阅读说明：本技术 生物反应器装置和方法 (Bioreactor apparatus and method ) 是由 J·P·梅尔乔里 于 2020-05-04 设计创作，主要内容包括：提供了一种用于产生生物质或生物产物的设备,所述设备包括：至少一个细长生物反应器,所述生物反应器包括至少一个外膜片层,所述膜片层限定能够被填充有液体或凝胶的大体上管状的隔室,其中所述膜片层包括可渗透以跨所述膜片层气体转移的材料。提供了腔室,所述腔室包括将气体气氛限定和包封在内部的壁。所述生物反应器的至少一部分位于所述腔室内部。控制系统控制所述腔室内的所述气氛的成分并且气体转移在所述管状隔室与包括在所述腔室内的所述气氛之间跨所述生物反应器的膜片层发生。也提供了使用所述设备以便制造生物质的方法。(There is provided an apparatus for producing biomass or bioproducts, the apparatus comprising: at least one elongate bioreactor comprising at least one outer membrane layer defining a substantially tubular compartment capable of being filled with a liquid or gel, wherein the membrane layer comprises a material permeable to gas transfer across the membrane layer. A chamber is provided that includes walls that define and enclose a gas atmosphere within. At least a portion of the bioreactor is located inside the chamber. A control system controls the composition of the atmosphere within the chamber and gas transfer occurs between the tubular compartment and the atmosphere included within the chamber across the membrane layer of the bioreactor. Methods of using the apparatus for making biomass are also provided.)

1. An apparatus for producing biomass or bioproducts, the apparatus comprising:

at least one elongate bioreactor comprising at least one outer membrane layer defining a substantially tubular compartment capable of being filled with a liquid or gel, wherein the membrane layer comprises a material permeable to gas transfer across the membrane layer;

a chamber comprising walls defining and enclosing a gas atmosphere inside;

wherein at least a portion of the bioreactor is located inside the chamber; and

a control system that controls a composition of the atmosphere within the chamber;

Wherein gas transfer occurs across a membrane layer of the bioreactor between the tubular compartment and the atmosphere comprised within the chamber.

2. The apparatus of claim 1, wherein the chamber is in the form of a tank, container, bucket, tent, warehouse, inflatable structure, or room.

3. The apparatus of any one of claims 1 to 2, wherein the atmosphere within the chamber can be elevated to a pressure greater than or less than atmospheric pressure.

4. The apparatus of any one of claims 1 to 3, wherein the control system is configured to modify the atmosphere composition of the chamber by:

(i) introduction of oxygen-containing compounds₂Of (2) a gas

(ii) CO depletion₂Concentration; and/or

(iii) Steam is introduced.

5. The apparatus of any one of claims 1 to 4, wherein the chamber further comprises:

(i) a sterilization system;

(ii) a gas circulation device; and/or

(iii) An illumination source, optionally wherein the illumination source emits visible and/or UV light.

6. The apparatus of any one of claims 1 to 5, wherein at least one wall or a portion of one wall of the chamber allows visible light to be transmitted therethrough into the interior of the chamber.

7. The apparatus of any one of claims 1 to 6, wherein the chamber comprises an assembly for supporting the at least one elongate bioreactor inside, preferably wherein the assembly comprises a plurality of armatures arranged in a horizontal or vertical parallel or anti-parallel array.

8. The apparatus of claim 7, wherein the assembly comprises at least one support configured to support the at least one elongated bioreactor.

9. The apparatus of claim 8, wherein the scaffold substantially encloses all or a portion of the elongated bioreactor, preferably wherein the scaffold comprises a mesh or perforated sheet such that atmospheric circulation may be allowed via the perforations of the sheet.

10. The apparatus of any one of claims 1 to 9, wherein the elongated bioreactor comprises one or more hose portions, wherein each hose portion comprises a gas permeable polymeric membrane.

11. The apparatus of claim 10, wherein the gas permeable polymeric membrane is selected from the group consisting of: silicones, polysiloxanes, Polydimethylsiloxane (PDMS), fluorosilicones, silicones, cellulose (including plant cellulose and bacterial cellulose), cellulose acetate (celluloid), nitrocellulose, and cellulose esters.

12. The apparatus of claim 11, wherein the diaphragm has:

(i) an oxygen permeability of at least 350 barrer, at least 400 barrer, at least 450 barrer, at least 550 barrer, at least 650 barrer, at least 750 barrer, suitably at least 820 barrer;

(ii) a carbon dioxide permeability of at least 2000 barrers, at least 2500 barrers, at least 2600 barrers, at least 2700, at least 2800 barrers, at least 2900 barrers, at least 3000 barrers, at least 3100 barrers, at least 3200 barrers, at least 3300 barrers, at least 3400 barrers, at least 3500 barrers, at least 3600 barrers, at least 3700 barrers, at least 3800 barrers, suitably at least 3820 barrers; and/or

(iii) Water vapor permeability of at least 5000 barrer, at least 10000 barrer, at least 15000 barrer, at least 20000 barrer, at least 25000 barrer, at least 30000 barrer, at least 35000 barrer, at least 40000, at least 60000 barrer, and typically at least 80000 barrer.

13. The apparatus of any one of claims 1 to 12, wherein the membrane has a thickness of at least 10 μ ι η and at most 1mm, suitably at least 20 μ ι η and at most 500 μ ι η, optionally at least 20 μ ι η and at most 200 μ ι η.

14. The apparatus of any one of claims 10 to 13, wherein the one or more hose portions are engaged by one or more connectors that facilitate fluid communication between the one or more hose portions.

15. The apparatus of claim 14, wherein the one or more connectors comprise a valve operable to reduce or stop fluid communication between the one or more hose portions.

16. The apparatus of any one of claims 1 to 15, wherein the bioreactor is in fluid communication with an auxiliary system.

17. The apparatus of any one of claims 1 to 16, wherein the one or more bioreactors comprise liquid cell growth medium.

18. The apparatus of claim 17, wherein the one or more bioreactors comprise microorganisms or algae selected from: photoautotrophic organisms, chemotrophic organisms, and mixotrophic organisms.

19. The apparatus of claim 18, wherein the organism is selected from one or more of the following: cyanobacteria, prophylobacter, spirochetes, gram-positive bacteria, chloromycetes such as the phylum Chlorophytum, Phycomycota, Bacteroides Cytophaga, Thermotoga, Aquifex, halophilic bacteria, Methanosarcina, Methanococcus, Thermococcus celer, Thermobacter, Pyrenophora, Enamantaria, Myxomyces such as Myxomyces, ciliate, Trichomonas, Trichosporon, Trichomonas, archaea, Proteus, Giardia, Porphyromonas, Radioacetic, Diatoma, Protozoa, Brown algae, Red algae, Green algae, snow algae, Dinophyta, Crypthecophytes, vesicle algae, Gray algae, plankton, Perolor, rotifer, and cells or whole organisms from animals, fungi, or plants.

20. The apparatus of claim 17, wherein the bioreactor comprises a eukaryotic cell culture; suitably animal or plant cell cultures; optionally mammalian cell culture.

21. The apparatus of claim 18, wherein the bioreactor comprises a human cell culture.

22. The apparatus of any one of claims 1-21, wherein the control system is further configured to control a temperature of the atmosphere within the chamber.

23. A method for producing biomass, the method comprising:

providing an apparatus, the apparatus comprising:

at least one elongate bioreactor comprising at least one outer membrane layer defining a substantially tubular compartment capable of being filled with a liquid or gel, wherein the membrane layer comprises a material permeable to gas transfer across the membrane layer;

a chamber comprising walls defining and enclosing a gas atmosphere inside, wherein at least a portion of the at least one bioreactor is located inside the chamber;

a control system that controls a composition of the atmosphere within the chamber;

the at least one elongated bioreactor comprises a liquid cell growth medium and a microorganism or algae selected from chemotrophs and mixotrophic organisms, and/or a eukaryotic cell culture;

Culturing the organism or cell culture in the one or more bioreactors; and

separating at least a portion of the biomass present within the liquid culture medium.

Technical Field

The present invention is in the field of biomass production, in particular via the use of microbial or cellular bioreactors.

Background

Organisms that perform aerobic respiration consume oxygen and produce carbon dioxide and heat. In a high density, high growth environment, it is necessary to provide oxygen to the microorganisms, as well as to remove CO ₂Metabolic waste products and excess heat in order to promote maximum growth rate.

Chemoheterotrophic microorganisms such as yeast, which cannot fix carbon to make organic compounds and must consume organic material from external sources, have been growing for centuries in the same manner, i.e., in large tanks and more recently in batch fermenters. However, fermenters are primarily designed to allow fermentation, a specific metabolic process that works in the absence of oxygen, while the intended product for the market is typically a fermentation by-product (e.g., alcohol produced by fermentation of yeast).

When market needs for the entire biomass of microorganisms or products contained within their cells (i.e. beyond just their fermentation by-products) appeared in the 20 th century, existing fermenters were modified in which an aerator was installed on the bottom of the tank in order to deliver oxygen or oxygen-containing gas. This enables the contained microorganisms to perform cellular aerobic respiration within the fermenter. In addition, modifications are sometimes made in view of such aeration, for example to make aerated fermenters tall and thin to increase the hold time of oxygen bubbles as they travel vertically to the top of the liquid growth medium or broth.

Since such adaptations achieve aerobic respiration within the tank previously intended for fermentation use, conventional designs result in inefficient, complex, and expensive production of biomass or cell products for at least the following reasons:

energy costs, equipment requirements and related complexity are high due to the need to sterilize the inlet air for aeration.

Energy costs, equipment requirements and complexity are high due to the need to compress and deliver oxygen (usually in the form of air).

Energy costs, equipment requirements and complexity are high due to the need to mix the liquid medium especially at high cell densities (i.e. stirrers and stirring mechanisms).

Capital costs of the air compressor, filters and other equipment required.

Foam formation by aeration due to biomass loss in the generated foam, the cost of the antifoaming agent increases, and production quality may decrease.

Difficulty in controlling the temperature inside the tank; since they are solid and sealed, they typically require a cooling water jacket, meaning that the capital and energy costs of chilled water are higher.

The risk of contamination is due to the large number of air injection nozzles, valves, sensor ports, paddles, inlets, agitator housings, etc., which are sites of high risk of contamination and are difficult to clean and sterilize.

Because of the need for continuous aeration, there is a risk of introducing contaminants such as fungal spores and bacteria, regardless of the filtration of the input air. Estimates by industry experts indicate that up to 30% of the total biomass in an industrial fermentor can be affected by contamination, quality degradation, and final product yield.

Cleaning costs are expensive due to the ease of biofilm formation on stainless steel and the necessary aeration related features, which is difficult to remove with steam alone, requiring increased labor costs in some cases.

In most cases it is necessary to operate in a batch procedure, resulting in a reduction in annual production due to the downtime required for cleaning and subsequent regrowth to the desired density.

The transfer of gas into the bioreactor is typically achieved by using aeration techniques, such as by compressing CO₂、O₂Or air and delivering the compressed gas into the liquid medium through a nozzle or by bubbling or spraying the gas into the liquid medium (see e.g. US2015/0230420, WO 2015/116963). These techniques can be used to add a desired gas or can also be used to remove unwanted excess gas (see e.g. US 2015/0093924).

This technique can be disadvantageously inefficient in terms of energy requirements and infrastructure costs. When a soluble gas is bubbled through a liquid, only a small proportion of the gas will successfully dissolve; the remaining gas is wasted, resulting in energy waste and inefficient gas uptake. Gas removal by this technique is limited by the gas that may be trapped in the generated bubbles, which provide only a limited surface area for efficient gas exchange.

For example, aerobic stirred fermenters are commonly used, which have a high aspect ratio (about 3 to 1), and use a gas bubbling at the bottom of the tank to deliver oxygen and remove carbon dioxide, and also require the use of active stirring and heat exchange cooling methods.

Similarly, gas-lift fermenters of the common internal circulation type have a very high height to diameter ratio (about 5 to 1), where mixing is provided by moving liquid and gas up a central cylinder, where the liquid is returned in a downward flow in the surrounding annular space to deliver oxygen to remove carbon dioxide, and to allow a heat exchange cooling method, as the mass of the downward flowing liquid impedes transfer from the central core. Both of these methods have high operational and capital costs and have a significant risk of contamination from the gas inlet (despite sterilization of the input gas).

WO 2005/100536 a1 describes an incubator and incubation method that can simultaneously incubate multiple cells that prefer different gas concentrations without the need for multiple incubators. The incubator is not suitable for continuous flow loops containing media, but looks like a static incubator that moves cells within a fixed volume of media by agitation or rotation. No system for automatically harvesting biomass is described, nor is any particular reasonable suitability for cell or microorganism types described. No details are described regarding the nature of the materials required for the device, including for example in terms of gas permeability, gas pressure or structural arrangement for improving gas transfer.

The present invention solves the problems in the prior art that are of considerable importance in the production of valuable products from biomass and cellular material and provides a simple and cost-effective solution to the problems posed by the cultivation of large quantities of organisms to provide them with sufficient oxygen and/or other required gases and to produce biomass. These and other uses, features and advantages of the present invention should be apparent to those skilled in the art from the teachings provided herein.

Disclosure of Invention

In one aspect, there is provided an apparatus for producing biomass or bioproducts, the apparatus comprising: at least one elongate bioreactor comprising at least one outer membrane layer defining a substantially tubular compartment capable of being filled with a liquid or gel, wherein the membrane layer comprises a material permeable to gas transfer across the membrane layer. The apparatus also includes a chamber including walls defining and enclosing a gaseous atmosphere within an interior, wherein at least a portion of the bioreactor is located within the chamber interior. Additionally included is a control system that controls a composition of the atmosphere within the chamber. In use, gas transfer occurs across the membrane layer of the bioreactor between the tubular compartment and the atmosphere comprised within the chamber.

The walls of the chamber may be substantially rigid or flexible. The chamber may be in the form of a tank, container, bucket, tent, warehouse, inflatable structure, or room. The atmosphere within the chamber may be elevated to a pressure greater than or less than atmospheric pressure. Substantially all of the bioreactor may be located within the chamber. The chamber may further comprise a sterilization system, a gas circulation device, and/or an illumination source, optionally wherein the illumination source emits visible and/or UV light. Such illumination sources may be sporadic or intermittent. In some embodiments, at least one wall or a portion of one wall of the chamber allows visible light to be transmitted therethrough into the interior of the chamber.

In some embodiments, the control system is configured to modify the atmosphere composition of the chamber by one or more of: e.g. in the form of atmospheric air, suitably pre-filtered air₂Introduction of (2); CO 2₂Depletion of concentration; and the introduction of steam.

In some embodiments, the chamber comprises an assembly for supporting the at least one elongated bioreactor inside. The assembly may include a plurality of shelves arranged in a horizontal or vertical parallel or anti-parallel array. The shelf may include a support configured to support the at least one elongated bioreactor. The scaffold may substantially encapsulate all or a portion of the elongated bioreactor. The scaffold may comprise a mesh and/or a perforated sheet such that atmospheric air circulation may be allowed via the perforations of the sheet. The support may be planar or curved. In some embodiments, the scaffold may be a solid sheet without holes or perforations and made of any suitable material (e.g., metal, aluminum, steel, and/or polymer/plastic) capable of providing support to the bioreactor. In one embodiment, the base of the chamber is integrated into a support structure for supporting the elongate bioreactor, in which case the base of the chamber suitably comprises a solid shaped or moulded sheet of any suitable material as shown in figure 15.

In some embodiments, the elongated bioreactor comprises one or more hose portions, wherein each hose portion comprises a gas permeable polymeric membrane. In some embodiments, the breathable polymeric membrane comprises a material selected from: silicones, polysiloxanes, Polydimethylsiloxanes (PDMS), fluorosilicones, silicones, vinyl methyl siloxanes (VMQ), phenyl vinyl methyl siloxanes (PVMQ), silicon oxide polymers, sulfonated polyether ether ketones (SPEEK), poly (ethylene oxide), poly (butylene terephthalate) or poly (ethylene oxide), poly (butylene terephthalate) block copolymers (PEO-PBT), cellulose (including plant cellulose and bacterial cellulose), cellulose acetate (celluloid), nitrocellulose, and cellulose esters. The membrane may be an elastomer. In some embodiments, the membrane has an oxygen permeability of at least 350 Barrer (Barrer), at least 400 Barrer, at least 450 Barrer, at least 550 Barrer, at least 650 Barrer, at least 750 Barrer, suitably at least 820 Barrer. The membrane may have a carbon dioxide permeability of at least 2000 barrers, at least 2500 barrers, at least 2600 barrers, at least 2700, at least 2800 barrers, at least 2900 barrers, at least 3000 barrers, at least 3100 barrers, at least 3200 barrers, at least 3300 barrers, at least 3400 barrers, at least 3500 barrers, at least 3600 barrers, at least 3700 barrers, at least 3800 barrers, suitably at least 3820 barrers. The membrane may have a water vapor permeability of not less than about 5000 butler, suitably not less than about 10000 butler, about 15000 butler, about 20000 butler, 25000 butler, about 30000 butler, about 35000 butler, about 40000 butler, about 60000 butler and typically at least about 80000 butler.

The membrane may have a thickness of at least 10 μm and at most 1mm, suitably at least 20 μm and at most 500 μm, optionally at least 20 μm and at most 200 μm.

In some embodiments, the one or more hose portions are joined by one or more connectors that facilitate fluid communication between the one or more hose portions. The one or more hose portions may be formed with a variable membrane thickness such that a portion of the membrane proximal to the one or more connectors is thicker than a portion of the membrane distal from the one or more connectors. The apparatus may comprise a plurality of hose portions joined by one or more connectors facilitating fluid communication between the plurality of hose portions, and wherein the thickness of the membrane between hose portions is dependent on the vertical positioning of the hose portions within the chamber. The connector used in the device may include a valve configured to selectively prevent or allow liquid culture medium to pass through the connector.

The bioreactor of the present invention may be in fluid communication with an auxiliary system. The one or more bioreactors may contain cell growth media. The one or more bioreactors may comprise microorganisms or algae selected from: chemotrophic organisms and mixed trophic organisms. The bioreactor may comprise organisms selected from one or more of the following: cyanobacteria (Cyanobacteria), prophytic bacteria (Protobactria), spirochetes (Spirochaetes), gram-positive bacteria, Chloromycetes such as the phylum Chlorotrichum (Chloroflexia), Phycomycetes (Planctomycetes), Bacteroides Cytophaga (Bacteroides cytophaga), Thermotoga (Thermotoga), Aquifex (Aquifex), Halobacterium (halophiles), Methanosarcina (Methanosarcina), Methanobacterium (Methanobacterium), Methanococcus (Methanococcus), Thermococcus (Thermococcus cell), Thermopsis (Thermoproteus), Pyrenophora (Pyrococcus), Amidobacterium (Entamoebaceae), Myxomyces such as Myxomyces (Myctozozoxa), Diliota (Ciliaceae), Trichosporon trichomonas (Trichloromonas), Microsporum (Microsporum), Microcystis sp.sp.sp.sp.sp.sp.e), Rhodococcus (Tetragenospora), Rhodophyta (Pyrococcus), Microchaeta (Pyrococcus, Microchaeta), Microchaeta (Phaeophyta (Pyrococcus), Microchaeta), Microchaetes (Pyrococcus), Microchaetes (Pyrococcus, Microchaetes (Pyrococcus), Microchaetes (Pyrococcus), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaetes), Microchaetes (Microchaete, Dinoflagellate (Haptophyta), Crypthecodinium (Cryptophyta), Heteroclada (Alveolata), Gracilaria (Glaucophytes), Phytoplankton (Phytoplankton), Phytoplankton (Phytophyton), Percolozoa (Percolozoa), Rotifera (Rotifera), and cells or whole organisms from animals, fungi, bacteria or plants.

In some embodiments, the bioreactor comprises a eukaryotic cell culture; suitably animal or plant cell cultures; optionally mammalian cell culture. The animal cell culture may comprise cells selected from one or more of: muscle cells, adipocytes, epithelial cells, myoblasts, satellite cells, side population cells, muscle-derived stem cells, mesenchymal stem cells, myogenic pericytes, or hemangioblasts. The bioreactor may comprise a human cell culture.

In another aspect, there is provided a method for producing biomass, the method comprising providing an apparatus as described above. In particular, the device comprises: at least one elongate bioreactor comprising at least one outer membrane layer defining a substantially tubular compartment capable of being filled with a liquid or gel, wherein the membrane layer comprises a material permeable to gas transfer across the membrane layer. The apparatus further comprises: a chamber comprising walls defining and enclosing a gas atmosphere inside, wherein at least a portion of the at least one bioreactor is located inside the chamber; and a control system that controls the composition of the atmosphere within the chamber. The at least one elongated bioreactor comprises a liquid cell growth medium and a microbial or algal organism selected from a chemotrophozoite and a mixotrophic organism, and/or a eukaryotic cell culture. The method comprises the following steps: culturing the organism or cell culture in one or more bioreactors of the apparatus; and separating at least a portion of the biomass present within the liquid culture medium.

Drawings

The invention is further illustrated by reference to the accompanying drawings, in which:

FIGS. 1A and 1B are diagrams showing a cross-section of an apparatus according to an embodiment of the present invention having a linear bioreactor with an inlet and an outlet on opposite sides, the linear bioreactor being disposed within a plenum chamber also provided with an inlet and an outlet.

