Figure 5, the envelope 15a is shaped to define a terminal receptacle 3a which functions as a culture medium holding vessel . The envelope 15a includes a flowbed portion 16c and a base portion 16d defining the lower surface of the holding receptacle 3a. Upstream of the flowbed portion 16c the envelope 15a is shaped to define a trough 22. Culture medium is introduced to the upstream end of the envelope 15a, the culture medium typically collecting m the trough 22. Culture medium overflowing from the trough 22 beneficially forms a thin uniform film along the flowbed portion 16c of the envelope 15a, and then collects m the holding receptacle 3a.

The positive air/gas pressure internally of envelope 15a effectively inflates the holding receptacle 3a and trough 22. In this embodiment, the holding receptacle portion 3a of the envelope 15a effectively replaces the holding vessel 3 of the embodiment shown m Figure 1. Thus, algae respiration can occur m the holding receptacle portion 3a when the bioreaction system 1 is used as a photobioreactor .

Any or all of the parallel arrangement of Figure 3, the cascade arrangement of Figure 4 and the arrangement of Figure 5, may, of course, be combined in the same system.

Claims

Claims :

1. A method of cultunng microorganisms m a bioreaction system, which method comprises providing at least one substantially liquid impermeable flexible elongate tubular envelope having an inlet and an outlet, said outlet being spaced downstream from said inlet, said envelope comprising a longitudinally extending flowbed and a longitudinally extending roof connected to said flowbed; providing a gaseous atmosphere between the flowbed and the roof; introducing culture medium to said flowbed; and allowing said culture medium to flow along said flowbed from upstream to downstream.

2. A method according to claim 1, wnerem said flowbed is inclined such that culture medium flows under gravity from upstream to downstream.

3. A method according to claim 1 or 2 , wherein said envelope is substantially gas impermeable.

4. A method according to any of claims 1 to 3 , wherein gas is introduced into said envelope so as to provide internal positive pressure within said envelope.

5. A method according to claim 4, wherein said introduced gas is substantially non-contaminating and/or filtered.

6. A method according to claim 4 or 5, wherein said introduced gas is provided relatively warmer or cooler than the ambient air temperature .

7. A method according to any preceding claim, wherein said culture medium is introduced together with nutrients into said envelope, such that said culture medium with said nutrients therein flows from upstream to downstream.

8. A method according to any preceding claim, wherein said envelope has been previously sterilised.

9. A method according to any preceding claim, wherein said envelope comprises plastic sheeting material .

10. A method according to claim 9, wherein said envelope is of blown plastic sheeting, said envelope having longitudinally spaced first and second ends.

11. A method according to claim 10, wherein said envelope is seamless m at least one of a longitudinal direction and a transverse direction.

12. A method according to claim 11, wherein said envelope is seamless both m the longitudinal direction and in the transverse direction.

13. A method according to any preceding claim, wherein said flowbed is a substantially planar bed surface.

14. A method according to any preceding claim, wherein said flowbed rests on a support surface on which said flowbed conforms and lies flat.

15. A method according to any preceding claim, wherein the roof of said envelope is supported by support members .

16. A method according to any preceding claim, wherein the flowbed of said envelope is substantially non- πgid and the roof of said envelope is more rigid than said flowbed.

17. A method according to any preceding claim, wherein said inlet communicates with an upstream part of a flowpath, and said outlet communicates with a downstream part of said flowpath.

18. A method according to any preceding claim, wherein said bioreaction system includes a trough, said trough connect with, and upstream of, said flowbed.

19. A method according to claim 18, wherein said trough is formed integrally with said flowbed, said trough comprising a flexible walled enclosure portion of greater depth dimension than said flowbed.

20. A method according to any preceding claim, wherein said bioreaction system includes a fluid circuit for recycling at least some of said culture medium from downstream of said envelope to upstream of said envelope .

21. A method according to claim 20, wherein said recycled culture medium is pumped around said fluid circuit.

22. A method according to claim 20 or 21, wherein an external atmosphere is substantially barred from said fluid circuit .

23. A method according to any preceding claim, m which a vessel connects with the flowbed of said envelope, said vessel holding a volume of said culture medium.

24. A method according to claim 23, wherein said culture medium is drawn from said vessel, passed along said flowbed and subsequently returned to said vessel.

25. A method according to claim 23 or 24, wherein said vessel is formed integrally with, and downstream of, said flowbed.

26. A method according to claim 25, wherein said vessel comprises a flexible walled enclosure portion of greater depth dimension than said flowbed.

27. A method according to any preceding claim, wherein said bioreaction system includes a plurality of said envelopes .

28 A method according to claim 27, wherein sa d plurality of envelopes are arranged hydraulically m parallel .

29. A method according to claim 28, wherein said parallel envelopes are connected to one another by respective inlet and outlet manifold arrangements.