Fig. 2 shows a cross section of an arrangement according to another embodiment of the invention, wherein two bioreactors are connected directly in series.

Fig. 3a and 3b show cross-sections of an arrangement according to another embodiment of the invention, wherein two bioreactors are directly connected in series, wherein each bioreactor is contained within a chamber.

Fig. 4 shows a cross section of an arrangement according to another embodiment of the invention, wherein five pairs of bioreactors are connected in series.

Fig. 5 shows a cross section of an arrangement according to another embodiment of the invention, wherein five pairs of bioreactors are connected in parallel.

Fig. 6a to 6d show an arrangement of an array of bioreactors that may be used in some embodiments of the present invention.

Fig. 7a and 7B show planar sections a and B by representations of an apparatus according to some embodiments of the invention.

Fig. 8a and 8b illustrate additional features that may be included within a connector or catheter of a system according to some embodiments of the present invention.

Figure 9 illustrates a suitable system of an embodiment of the present invention, including any embodiment of one or more bioreactors and associated ancillary systems.

Fig. 10 shows a cross-section of a support member for use with an apparatus according to an embodiment of the invention.

Figure 11 shows a cross-section of an apparatus according to an embodiment of the invention comprising a bioreactor supported on a support member.

Fig. 12 shows a perspective view of a support member for use with a device according to an embodiment of the invention.

Figure 13 shows a cross-section of an apparatus according to an embodiment of the invention comprising a convexly curved upper chamber wall to encourage water, snow, sand and other matter that may be deposited on an internal or external surface to run off under gravity.

Fig. 14a to 14d show views of a bioreactor supported on a support structure and/or bioreactor support structure according to some embodiments of the present invention. Fig. 14a and 14b show cross-sections of an array of bioreactors supported on a shelf-like support structure. FIG. 14c shows a perspective view of an embodiment of a bioreactor supported, contained and reinforced by surrounding mesh. Figure 14d shows a side view of an array of bioreactors supported on a shelf-like support structure.

Figure 15a shows an array of bioreactors supported on a planar support structure that also defines the base of the chamber and a cross section of the convexly curved upper chamber wall to increase its structural strength and encourage material that may be deposited on internal or external surfaces to flow under gravity, in accordance with some embodiments of the invention.

Figure 15b shows a cross-section of an array of bioreactors and integrated lighting devices supported on a planar support structure defining the base of a plurality of chambers, in accordance with some embodiments of the present invention. The integrated lighting may be used for growth of phototrophic and/or mixotrophic organisms.

Figure 15c shows a cross-section of an array of bioreactors supported on a planar support structure according to some embodiments of the present invention.

Figures 16a to 16c show cross sections of a bioreactor formed from a single sheet of membrane folded to form an elongate seam and joined on itself. Figure 16a shows how a single membrane layer can be folded before joining the two edges to define the bioreactor inside. Figure 16b shows a bioreactor formed from a single membrane layer folded and glued onto itself. Figure 16c shows a bioreactor formed from a single membrane layer folded and bonded to itself and in which the bonded portions also provide additional structural reinforcement at the underside of the bioreactor in contact with the planar support structure.

FIG. 17a shows a perspective view of an example bioreactor with end stiffeners.

FIG. 17b shows a perspective view of an example bioreactor with two end stiffeners and a continuous lower stiffening structure.

Figure 18 shows a suitable system for one embodiment of the invention for the experiment described in example 1, including a bioreactor system and associated ancillary systems.

Figure 19 shows a suitable system for one embodiment of the invention for the experiment described in example 2, including a bioreactor system and associated auxiliary systems including illumination sources (natural or artificial).

Figure 20 shows the results of example 1 in the form of a graph of optical density in liquid medium for two experimental runs (run a and run B).

FIG. 21 shows the results of example 1 in the form of a graph of the temperature in the liquid medium.

FIG. 22 shows the results of example 2 in the form of a graph of optical density in liquid medium.

FIG. 23 shows the results of example 2 in the form of a graph of the temperature in the liquid medium.

Detailed Description

All references cited herein are incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present inventors have developed a gas permeable bioreactor apparatus suitable for producing biomass that is included within a chamber. Advantageously, the atmosphere within the chamber can be controlled so as to supply the bioreactor apparatus with a gas feed of a specified composition and to remove the exhaust gas. Embodiments of the present invention allow for a given device to include an atmosphere that is optimized to improve or maximize biological survival, biological growth rate, and/or biomass production within a bioreactor. Alternative embodiments of the invention allow specifying means comprising controlling the growth of the microorganisms comprised in the bioreactor or regulating the atmosphere of biomolecule synthesis by the microorganisms comprised in the bioreactor. These and other embodiments of the present invention are described in more detail below.

Before further elaborating the invention, numerous definitions are provided that will aid in understanding the invention.

As used herein, the term "comprising" means that any of the recited elements are necessarily included, and that other elements may also be optionally included. "consisting essentially of means that any recited elements are necessarily included, excluding elements that would materially affect the basic and novel characteristics of the recited elements, and that other elements may optionally be included. "consisting of …" means that all elements except the listed elements are excluded. Embodiments defined by each of these terms are within the scope of the invention.

As used herein, the terms "autotroph," "autotroph," or "autotrophic" refer to organisms and processes capable of producing complex organic molecules from inorganic chemicals in their environment. In particular, this means fixing carbon, typically carbon dioxide, into organic compounds. The energy required for this can come from light or from chemical reactions. Photosynthesis is an example of a (photo) autotrophic process. Chemoautotrophs, defined below, use the energy obtained from chemical reactions to fix inorganic carbon (e.g., from carbon dioxide) into organic compounds.

As used herein, the term "heterotrophic organism", "heterotrophic" or "heterotrophic" refers to organisms and processes that are incapable of fixing carbon to form organic compounds, that is, they consume organic matter from the surrounding environment and convert them into organic molecules for their own use.

As the skilled person will know, the term "photosynthesis" refers to the biochemical processes that occur in green plants and other photosynthetic organisms, including photosynthetic microorganisms including algae and cyanobacteria. The process of photosynthesis utilizes electromagnetic waves (light) as an energy source by photon capture to convert carbon dioxide and water into metabolites and oxygen. As used herein, the term "photosynthetic microorganism" refers to any microorganism capable of photosynthesis. As used herein, the relative terms "photosynthetic" and "photosynthesis" are synonymous with "photosynthetic" and the two terms can be used interchangeably herein.

As used herein, the term "phototrophic organism", "phototrophic" or "phototrophic" refers to any organism or process that is capable of capturing energy from light for any purpose, particularly organisms and processes that generate energy by photon capture and/or use energy from electromagnetic waves (light) to generate organic compounds. As mentioned above, the production of organic compounds by fixing inorganic carbon using energy from light is called photosynthesis. The term "photoautotrophic organism" as used herein is another term for an organism that is capable of utilizing energy from light to produce organic compounds from carbon dioxide. As described below, photosynthetic and photoautotrophic organisms are not limited to using photosynthesis alone, and many organisms can use photosynthesis or can photosynthesize. In addition, some organisms use light to provide cellular energy (e.g., in the form of ATP), but are not necessarily capable of fixing carbon to produce organic compounds. The term "photo-heterotrophic organism" as used herein refers to an organism that is capable of producing cellular energy from light but is unable to fix (sufficient) inorganic carbon to supply its needs.

As used herein, the term "chemotrophic organism," "chemotroph," or "chemotroph" refers to organisms and processes that obtain energy by oxidizing electron donors in their environment. These molecules can be organic (chemoorganotrophozoites) or inorganic (chemoanotrophozoites). Chemotrophic organisms may be autotrophic or heterotrophic. For example, organisms that consume organic carbon compounds from their environment and oxidize these compounds to produce ATP are chemotrophic organisms. "chemoheterotrophic organism", a term that includes most animals and fungi, refers to an organism that consumes organic compounds from an external source and utilizes them to form their own organic compounds rather than directly fixing carbon to make the organic compounds. "chemoautotrophs" refers to organisms that are capable of using the energy obtained from chemical reactions to fix inorganic carbon (e.g., from carbon dioxide) into organic compounds. Examples of such chemical energy sources include hydrogen sulfide, elemental sulfur, ferrous iron, molecular hydrogen, and ammonia. Many chemoautotrophic organisms are extreme microorganisms, bacteria or archaea that live in harsh environments, and are the major producers in such ecosystems. Chemoautotrophs usually fall into several groups: methanogens, halophilics, sulfur oxidants and reductants, nitrifying bacteria, anammox bacteria, thermophilic acidophilic bacteria, manganese oxidants, iron oxidants and hydrogen oxidants. For example, hydroxide bacteria can use oxygen as the last electron acceptor to oxidize hydrogen as an energy source. Similarly, methanogens are microorganisms that produce methane as a metabolic byproduct under hypoxic conditions, and some use hydrogen to reduce carbon dioxide to methane and water.

As used herein, the term "mixotrophic organism", "mixotrophic" or "mixotrophic" refers to an organism and process that is capable of using more than one energy source and/or organic compound. Most often, this refers to organisms that are capable of harvesting or producing energy and/or organic compounds using a mixture of light and chemical inputs. Mixotrophic organisms exist in a range between fully obligate chemoheterotrophs and fully obligate photoautotrophs. The use of such a source mixture may be obligatory, in which the organism must use the source mixture to live, or facultative, in which the organism preferentially uses one source and another in certain situations, for example using a chemical energy source in light-limited situations. Thus, a "mixotrophic organism" is both a photoautotrophic and a chemotrophic organism, and may be a photoautotrophic, chemoautotrophic, a photoheterotrophic or a chemoheterotrophic organism.

The skilled person will also know of the CO in the liquid₂Reference to concentration or percentage of (carbon dioxide) refers to the Dissolved Inorganic Carbon (DIC), i.e. dissolved CO, of the solution₂And related inorganic substance H₂CO₃(Carbonic acid), HCO₃ ^-(bicarbonate ion) and CO ₃ ^2-(carbonate ion) concentration. Similarly, references herein to "gas concentration" and the like are intended to include any and all ionic species or compounds formed from a gas in a liquid or aqueous context, such as ammonium ions (NH) as a result of ammonia gas₄ ⁺) Or as sulfurSulfuric acid (H) as a result of oxidation₂SO₄)。

As used herein, the term "translucent" has its ordinary meaning in the art and refers to a light transmissive material that allows light to pass through, resulting in random internal scattering of light. The term is synonymous with "translucent".

As used herein, the term "transparent" has its ordinary meaning in the art and refers to a material that allows visible light to pass through it so that objects can be clearly seen on the other side of the material, in other words it can be described as "optically clear". All of the membrane and non-membrane materials, chamber walls, additional components, control structures, coatings, and other materials described herein may be substantially translucent or substantially transparent.

As used herein, the term "permeable" or "breathable" means allowing gases, particularly oxygen (O)₂) Carbon dioxide (CO)₂) Nitrogen (N)₂) Water vapor (H) ₂O) and optionally methane (CH)₄) And/or sulfur dioxide (SO)₂) Some or all of which are transferred from one side of the material to the other in either or both directions. As used herein, the relative terms "inhalable" and "semi-permeable" are synonymous with "permeable" and the two terms can be used interchangeably herein. Typically, the material is in the form of a sheet, film or membrane. Permeation is directly related to the concentration gradient of the permeate (such as a gas), the intrinsic permeability of the material, and the diffusivity of the permeating substance in the membrane material.

The permeability of a gas through a particular material is measured herein using the barrer. The basiler measures the rate of gas flow through a region of material having a certain thickness driven by a given pressure. The basil is defined as:

it will be appreciated that barrer is the most common measure of gas permeability in current use, particularly in relation to breathable membranes, however permeability may also be defined by other units, examples of which include kmol.m.m.m-2. s-1.kPa-1, m3.m.m-2.s-1.kPa-1 or kg.m.m-2.s-1. kPa-1. ISO 15105-1 specifies two methods for determining the gas transmission rate of monolayer plastic films or sheets and multilayer structures at differential pressure. One method uses a pressure sensor and the other method uses a gas chromatograph to measure the amount of gas permeated through the test sample. Other equivalent measurements of gas permeability are known to the skilled person and will be readily equivalent to the basil measurements described herein.

As used herein, the term "biomass" refers to any living or dead microorganism, including any portion of a microorganism (including metabolites and byproducts produced and/or excreted by the microorganism).

As used herein, the term "device" may include one "unit" or may include an array or combination of multiple "units".

As used herein, the term "chamber" also refers to a "gas chamber" and the two terms can be used interchangeably herein.

As used herein, the term "fluid" refers to a flowable material, typically a liquid and suitably a liquid medium, which is included in the unit, and thus in the device of the invention. "fluid" may also be used to describe a gas, such as the atmosphere contained within the chamber of the present invention.

As used herein, the term "liquid medium" has its usual meaning in the art and is a liquid used to grow and contain organisms. The liquid medium can include one or more of the following: fresh water, brackish water, salt water, brine, seawater, wastewater, sewage, nutrients, phosphates, nitrates, vitamins, minerals, micronutrients, macronutrients, metals, digests, fertilizers, microbial growth media, BG11 growth media, PYGV media, and organisms. The liquid medium can in particular also comprise a carbon source of the included organism; often these are sources of glucose. Such suitable carbon sources can include lignin, cellulose, hemicellulose, starch, xylan, polysaccharide, xylose, galactose, sucrose, lactose, glycerol, molasses or glucose or derivatives thereof. Since high densities of microorganisms can be supported in the device of the invention, the term liquid medium is intended to encompass a wide variety of viscosities, including substantially gel-like or semi-solid compositions.

As used herein, terms relating to the orientation of the device of the present invention are generally used in their commonly held meanings, but are also intended to vary as appropriate depending on the particular intent or configuration of the invention. Thus, terms such as upper, top and above may refer to directions away from the earth's gravity. Similarly, terms such as lower, bottom and below refer to the direction toward the earth's gravity.

The present invention uses the general class of breathable membrane bioreactors described in WO2017/093744 and WO2018/100400 for culturing photosynthetic organisms but further adapted to provide applications to organisms with various nutritional capabilities. This method overcomes several problems seen with existing bioreactor systems because it enables, in part (including on a large scale), much less energy intensive gas transfer control in the liquid culture medium and provides greater versatility compared to systems requiring means for controlling aeration and compression of feed gases directly dosed to the liquid culture medium. The operational complexity and extra weight associated with compression and aeration techniques are also avoided. Due to the nature of the invention, the natural expansion properties of the gas means that the supplied gas can be easily supplied and expanded to rapidly change the composition of the entire chamber. This provides a further benefit in that the gas concentration within the chamber can be controlled relatively easily on a large scale and by extension, the gas concentration in the liquid culture medium can be controlled on the same scale.

In the case of high growth rates of the cultured organisms or in other cases where the bioreactor is exposed to sunlight or any other heat source (natural or artificial), a large amount of excess heat may be generated and/or collected in the bioreactor, which may damage or kill the organisms contained within the bioreactor. The membrane of the bioreactor of the invention is in some embodiments permeable to water vapour and dissipation of this vapour represents an efficient method of dissipating heat from the liquid culture medium, thereby further improving thermal control. Furthermore, the large surface area provided by the membrane of the bioreactor in contact with the atmosphere within the chamber and the thin wall thickness of the membrane layer of the bioreactor also provide for efficient heat transfer by contact with the ambient gas atmosphere in the chamber. Thus, the present invention is able to control the liquid temperature by controlling the temperature of the gas atmosphere within the chamber. This particular method enables a constant heat exchange to be achieved over the entire length of the bioreactor and allows a substantially uniform liquid culture medium temperature to be maintained over the entire length of the bioreactor, regardless of its length; in contrast, conventional heat exchange methods (utilized by standard bioreactors) alter the temperature of the liquid culture medium only in specific portions of the bioreactor system. This is suitable for single vessel bioreactors, but may be problematic for elongated bioreactors (e.g., based on tubular liquid circuits as described herein) because they are not able to maintain a uniform liquid culture medium temperature throughout the entire bioreactor length. This is due to the fact that: after the liquid culture medium travels through the heat exchanger and its temperature is modified, its temperature will change constantly during its circulation through the bioreactor system. The thickness of the membrane layer of the bioreactor can be suitably modified to increase or decrease the heat transfer rate (i.e., heat transfer coefficient) and gas transfer rate between the liquid culture medium and the gaseous atmosphere within the chamber.

Another benefit of the present invention is in improving the robustness and environmental tolerance of the bioreactor included in the assembly. The walls of the chamber may be configured to provide thermal insulation against external factors such as changing environmental or seasonal conditions. This insulation also reduces the energy necessary to maintain the temperature of the liquid culture medium contained within the bioreactor. Physical protection of the potentially fragile membranes of the bioreactor is also provided against factors such as weather, wind or hail or animal damage. The provision of the additional barrier also serves to contain the overflow from the bioreactor into the environment.

Furthermore, the nature of the device of the present invention means that the cleaning and sterilization process can be performed efficiently and effectively. According to one embodiment of the invention, the tubular configuration of the membrane comprising and containing the liquid culture medium allows to achieve the removal of dead ends, corners, edges, seams and other cracks by enabling a substantially uniform cross section of the bioreactor. The present invention allows for quick and effective cleaning as such features provide areas where unwanted microorganisms and biofilm can attach or where debris, spent liquid media or other debris can accumulate and be difficult to clean effectively. The unnecessary gas sparging or spraying techniques also means that the nozzles, outlets and inlets required for such techniques will not be in contact with the liquid medium or the organisms and therefore will not have to be cleaned. Such features can be difficult to clean and are often areas of microbial growth or debris collection, and may even be a source of contamination by itself through the introduction of contaminants to the input gas. Thus, the present invention allows for increased sterility and flexibility in process setup and shutdown, as cleaning before and after use may be more effective.

Bioreactor

According to one embodiment of the invention, a bioreactor of a device is provided, the bioreactor comprising at least one outer layer as a membrane layer. One or more of the membrane layers may be flexible. The transmission of at least a portion of one of the membrane layers and optionally substantially all of the permeable gas of each membrane layer across the membrane. As used in this context, the phrase "at least a portion" means a region of the layer having sufficient size to allow gas to pass through the outer layer of the bioreactor. The gases are typically, but not limited to, oxygen, carbon dioxide, and water vapor, and may include nitrogen, nitrogen oxides, sulfur oxides, hydrogen, and/or methane.

The permeability coefficient for oxygen through the membrane sheet may be not less than about 100 barrers, suitably not less than about 200 barrers, about 300 barrers, about 400 barrers, about 500 barrers, about 600 barrers, about 700 barrers, about 800 barrers, about 900 barrers, about 1000 barrers, about 1250 barrers, about 1500 barrers, and typically not less than about 2000 barrers.

The permeability coefficient of carbon dioxide through the membrane sheet may be not less than about 100 barrers, suitably not less than about 200 barrers, about 400 barrers, about 600 barrers, about 800 barrers, about 1000 barrers, 1500 barrers, about 2000 barrers, about 2500 barrers, about 3000 barrers, about 3500 barrers, about 4000 barrers, about 4500 barrers, about 5000 barrers, about 7500 barrers, and typically not less than about 10000 barrers.

The permeability coefficient of water vapor through the membrane may be not less than about 5000 barrers, suitably not less than about 10000 barrers, about 15000 barrers, about 20000 barrers, 25000 barrers, about 30000 barrers, about 35000 barrers, about 40000 barrers, about 60000 barrers, and typically not less than about 80000 barrers. The water vapor permeability can also be measured in g/m²Measurement is carried out for 24 h. In these aspects, a suitable water vapor permeability through the membrane sheet may be about 3200 at a membrane sheet thickness of 20 μm, 1200 at a thickness of 50 μm and 800 at a thickness of 100 μm.

Permeable to methane (CH) in the membrane₄) In the case of (a), the permeability coefficient of methane through the membrane sheet may be no less than about 100 barrers, suitably no less than about 250 barrers, about 500 barrers, about 600 barrers, 700 barrers, about 800 barrers, about 900 barrers, about 1000 barrers, about 1500 barrers, and typically no less than about 5000 barrers.

When the membrane is permeable to sulfur dioxide (SO)₂) The permeability coefficient for sulfur dioxide through the membrane may be no less than about 1000 barrers, suitably no less than about 2500 barrers, about 5000 barrers, about 6000 barrers, about 7000 barrers, about 8000 barrers, about 9000 barrers, about 10000, about 12000, about 14000 barrers, and typically no less than about 16000 barrers. Typically, the permeability of sulfur dioxide is about 12500 barrer.

When the membrane is permeable to hydrogen sulfide (H)₂S), the permeability coefficient of hydrogen sulfide across the membrane sheet may be not less than about 1000 barrers, suitably not less than about 2500 barrers, about 5000 barrers, about 6000 barrers, about 7000 barrers, about 8000 barrers, about 9000 barrers, about 10000 barrers, and typically not less than about 12000 barrers. Typically, the permeability of hydrogen sulfide is about 8400 barrer.

When the membrane is permeable to molecular hydrogen (H)₂) When used, the permeability coefficient of molecular hydrogen through the membrane sheet may be no less than about 100 barrers, suitably no less than about 250 barrers, about 500 barrers, about 600 barrers, 700 barrers, about 800 barrers, about 900 barrers, about 1000 barrers, about 1500 barrers, and typically no less than about 2000 barrers. Typically, the permeability of molecular hydrogen is about 550 barrers.

When the membrane is permeable to molecular nitrogen (N)₂) When used, the permeability coefficient of molecular nitrogen through the membrane sheet may be no less than about 50 barrers, suitably no less than about 100 barrers, about 200 barrers, about 300 barrers, 500 barrers, about 700 barrers, about 900 barrers, about 1000 barrers, about 1500 barrers, and typically no less than about 2000 barrers. Typically, the permeability of molecular nitrogen is about 200 barrers.

The bioreactor may be exposed to illumination sources from a single direction or from multiple directions, whether artificial or natural. If the bioreactor is positioned such that it receives light primarily from a single direction and one (first) membrane layer is less transparent or less translucent than the other (second) membrane layer, the first membrane layer may be located on the side of the bioreactor facing the primary light source. It is contemplated in some cases that the film layer may be substantially opaque or opaque to visible light and may not include or be intended as a light source. Typically, the membrane layer is at least translucent, and suitably substantially transparent to allow visual inspection of the contents of the bioreactor.

Typically, the film layer comprises one or more breathable materials. It is important that the gas permeable material is liquid impermeable to prevent the liquid culture medium inside the bioreactor from leaking to the outside. The air permeable material may be porous (including microporous structure air permeable materials) or non-porous. Gas permeable materials are referred to as porous materials if the gas particles are able to migrate through the microporous structure by moving directly. If the breathable material is porous, it is important that it is substantially liquid impermeable. Suitably, the gas permeable material is non-porous, which also avoids penetration of liquid through the gas permeable material and avoids lower transparency that may be associated with the porosity of the material.

The breathable material may be a polymer, such as a chemically optimized breathable polymer. Chemically optimized polymers may be preferred over corresponding unmodified polymers because they may be cheaper, more tear resistant, hydrophobic, antistatic, more transparent, easier to make, less brittle, more elastic, more gas permeable, and selectively permeable to a particular gas. Chemical modifications can be performed on the polymer in any manner that will be known to the skilled artisan, such as by modifying the chemical composition of the monomers, backbone, side chains, end groups, and/or by processes that use different curing agents, crosslinking agents, fillers, vulcanization, manufacturing, fabrication, and other methods.

The film layer can comprise any suitable breathable material, including but not limited to: silicones, polysiloxanes, Polydimethylsiloxanes (PDMS), fluorosilicones, silicones, VMQ (vinyl methyl siloxane), PVMQ (phenyl vinyl methyl siloxane), silica polymers, sulfonated polyether ether ketone (SPEEK), poly (ethylene oxide), poly (butylene terephthalate) or poly (ethylene oxide), poly (butylene terephthalate) block copolymers (PEO-PBT) (e.g., 1000PEO40PBT60), cellulose (including plant cellulose and bacterial cellulose), cellulose acetate (celluloid), nitrocellulose, and cellulose esters. Porous materials, in particular nanoporous silicon, porous silicon nanostructures are also envisaged for use.

In suitable embodiments, the membrane layer comprises a polysiloxane, optionally an optimized polysiloxane. The polysiloxane may be chemically or mechanically modified. Typically, the membrane layer comprises a silicone elastomer. It has been found that polysiloxanes are good candidates for breathable membranes due to Si-O bonds in the polymer structure that promote higher bond rotation, thereby increasing chain mobility and thereby increasing the level of permeability. Silicone elastomers (such as silicone rubber) are also flexible, UV radiation resistant and elastomeric materials.

In an embodiment, the membrane layer comprises Polydimethylsiloxane (PDMS), suitably optimized polydimethylsiloxane. Typically the membrane layer comprises a Polydimethylsiloxane (PDMS) elastomer. Polydimethylsiloxane (PDMS) can take the form of an elastomer, resin, or fluid. PDMS elastomers can be formed by using cross-linking agents, by UV curing techniques, and other methods. PDMS is a typical breathable material due to its very high permeability to oxygen, carbon dioxide and water vapor, its optical transparency and its resistance to UV radiation. These elastomers generally do not support microbial growth on their surface, so uncontrolled biofilm growth and/or biofouling is avoided, which may reduce the efficacy of the device to produce biomass (shield light). Biofilm growth can optionally be promoted by the use of a biosupport and/or additional components as described below. Additionally, Polydimethylsiloxane (PDMS) elastomers are flexible and elastomeric materials.

Polydimethylsiloxane (PDMS) may be chemically or mechanically modified to increase its gas permeability and/or alter its properties. The PDMS elastomer typically has an oxygen permeability of at least 350 barrers, at least 400 barrers, at least 450 barrers, at least 550 barrers, at least 650 barrers, at least 750 barrers, suitably at least 820 barrers. Suitably the PDMS elastomer has a carbon dioxide permeability of at least 2000 barrers, at least 2500 barrers, at least 2600 barrers, at least 2700, at least 2800 barrers, at least 2900 barrers, at least 3000 barrers, at least 3100 barrers, at least 3200 barrers, at least 3300 barrers, at least 3400 barrers, at least 3500 barrers, at least 3600 barrers, at least 3700 barrers, at least 3800 barrers, suitably at least 3820 barrers. The performance of PDMS used in embodiments of the present invention can be optimized by chemical, mechanical and process driven interventions related to, but not limited to: molar mass of the Polymer chain (M) _m) Dispersibility in the polymer (dispersibility is the ratio of weight average molar mass to number average molar mass), temperature and duration of heat treatment during curing, ratio of crosslinker to PDMS, crosslinker chemistry, different end groups (such as methyl-, hydroxyl-, and vinyl-terminated PDMS) that may affect the way end-linked PDMS structures are formed during crosslinking.

Alternatively, the nanocomposite may be used to make highly breathable film materials. Nanomaterials and nanostructures mixed with the membrane material can be used to increase the permeability of the membrane material. Nanoclay-filled siloxanes and more specifically nanoclay-filled poly (dimethylsiloxane) PDMS are examples that can be used in the present invention. It was found that nanoclays (nanoparticles of layered mineral silicates) provide substantial polymer reinforcement, but the gas permeability of the nanocomposites is still high, regardless of the large nanolayer aspect ratio. The random orientation of clay nanolayers in a polymer matrix results in a lack of effective gas barrier properties, thereby increasing its gas permeability properties.

In another embodiment, the membrane layer comprises bacterial cellulose. Although bacterial cellulose has the same molecular formula as plant cellulose, it has significantly different macromolecular properties and characteristics. In general, bacterial cellulose is more chemically pure and contains no hemicellulose or lignin. Furthermore, due to the high plasticity during formation, bacterial cellulose can be produced on a variety of substrates and can grow into almost any shape. Additionally, bacterial cellulose has a more crystalline structure than plant cellulose and forms characteristic thin ribbon-like microfibrils, which are significantly smaller than microfibrils in plant cellulose, so that the porosity of bacterial cellulose becomes much higher. For example, the skilled person will be aware of many bacterial systems which have been monitored to optimise cellulose production, such as cellulose biosynthesis systems of Acetobacter species (Acetobacter sp.), azotobacterium species (Azotbacter sp.), Rhizobium species (Rhizobium sp.), Pseudomonas species (Pseudomonas sp.), Salmonella species (Salmonella sp.), and Alcaligenes species (Alcaligenes sp.) which are capable of being expressed in e. The bacterial cellulose can be treated such that its surface provides a chemical interface to enable binding to molecules.

The other layers of the bioreactor may also be membrane layers-i.e. gas permeable layers-as defined above, or they may comprise non-membrane layers, comprising any suitable material, such as natural or synthetic materials. Suitably, the layers are at least translucent, and typically transparent. These layers are suitably smokable. In typical embodiments, all layers of the bioreactor are gas permeable membrane sheets as defined herein. In other embodiments, the membrane bioreactor comprises a single layer, such as a tube formed from a continuous layer or a single membrane or a single layer folded over itself and sealed to itself in one or more places to create a bioreactor. For example, as shown by the cross-sections of fig. 16a and 16b, the monolayer is folded over on itself to form the bioreactor (60) and the regions (152) where the two edges of the same layer overlap are sealed together with a glue adhesive to form the seam (150).

The membrane layer may be made substantially entirely of breathable material or may contain additional material. In particular, the membrane layer may have one or more integral ribs, or may include an internal mesh, which may be made of a support material that is typically strong and rigid or semi-rigid, and may be flexible and/or resilient. Suitably, the support material may be flexible rather than elastic, for example to allow the bioreactor to be shaped in a particular manner. These structures can provide improved strength to the bioreactor and/or help the bioreactor maintain its shape, and are arranged such that the membrane as a whole is still permeable to gas. Such an interior material may, for example, be the result of a co-extrusion of the breathable material and the support material.

Suitably, the bioreactor comprises a tube, pipe or hose, typically having an axial length exceeding its lumen width (i.e. diameter), comprising a single continuous membrane of gas permeable material, which may be made by extrusion, moulding, injection moulding, from a single membrane layer folded over itself and sealed to itself and rotomoulding or by any other suitable process. Typically, such a tube or hose arrangement has a substantially uniform cross-sectional aperture across at least a majority of its length, optionally for its entire length. This cross-sectional profile may be, but is not necessarily, round or circular, or may be elliptical, oval, or have the shape of a rounded polygon, such as a square or rectangle. Suitably, the cross-section lacks interior dead ends, sharp corners, edges, seams and other crevices. In other words, the internal profile of the orifice of the bioreactor is substantially uniform due to the smooth surface for at least a majority of the length of the bioreactor. The end stiffener (144) can be used to stiffen the terminal portion of the diaphragm hose portion (fig. 17a and 17b) by attaching thicker walls or stronger material. This is to reinforce the area where the hose is in contact with the connector to connect it to an adjacent hose portion. A similar reinforcement (bottom reinforcement) can be applied along the underside of the hose portion (149), especially if the hose rests on a flat or planar surface, stand or support mesh (fig. 17b and cut-out portion fig. 16 b). This is to strengthen the underside seam and avoid tearing and puncturing while contacting the support surface and during installation. In other embodiments, the stiffener underside (149) can coincide with the seam location, where the single film sheet layer is folded over itself and sealed to itself (152 in fig. 16 a); suitably the reinforcement underside (149) comprises an adhesive for sealing the single membrane layer to itself to form the elongate flexible tube bioreactor (figure 16 c). This reinforcement can be done in any suitable way, for example by attaching a thicker layer of the same membrane material (using an adhesive method), or attaching a stronger and/or thicker material, such as a flexible non-elastic polymer or a thicker mesh, or by using more layers of heat-curable silicone adhesive tape, or by using more layers of self-curable (or UV-curable) silicone glue.

In suitable embodiments, the first and second layers or the monolayer folded over on itself to form the bioreactor (suitably a hose bioreactor) are bonded by adhesion and/or heat pressing. Hot pressing utilizes the application of heat and pressure for a predetermined period of time to form a weld. Those skilled in the art will be familiar with suitable hot pressing techniques for this application. The precise temperature and duration required to bond the portions of the first and second layers together will depend on the particular materials contained in the two layers. Alternatively or additionally, an adhesive interface can be used to bond portions of two layers together or to bond a single layer folded over itself; once applied to each layer or to a single layer, the adhesive interface can be cured using a hot-pressing technique, or can cure spontaneously at room temperature, or can cure spontaneously at a particular temperature, or can cure after irradiation with UV light (including ultraviolet wavelengths) or other suitable wavelengths of light, or can cure using heat or pressure alone. As used herein, the term "glue interface" also includes the use of a non-crystalline (non-vulcanized) polymer that is capable of bonding two layers with hot or wet pressing. As used herein, the relative terms "glue interface", "adhesive" and "adhesive interface" are synonymous and the three terms can be used interchangeably herein.

The glue interface thickness varies depending on its composition, material and layer material. Suitably, the glue interface thickness is not less than: 1 μm, optionally 10 μm, suitably 20 μm, typically 50 μm. Typically, the glue interface thickness is no more than 20mm, no more than 10mm, no more than 5mm, no more than 2mm, optionally 1mm, suitably 600 μm, typically 200 μm.

More specifically, if the first and second layers or the single layer folded on itself comprise polysiloxane and/or dimethylpolysiloxane (PDMS), the two layers can be bonded together by using a silicone adhesive which can be in liquid form, viscous liquid gel form, layer tape form, and/or can comprise all types of silicone adhesives which can be cured below or above 22 ℃ or can be cured with pressure or can be cured after irradiation with UV light (including light of ultra violet wavelength) or other suitable light wavelengths. After applying the silicone adhesive on the two layers or on the single layer folded on itself, the bonding area is generally pressed within a determined period of time as specified by the type of silicone adhesive and, if the type of silicone adhesive used also requires thermal curing, it is heated at a determined temperature and within a determined period of time as specified by the type of silicone adhesive utilized.

Types of possible silicone adhesives include, but are not limited to, silicone gums and silicone adhesive layers, such as VVB Birzer ADT-X (which is between 1N/cm) with a thickness between 0.20mm and 0.60mm²And 15N/cm²Pressure between 140 ℃ and 180 ℃ and thermocompression bonding for 30 to 60 seconds), adhesive Research with a thickness between 25 μm and 100 μmIS-7876 Silicone transfer Adhesives (which are pressure sensitive Adhesives bonded with pressure and temperature greater than-5 ℃), cured when exposed to atmospheric moisture at room temperatureRTV10533 one-component silicone adhesive.

Alternatively, the silicone adhesive interface can be composed of a thin layer of uncured polysiloxane and/or dimethylpolysiloxane (PDMS) that can be mixed with its cross-linking agent and quickly applied over the predetermined bonding area on each layer, then pressed and heated to cure, thereby bonding the two layers together.

In some embodiments, a "glue interface" and/or a silicone adhesive can be used to bond two layers together or to bond a single layer folded over itself in the region where the fluid conduit is typically located. This combination will create a control structure to control the flow of liquid media to divide or divert the fluid conduits among the multiple conduits.

Advantages of embodiments having one or more bioreactors in the shape of a tube or hose include a reduction in the sites within the bioreactor where liquid media, cells, and/or contaminants may accumulate due to the generally uniform cross-section and lack of internal edges, seams, cracks, etc. In a confined interior space such as an interior seam, the flow rate may be reduced and solid objects such as cells or contaminants may become trapped or otherwise accumulate. Such confined areas are also difficult to clean effectively because cells, debris and contaminants can become trapped. This may lead to cell breakdown and further contamination of the bioreactor contents.

The tube or hose arrangement is also space efficient and the multiple tube bioreactors can be arranged in series (with the outlet of one bioreactor flowing into another bioreactor connected thereto (see e.g. fig. 4)), in parallel (see e.g. fig. 5) or in a combination of these methods in a single chamber. For example, a multi-tube bioreactor may be arranged in series such that the flow within each bioreactor runs in an anti-parallel direction to the previous bioreactor, such that the liquid culture medium takes a serpentine path through several bioreactors. Where two or more bioreactors are connected so as to be in fluid communication with each other, the connector or conduit joining them may be a separate component which need not contain any gas permeable material. Connectors may also be used to connect the bioreactor to auxiliary systems or to outlets or inlets. The connector may comprise a valve, typically a solenoid valve or diaphragm valve, which acts to prevent or allow fluid to pass through the connector, for example between one bioreactor and the next. Advantageously, this can allow several "choke points" to be achieved within a system comprising a plurality of bioreactors arranged in series. This enables any hydrostatic pressure stress from sudden pauses in flow within the system to be shared between adjacent bioreactors and prevents pressure waves from propagating throughout the connected bioreactors. Otherwise, if the flow suddenly stops, for example due to a pump failure, the "water hammer" effect may place excessive stress on certain components within the system, with all bioreactors remaining fluidly connected. Any means of mitigating such effects may be used in the system according to the present invention as appropriate, such as pressure regulators, slow-closing valves, flow diverters, shock absorbers, dampers, and the like.

It is envisaged that features may be introduced which allow improved mixing of the liquid culture medium as it flows through the bioreactor or bioreactor array. In this regard, a static mixer can be installed in the bioreactor (either inside the membrane bioreactor itself, or inside one or more connectors between the membrane bioreactors) to increase turbulence in the bioreactor and promote mixing of the liquid culture. These mixers are static and designed to mix fluids passing through them in motion. For example, the static mixer can include a helical structure that disrupts the flow of the liquid medium.

The breathable film sheet may have a thickness of no more than about 2000 μm, a thickness of no more than about 1000 μm, suitably no more than about 800 μm, about 600 μm, about 500 μm, about 400 μm, about 200 μm and generally no more than about 100 μm, optionally no more than about 50 μm, suitably no more than 20 μm, suitably no more than 10 μm or less. The breathable film sheet may be at least 10 μm thick, at least 20 μm thick, suitably at least 50 μm, at least 100 μm, at least 200 μm and optionally at least 500 μm thick. The thickness of the bioreactor membrane may vary across its length, for example in the case of a bioreactor connected to another bioreactor or another object by a connector, the thickness may increase in a portion of the membrane proximal to the connector as compared to a membrane distal to the connector. The membrane thickness can also vary depending on the location of the bioreactor in the array, e.g., a bioreactor in a lower vertical location may be thicker to provide more protection against bulging under pressure.

The diameter of the bioreactor of the invention (i.e., the maximum diameter of the cross-section of the bioreactor perpendicular to the direction of flow of the liquid culture medium) may be no more than about 20cm, no more than 15cm, 10cm, 9cm, 8cm, 7cm, 6cm, 5cm, 4cm, 3cm, 2cm, or no more than about 1 cm. The diameter may be no less than about 0.5cm, no less than about 1cm, 2cm, 3cm, 4cm, 5cm, 8cm, or no less than about 10 cm. Typically between 8cm and 2cm in diameter, typically between 7cm and 2cm, suitably between 5cm and 3 cm. The diameter may typically be less than 5cm for chemoheterotrophs and less than 10cm for photoautotrophs.

The length of the bioreactor, i.e. the distance between the inlet and the outlet of an individual bioreactor, may be no more than about 100m, optionally no more than about 75m, about 50m, about 25m, about 10m, about 9m, about 8m, about 7m, about 6m, about 5m, about 4m, about 3m, about 2m, about 1m, about 0.5m, typically no more than about 0.1 m. Typically the length of a single bioreactor is between about 10m and about 1m, suitably between 5m and 1m, and in embodiments between 3m and 1 m.

As discussed, the plurality of bioreactors can be connected in series and can be arranged such that the flow direction of one bioreactor is opposite to the flow direction of the previous bioreactor. The continuous bioreactor can be arranged to run a length of perhaps no more than about 2000m, 1500m, 1000m, 750m, 500m, 400m, 300m, 250m, 200m, 100m, 80m, 60m, 40m, 20m, 10m, 5m, 1m or less before such a change in direction occurs. Suitably this length is between about 1000m and about 50m, typically between about 800m and about 150m, suitably between about 400m and about 200m, optionally between about 300m and about 100 m. Typically, this length is chosen to be as long as possible before the change in direction occurs (as this increases the pressure), but without causing undue difficulty in maintenance.

In the case where a plurality of bioreactors are arranged horizontally, the plurality of bioreactors are arranged in parallel or otherwise, as the bioreactors connected in series change in direction, so the horizontal (width) dimension of the array of bioreactors (see fig. 6D) may be no more than about 200m, 150m, 100m, 75m, 50m, 40m, 30m, 25m, 20m, 15m, 10m, 9m, 8m, 7m, 5m, 4m, 3m, 2m, suitably no more than about 1m or less. Suitably this dimension is between about 75m and about 1m, typically between about 40m and about 5m, optionally between about 30m and about 5m, and suitably between about 20m and about 8 m. The minimum horizontal dimension may obviously be no smaller than the horizontal diameter of a single bioreactor. This width dimension should be chosen to allow for the inclusion of a sufficient volume of liquid culture medium, but not so wide as to create excessive pressure by requiring multiple changes in flow direction.

Similarly, multiple bioreactors can be arranged or "stacked" vertically. The minimum height of the array of bioreactors can obviously be no less than the height of the individual bioreactors. The total height of the array (see fig. 6D) may be no more than about 100m, 50m, 25m, 20m, 10m, 9m, 8m, 7m, 6m, 5m, 4m, 3m, 2m, 1m, 0.5m, 0.4m, 0.3m, 0.2m, typically no more than about 0.15 m. Typically, this dimension is between about 10m and about 0.15m, suitably between about 5m and about 0.5m, optionally between about 3m and about 0.5m, alternatively between about 2m and about 1 m. The height should be chosen to allow for the inclusion of a sufficient volume of liquid culture medium, but not so high as to create excessive pressure and/or cause difficulties in maintenance.

When a plurality of bioreactors are arranged side by side or vertically, the gap left vertically or horizontally between them (see fig. 6D) may be at least about 1mm, about 5mm, about 10mm, about 50mm, or at least about 100 mm. Typically, the gap is about 10mm horizontally and suitably about 50mm vertically. In some cases, no gaps may be left (i.e., adjacent bioreactors may be in contact). Generally, the gap size is selected to allow for efficient circulation of gas between the bioreactors.

The volume included within the bioreactor or array is not intended to be particularly limited except by the capacity of the bioreactor and other parts of the system.

Chamber

The chamber is generally defined by one or more exterior walls and contains a gas that may include O₂Such as, for example, the atmosphere. O in gas mixture₂May be higher than O in the liquid medium included in the bioreactor₂Thereby increasing the concentration difference between the liquid culture medium and the ambient atmosphere within the chamber. In this way, O₂The rate of gas transfer through the membrane into the liquid medium is increased.