30. A method according to claim 27, wherein said plurality of envelopes is m a cascade or series formation .

31. A method according to any preceding claim wherein said envelope comprises translucent and/or transparent material .

32. A method of cultunng microorganisms substantially as described herein with reference to the accompanying drawings .

33. A bioreaction system for cultunng microorganisms including at least one substantially liquid impermeable flexible elongate tubular envelope having an inlet and an outlet, said outlet being spaced downstream from said inlet, said envelope comprising a longitudinally extending flowbed and a longitudinally extending roof connected to said flowbed; wherein said envelope is arranged to provide a gaseous atmosphere between said flowbed and said roof, and said flowbed is arranged such that culture medium introduced to said flowbed can flow along said flowbed from upstream to downstream.

34. A bioreaction system according to claim 33, wherein said flowbed is inclined such that culture medium can flow under gravity from upstream to downstream.

35. A bioreaction system according to claim 33 or 34, wherein said envelope is substantially gas impermeable .

36. A bioreaction system according to any of claims 33 to

35, including means for introducing gas into said envelope so as to provide internal positive pressure within said envelope.

37. A bioreaction system according to any of claims 33 to

36, wherein said envelope is susceptible to sterilisation.

38. A bioreaction system according to any of claims 33 to 37, wherein said envelope comprises plastic sheeting material .

39. A bioreaction system according to claim 38, wherein said envelope is of blown plastic sheeting, said envelope having longitudinally spaced first and second ends.

40. A bioreaction system according to claim 39, wherein said envelope is seamless m at least one of a longitudinal direction and a transverse direction.

41. A bioreaction system according to claim 40, wherein said envelope is seamless both m the longitudinal direction and m the transverse direction.

42. A bioreaction system according to any of claims 33 to 41, wherein said flowbed is a substantially planar bed surface.

43. A bioreaction system according to any of claims 33 to 42 , wherein said flowbed is arranged to conform to and lie flat when resting on a support surface.

44. A bioreaction system according to any of claims 33 to 43, wherein the roof of said envelope is supported by support members .

45. A bioreaction system according to any of claims 33 to

44, wherein the flowbed of said envelope is substantially non-rigid and the roof of said envelope is more rigid than said flowbed.

46. A bioreaction system according to any of claims 33 to

45, wherein said inlet communicates with an upstream flowpath defining structure, and said outlet communicates with a downstream flowpath defining structure .

47. A bioreaction system according to any of claims 33 to 46, including a trough, said trough connecting with, and upstream of, said flowbed.

48. A bioreaction system according to claim 47, wherein said trough is formed integrally with said flowbed, said trough comprising a flexible walled enclosure portion of greater depth dimension than said flowbed.

49. A bioreaction system according to any of claims 33 to 48, including a fluid circuit for recycling at least some of said culture medium from downstream of said envelope to upstream of said envelope.

50. A bioreaction system according to claim 49, wherein a pump means is provided for pumping said culture medium around said fluid circuit .

51. A bioreaction system according to claim 49 or 50, wherein said fluid circuit is substantially entirely enclosed from an external atmosphere.

52. A bioreaction system according to any of claims 33 to 51, including a vessel connected with the flowbed of said envelope, said vessel being arranged to hold a volume of said culture medium.

53. A bioreaction system according to claim 52, wherein said vessel is formed integrally with, and downstream of, said flowbed.

54. A bioreaction system according to claim 53, wherein said vessel comprises a flexible walled enclosure portion of greater depth dimension than said flowbed.

55. A bioreaction system according to any of claims 33 to

54, including means permitting harvesting of culture from said system.

56. A bioreaction system according to any of claims 52 to

54, wherein said vessel is connected to means permitting harvesting of culture from said system.

57. A bioreaction system according to claim 55 or 56, wherein said harvesting means is arranged to permit batch and/or continuous harvesting of said culture.

58. A bioreaction system according to any of claims 33 to 57, including means permitting entry of microorganism culture into said system.

59. A bioreaction system according to any of claims 33 to

58, including means permitting entry of nutrients into said system.

60. A bioreaction system according to any of claims 33 to

59, including a plurality of said envelopes.

61. A bioreaction system according to claim 60, wherein said plurality of envelopes is arranged hydraulically m parallel .

62. A bioreaction system according to claim 61, wherein said parallel envelopes are connected to one another by respective inlet and outlet manifold arrangements.

63. A bioreaction system according to claim 60, wherein said plurality of envelopes is arranged m a cascade or series formation.

64. A bioreaction system according to any of claims 33 to 63 wherein said envelope comprises translucent and/or transparent material .

65. A bioreaction system for cultunng microorganisms substantially as described herein with reference to the accompanying drawings .