Due to O in the liquid medium₂Is consumed by the cells contained therein and has more O ₂From the atmosphere in the chamber to the liquid medium across the membrane of the bioreactor, so O₂The gas transfer rate will decrease over time as the concentration difference stabilizes to equilibrium. To overcome the tendency towards equilibrium, the delivery of the oxygen-containing gas through the gas chamber inlet can be continuous or intermittent₂And a similar volume of gas can be removed through the outlet, typically using a controlled valve such as a solenoid valve and/or a pressure sensitive valve. Optionally, the valve can be closed and/or restricted when delivering the gas mixture to pressurize the gas chamber above ambient standard atmospheric pressure and so further increase the gas transfer rate across the gas permeable membrane of the bioreactor.

The gas mixture introduced into the gas chamber may also include CO at a concentration lower than that found in the liquid culture medium of the bioreactor or at a concentration lower than atmospheric₂Low level of CO₂To increase CO from the liquid medium₂The rate of depletion. Or, can be introduced by introducing an inert gas such as nitrogen, helium, argon or methane and/or O₂In the gas chamber to remove CO from the liquid medium₂So as to increase CO between atmosphere and liquid medium₂The concentration difference. It may also be desirable to increase the CO in the gas mixture ₂The concentration of (c). For example, CO₂Or other gases may be used to alter the pH level of the liquid medium. This may be advantageous to encourage the growth of organisms that prefer low pH, such as so-called extreme microorganisms, some of which are capable of growing in environments with a pH between 2 and 4. Additionally, certain organisms respond to stress in low pH environments by altering their behavior and/or biomass production, and it may be desirable to stimulate the production of specific stress-induced products.

Other organisms may need to be supplied with different gases and the chamber atmosphere can be controlled accordingly, for example CO can be supplied where the organisms are autotrophic₂Methane can be supplied in the case where the organism is methanotrophic, or hydrogen can be supplied in the case where the organism is hydrotrophic or hydrogentrophic. Certain hydroxides are defined by the ability to use gaseous hydrogen as an electron donor while using oxygen as an electron acceptor and to fix carbon dioxide. As a result, the chamber containing hydrogen, carbon dioxide and O can be used₂The chamber atmosphere of the mixture of (a). These "depend on CO₂In contrast to organisms that also oxidize hydrogen gas under aerobic conditions but are unable to undergo autotrophic carbon dioxide fixation (such as acetobacter, azotobacter, enterobacteriaceae, etc.). In the case of hydrogen supply, it is envisaged that electrolysis can be carried out in an auxiliary system, for example directly inside the liquid medium or in a water tank in or beside the chamber, to produce hydrogen from water, which would avoid pumping hydrogen into the gas chamber, which could have a safety hazard.

Likewise, certain organisms, such as certain hydroxidizing organisms and methanogens, may prefer anaerobic conditions. In this case, the chamber atmosphere can be controlled to be deficient in oxygen or any gas that may be detrimental to growth and/or survival.

In some embodiments, the gas chamber may be divided into two or more sections, referred to herein as a first chamber and a second chamber, etc., into which different gases or gas mixtures can be introduced. For example, the first chamber can contain rich O₂However the second chamber may contain depleted CO₂Such as for efficient removal of CO₂Is rich in N₂A gas. In certain embodiments of the invention, the bioreactor provides an intermediate barrier between the first chamber and the second chamber (and additional chambers if desired). Thus, in this embodiment of the invention the first and second chambers are defined by the outer walls of the chambers in combination with the membrane wall of the intermediate bioreactor.

The gas being capable of expanding by the gas or reducing O by use thereof₂A low energy means of feed delivery cost (or any other suitable gas) such as a fan, turbine or other paddle passively moves inside the chamber. Alternatively, the gas can be compressed prior to introduction into the gas chamber. It is contemplated that the pressure inside the chamber can be controlled by the introduction or removal of a gas. For example, the pressure inside the chamber may be higher than the atmospheric pressure outside the chamber, or the pressure inside the chamber can be reduced compared to the atmospheric pressure outside the chamber.

The internal environment of the chamber can be controlled internally or by controlling the gas supply and/or gas exhaust. The humidity of the atmosphere within the chamber can be controlled, for example, by introducing a gas mixture having a reduced or increased humidity compared to the chamber atmosphere, or by the presence of a desiccant or humidifier installed in the gas inlet, or by a desiccant or humidifier or material or coating placed inside the chamber itself or within the attachment assistance system. Most often, the chamber atmosphere needs to be dried due to the passage of water vapor from the liquid medium through the bioreactor membrane into the chamber atmosphere. For example, the chamber atmosphere can be circulated to a desiccant for drying before being returned to the chamber; typically the desiccant may be in the form of a honeycomb wheel. The temperature of the chamber atmosphere can be controlled, for example, by introducing a gas mixture having a reduced or increased temperature compared to the surrounding chamber atmosphere, or by the presence of cooling or heating means installed in and/or before the gas inlet. For example, the chamber atmosphere can be circulated to an air conditioning unit and/or an air heating unit before returning to the chamber. In some cases, the gas mixture in a chamber can be recirculated in the same chamber, or transferred to the next chamber in the case of a series arrangement of multiple chambers. The gas can be dried, cooled, heated, filtered, cleaned and/or supplemented with an appropriate amount of the desired gas to adjust its composition and/or be further cooled, heated and/or dried prior to returning the gas mixture to the chamber.

The internal chamber temperature can also be controlled or influenced by controlling the temperature of the gas introduced into the chamber. For example, heated or cooled gases that can control the chamber atmosphere and even the temperature of the liquid medium of the bioreactor can be introduced. The heating unit and/or the cooling unit can be comprised by or contained within the chamber itself, which enables a more direct control of the temperature of the atmosphere already within the chamber.

At least a portion of the walls defining the chamber material may be transparent or translucent to allow efficient transmission of light so that when the cells included within the bioreactor are phototrophic or mixotrophic, they can use the light for energy production or inorganic carbon fixation. Such transparency may be useful even in cases where the cells do not require light, for example to enable an operator to directly and locally inspect the interior of the chamber. In some embodiments, at least a portion of one or more of the walls, e.g., the wall furthest from the light source, is reflective in order to increase the passage of light through the bioreactor. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the area of the wall can be light transmissive.

"switchable glass", "smart glass" or similar materials may be used in the present invention. These are materials that alter their light transmission properties when voltage, light or heat is applied (which may be, but is not limited to, rigid like glass, flexible like polymer films or coatings). These may be particularly useful in areas with high light exposure, for example to reduce damage to materials or microorganisms due to particularly high light. Typically, a material changes from being substantially translucent and/or substantially transparent due to reflective optical properties (similar to a mirror finish), changing from blocking some (or all) wavelengths of light to passing light. Examples of technologies that may be used in pursuit of the foregoing include, but are not limited to, electrochromic, photochromic, thermochromic, aerosol, micro-blind, and polymer dispersed liquid crystal devices.

Suitably, the walls of the chamber are substantially gas impermeable and the chamber is substantially gas tight as a whole to prevent loss or contamination of the controlled atmosphere contained therein. The chamber need not be completely gas-tight, as long as it meets the purpose of allowing the internal atmosphere to be controlled to some extent in terms of gas composition, temperature, humidity, pressure, or other aspects.

The walls of the chamber can be composed of or defined by structures or body components of vehicles, industrial machines, ships, spacecraft or spacecraft, submersibles, wall cavities, containers, greenhouses, basements, building structures, building rooms and/or switch rooms.

In these and/or other cases, the chamber walls can comprise opaque/translucent materials. In such cases an auxiliary light source inside the chamber may be used. These secondary light sources may be LEDs/OLEDs or fluorescent tubes, or may be natural light guided by optical fibers and/or optical components. Such auxiliary light sources may be used similarly in cases where the chamber walls are translucent/transparent but the device is located inside or away from natural light. In some cases, at least a portion of the inner chamber wall may be or may contain a reflective material. This may increase the efficiency of the light supply to the cells in case an internal light source is used. In some cases, for example where an external light source is used, a mixture of translucent/transparent and reflective materials may be used. In some such cases, a portion or all of the interior wall or walls furthest from the light source may be reflective to increase the efficiency of use of the supplied light. In embodiments where the mixed vegetative organisms are cultured, the light source may supply the light necessary for their growth. The light source may be configured to provide sporadic and/or intermittent illumination depending on the requirements of the implementation of the invention and/or the living being used.

Any translucent/transparent portion that allows light to be transmitted into the chamber can be comprised of any suitable translucent/transparent material. The chamber can be composed entirely of translucent/transparent material or can be supported on a support structure such as a scaffold or frame as described below.

Suitably the chamber comprises a substantially gas impermeable material that is strong, light and may possess good thermal insulation properties. Optionally the material is provided as a sheet and/or film. In some embodiments the material is inflexible, inelastic, transparent and strong, including for example glass, high performance glass, low iron glass with very high solar energy transmission (Pilkington sun plus)^TM) Glass composites, strengthened glass composites with increased strength, impact resistant glass composites, low reflectivity glass, high light transmittance glass, double and/or triple layer hollow glass with or without vacuum/argon/air in between, or glass composites made of several layers of different materials to increase strength and/or light transmittance, or electrically switchable smart glass. Alternatively, the chamber may comprise a metal or metal alloy, such as aluminium or steel, or a composite material, such as a carbon fibre composite, a glass fibre or wood fibre material (e.g. MDF), concrete, stone, clay, tile, mortar, a plastics polymer.

In other embodiments, the chamber wall material is flexible and resilient, including, for example, Ethylene Tetrafluoroethylene (ETFE), acrylic/PMMA, polycarbonate, and/or other plastics and plastic composites. Suitably, the chamber wall material comprises polyvinyl chloride (PVC), polyurethane, vulcanized rubber, silicone, polyethylene and/or nylon, textile reinforced polyurethane plastic, woven fabric coated with a polymer such as PVC, nylon, PC, silicone, rubber.

Suitable properties of ETFE include its translucency and/or transparency, very high light transmittance, and resistance to ultraviolet light. ETFE is also advantageously recyclable, easy to clean (due to its non-stick surface), elastic, strong and light, while having good thermal insulation, high corrosion resistance and strength over a wide temperature range. With heat welding, tears can be repaired with a patch or multiple sheets assembled into a larger panel.

Acrylic is suitable as a chamber wall material due to its strength, high transparency and weatherability and resistance to ultraviolet radiation.

In a particular embodiment of the invention, the use of a flexible and/or elastic material allows inflating the chamber by supplying an atmosphere within the chamber that has a relatively positive pressure compared to the surrounding atmosphere outside the device. Alternatively, the expansion of the gas within the chamber due to the increase in temperature may also cause a corresponding increase in the relative positive pressure. In some embodiments, the pressure in the chamber may even be negative compared to the surrounding atmosphere outside the device, e.g. the action of removing gas from the chamber by a fan or blower. The chamber can be fully inflated from a collapsed (uninflated) state and/or can be built around or otherwise supported by rigid or semi-rigid scaffolding, which may be internal or external to the chamber itself, and may be integral with the chamber, or may be separate therefrom. The chamber wall material can be reinforced by an integral skeleton comprising components of rigid or semi-rigid scaffolding and/or by using reinforcing seams made of the same or similar material as the chamber wall. These stiffeners can also be used to control the shape and structure of the chamber when constructed and inflated. Such an arrangement allows systems according to some embodiments of the present invention to be easily and quickly constructed, dismantled and/or transported in their collapsed (uninflated) form. Weight can also be reduced by using such embodiments, increasing suitability for transportation as well as for temporary and/or remote use, such as in space, polar research stations, or other inaccessible locations. Such portable structures can also be placed inside a warehouse or any kind of structure or chamber, such as a basement or tunnel, to create multiple independent chamber modules inside the structure that provides a protected environment. These aeration chambers can be easily changed, disassembled or moved to renew the array of bioreactors without compromising the structure of the building.

In certain embodiments of the invention, the use of a flexible and/or elastic material will allow for the creation of a convex, domed, arched or otherwise protruding shape (relative to the position outside the chamber) to the upper wall of the chamber as a result of a positive pressure inside the chamber relative to the surrounding atmosphere (i.e. the chamber is inflated by the supplied gas) or by using auxiliary structures attached to the walls of the chamber to create the convex shape. This may help to avoid "puddles" of rain, snow, leaves, powder, sand or other debris if the equipment is deployed in the field. Furthermore the convex shape will facilitate self-cleaning of the material when it rains and/or facilitate manual/automatic cleaning performed by a plant operator or an automatic cleaning system. For similar reasons, any upper surface of the chamber may be slightly inclined with respect to the horizontal in other embodiments of the invention, for example by the side walls of the chamber having different heights.

Another advantage of such an arrangement is that a measure is activated to control the humidity of the interior chamber-moisture in the chamber atmosphere may condense on the interior of the chamber walls, especially if the interior of the chamber is warmer than the outside atmosphere. With a convex or sloping upper wall, any condensate can be encouraged to run away from the upper wall of the chamber, thereby reducing the interference with light transmission that may occur.

Graphene coatings can be used to reinforce materials, provide an antimicrobial growth coating, and provide electrical conductance that can then help detect breakage (e.g., tearing) of the material. Coatings, treatments, paints or films for reducing mold, bacteria and fungal growth can also be applied to the interior surfaces of the chamber. A specific material intended to prevent the growth of moulds or any microorganisms can be used as a component of the chamber. The material can also include graphene, carbon nanotubes and/or graphite for reinforcement or to enable the use of thinner and lighter wall materials.

It is conceivable that the interior of the chamber may be easily accessed for maintenance purposes by completely or partially removing one or more of the walls comprising the chamber.

The minimum size of the chamber is primarily dictated by the size of the bioreactor or bioreactor array contained. In some embodiments, sufficient additional space may be left between the outermost edge of the bioreactor or bioreactor array and the chamber walls to allow access for maintenance personnel or equipment (see fig. 6D).

Biological organisms

The devices and methods of these inventions may be used to culture any microorganism, cell or small organism taken from the field of bacteria, archaea or eukaryotic taxonomy, provided it can be supported in a suitable liquid culture medium. Such cells and organisms may be heterotrophic or mixotrophic. Additionally, the devices and methods of these inventions are suitable for culturing phototrophic organisms, including photoautotrophic organisms.

More specifically, the cells and/or organisms may be part of a taxonomic group and other defined groups including: cyanobacteria, propterium, spirochete, gram-positive bacteria, chloromycete such as the phylum Chlorophytum, Phycomycota, Bacteroides Cytophaga, Thermotoga, Aquifex, halophilum, Methanosarcina, Methanococcus, Thermococcus, Thermobacter, Neamoeba, Myxomyces such as Myxomyces, ciliate, Dinoflagellates (Dinoflagellates), Dinophyceae (Dinophyceae), Trichomonas, Microsporozoa, Trichomonas, archaea, Proteus, Trichophyton, Fomitochoda, Porphyra, Radioach, Diatoma, Protozoa, Brown algae, Red algae, Green algae, snow algae, Dinophyta, Crypthecodinium, Heterophyta, Gray algae, plankton, Phytophyta, Trypanosoma, and cells or whole organisms from animals, fungi or plants.

Suitable bacteria may include Escherichia coli, Escherichia coli BL21(DE3), Escherichia species (Escherichia sp.), Acetobacter species (Acetobacter sp.), Acetobacter xylinum (Acetobacter xylinum), Sarcina gastri (Arcina ventricus), Zymomonas mobilis (Zymomonas mobilis), Gluconobacter xylosus (Gluconobacter xylinum), Pseudomonas species #142, Microbacterium laeviformis (Microbacterium laeviformes), Bacillus polymyxa (Paenibacillus polymyxa), Bacillus licheniformis (Bacillus licheniformis), Bacillus subtilis (Bacillus subtilis), Bacillus macerans (Bacillus cererans), Streptococcus salivarius, Salmonella salivarius, Leuconobacter mesenteroides (Leuconostoc), Microbacterium lutescens (Lactobacillus), Microbacterium strain (Bacillus cereus), Bacillus subtilis), Bacillus macerans (Bacillus cereus), Bacillus cereus, Clostridium sp.sp., Lactobacillus paracoccus (Lactobacillus plantarum), Pseudomonas sp., Lactobacillus strain (Lactobacillus strain, Bacillus cereus), Pseudomonas strain (Bacillus cereus), Pseudomonas sp., Lactobacillus strain, Bacillus cereus strain, Bacillus cereus strain, Bacillus cereus, agrobacterium tumefaciens (Agrobacterium tumefaciens) and Brevundimonas diminuta (Brevundimonas diminuta). Other suitable bacteria can include Deinococcus species (Deinococcus sp.), Deinococcus radiodurans (Deinococcus radiodurans), Deinococcus georgis geothermalis (Deinococcus geothermalis), Deinococcus cellulolyticus (d.cellulolyticus), Deinococcus fujiensis (d.radiodurans), Deinococcus proteolicus (d.proteoliticus), Deinococcus radiodurans (d.radiodurans), Deinococcus radiophilis (d.radiophilis), Deinococcus macrorrhalis (d.grandis), Deinococcus indicus (d.indeicus), Deinococcus aerogenes (d.fregnus), Deinococcus lithospermica (d.saxicola), Deinococcus przewalensis (d.marica), Deinococcus marylactis marmoratus (d.marmoratus), Deinococcus deserticola (d.aetiorrhalis), Deinococcus aegyptis, Deinococcus Deinococcus aquaticus (d.aquaticus), deinococcus aquaticus (d.aquatilis), deinococcus aquilicola, deinococcus aquaticus (d.aquivivus), deinococcus natans (d.caeni), deinococcus kei (d.claudionis), deinococcus fici (d.ficus), deinococcus goviensis (d.gobiensis), deinococcus homani (d.hokamensis), deinococcus hopenis, deinococcus mineralocoris (d.misense), deinococcus nagawamori (d.navajonensis), deinococcus papagomensis, deinococcus maritima (d.perariensis), deinococcus pimentalis, deinococcus pisciosus (d.piscis), deinococcus radiomollis, deinococcus roseoflavris (d.rosea), deinococcus uniensis (d.uniensis), deinococcus versicolor (d.yuensis), deinococcus terreus. Specifically, contemplated species include Escherichia coli, Escherichia species, Acetobacter species, Zymomonas mobilis, Gluconobacter xylosus, Pseudomonas species, Microbacterium levansgenes, Bacillus polymyxa, Bacillus licheniformis, Streptococcus salivarius, Leuconostoc mesenteroides, Aerobacter levanserina, gamma-and alpha-Proteobacteria, Vibrio species, Pseudomonas fluorescens, Bacillus crescentus, Agrobacterium tumefaciens, Brevundimonas diminuta, Thermus rhodochrous (Meiothermus ruber), and Thermus profundus.

Pathogenic organisms can also be cultured in the device according to the invention, for example for use in vaccine production. Other bacteria which may be of interest include Bacillus subtilis, Corynebacterium glutamicum (Corynebacterium glutamicum), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Zymomonas mobilis, Agrobacterium tumefaciens, Sinorhizobium meliloti (Sinorhizobium meliloti), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Paracoccus mutans (Paracoccus versitus), Pseudomonas fluorescens, Pseudomonas putida (Pseudomonas putida), Salmonella enterica (Salmonella enterica), Escherichia fergusonii (Escherichia fergusonii), Yersinia pestis (Yersinia pestis), Yersinia pseudotuberculosis (Yersinia pseudouberculosis), Yersinia enterocolitica (Yersinia enterocolitica), Shigella flexneralis (Shigella Shigella), Shigella Shigella (Shigella Shigella), Shigella dysenterica (Pectia), Shigella pectinifera (Pectinospora), Shigella pectinifera (Pectia), Shigella dysenterica (Pectia), Shigella pectinifera (Pectii), Shigella Shigella (Pectii), Shigella pectinifera), Pectii (Pectia), Shigella (Pectinospora pectinifera), Shigella (Pectii), Shigella (Pectina), Shigella (Pectii), Pectii (Pectina), Pectina (Pectina ), Shigella (Pectina), and Pectina (Pectinatum pectinifera (Pectinatum pectinifera (Pectinatum) and Shigella (Pectinatum) of the method (Pectinatum) of the same), and Shigella (Pectinatum) of the method (Pectinatum, Shigella (Pectinatum) of the same), and the method (Pectinatum, Shigella (Pectinatum) of the method of the same), and the method of the same, Shigella (Pectinatum, Shigella (Pectinatum) of the same), and the method of the same, Shigella, and the same, and the method of the same, and the, Erwinia pyretophysa (Erwinia pyrifolia), Erwinia pyretophysa (Erwinia amylovora), Erwinia bivalia (Erwinia biclinae), Coccus aphidicola (Buchnera aphiicola), Enterobacter species 638, Enterobacter cloacae (Enterobacter cloacae), Enterobacter albugineus (Enterobacter asburrae), Enterobacter aerogenes (Enterobacter aegerens), Cronobacter sakazakii (Cronobacter sazakii), Cronobacter sullii (Cronobacter terreus), Klebsiella pneumoniae (Klebsiella pneumoniae), Klebsiella varicola (Klebsiella viridans), Klebsiella oxytoca (Klebsiella pneumoniae), Klebsiella viridifra (Klebsiella viridans), Klebsiella viridogrisra (Escherichia), Klebsiella viridifra strain (S), Klebsiella viridans (S Bacterial sweetpotato stem rot (Dickeya dadantii), dikayama (Dickeya zeae), Pantoea ananatis (Pantoea anantis), Pantoea species (Pantoea sp.) At-9b, Pantoea ubiquitina (Pantoea vagans), Lalenella species (Rahnella sp.) Y9602, Haemophilus parasuis (Haemophilus parakuaisi), Haemophilus parainfluenzae (Haemophilus parainfluenzae), Pasteurella multocida (Pasteurella multocida), Microbacterium acidophilum (Agegregabacter aphylporus), Actinobacillus actinomycetemcomitans (Aggregatobacter actinomycetemnum), Vibrio cholerae (Vibrio cholerae), Vibrio vulnificus (Vibrio vjinicus), Vibrio vulnificus parahaemolyticus (Vibrio parahaemolyticus), Vibrio parahaemolyticus, Vibrio parahaemophilus, Vibrio parahaemolyticus, Vibrio parahaemophilus, Vibrio parahaemolyticus, Vibrio parahaemophilus, Vibrio parahaemophilus, Vibrio parahaemophilus, Vibrio parahaemophilus, Vibrio parahaemophilus, shewanella borrelia (Shewanella brackica), Shewanella pv (Shewanella loihica), Shewanella species ANA-3, Shewanella species MR-7, Shewanella putrescentiae (Shewanella putrefensis), Shewanella sedimentata (Shewanella sediminis), Shewanella species MR-4, Shewanella species W3-18-1, Shewanella warfarensis (Shewanella woodynia), Salmonella lngomeretrica (Psychromonas ingensis), Iridago ariella (Ferroomonas basilica), Aeromonas hydrophila (Aeromonas hydrophila), Aeromonas campestris (Aeromonas campestris), Pseudomonas sp, Pseudomonas aeruginosa (Pseudomonas sp), Bacillus cerealis (Pseudomonas sp), Bacillus cereus (Bacillus cereus), Bacillus cereus sp), Bacillus cereus (Bacillus cereus sp), Bacillus cereus (Bacillus cereus, Bacillus cereus sp), Bacillus cereus (Bacillus cereus, Bacillus cereus sp), Bacillus cereus (Bacillus cereus, Bacillus cere, Bacillus cytotoxics (Bacillus cytotoxins), Bacillus thuringiensis (Bacillus thuringiensis), Bacillus westernensis (Bacillus wehenstephaniensis), Bacillus pseudochinensis (Bacillus pseudorhizogenes), Bacillus megaterium (Bacillus megaterium), Staphylococcus aureus (Staphylococcus aureus), Microbacterium siberium (Exiguobacterium sibiricum), Microbacterium species ATlb, Micrococcus casei (Micrococcus caseii), Bacillus polymyxa (Paenibacillus polymyxa), Streptococcus pyogenes (Streptococcus pyenes), Streptococcus pneumoniae (Streptococcus pyogenes), Streptococcus dysgalactiae (Streptococcus mutans), Streptococcus mutans (Streptococcus mutans), Streptococcus thermophilus (Streptococcus mutans), Streptococcus pyogenes (Streptococcus pyococcus (Streptococcus pyogenes), Streptococcus mutans (Streptococcus lactis), Streptococcus dysgalactiae (Streptococcus pyogenes), Streptococcus mutans (Streptococcus pyogenes), Streptococcus pyogenes (Streptococcus lactis), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus lactis (Streptococcus pyogenes), Streptococcus pyogenes (Streptococcus lactis), Streptococcus pyogenes), Streptococcus lactis (Streptococcus pyogenes), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus lactis (Streptococcus pyogenes), Streptococcus lactis (Streptococcus pyogenes), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus pyogenes), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus pyogenes), Streptococcus lactis (Streptococcus pyogenes), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus, Streptococcus pseudopneumoniae (Streptococcus pseudopneumoniae), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus gasseri (Lactobacillus gasseri), Enterococcus faecalis (Enterococcus faecalis), Aerococcus urealyticus (Aerococcus urensis), Carnobacterium species (Carnobacterium sp.)17-4, Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium perfringens (Clostridium perfringens), Clostridium tetani (Clostridium tetani), Clostridium norbomianum (Clostridium novyi), Clostridium botulinum (Clostridium bornatum), Clostridium reductium (Desulfuricum) and Clostridium difficile (Clostridium urens), Clostridium bradycardia (Clostridium), Rhodococcus rhodochrous (Clostridium), Mycoplasma gallisepticum (Mycoplasma gallisepticum), Mycoplasma pneumoniae (Mycoplasma pneumoniae), Mycoplasma uremia Mycoplasma gallisepticum, Mycoplasma uremia, Mycoplasma uremia, Mycoplasma uremia, Mycoplasma, Mycopla, Mycoplasma lechicii, Mesoplasma florum, Propionibacterium acnes, Mesorplasma polycistrita (Nakamurella multipartita), Borrelia burgdorferi (Borrelia burgdorferi), Borrelia gariae (Borrelia gariii), Borrelia aryabhatii (Borrelia afzelii), Prochloranthus oceanicus (Prochlorococcus marina), Bacillus Lysinibacillus sphaericus (Lysinibacillus sphaericus), Pyrenophora borealis (Rhodopirillum boreale), or combinations thereof. In particular, lactobacillus johnsonii and clostridium acetobutylicum are contemplated.

Methanotrophic organisms are capable of metabolizing methane as a source of carbon and energy. The use of such organisms may be useful in treating methane-containing gases in the apparatus according to the invention, and can therefore have applicability against global warming, since methane is a powerful greenhouse gas. It is noted that the growth of some methanotrophic organisms may also require the provision of carbon dioxide in the liquid medium in order to favor a particular metabolic pathway and thus growth. In this case, the atmosphere maintained within the chamber can be adapted to meet the needs of the cultured organisms, for example by providing carbon dioxide at a level above normal atmospheric levels. Suitable methanotrophic or archaebacteria can include Methylomonas 16a ATCC PTA 2402, Methylobacterium species (Methylobacterium sp.), Methylobacterium extorquens (Methylobacterium exiaques), Methylobacterium radiodurans (Methylobacterium radiodurans), Methylobacterium extremum (Methylobacterium populi), Methylobacterium chloromethylatum (Methylobacterium chloromethanicum), or Methylobacterium nodosum (Methylobacterium nodulans), Methylobacterium methylotrophicum species (methylomicron sp.), Methylobacterium methylotrophicum OB3B (NRRL B-11,196), Methylobacterium sporogenes (Methylobacterium sporogenes) (NRRL B-11,197), Methylobacterium parvum species (Methylobacterium sp), Methylobacterium parvum (Methylobacterium methylotrophicum sp), Methylobacterium parvum (nrb-11,198), Methylobacterium sp), Methylobacterium albonum (nrb-3511), Methylobacterium sp, Methylobacterium albonum 11,199, Methylobacterium sp, methyl, Methylococcus capsulatus, Methylobacillus sp., Methylobacillus capsulatus Y (NRRL B-11,201), Methylococcus capsulatus (NCIMB 11132), Methylobacillus organophilus (Methylobacillus organophilus), Methylobacillus organophilus (ATCC 27,886), Methylomonas bacterium AJ-3670(FERM P-2400), Methylomicrobium sp. (Methylomicbium sp.), Methylobacillus alcaliphilus (Methylobacillus alcaliphilus), Methylobacillus methylotrophus (Methylocella sp.), Methylobacillus laterosporus (Methylocella Silvestris), Methylobacillus acidophilus (Methylophilus inrichum), Methylobacillus acidophilus (Methylobacillus sp.), Methylobacillus acidopileus sp., Methylobacillus acidophilus (Methylobacillus sp.,) or Methylobacillus capsulatus (Methylobacillus. In particular, Methylococcus species, Methylobacter species, Methylomonas species, Methylococcus capsulatus, and Methylococcus pekinensis are contemplated.

So-called probiotics, archaebacteria and fungi as organisms intended to be consumed live to provide health effects include especially lactobacillus, bifidobacterium, saccharomycetes, enterococcus, streptococcus, pediococcus, leuconostoc, bacillus and can include escherichia coli, lactococcus, enterococcus, oenococcus, pediococcus, streptococcus and leuconostoc species, lactobacillus species can include lactobacillus plantarum, lactobacillus johnsonii, lactobacillus acidophilus, lactobacillus sake, lactobacillus bulgaricus, lactobacillus salivarius, lactobacillus acidophilus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus bulgaricus, lactobacillus brevis, lactobacillus johnsonii, lactobacillus plantarum and lactobacillus fermentum. Other predetermined species include Saccharomyces boulardii, Bifidobacterium bifidum, Bacillus coagulans, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp lactis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, lactococcus lactis, enterococcus faecium, enterococcus durans, and Streptococcus thermophilus, Bacillus subtilis, and Bacillus cereus. In particular, the genera lactobacillus, bifidobacterium bifidum, bacillus coagulans, bifidobacterium infantis, bifidobacterium adolescentis, bifidobacterium bifidum and bacillus coagulans, bifidobacterium infantis, enterococcus faecium and streptococcus thermophilus are envisaged.

The groups and genera of archaea which can be used in the present invention include in particular the phylum Fangxiella (Crenarchaeota), the phylum eurycota (Euryarchaeota), the order Thiococcales (Desulfurococcales), the order sulfolobules (sulfolobules), the order Archaeoglobulinales (Archaeoglobulines), the order Halobacteriaceae (Halobacteriales), the order Methanobacteriales (Methanobacteriales), the order Methanococcales (Methanococcus methanolica), the order Methanopyrum (Methanopyrales), the order Pyrococcales (Thermocales), the genus Thermoplasma (Thermoplasma), the order Aeropyrum pernix (Aeropyrum pernix), the genus Sulfolobus solfataricus (Sulfolobacter), the genus Thermomyces (Methylococcus thermophilus), the genus Thermomyces (Methanococcus thermophilus), the species Methanophorus (Methanophorus), the strain (Methylobacillus thermophilus), Methylophilus thermophilus (Methylophilus), Methylophilus thermoascus (Methylophilus), Methylophilus thermoascus taurocarbinonotus, Methylophilus), Methylophilus (Methylophilus), Methylophilus taurocarbinonotus, Methylophilus (Methylophilus taurocarbinonotus), Methylophilus sulfide, Methylophilus, Meth, Pyrococcus horikoshii (shinkaj), Pyrococcus pasteurianus (Pyrococcus abyssi), Pyrococcus furiosus (Pyrococcus furiosus), Pyrococcus ritonae (Thermococcus litoralis), Thermococcus pasteurianus (Thermococcus barosiii), Thermoplasma acidophilus (Thermoplasma acidophilum), Thermoplasma volcanium (Thermoplasma volcanium), Halobacterium species NRC-1, Methanococcus jannaschii DSM 2661, Pyrococcus pasteurianus GE5, Thermoplasma acidophilus DSM 1728 and Thermoplasma volcanium GSS 1.

The device according to the invention can also be used for the cultivation of hydroxidizing organisms that oxidize oxygen to an energy source while using oxygen as the last electron acceptor. This is achieved bySome of these organisms preferably grow under microaerophilic conditions, i.e., in an environment having oxygen levels that are lower than those present in the normal atmosphere. As a result, less than 21% O can be maintained₂Typically about 2 to 10% O₂The chamber oxygen concentration of (a). For example, a mixture of hydrogen, carbon dioxide and oxygen can be supplied. These organisms can include, but are not limited to, hydrogenobacterium species (Hydrogenobacter sp.), hydrogenobacterium thermophilum (Hydrogenobacter thermophilus), marine aquaporin bacteria (Hydrogenobacter marinus), Helicobacter pylori species (Helicobacter sp.), Helicobacter pylori (Helicobacter pylori), hydrogenotrophus species (hydrogenotrophus sp.), Bacillus hookeri (cuprinus greediligena), Rhodococcus rhodochrous (Rhodococcus rhodochrous), Alcaligenes species (Alcaligenes sp.), Alcaligenes eutrophus (Alcaligenes eutrophus), Alcaligenes eurinogenes (Alcaligenes eutrophus), Alcaligenes latus (Alcaligenes disporhius), Pseudomonas paradoxorum (Pseudomonas protovorans), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas paradoxorhii), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Bacillus acidovorax), Pseudomonas aeruginosa (Bacillus subtilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Bacillus subtilis) High temperature water bacteria (Pseudomonas hydrogenophila), Pseudomonas parvum (Pseudomonas pallonii), Pseudomonas saccharophila (Pseudomonas saccharophila), Pseudomonas thermophila (Pseudomonas thermophila), Serissa hydrocarbonate (Seliberia carboxyhydroyena), Flavobacterium thermophilum (Flavobacterium thermophilum), Paracoccus denitrificans (Paracoccus densificans), Flavobacterium autotrophus (Xanthobacter autotrophus), Flavobacterium autotrophicum, Arthrobacter sp. (1IX, RH 12), Mycobacterium gordonii (Mycoterium gordonae), Nocardia autotrophicum (Nocardia autotrophica) and Nocardia nigrella (Candida opaea). Some contemplated organisms utilize hydrogen gas (such as Desulfovibrio (Desulfovibrio), Clostridium acetate (Clostridium aceticum), Acetobacter xylinum (Aceto-bacterium)) under anaerobic conditions with sulfate or carbon dioxide as hydrogen acceptors wood) and Methanobacterium thermoautotrophicum (Methanobacterium thermoautotrophicum)).

The yeast species that can be used in the present invention include in particular Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces boulardii. Other suitable yeast species include saccharomyces, pasteuria, carlsbergia, leucosporum, crymodorosporium, geotrichum, candida, rhodotorula, trichosporon, schizosaccharomyces, lochiosporium, sporobolomyces, candida tropicalis, a group consisting of phaffia, kluyveromyces lactis, hansenula, metschnikowia, and any combination thereof. Among these, particular species of Saccharomyces, Asparagus, Rhodotorula, Trichosporon, Schizosaccharomyces, Spodoptera, Sporobolomyces and Candida tropicalis are contemplated.

Fungi that may be used in the devices and methods of the invention include filamentous fungi such as Aspergillus japonicus, Aspergillus niger, Aspergillus foetidus, Aspergillus oryzae, Aureobasidium pullulans, Sclerotinia sclerotiorum, and Aureobasidium pullulans. The species of mold includes members of a community including Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Vitis, Trichoderma reesei, Trichophyton, Aspergillus oryzae, Monascus, Penicillium, Fusarium, Geotrichum, Neurospora, Rhizomucor miehei, Rhizopus oligosporus, Rhizopus, Neurospora, Rhizomucor, Uvularia, and Sphaerotheca. Among the moulds, particular mention may be made of Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Botrytis, Trichoderma and Trichophyton.

Myxomycete refers to many communities of facultative multicellular eukaryotes. Suitable examples for use in the present invention include polychitozoon (Physarum polycephalum), Fungium (Fuligo septica), Hymenopile species (Fuligo sp.), Dicidophyte (Stemonitis fusca), Trichosporon sp, Dichodesmus (Diachypodia), Trichosporon sp, Trichosporon (Trichiya sp), Trichosporon (Dictyostyle), Dictyosium sp, Dictyosium purpureum (Dictyosidium sp), Trichosporon nuciferum (Dictyosidium sp), Trichosporon (Dictyosium co-deum), Myxophytes (Myxophyceae), Trichosporon (Dictyosidium sp), and Actyosidium (Actyosidium sp), and especially Phyllophora species, Phyllotreta, and Phyllotreta (Phyllotreta), and Phyllotreta species of Myxomycota, and Phyllotreta, especially, Phyllotreta, Phyllophora sp.

Photosynthetic microorganisms can also be used in the device according to the invention. Such potential organisms include members of communities such as pediococcus, chlorella, prototheca, chlorella and scenedesmus. Other possibilities include the species Coccomys orientalis (Achnanthes orientalis), the genus Argomyces (Agmenellum), Coccomys hyaloides (Amphioria hyalina), Coccomys coffeiformis (Amphioxus coffeiformis), Coccomys coffei (Amphiobacillus coffei) Linea, Coccomys coffei (Amphiobacillus coffei) Punctata, Coccomys coffei (Amphiobacillus taylori), Coccomys coffei (Amphiobacillus coffei) Leptomys (Amphiobacillus coelicoides), Coccomys coffei (Coccomys coenosus), Coccomys coffei (Bochys), Coccomydia sp.) Tenuii (Anabaena), Coccomydia sp), Anabaena (Anabaena, Coccomydia sp), Coccomydia sp (Bochys sp), Bochys sp (Bochys strain Bochys), Bochys strain Bochyuroides (Bochys), Bochys strain Bochys (Bochys), Bochys) Bochys (Bochys) Bochys, Bochylotus strain Bochyloti (Bochys), Bochys strain Bochys, Bochyloti (Bochys) Bochys, Bochyloti, Bochys, Bochylotus Bochyloti (Bochylotus) Bochylotus Bochys, Bochylotus Bochys, Bochylotus, Bochys, Bochylotus strain Bochylotus Bochys, Bochylotus strain Bochylotus, Bochys, Bochylotus (Bochys, Bochylotus Bochys, Bochylotus, Bochys, Bochylotus strain Bochys, Bochylotus strain Bochys, Bochylotus strain (Bochylotus, Bochys, Bochylotus strain Bochys, Bochylotus, Bochysolen strain Bochysolen, Bochysolen strain (Bochysolen, Bochysolen strain, Bochysolen strain, Bochysolen strain, Bochysolen, verbena spp (Carteria), Chaetoceros gracilis, Chaetoceros mularia (Chaetoceros muelleri), Chaetoceros gracilis (Chaetoceros muelleri), Chaetoceros europaea wide-salt variety (Chaetoceros muelleri subsalsum), Chaetoceros species (Chaetoceros sp.), Chlorella anomala (Chlorella anatata), Chlorella Antarctica (Chlorella anatipes), Chlorella lutescens (Chlorella aureovirginosa), Chlorella viridis (Chlorella viridans), Chlorella viridis (Chlorella viridescens), Chlorella viridis (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis water Chlorella viridis, Chlorella viridis (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis strain 35chlorella strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella viridis strain (Chlorella viridis), Chlorella strain (Chlorella, Chlorella luteoviridis (Chlorella luteoviridis var. aureoviridis), Chlorella lutescens (Chlorella lutescens var. lutescens), Chlorella rubra (Chlorella lutescens), Chlorella minutissima (Chlorella lutescens), Chlorella abortus (Chlorella minutissima), Chlorella foeniculis (Chlorella lutescens), Chlorella nocturna (Chlorella nocarpa), Chlorella ovale (Chlorella ovalis), Chlorella miniata (Chlorella minutissima), Chlorella phototrophylla (Chlorella lutescens), Chlorella procumbens (Chlorella privilens), Chlorella procumbens (Chlorella pringshmimi), Chlorella protothecoides (Chlorella protothecoides) (including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25), Chlorella protothecoloris (Chlorella protothecoides), Chlorella acidifera strain (Chlorella viridis), Chlorella vulgaris (Chlorella viridis ), Chlorella viridis (Chlorella viridis), Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella viridis, Chlorella, chlorella salina (Chlorella salina), Chlorella simplex (Chlorella simplicissima), Chlorella thermotolerans (Chlorella sorokiniana), Chlorella species, Chlorella globosa (Chlorella sphaerica), Chlorella stetoglosa (Chlorella stigmatophora), Chlorella vannieri (Chlorella vannielii), Chlorella vulgaris (Chlorella vularia vulgaris), Chlorella vulgaris var vulgaris (Chlorella vularia vulgaris var. vulgaris), Chlorella vulgaris var. vulgaris (Chlorella vulgaris var. vulgaris), Chlorella vulgaris var. vulgaris (Varioviridis), Chlorella vulgaris (Chlorella vulgaris ), Chlorella vulgaris var. vulgaris (Chlorella vulgaris), Chlorella vulgaris (Chlorella vulgaris, Chlorella viridans), Chlorella vulgaris (Chlorella viridula vulgaris, Chlorella viridula vulgaris (Chlorella vulgaris) Chlorella (Chloromonium), Crypthecodinium sp (Chromomonas sp.), Chlorococcus species (Chrysosporia sp.), Coccomydia species (Cricosphaera sp.), Cryptococcus species (Cryptococcus sp.), Cryptococcus sp., Cycotina (Cycothecodinium cohnii), Cryptomonas species (Cryptomonas sp.), Cyclotella sp., Cycotina (Cycotina crystica), Cyclotella (Cycotina) mermaniana (Cycotina), Cyclotella sp., Dunaliella bardawil (Dunaliella viridis), Dunaliella biova (Dunaliella viridis), Dunaliella viridis (Dunaliella viridis), Dunaliella viridis, Dunaliella, Cryptoalgae (Dunaliella viridis, algae, Cryptoalgae (Dunaliella viridis, Cryptoalgae (Dunaliella viridis, Cryptoalgae (Dunaliella viridis, Cryptoalgae (Cryptoalgae, Cryptococcus algae, Cryptococcus algae, Cryptococcus algae, Cryptoalgae, Cryptococcus algae, Cryptococcus algae, algae, Monococcus species (Eremosphaera sp.), Elliparis species (Ellipsiodon sp.), Euglena (Euglena), Volvia species (France sp.), Clostridia (Fragilaria cromonensis), Fragilops species (Fragilaria sp.), Gleococcus species (Gleoocapsa sp.), Gloenopsis species (Gloeocapsa sp.), Gloenopsis species (Gloethomonas sp.), Hymenospora species (Gloenoprion sp.), Hymenophora species (Hymenomonnas sp.), Rhodococcus pluvialis (Hautococcus pluvialis), Rhodococcus species (Hannococcus sp.), Isochrysis chrysosporium (Isochrysis galbana), Isochrysis globulus (Isochrysis galbana), Isochrysis galbana (Monochrysosporium sp.), Microchrysosporium sp (Monocauliflora sp.), Microchrysosporium sp), Microchrysosporium species (Micrococcus species (Microcaulon sp.), Microcystis (Microcauliflora), Microcystis sp.), Microchrysosporium (Microcystis sp), Microchrysosporium sp Navicula pisifera (Navicula bisskantrae), pseudonavicula pisifera (Navicula pseudochinensis), Navicula pisifera (Navicula saprophylla), Navicula pisifera (Navicula saprophylla), Navicula pisifera (Navicula sp.), Verbenaria pisifera, Phyllostachys nigra pisifera, Phyllonita pisifera, pisifera, pisifera, pisifera pis, Fucus vesiculosus (Ocystis parva), follicle algae (Ocystis pusilla), Oocystis species (Ocystis sp.), Oscillatoria lacustris (Oscilatoria limnetica), Oscillatoria species (Oscilatoria sp.), Oscillatoria subulata (Oscilatoria subsbergrevis), Chlorella Kelvinea (Parachlorella kesleri), Pachyrhinella Acidophila (Pasperita acidophilus), Parathrina species (Pavlova sp.), phage species (Phagus), Phormidium species (Phormidium sp.), Platyphus species (Platyphus sp.), Particillus karyococcus catarrhalis (Pleuromyces carotovora), Coccocus (Phormidis sp.), Phormidis species (Platyphus sp.), Particillium sp), Coccocus carinatus (Pleurophycus carotovora), Coccocus (Phosphaera), Coccidium sp), Phormidium sp, Moricoccus sp, Coccus sp (Protovorax sp), Phormidis sp, Phormidium sp, Morinum (Proteus sp), Morus (Proteus sp), Morinum, Morus sp), Morus (Phormidis), Morus sp), Morus (Phormidium sp), Morus (Phormidis (Phormidium sp), Morus (Phormidium sp), Morus (Phormidis), Morus (Phormidium sp), Morus (Phormidis), Morus (Phormidium sp), Morus (Phormidis), Morus (Phormidium sp), Morus (Phoenium (Phormidium sp), Morus (Phoenium), Morus (Phormidium sp), Morus (Phoenium (Phormidium sp), Morus (Phormidium sp), Morus (Phoenium), Morus (Phormidium sp), Morus (Phoenium (Morus (Phoenium), Morus, Chrysophyceae capsulata (Sarcochytophycete), Scenedesmus armatus (Scenedesmus armatus), Schizochytrium (Schizochytrium), Gossypium (Spirogyra), Spirulina (Spirolinia plastenis), Schizochytrium species (Stichococcus sp.), Synechococcus species (Synechococcus sp.), Tetragonium species (Tetraedron), Tetraselmis sp., Thalassimus wessoensis (Thalassisi), and Microcystis frena (Viridulariaceae), Euglenophyceae, Chlorophyceae (Prasinophyceae), Rhodophyceae (Phaeophyceae), Porphyceae (Porphyceae), Porphyceae (Porphyceae), and/or (Porphyceae), Porphyceae (Porphyceae), Porphyceae (Porphyceae), and/or (Porphyceae), Porphyceae (Porphyceae), and/or (Porphyceae), Porphyceae (Porphyra), Porphyra (Porphyra), Porphyra (Porphyra), Porphyra (Porphyra), Porphyra (Porphyra), Porphyra (Porphyra), Rhodophyra (Porphyra), Rhodophyra (Phaeophyra (Porphyra), Porphyra (Phaeophyra (Porphyra), Rhodophyra (Porphyra), Porphyra (Phaeophyra (Porphyra), Rhodophyra (Phaeophyra), Porphyra), schizotrium species, Crypthecodinium sp, Phaeodactylum species (Phaeodactylum sp), and Scutellaria species (Odonella sp.), Graptospira longata (Odonella aurantiaca), Botryococcus species, Stachys botrytis, Botryococcus braunii, Chlamydomonas sp, Chlamydomonas coccinea (Chlamydomonas sp), Chlamydomonas coccinea (Chlamydomonas unicaudata), Chlamydomonas aethiopica (Chlamydomonas ehrenbergii), Chlamydomonas euthana (Chlamydomonas monergensis), Chlamydomonas Chlamydomonas, Chlamydomonas maxima (Chlamydomonas moascus), Chlamydomonas auromonas arctii, Chlamydomonas ovansis, Chlamydomonas maxima (Chlamydomonas oculatum), Chlamydomonas nivais, Chlamydomonas nigella, Chlamydomonas nivais, Chlamydomonas nigella, Chlamydomonas nivais, Chlamydomonas nikola, Chlam, Chlamydomonas nikola, Chlamydus nikola, Chlamydomonas nikola, Chlamydorum nikola, Chlamydus nikola, Chlam, Chlamydomonas nikola, Chlamydorum nikola, Chlamydorum nikola, Chlamydus, Chlamydorum nikola, Chlamys, Chlamydorum nikola, Chlamys, Chlamydorum nikola, Chlamydorum niko. In some embodiments, such organisms may be one or more of the following: rhodococcus species, Haematococcus pluvialis, Chlorella species, autotrophic Chlorella (Chlorella autotrophica), Chlorella vulgaris, Scenedesmus species (Scenedesmus sp.), Synechococcus species (Synechococcus sp.), Synechococcus elongatus (Synechococcus elongatus), Synechocystis species (Synechocystis sp.), Arthrospira species (Arthrospira sp.), Arthrospira species (Arthrospira sp.), Geotrichum species (Geithromyces sp.), Lythrospira species (Spirothrix sp.), Lyophyllum species (Geithromyces sp.), Lyophyllum species (Lyophyllum sp.), Chromococcus species (Micrococcus sp.), Micrococcus sp), Micrococcus species (Geithromyces sp.), Micrococcus sp., Micrococcus, Nostoc sp, Nannochloropsis sp, Colletoceros sp, Phaeodactylum tricornutum, Dunaliella salina, Arthrospira platensis, Nannochloropsis sp, and Synechococcus ocellatus sp. In particular, Chlorella (Prototheca), Chlorella, Pseudochlorella, Chlorella (Pseudochlorella), Scenedesmus, species of the genus amateus (Amphora sp.), Chlorella aureoviridis (Chlorella aureoviridis), Chlorella vulgaris, species of the genus Dunaliella, Dunaliella baillonii (Dunaliella bardawil), Dunaliella salina, euglena, haematococcus pluvialis, species of the genus rhodococcus erythropolis, nannochloropsis salina, species of the genus micrococcus, species of the genus trichomonas vulgaris, species of the genus gadiformis, Scenedesmus sp, Schizochytrium (Schizochytrium), gossypium aquaticum (Spirogyra), Spirulina platensis (Spirulina platensis), species of the genus schizochytrirnula, Chlorella sp, Chlamydomonas sp, chlamydia.

Diatom species can include cold rhombohedral (n.frigida), krylon rhombohedral (Nitzschia berguensis), Nitzschia gongylus (n.lacuum) and in particular phaeodactylum species, phaeodactylum tricornutum, dactylum species and cerinus microcystis (Cyclotella meneghiniana) as well as diatoms like diatoms (Bacillariophyceae), synechophyceae (cospinodicoccaceae) and navicula (Naviculales).

Rotifers, a group of microscopic and near microscopic animals, may also be used.

Capnophilic bacteria are also contemplated for use. These microorganisms thrive in the presence of high concentrations of carbon dioxide and can be used particularly in applications where high carbon dioxide sequestration is desired.

Extreme microorganisms refer to many groups of organisms capable of withstanding the extreme conditions unusual in environments, typically high or low temperatures, extreme pH, salinity, dryness and/or radiation levels. Particularly contemplated examples that may be used in the apparatus and method according to the present invention include the following members of the order: red algae in the warm spring (Cyanidiales), the galenical family (Galdieiaceae), the species of the genus Primulina (Cyanidioschyzon sp.), the class Rhodophyceae in the warm spring (Cyanidiophyceae), the species of the genus Rhodophyceae in the warm spring (Galdieria sp.), the unicellular species Primula (Cyanidioschyzon merolae) DBV201, the daedalium cyanide (cyanium dadium), the largest cyanide alga Cyanidium maxium maximum, the Particidium cyanide (Cyanidium partitum), the bursting cyanide (Cyanium rumpenensis), the daedala red algae (Galdiia daedala), the largest hot spring red algae (Galdiia maxilla), the Viola red algae (Galdiia partita) and especially the species Rhodophydium sulfate (Galdiia suvialis), the hot spring red algae (Galdii suvialis) and the original red algae (Galdii) are.

Plant species, in particular aquatic plant species including green algae, may be cultivated in the apparatus and method according to the invention. The whole plant organism can be used under appropriate circumstances. Suitable classes can include members of: the species Lemnaceae (duckweed), Araceae (Araceae), Wolff duckweed (spoteless watermeil), Arthrona minor (rootless duckweed), Lemnaceae (Lemnaceae), Lemna thalli (Lemna thalli), Lemna trisulca (Lemna trisulca), Spirulina species, Arthrobacter species, Lemna minor (Lemna minor), Lemna minor (Lemna aenoquinalis), Valdilla minor (Lemna valdiiana), European Brussia minor (Lemna obscura), Spirodela polyrhiza (Spirodella polyrhiza), Wolfia minor (Wolffa), species of Wolflamna, and species of Spirodela. In particular, lemnaceae, wolffia europaea and wolffia species are envisaged.

Plankton is a general term for marine micro-animals and microbial communities. Examples for use in the present invention include coccolithosiphon, dinoflagellate, metaplankton and protplankton, and in particular the genus coccolithosiphon such as marine coccolithosiphon.

Amoebae refers to various cells or single-cell biological groups capable of changing their shape by the extension of pseudopodia. Examples of such organisms for use in the present invention include varroa carinata (Chaos carinatus), zoomorpha zoonotic (Chaos Diffluens), zoomorpha species (Chaos sp.), gongylus species (Naegleria sp), formosan gomorpha (Naegleria fowleri), Entamoeba species (Entamoeba sp.), filariasis amoeboides (Cercozoan amoeboids), foscarnea species (euglyha sp.), long squamosa (euglyha rotiana), and podophyllum species (Gromia sp.), round spongiopsis (gromura sphaerica), foramen species (foramiria sp.), poly-mularia (massternesia), massonian (massierella sp.), massierella sp), plasmodium sp, and plasmodium fragrans (plasmodium fragrans).

In addition, the present invention may be used to culture cells from multicellular organisms. In particular, animal cells from animals such as domestic animals including chickens, ducks, turkeys and poultry that are capable of growing in the apparatus and method according to the present invention; fish, bovine or porcine cells, hunting or aquatic animal species and insect cells include muscle cells, adipocytes, epithelial cells, myoblasts, satellite cells, side population cells, muscle-derived stem cells, mesenchymal stem cells, myogenic pericytes or angioblasts. A myogenic cell as used herein refers to a cell from an embryonic stem cell line, induced pluripotent stem cell line, extra embryonic cell line or somatic cell modified to express one or more myogenic transcription factors. In particular, muscle cells or similar cells may be grown for use in the production of so-called laboratory grown meat for nutrition of humans or other animals. Totipotent cells from human embryonic cells and human embryos were excluded.

Some organisms, whether native strains or genetically modified or engineered strains, can have the ability to ingest air pollutants such as: NO₂(and such as NO, N)₂O₂、N₂O₃、N₂O₅Other NOx), SO₂(and e.g. S)₂O₂、SO、SO₃Other SOx), VOC, NH₃Or such as N₂CO removal of O₂Other "greenhouse" gases. If so, these gases can be pumped in the gas chamber to then be transferred in the liquid medium. These gases may also come from exhaust gases.

In this regard, sulfur-bearing organisms can also be grown in the apparatus. These organisms perform sulphur oxidation to produce energy. Some inorganic forms of reduced sulfur, mainly sulfides (H)₂S/HS^-) And elemental sulfur (S)₈) Capable of being oxidized by chemically non-trophic sulfur oxidizing prokaryotes, usually with oxygen (O)₂) Or Nitrate (NO)₃ ^-) Reductive coupling of (2). Most of these sulfur oxidizing agents are capable of using reduced sulfur species as a source for carbon dioxide (CO)₂) An immobilized electron donor. The bioenergy is used in the chamber to contain CO₂And another sulfur-containing gas to deliver the desired sulfur species into the liquid medium, particularly where the membrane is permeable to such gas. Or the sulfur-containing molecules can be added directly to the liquid medium in gaseous or liquid (water) form via a nozzle. Forms of sulfur that can be used in the chamber (or by direct addition) include H ₂S or using a catalyst such as NaHS or Na₂H of S₂An S donor compound. The related organisms include Thiobacillaceae (Beggiatoaceae), Thiobacillaceae (Thiobacillaceae), sulfolobules (sulfolobus order), sulfolobus (sulfolobus) genus, Acidienus (Acidianus) genus, Vibrio kluyveri (Hydrogenovirino crurogens) and Desulobulbaceae (Desulfobulbocaceae). Relatedly, some anaerobic sulfur-oxidizing organisms may be photoautotrophic organisms that derive energy from sunlight but use reduced sulfurationThe compound replaces water as an electron donor for photosynthesis.

In some embodiments, the organisms of the bioreactor are genetically modified to possess a specific trigger that is activated by exposure to a gas or vaporized stimulant that can be delivered to the atmosphere included within the chamber. When this stimulant is introduced into the chamber, it diffuses across the membrane of the bioreactor and is delivered into the liquid medium. The stimulant acts as a trigger and induces the organism to respond in a predetermined manner as intended by genetic intervention. For example, a stimulant may induce production or stop production of a particular metabolite and/or may alter the rate of production of a particular metabolite.

With respect to providing rich O in the chamber₂And/or depletion of CO₂The above description of the atmosphere of (a) applies to all other suitable gases, the control of which can be used for various purposes.

A gas can be introduced into the chamber to control the pH of the liquid medium included within the bioreactor. According to a particular embodiment of the invention, the CO in the atmosphere₂And/or ammonia (NH)₃) Can be used to control the pH of the liquid medium.

As described above, living organisms can be modified (or have natural abilities) to respond to the presence or absence of certain gases by altering their physiological processes, and can control the mixture of gases supplied to the atmosphere included within the chamber to provide or remove such gases.

Atmosphere of the chamber

The composition and/or amount of the gas mixture supplied to the device may be controlled and adjusted in response to changes in one or more parameters measured within the liquid culture medium within the bioreactor, and/or in response to metabolic or other physiological states of the cells included within the bioreactor. For example, a change in a parameter including a change in pH in a liquid medium may result in the provision of a gas (e.g., CO) that affects pH₂) The supply of (2). Alternatively, low O is detected in the liquid medium ₂The concentration may result in supplying an increased level of O in the input gas₂. Can be used forTo monitor the state of the liquid medium and/or the cells by means of an auxiliary system of the control device (see below).

The input gas may need to be pre-treated before it is delivered to the gas chamber, for example to remove substances that may be toxic to the cells or that may affect the cleanliness or transparency of the bioreactor or chamber surfaces. The pretreatment of the chamber gas feed may include any suitable technique or strategy, such as High Efficiency Particulate Air (HEPA) filters and/or activated carbon filters, and is operable to remove specific air pollutants, Volatile Organic Compounds (VOCs), various grades (e.g., PM1, PM2,5, PM10) of particulate matter, soot, and any other undesirable or toxic substances.

According to a particular embodiment of the invention, the feed gas can be delivered in the chamber in a direction opposite to the general direction of flow of the liquid culture medium in the bioreactor. In this way a counter-current arrangement can be established, wherein (due to the consumption of O occurring during the flow of the liquid culture medium through the bioreactor system)₂Process) can be made to have the highest O ₂Concentration of feed gas with lowest dissolved O₂Liquid medium contact at a concentration and likewise with minimal CO₂Gas contact with highest concentration of dissolved CO₂Liquid medium of concentration. This increases the concentration difference of the gas and therefore improves the gas transfer efficiency. In another embodiment, the CO consumption occurs (due to the liquid culture medium flowing through the bioreactor system)₂Process) can be made to have the highest CO₂Concentration of feed gas with lowest dissolved CO₂Liquid medium contact of concentration and likewise with minimum O₂Gas contact of concentration having highest dissolved O₂Liquid medium of concentration.

Support structure and auxiliary system

The apparatus can include a support structure including frames, scaffolding and/or manifolds for elevating and/or supporting bioreactors within the chamber-and supporting arrays of bioreactors included within a chamber or chambers within the apparatus. The support structure may also or alternatively maintain the shape and structure of the chamber itself, and/or in directing the flow of the gas atmosphere around the bioreactor included within the chamber. Additionally or alternatively, the support structure may further assist in attaching the device to a mount or other surface, and assist in providing stability of the device as a whole.

In certain embodiments of the invention, the support structure can comprise an extrusion of a rigid solid material, and is preferably lightweight, as described in the exemplary apparatus below. The support structure need not be transparent, even in embodiments where part or all of the chamber walls are transparent, but it can be and can be made of any suitable material, typically a strong, light and non-toxic material, with high resistance to oxidation, corrosion, extreme temperatures and ultraviolet radiation. The support structure can comprise a substantially solid material, or can comprise a porous structure to reduce its weight while maintaining strength.

In particular, it is envisaged that the support structure may be used to support the bioreactor itself in order to help them bear the weight of the liquid culture medium and the cells contained within them. Particularly towards the middle of a portion of the bioreactor, the weight of the contents may cause the material comprising the bioreactor to sag, stretch or weaken. In addition, clogging or excessive pressure of the liquid culture medium within the bioreactor may cause ballooning, which may result in costly and inconvenient damage or rupture of the membrane comprising the bioreactor. Thus, one or more bioreactor support structures or support assemblies contacting the underside of the bioreactor may be used.

Such a bioreactor support structure may comprise fins, slots or brackets in which the bioreactor is located, which may be protrusions of the lower inner wall of the chamber and/or any other inner wall. The bioreactor support structure may be a net or a series of ropes, strings or cables attached to the side inner walls of the chamber and/or to any other inner wall of the chamber. The bioreactor support structure may advantageously be discontinuous, i.e. comprise gaps, to enable gas from the chamber atmosphere to contact the membrane of the bioreactor. Suitably, the bioreactor support structure may be flexible, or generally a rigid or semi-rigid mesh having a plurality of perforations or holes capable of supporting the bioreactor while still allowing gas access to the membrane of the bioreactor for effective gas exchange, even if it contacts the support structure. Indeed, it is envisaged that in some arrangements the bioreactor support structure may contact not only the underside of the bioreactor, but also the sides and top. This may also help to prevent bulging (radial expansion) of the bioreactor, thereby protecting against bursting. In some embodiments, the bioreactor support structure comprises a flexible, semi-rigid, or rigid mesh substantially surrounding a cross-sectional circumference of at least a portion of the bioreactor. In other embodiments, the mesh surrounds the entire cross-sectional circumference of the bioreactor to prevent bulging (radial expansion) of the bioreactor to protect against rupture, and to control the cross-sectional shape of the bioreactor (e.g., control the diameter when the bioreactor is in a tubular shape). The mesh may enclose all or a portion of the elongated bioreactor. The density of pores or pore sizes within the mesh may vary depending on the location and need for support. For example, the mesh around the underside of the bioreactor may have smaller, fewer and/or more widely spaced holes to provide more support, whereas the mesh around the top of the bioreactor may have larger, more and/or more closely spaced holes to facilitate gas access to the bioreactor. The mesh can be made in any suitable manner, and it can be made of connected strands, strings, wires or cables; it may be made of perforated or otherwise perforated sheet material, or of woven or knitted fabric. The mesh may be of any suitable material, for example a plastics polymer, typically a plastics polymer containing a UV stabiliser. The mesh may have any suitable thickness, it may be no less than 0.1mm and no more than 3mm thick, typically 1mm thick or less. The apertures of the mesh may be of any shape and size, they may be no less than 0.1mm and no more than 10cm wide, suitably no more than 10mm, no more than 5mm, typically no more than 3 mm.

These supports may also advantageously allow the bioreactor to be suspended above the lower inner wall of the chamber, which can allow gas from the chamber atmosphere to enter portions of the bioreactor membrane other than the top exposed portions, and can also allow a vertical arrangement (or "stack") of multiple bioreactors to be arranged in the same chamber. Suitably, the support assembly may be arranged as a series of shelves or armatures arranged to support a three-dimensional array of bioreactors. The shelves, which may be any of the support structures discussed, can be arranged in horizontal and/or vertical, parallel and/or anti-parallel arrays.

Support structures may also be present on the interior of the bioreactor to provide, support or maintain the shape of the bioreactor, or may be included within the membrane of the bioreactor itself. In particular, the membrane may be a composite material that includes internal membranes, meshes, ribs, or other structures to help the bioreactor maintain shape and strength while preserving sufficient gas permeability. Such composites can be produced using coextrusion manufacturing techniques.

Suitably, the support structure can comprise: plastics, such as bioplastics, thermoplastics, thermoset polymers, amorphous plastics, crystalline plastics; synthetic polymers such as acrylics, polycarbonates, polyesters, polyurethanes, carbon fiber composites, kevlar composites, carbon fibers and kevlar composites, or glass fibers; a metal or metal alloy such as steel, mild steel, stainless steel, aluminum or titanium; natural materials such as wood or coated wood; or carbon-based materials such as graphene, carbon nanotubes, or graphite.

The bioreactor of the device may be connected to an auxiliary system that controls the supply and conditions of the gas and/or liquid culture medium used. Depending on the application of the device, the auxiliary system may be of any degree of complexity and consist of any kind of auxiliary components.

In a suitable embodiment of the invention, the device is connected to an auxiliary system consisting essentially of conduits for gas and for liquid medium, a water tank, a gas or metal tank, pumps for gas and liquid medium, valves, a biomass separator, an artificial lighting system (especially in the absence of natural light), a water temperature control system, sensors and a computer. One, more or all of the components of the auxiliary system can be arranged inside or outside the chamber. The different features of the auxiliary system need not all be included together but may be dispersed in different parts of the system as a whole. For example, biomass separators, gas outlets and/or inlets for nutrients may be included in the connectors between the individual bioreactors.

The conduit and reservoir (water tank) may be of any type and of any suitable material.

The pump may also be of any type; usually the liquid pump is a peristaltic pump which is able to reduce the risk of contamination of the liquid medium and the rupture of the cells used, since the peristaltic tube is the only component in contact with the liquid medium. In some embodiments, a diaphragm pump (also referred to as a diaphragm pump) can be used. The patch pump creates relatively little friction with the liquid medium and may therefore be advantageous in reducing the risk of cell rupture and contamination. In some other embodiments, progressive cavity pumps, screw pumps and gear pumps can be used. The screw pump generates relatively little friction with the liquid medium and therefore may have advantages in reducing cell disruption while being able to pump liquid at high flow rates.

The biomass separator may be of any type known to the skilled person; suitably the biomass separator is a centrifugal bio-separator, a filtration system comprising small pore size mesh, sieve and/or micro/nano filtration device and/or settling device and/or clarification process. Multiple biomass separation devices can be installed in series, such as an initial clarification process or a micron filtration device followed by a centrifuge.

The liquid medium temperature control may be of any type known to the skilled person; generally, the liquid medium temperature is controlled by controlling the temperature of the gas atmosphere within the chamber. The temperature of the gaseous atmosphere within the chamber can be heated and/or cooled by any suitable means; typically, it is cooled by an air conditioning unit within the chamber or connected to the chamber through an inlet and an outlet. In other embodiments, the liquid culture medium temperature control includes heating or cooling components that may be suitably mounted around or within portions of the conduit, around portions of the bioreactor, before the gas inlet of the chamber, and/or around or within the reservoir. The transmission of infrared light to a transparent or translucent conduit may also be a means of heating the liquid medium. The heating means may be of any type and suitably can comprise a heat exchange mechanism. Excess heat from the liquid medium generated by physiological processes or high ambient temperatures can be used to heat water for domestic or industrial purposes, or water from sources such as drainage, rain water, sewage, and/or grey water can be used to remove excess heat. Likewise, heat or cold generated from domestic or industrial sources can be used to heat or cool the liquid medium as necessary. In some embodiments, the heat may be generated by an electric heater that converts electric current into heat. In some other embodiments, the heating and/or cooling component may be any suitable type of heat exchange device, such as a liquid-to-gas heat exchanger, a two-liquid heat exchanger, a two-gas heat exchanger, an air conditioning unit (AC), a dual tube heat exchanger, or a plate heat exchanger. The air conditioning of the atmosphere within the chamber is carried out within the chamber or in the location of the auxiliary system before the gas mixture arrives in the chamber. The heat exchange between the two liquids is suitably carried out in the place of an auxiliary system before the liquid culture medium reaches the bioreactor.

Artificial lighting systems of any kind including any artificial light source known to the skilled person can be used, suitably the lighting system comprises LEDs, typically the artificial light source is designed and/or controlled to emit electromagnetic radiation (light) of a specific wavelength corresponding to the Photosynthetic Active Radiation (PAR) requirements of any photosynthetic microorganism contained in the device and/or to promote a specific biological activity, thereby increasing the production of specific products in the biomass, e.g. by using LEDs emitting specific wavelengths. For example, LED-based light sources are capable of emitting wavelengths between about 620nm and 750nm (red light) to facilitate the production of some organisms of pigments that primarily absorb red light, such as the pigment phycocyanin. The artificial lighting system may be comprised within a support structure comprising an array or strip of LEDs or optical fibers. The intensity and quality of the light emitted by the lighting system can be automatically controlled (following input from any kind of sensor, e.g. PAR sensor, humidity sensor, temperature sensor, chemical sensor, pH sensor, etc.) to facilitate specific microbial physiological activities and/or to respond to environmental changes and/or to increase or modify biomass production. The amount of light transmission (natural or artificial) through the "switchable" or "smart glass" material as discussed above can similarly be automatically controlled for similar reasons.

In some embodiments, the artificial lighting system may provide a wavelength of light that can be used to sterilize or disinfect some or all of the bioreactors and/or chambers of the present invention. This can serve as or supplement a cleaning, disinfecting or sterilizing process as discussed below. In particular, such lighting systems may generate Ultraviolet (UV) radiation that can kill or damage bacteria and other unwanted contaminant organisms. Suitably, the UV radiation is short wavelength UV, sometimes referred to as UVC. The UV radiation source in such systems may typically be a UV lamp, suitably a UV generating LED. The wavelength of the UV radiation may include wavelengths between 260nm and 270 nm. Suitably, wavelengths below about 254nm may be excluded or blocked to reduce ozone generation. In some applications, ozone generation may be desirable to obtain additional disinfecting properties thereof, and the wavelength of the UV radiation may be selected to encourage this.

Since UV radiation may be harmful to humans, in particular to the skin and eyes, such UV disinfection systems can suitably be in embodiments where the walls of the chamber are substantially opaque or at least not transparent to the UV wavelengths used. Alternatively, the chamber can be covered or coated with such an opaque or UV-opaque layer prior to activation of the UV disinfection system. Additionally, since UV radiation may age or damage many types of materials, such as several polymers, any delicate material (which may include a bioreactor) may be removed from the chamber prior to activating the UV system, or a system or device may be arranged to protect the delicate material from UV radiation.

According to a particular embodiment of the invention, when the concentration of biomass in the liquid medium comprised in the bioreactor reaches a desired level, the three-way valve directs the flow into a biomass separator that separates at least a portion of the biomass from the liquid medium, the sequestered biomass proceeds to the vessel for additional treatment while the liquid medium is directed back into the reservoir. It may be necessary to regenerate the liquid culture medium before returning it to the bioreactor. In some cases the liquid medium will comprise metabolites produced by the cultured organism; it may be desirable to destroy these metabolites to maintain an optimal growth rate, since in many cases the presence of excess of such metabolites causes a decrease in growth. Such metabolites can be removed using a filtration system, UV treatment, and/or chemical treatment. Alternatively, the filtered liquid culture medium from the biomass separation process can be discarded. This action of directing flow into the biomass separator can be performed periodically and for a predetermined period of time before the valve again changes the flow path into the reservoir. This timing can be optimized with respect to each application, microorganism used, surrounding environment and physical location of the device. In another embodiment instead of a binary switch, the valve can change the pore size of the channel, thereby controlling the flow rate and amount of liquid media delivered to the biomass separation process.

Nutrients can be periodically introduced directly into the reservoir in the system. Water and/or microorganisms can similarly be introduced into the liquid medium or cleaning fluid.

All kinds of other system components can be utilized, as an example a controllable pressure valve or a pressure regulator can be placed in the system, in which case the pressure valve can control the volume change of the device by the influence of a liquid or gas pressure change. Some valves can control the flow rate into the cell.

If desired, make-up air and/or O-enriched air can optionally be introduced into the main bioreactor supply conduit₂And/or air as another gas. VentilationPorts can be installed in the conduits to remove gas that has accidentally entered the hydraulic system, for example during installation of the system, and are typically located in the uppermost position of the system to facilitate the venting of undesirable gases.

Sensors including transparent/translucent conductive materials and/or any other conductive materials can be disposed on any surface of the chamber (either inside or outside the chamber) to monitor conditions such as irradiance levels, temperature, humidity, or other environmental conditions. These or similar sensors, when located inside the chamber, may be used to detect gas concentration levels, humidity, and/or temperature in the chamber.

Embodiments of the invention and/or auxiliary systems can include embedded sensors that can be used, for example, to monitor, for example, CO in a liquid medium and/or atmosphere₂Concentration and/or O₂Chemical concentration of concentration; and/or for monitoring temperature and other environmental and biological parameters such as toxicity levels, and/or for monitoring biomass concentration and/or total cell density and/or viable cell density and/or activity of microorganisms in a liquid culture medium.

The sensor can be fully or partially embedded in the bioreactor or chamber, in a secondary system of tanks or conduits, and/or in a control or support structure and/or attached to the inside or outside of an external layer or on the surface of an internal add-on component.

The sensor can allow monitoring of the environment inside the bioreactor of the device, so as to enable control of parameters including, but not limited to: liquid media flow rate, liquid media quality, nutrient level, temperature, biomass extraction rate, gas mixture, gas flow rate, gas chamber pressure, and illumination intensity (and/or light shielding such as provided by "smart glass"). The purpose of this control is to optimize the metabolic efficiency of the cells contained within the device, and/or to stimulate specific metabolic/microbial activities and thus to optimize the production efficiency of the biomass and/or to modify its composition.

Similarly, the sensors can allow monitoring of the environment inside the chamber of the device in order to enable control of parameters including, but not limited to, gas flow rate, quality, composition, temperature, optical transparency, and humidity.

Cleaning and sterilizing

A cleaning procedure can be initiated to clean and/or sterilize the bioreactor unit and/or the conduits and/or the water tank and/or all auxiliary systems and/or chambers. Cleaning is performed when it is necessary to flush the system, collect all biomass in the system, or for temporary shut down. The "cleaning fluid" can be made of any compound known to the skilled person. It may include hydrogen peroxide, ethanol, water, brine, detergents, bleaches, surfactants, alkalis, it may be CIP100 or CIP150 from Steris or any other suitable cleaning composition. The cleaning fluid can enter the system through a specific conduit (inlet) in any point of the system and can exit at any point of the system (outlet) to allow cleaning only in a specific location as required, rather than cleaning the entire system. Typically, cleaning liquids like CIP100 are heated to a desired temperature, typically exceeding 30 ℃, and maintain turbulence for a determined period of time. The cleaning fluid may also be gaseous in nature and can comprise steam, heated air or water vapour, suitably supplied at a temperature above 120 ℃.

The sterilization procedure aims at destroying and removing any and all organisms within the system to obtain a permanent shut-down, decontamination. The method may include pumping a fluid, such as a vapor of hydrogen peroxide or a low temperature dry vapor, into the system. Sterilization may also include the use of electromagnetic radiation, typically UV radiation, to sterilize any of the components of the present invention as discussed above. An advantage of the dry hydrogen peroxide vapor is that it does not require high pressures for effective sterilization. When it is necessary to pressurize a sterilization fluid, such as steam, for effective sterilization, it may be advisable to first pressurize the chamber atmosphere and subsequently pressurize the interior of the bioreactor in order to avoid damage or bursting of the bioreactor.

In some embodiments (as shown in fig. 18), a series of valves (140, 141, 142), drain outlets (145), and auxiliary inlets (146) may be used during cleaning, sterilization, priming, inoculation, liquid media removal, biomass harvesting, and/or growth media introduction procedures of the system. For example, to replenish contaminated cleaning liquid previously used to clean the bioreactor with fresh sterile solution, the central valve (141) will be closed, the other two valves (140, 142) will be opened and the pump (72) will continue to operate to allow contaminated cleaning liquid to be discharged from the discharge outlet (145) and new fresh sterile solution to be introduced in the system from the auxiliary inlet (146).

Biomass collection

An advantage of some embodiments of the invention is that biomass can be produced continuously within a unit and harvested on a continuous basis.

The biomass that can be collected from some embodiments of the invention varies depending on the setup and conditions of the apparatus of the invention, the cells included within the bioreactor, the desires of the user of the invention, and the nature of the separation and processing of the biomass. General types of biomass that can be collected from the present invention in various embodiments can include, but are not limited to: a metabolite of the cell; secreted proteins and other cellular products; products of photosynthesis, aerobic respiration, and/or anaerobic respiration; cell contents including organelles, cell membranes, cell walls; macromolecules including polysaccharides such as starch and cellulose, fats, phospholipids, proteins, glycoproteins, glycolipids, and/or nucleic acids; carbohydrates such as mono-, di-and/or oligosaccharides; fatty acids and/or glycerol; whole organisms including cells, clumps and/or colonies of single cell organisms or whole multicellular organisms or portions thereof.

Applications of biomass produced by embodiments of the invention can include: a food; feed for animals, plants or any organisms; feeds suitable for aquatic animals or other organisms; a pharmaceutical; a cosmetic; a fuel; a biochemical; an oil; substitutes for mineral oils and mineral oil products; manufacturing oil; and vaccines.

Biomass accumulates in the liquid medium within the bioreactor. Biomass can be harvested directly from the liquid medium. Biomass is produced mainly in liquid culture mediumFormed in the system during the travel of the reactor, as this is where it takes the most time and is supplied with O₂. To release the biomass, liquid medium enters the device via one or more inlets, passes through one or more channels, and exits the device via one or more outlets along with the biomass carried in the flow. The outlet can be connected to a suitable container for receiving the harvested biomass.

A particular advantage of the present invention is the ability to harvest product on a continuous, semi-continuous or batch basis due to the ability to circulate liquid medium continuously through the system. Harvesting may occur, for example, when a particular cell density is reached that can be expressed in grams per liter, such as at least about 1g/l, at least about 2g/l, about 5g/l, about 10g/l, about 20g/l, about 30g/l, about 50g/l, about 75g/l, or at least about 100 g/l. For example, continuous harvesting can be achieved if a percentage of the liquid culture medium that passes through the auxiliary system is continuously harvested after flowing through the bioreactor and the liquid culture medium is added to the system to replace it. Any suitable amount can be harvested depending on the organism being cultured, the volume of the bioreactor system, and the time it takes for the liquid culture medium to flow through the entire system. For example, 100% of the liquid culture medium can be harvested by the auxiliary system, or the harvest can achieve no more than 90%, no more than 70%, 50%, 30%, 20%, 10%, 5%, 1%, or no more than 0.5% of the liquid culture medium as it exits the bioreactor.

Alternatively, biomass can be harvested intermittently on a semi-continuous basis. For example, a certain percentage of biomass can be harvested from the apparatus of the invention on an hourly, daily or weekly basis, frequently. For example, harvesting may be performed weekly, daily, every 12, 6, 4, or 2 hours, or hourly. The harvested volume can be replaced by adding liquid medium (with or without additional organisms) and additional nutrients. Harvesting may be regular, after a set period of time, or can be triggered by reaching a certain biomass density or biomass concentration or a predetermined product concentration. As above, the amount taken can be appropriately changed based on living beings and systems. For example, harvesting during semi-continuous operation can achieve no more than 98%, no more than 95%, 90%, 70%, 50%, 30%, 20%, 10%, 5%, 1%, or no more than 0.5% of the liquid culture medium as it exits the bioreactor.

Such continuous or semi-continuous processes have the benefit of predictable and continuous production of biomass, do not require the introduction of new or additional organisms into the bioreactor after harvesting, and can allow for reduced product variability as compared to batch processes that are more common in the case of standard fermenters. In a fermenter setting, the risk of contamination means that a continuous process is rarely suitable.

However, batch processes can also be used, and will involve harvesting the entire volume of liquid medium at a time after a set time has elapsed or a set density of organisms or biomass or products has been reached. This may involve emptying the entire system and/or flushing it with replacement fluid. This method can be used in conjunction with any continuous or semi-continuous process, for example when it is desired to clean the system or replace the cultured organisms.

In some embodiments (as shown in fig. 18), a series of valves (140, 141, 142), drain outlets (145), and auxiliary inlets (146) may be used during cleaning, sterilization, priming, inoculation, liquid media removal, biomass harvesting, and/or growth media introduction procedures of the system. For example, to replenish the growth medium consumed by the organism and to simultaneously remove liquid medium from the system, the central valve (141) will be closed, the other two valves (140, 142) will be opened and the pump (72) will continue to operate to allow liquid medium to be discharged from the discharge outlet (145) and to allow new liquid medium with growth medium to be introduced into the system from the auxiliary inlet (146).

Applications of

The apparatus of the invention can be used in many applications, primarily biomass production, but also for carbon dioxide production, nitrogen oxide or other gas sequestration, or where contaminant removal is required, or where wastewater treatment is required, or even for aesthetic or decorative applications such as municipal furniture or functional art devices. The device can be used at locations such as warehouses, breweries, industrial buildings, and the like. Similarly, the device can be used in conjunction with vehicles such as boats, airplanes, cars, trucks, and other road vehicles. The device can be used indoors and/or outdoors. In some embodiments, the device of the invention can provide carbon dioxide to a device intended to supply increased carbon dioxide to support the growth of photoautotrophic organisms, for example an air permeable patch bioreactor as described in WO2017/093744 and WO 2018/100400.

Applications suitable for the device of the present invention may be any indoor and/or outdoor construction applications, including but not limited to as part of a building facade, roof, awning, sun blind, window and/or indoor ceiling, indoor wall or indoor floor. The present invention can also provide thermal insulation to these buildings.

Additional suitable applications for the apparatus of the present invention may be intensive biomass production applications including, but not limited to, outdoor intensive biomass production plants, indoor intensive biomass production plants, such as in greenhouses, where natural light sources are primarily used. Biomass can comprise food ingredients and/or additives and/or can be used as a source of protein for human or animal consumption or for plant or other fertilizing purposes. Further suitable applications of the device of the invention can be with infrastructures including, but not limited to: urban infrastructure, freeways, bridges, industrial infrastructure, cooling towers, freeways, underground infrastructure, traffic sound barriers, silos, water towers or hangars.

Drawing (A)

Fig. 1A is a diagram showing a cross-section (see part a of fig. 7 a) of an apparatus (100) according to an embodiment of the invention comprising a linear bioreactor (60) comprising at least one inlet (3) and outlet (4) and at least one outer layer (5, 6) on opposite sides, which is partially or totally permeable to gas, and a liquid medium containing at least one cell (12) comprised within the bioreactor. The bioreactor is surrounded on substantially all sides by an atmosphere (1) defined by its housing within a chamber (50) comprising a wall (2), an inlet (7) and an outlet (8). The chamber (50) and the chamber wall (2) separate the atmosphere (1) from the external atmosphere (9). In some embodiments the chamber further comprises a chamber valve (22) for removing gas from the atmosphere (1). A potential transfer of gas (10) from atmosphere (1) to bioreactor contents (12) and a potential transfer of gas (11) from bioreactor contents to atmosphere (1) are shown.

FIG. 1B is a diagram of a similar apparatus in which the inlet and outlet of the bioreactor are connectors that can be clipped to the bioreactor. The bioreactor is in the shape of a tube. The bioreactor is supplied with liquid culture medium, for example from an auxiliary system, via a pipe system (3 ', 4'). An air inlet (7) introduces atmospheric air which has been cooled or heated and filtered as appropriate. In this arrangement, oxygen is shown as passing into the bioreactor, and carbon dioxide and water vapor are passing out.

Fig. 2 shows a cross-section of an arrangement according to another embodiment of the invention (see part a of fig. 7B), wherein two bioreactors (60) are connected directly in series such that their liquid culture media (12) are in fluid communication and the bioreactors are contained within a single chamber (50). In some embodiments more bioreactors may be connected within a single chamber.

Fig. 3a and 3b show cross-sections of an arrangement according to another embodiment of the invention, wherein two bioreactors (60) are directly connected in series, wherein each bioreactor (60) is contained within a chamber (50). The atmospheres (1) of the chamber (50) are in fluid communication with each other through apertures (23) in the chamber wall (2). The bioreactors may be connected via conduits (24).

Figure 4 shows a cross-section of an arrangement according to another embodiment of the invention in which five pairs of bioreactors are connected in series, with each successive pair arranged to run in an anti-parallel direction to the previous pair. The bioreactors are connected by connectors or conduits (24) which can simply connect one member of a bioreactor pair to the next, or can connect two pairs by using curved connectors or conduits, allowing anti-parallel flow directions to be set. Some or all of these connectors can contain valves (29) that can be automated, and can be, for example, solenoid valves or membrane valves to prevent the flow of liquid media when desired.

Fig. 5 shows a cross-section of an arrangement according to another embodiment of the invention, wherein five pairs of bioreactors (60) are connected in parallel. The piping system that supplies and recovers the liquid culture medium to and from the bioreactor is separated and connected to the end of the bioreactor with a connector. The views shown in fig. 4 and 5 may be cross-sections taken horizontally or vertically, that is to say a plurality of bioreactor pairs can be arranged next to one another in the horizontal plane or on top of one another in the vertical plane, respectively.

Fig. 6A and 6B show perspective views of arrangements of bioreactors that may be used in some embodiments of the present invention. 6A, wherein the bioreactors are arranged in pairs with each successive pair arranged to run in an anti-parallel direction to the previous pair. A plurality of layers is used so that the bioreactor is arranged in a three-dimensional space. In fig. 6B, the flow path is split into 5 parallel streams, which flow into different pairs of bioreactors. However, these flow paths also comprise a plurality of pairs of bioreactors arranged in series, again with each successive pair arranged to run in an anti-parallel direction to the previous pair.

Figure 6C shows another perspective view of a three-dimensional array of bioreactors that can be connected in any suitable manner.

Fig. 6D shows a cross-section of a three-dimensional array of bioreactors (60) included within chamber (50), with horizontal (110) and vertical (111) distances between adjacent bioreactors, the width (112) and height (113) of the bioreactor array, and the distance (114) between the outermost portion of the bioreactor array and the chamber itself, labeled.

Fig. 7A and 7B illustrate planar sections a and B by a representation of an apparatus according to some embodiments of the invention.

Fig. 8A and 8B illustrate additional optional features that may be included in any and all connectors or conduits of a system according to some embodiments of the present invention. Fig. 8A shows that the conduit (24) may have one or more vents (124) that may be used to remove any unwanted gases within the bioreactor system. The vent may also be used to allow gas to enter the bioreactor, for example, during maintenance or during the evacuation of all or part of the system. Fig. 8B shows that the conduit may have one or more inlets (121) for introducing a continuous or intermittent supply of glucose, nutrients and/or any other kind of liquid or gas mixture. The inlet can be supplied via a supply line (123) from a source (122) that can originate outside the chamber (50).

Fig. 9 illustrates a suitable system (70) of one embodiment of the present invention, including any embodiment of one or more bioreactors (60) according to the present invention as described herein within one or more chambers (50). Liquid medium (12) comprising cells in a reservoir (71) is delivered by a pump (72) through an inlet (3) into the bioreactor. One or more bioreactors (60) are enclosed within a chamber (50) which also encloses an atmosphere (1) controlled by the movement of gases through an inlet (7) and an outlet (8). The liquid culture medium is passed through one or more bioreactors, while gas transfer between the liquid culture medium in the bioreactor and the atmosphere (1) occurs through the membrane layer of a unit substantially as shown, for example, in fig. 1A. The liquid leaves the device through outlet (4) and reaches a three-way valve (74) which directs the liquid culture medium back into the reservoir (71), closing the circuit. Sensors (75) in the reservoir (71) measure the values of the culture parameters and send outputs to a computer which then controls the operation of the components of the auxiliary systems such as pumps, valves, artificial light systems (if used), temperature control systems, and biomass separators. The computer also controls the supply of gas to the chamber atmosphere (1) through the inlet (7) and the removal of gas through the outlet (8).

When the biomass concentration in the liquid medium reaches a desired level, the three-way valve (74) directs the flow into a biomass separator system (76) that separates the biomass from portions of the liquid medium, with the separated biomass continuing into a vessel (77) for additional processing while the liquid medium is directed back into the reservoir (71). This action of directing flow into the biomass separator can be performed periodically and for a predetermined period of time before the valve (74) again changes the flow path into the reservoir (71). This timing can be optimized with respect to each application, microorganism used, surrounding environment, and location of the device. Alternatively, the three-way valve (74) can regulate flow to the reservoir (71) and biomass separation system (76) to enable continuous harvesting of biomass while allowing for dynamic control of the amount of biomass removed from the system at a given time. For example, the valve (74) can deliver between 0% and 100% of all liquid media passing through the valve to the biomass separation system (76).

Nutrients can be periodically inserted (78) directly into the reservoir (71) in the system. Water and/or cells or cleaning fluids that can be similarly introduced into the liquid culture medium.

All kinds of other system components can be utilized, as an example a controllable pressure valve or pressure regulator (79) can be placed in the system, in which case the pressure valve can control the volume change of the device by the influence of the liquid pressure change. Some valves (82) are capable of controlling the flow rate into the cell.

In addition to the gas supplied to the chamber, supplementary air and/or air enriched with oxygen and/or other gases can optionally be introduced (81) in the main duct, if desired. Vents can be installed in the conduits to remove gases that may accidentally enter the hydraulic system, for example during installation of the system, and are typically located in the uppermost position of the system to facilitate the venting of undesirable gases.

A cleaning procedure can be initiated to clean and/or sterilize the unit and/or the conduit and/or the water tank and/or all auxiliary systems and/or the gas chamber. The cleaning procedure can be performed by using steam or heated air or water vapor as the cleaning medium. The "cleaning fluid" can be made of any compound known to the skilled person. It may comprise ethanol, water, hydrogen peroxide (H)₂O₂) Saline water, detergent, bleaching agent A surfactant, an alkali or any other suitable cleaning composition. The cleaning liquid can enter the system through a specific conduit in any point of the system and can exit at any point of the system to allow cleaning only in a specific location, as desired, rather than cleaning the entire system.

Fig. 10-13 show that the chamber assembly may comprise a support structure (90), which may comprise a metal structure and/or a plastic structure, e.g. an extruded structure, extending linearly (following the desired bioreactor array) on both sides. The structure may act as a structural support for the membrane bioreactor, in particular the upper and bottom surfaces. The structure may include housing means or fittings (91, 92, 93) to secure and/or hold the upper wall (92) of the chamber and the lower wall (93) of the chamber in place in the bioreactor (91). The ends on the modules can be enclosed by other support structure elements to create an enclosed chamber. Particularly in embodiments comprising an array of multiple chambers, the walls of the structure (see fig. 12) may comprise apertures (95) that enable gas to travel from one chamber portion to another. These structures may directly hold the bioreactor or may be connected to additional bioreactor support structures (96), such as ropes or mesh that hold the bioreactor. Fig. 11 and 13 show a cross-section across a bioreactor and chamber (see e.g. part B of fig. 7 a) and have a plurality of bioreactors positioned side by side, e.g. as seen in fig. 3 or fig. 4.

Fig. 13 illustrates an embodiment of the present invention adapted to prevent the collection of water or other substances on horizontal surfaces of the device such that light interference is reduced. In this figure, the upper wall of the chamber has a rounded convex shape so that water or other substances run off from this surface. The upper wall may be rigid and maintain its convex shape by its own strength, or it may be flexible and maintain its convex shape by inflation, i.e. the pressure inside the chamber above the outside. Another advantage of such embodiments is to encourage condensate on the interior of the upper wall to escape from a location directly above the bioreactor.

Fig. 14a and 14b show an alternative embodiment of a support structure that can hold bioreactors (60) in an array of shelves. In fig. 14a, a three-dimensional array of bioreactors is suspended on a plurality of shelves as shown including support structures (90), with the bioreactor support structures (96) suspending the bioreactors themselves. Fig. 14b shows an alternative embodiment, wherein the array of bioreactors is suspended by a support structure (90) comprising a shelf made of a plurality of racks, again wherein the bioreactors are suspended by bioreactor support structures (96). Fig. 14c shows that the bioreactor support structure (96) may be a retaining mesh (96) that may be perforated to allow gas to contact the bioreactor, and may substantially surround the entire circumference of the bioreactor. Fig. 14d shows a side view of a support structure (90) arranged as a plurality of shelves and supporting a plurality of bioreactors (60) on a bioreactor support structure (96).

Exemplary configurations of the present invention are suitable for growing chlorella in a fully heterotrophic mode for the production of high protein content biomass. In large warehouses of dimensions about 250m by 150m, there are many chambers containing air-filled tunnels constructed of materials shielded from light to have a substantially dark environment inside the chamber. Each chamber is approximately 100m long, 10m wide and 3m high.

Inside each chamber there are a plurality of bioreactor arrays, each bioreactor array comprising a plurality of tubular bioreactors defining a flow circuit. Each array of tubes is mounted on a shelving unit that supports the tubes in a number of vertical horizontal planes. Each shelving unit is approximately 70cm wide, 2.5m high and 90m long. Leaving a gap of about 70cm between each shelving unit to enable maintenance and ventilation. Seven shelves are arranged side by side in each chamber. For ease of maintenance, a space of about 5m is left between the outermost shelf and the chamber wall at each end.

Each tube bioreactor compartment was approximately 30mm in diameter and comprised a 50 μm thick polysiloxane membrane. Each bioreactor tube is about 5m in length and in each array 18 bioreactors are connected in series with a linear connector and then connected to subsequent bioreactors in adjacent rows using a curved connector. Each bioreactor array has 16 adjacent rows of bioreactors. In addition, at the end of each row of bioreactors, connectors are used to connect vertically to the bioreactors in adjacent stacks. There are 28 stacks in each bioreactor array. The arrangement of rows and stacks through each bioreactor array and the flow direction are similar to those shown in fig. 6A. Each bioreactor is surrounded on all sides by a mesh to provide support and maintain structural integrity. The support is further supported by a shelf unit fixed to the place where each tube sits and also comprises a mesh structure to allow the gases of the chamber atmosphere to enter the bioreactor membrane.

At one end of each chamber there is at least one air inlet connected to the filtration system and an impeller to direct external atmospheric air into the chamber, wherein this inlet air is maintained at around 17 ℃. At the opposite end of the tunnel there is an air purifier (outlet). The impeller creates a positive pressure inside the chamber compared to the atmosphere surrounding the chamber, thereby maintaining inflation of the chamber tunnel. The chamber tunnel is also attached to the ceiling of the warehouse in any suitable manner to prevent collapse in the event of an impeller failure.

The bioreactor compartments are connected in series and separated by a connector portion. Some connectors include access ports to allow for the introduction of glucose and other nutrients when necessary. The connector may also comprise a static helical mixer. Vents for removing unwanted gases within the bioreactor itself are located at the highest elevation point in the system and are suitably located on connectors linking bioreactors flowing in different directions.

The auxiliary system is mounted and connected to the bioreactor array and comprises a pump for flowing liquid culture medium through the bioreactor, a reservoir for cleaning the liquid culture medium and means for separating the biomass from the liquid culture medium, for inserting an initial inoculation of the organisms to be cultured, for introducing a cleaning fluid, for introducing a sterilization means and for monitoring the status of the system.

Chlorella is inoculated into a bioreactor system and grown to a cell density of 10-15 g/l. At the end of each growth phase (typically every 12 to 24 hours), between 80% and 90% of the biomass in the system is harvested and the filtrate regenerated and recycled. The harvested biomass is taken to a biomass vessel for further processing.

Related embodiments include a lighting system positioned between each shelving unit to deliver intermittent light and stimulate mixotrophic growth of mixotrophic microorganisms such as chlorella or galdiiria sp. Many eukaryotic microalgae are capable of mixed vegetative growth and can be grown either entirely photosynthetically or entirely heterotrophically or by using a combination of these methods. Chlorella is a notable example.

In another embodiment, no individual chambers are included, but rather the warehouse itself represents a single large chamber. Again, a gas, typically atmospheric air, is introduced into this chamber; suitably after filtration through a HEPA filter. This is especially contemplated where the organism used is completely heterotrophic and light does not induce a phototrophic pattern, or where the organism is an obligatory mixed trophic pattern and the light present in the warehouse is sufficient to achieve growth. Accordingly, a window may be provided to allow light to enter, and in some cases, the chamber may be substantially completely transparent, such as a greenhouse.

Examples

Example 1

An experimental setup was constructed to demonstrate a system according to an embodiment of the present invention. In particular, the apparatus demonstrates that it is capable of growing heterotrophic, chemoheterotrophic and/or mixotrophic organisms (which are contained in liquid culture medium inside a bioreactor of the type described herein) and that controlling the temperature of the gaseous atmosphere of a chamber containing a bioreactor of the type described herein results in control of the temperature of the liquid or gel contained in the bioreactor. This further demonstrates that efficient O generation occurs through the membrane layer of the bioreactor₂And CO₂Gas transfer to enable growth of heterotrophic, chemoheterotrophic and/or mixotrophic organisms in the liquid culture medium contained by the bioreactor. Furthermore, it also shows that the wall thickness of the membrane layer of the bioreactor is connected to the surrounding gas atmosphere via a connectionThe contact enables efficient heat transfer.

The setup is represented by a simplified diagram in fig. 18. This arrangement defines a system according to one embodiment of the invention. Referring to fig. 18, most of the features shown in this diagram are the same as those found in fig. 9. In addition, it is shown: an outlet (143) for extracting liquid culture medium from the apparatus (70) for sampling and analysis thereof or for collecting biomass; a series of elongated bioreactors (60) according to the present invention as described herein, having a tubular shape and end reinforcements (144) near the ends of each bioreactor section; a conduit and connector (24) connecting the bioreactor sections to each other and to the inlet (3) and outlet (4); a series of valves (140, 141, 142), drain outlets (145) and auxiliary inlets (146) for use during cleaning, sterilization, priming and inoculation procedures of the system. For example, to replenish the dirty cleaning liquid previously used to clean the bioreactor with fresh sterile solution, the central valve (141) will be closed, the other two valves (140, 142) will be opened and the pump (72) will continue to operate to allow the dirty cleaning liquid to be drained from the drain outlet (145) and to allow fresh sterile solution to be introduced in the system from the auxiliary inlet (146). Another embodiment is that to replenish the growth medium consumed by the organisms and to simultaneously remove liquid medium from the system, the central valve (141) will be closed, the other two valves (140, 142) will be opened and the pump (72) will continue to operate to allow liquid medium to drain from the drain outlet (145) and to allow new liquid medium with growth medium to be introduced into the system from the auxiliary inlet (146).

The bioreactor was made of 12 sections of patch hose connected in series to each other as shown in fig. 18. Each hose portion is composed of 200 μm thick, oxygen (O)₂) Has a permeability coefficient (ISO 15105-1) equal to about 400 Barler, carbon dioxide (CO)₂) Has a permeability coefficient equal to about 2100 barrer, nitrogen (N)₂) Has a permeability coefficient equal to about 200 barrer, hydrogen (H)₂) Has a permeability coefficient equal to about 550 and water vapor (H)₂O) has a permeability coefficient equal to about 30000 barrers. Each hose portion is shown by a cross-section through the hose in fig. 16BAs with VVB adt-x silicone adhesive folded over on itself and sealed to itself and heat pressed to produce a single membrane layer construction of a continuous flexible tube bioreactor section. Each portion of the membrane hose is completely enveloped by a fine transparent mesh to control the diameter of the hose to about 4.0cm, and it sits on the flat bottom surface of the chamber (50).

The bioreactor was filled to its normal working capacity with liquid medium containing growth medium, glucose and Chlorella vulgaris (UTEX 259). Chlorella vulgaris is known to be a mixed vegetative bacterium that can grow using multiple vegetative modes: growth in the absence of light and in the presence of an organic carbon source like glucose (in other words, chemoheterotrophic growth); or in the presence of light and CO ₂And in the absence of an organic carbon source (in other words, photoautotrophic growth); or in other heterotrophic or phototrophic modes. For this particular case study, chlorella vulgaris was grown in complete darkness during the entire duration of the experiment and in the presence of glucose in the liquid medium. The system is gas tight so that gas exchange between the liquid culture medium in the bioreactor and the atmosphere in the surrounding chamber occurs only through the silicone membrane layer of the bioreactor (60). Gases can be introduced or exhausted from the chamber via valves (7, 8) to control the pressure, humidity and gas mixture of the gas atmosphere in the chamber.

The chamber (50) is constructed from a steel chassis (box) with an open window on the upper surface glazed with a transparent ETFE layer of approximately 200 μm thickness. During the experiment, since the portion of the diaphragm hose was transparent, the opening window was completely covered with the aluminum panel to completely darken the interior of the chamber. The chamber was designed to accommodate some of the sensors used for this case study:

1. two temperature sensors (PT 100 from IFM),

2. humidity sensor (LDH 100 from IFM),

3. pressure transmitter with ceramic measuring element (IFM PA 9028).

The reservoir (71) is designed to accommodate a sensor (75). The sensors (75) used for this case study were:

a pH sensor ("EASYFERM PLUS PHI ARC 120" from Hamilton),

2. a turbidity sensor ("DENCYTEE UNIT 120" from Hamilton),

3. a temperature sensor (IFM TM4431 PT100),

4. pressure transmitter with ceramic measuring element (IFM PA 9026).

By controlling the temperature of the gas atmosphere within the chamber, the liquid medium temperature was maintained at 28 ℃ (with changes kept within + -0.2 ℃ oscillation range using PID control). The air atmosphere inside the chamber is heated to the desired temperature by an air heater device installed inside the chamber, which has to overcome the temperature of the air blown in the chamber (which is 21 ℃) and the temperature of the ambient air outside the chamber (which is also 21 ℃). Liquid media was pumped throughout the system by peristaltic pump "FMP 50" from Boyser. A valve can divert liquid media to an outlet (143) into the vessel for biomass collection and further liquid media sampling when needed.

The experiment was divided into two runs:

during run 1, air is constantly blown in through the inlet (7) in the chamber (50) and then exits from the outlet (8).

During run 2, both the chamber inlet (7) and outlet (8) are closed and the gas atmosphere within the chamber is sealed from other gases outside the chamber for the entire duration of the run.

During run 1, the optical density was seen to rise by approximately 4.8OD over 36 hours, then continued to increase after that; the optical density corresponds to the growth rate of the microbial culture, and it is represented by a solid line in the graph illustrated in fig. 20. In contrast, during run 2, the optical density decreased its rate of increase after 18 hours, it stopped increasing after 31 hours, and it started decreasing after 35 hours (indicated by the dashed line in the graph illustrated in fig. 20). The lower growth rate experienced in run 2 relative to run 1 is believed to be a consequence of the lower rate of oxygen exchange between the atmosphere in the chamber and the liquid culture medium inside the bioreactor. During run 2, the chamber is sealed from the outside atmosphere; thus, no fresh air can replenish the oxygen concentration in the chamber that permeates through the membrane bioreactor into the liquid culture medium and is consumed by the microorganisms.

This experiment shows that the technique works better when the level of oxygen in the chamber is controlled and maintained to a desired concentration in order to maintain a constant osmotic gas flow between the atmosphere in the chamber and the liquid medium in the membrane bioreactor. On the other hand, experiments also show that the technique does not perform well when the chamber is sealed, which replicates a non-membrane bioreactor sealed to any external gas atmosphere, in other words it replicates a non-gas permeable bioreactor (e.g. a gas impermeable tube or container bioreactor).

Furthermore, during the duration of the two runs, the temperature in the liquid medium was successfully maintained under the desired conditions (between 28.0 and 28.2, using PID control), demonstrating that the system was able to successfully control the liquid temperature by controlling the temperature of the gas atmosphere within the chamber. The liquid temperature during the duration of run 1 is shown by the graph illustrated in fig. 21.

Finally, this experiment shows that the technique is also effective for heterotrophic, chemoheterotrophic and/or mixotrophic organisms, which can control the temperature and concentration of certain gases, nutrients and metabolites in the liquid medium by controlling the gas atmosphere in the chamber.

Example 2

An experimental setup was constructed to demonstrate a system according to an embodiment of the present invention. In particular, the device demonstrates that it is capable of growing autotrophic and/or photoautotrophic organisms (which are contained in a liquid culture medium inside a bioreactor of the type described herein) and controlling the temperature of the gaseous atmosphere of a chamber containing a bioreactor of the type described herein (which may also be referred to as a 'photobioreactor' in this particular case) results in the control of the temperature of the liquid or gel contained in the bioreactor. This further demonstrates that efficient CO generation occurs through the membrane layer of the bioreactor ₂And O₂Gas transfer sufficient to enable containment by the bioreactorGrow autotrophic and/or photoautotrophic organisms in the liquid medium. Furthermore, it also shows that the wall thickness of the membrane layer of the bioreactor enables efficient heat transfer by contact with the surrounding gas atmosphere.

The case study setup is represented by the simplified diagram in fig. 19. This arrangement defines a system according to one embodiment of the invention. Referring to fig. 19, most of the features shown in this diagram are the same as those found in fig. 18. In addition, it is shown: a light source (147) shining light onto the bioreactor.

With reference to this experimental set-up, most of the features were identical to those of the experimental set-up used in example 1. The only differences are: LED lighting devices (VYPRx PLUS from fluent) designed to emit electromagnetic radiation (light) of a specific wavelength corresponding to the needs of the microorganisms and mounted over the open window of the chamber; an aluminum panel mounted over an open window of the chamber (50) is removed to allow sufficient light to pass through the window and illuminate the transparent diaphragm hose bioreactor portion inside the chamber.

The bioreactor is filled to its normal working capacity with a liquid medium comprising a growth medium and an artrospira platensis known to be capable of growing in the presence of light and CO only ₂The obligate photoautotrophic microorganism grown under (1). For this particular case study, spirochete was grown on a 16 hour light and 8 hour dark cycle for most of the duration of the experiment, with light intensity from about 50 μmol²The Photosynthetically Active Radiation (PAR) per second is gradually increased to approximately 300. mu. mol.m towards its end²And s. The liquid medium does not contain any organic carbon source. The system is gas tight so that gas exchange between the liquid culture medium in the bioreactor and the atmosphere in the surrounding chamber occurs only through the silicone membrane layer of the bioreactor (60). Gases can be introduced or exhausted from the chamber via valves (7, 8) to control the pressure, humidity and gas mixture of the gas atmosphere in the chamber.

Most of the sensors utilized in this experiment were identical to those used in example 1, with the addition of a PAR sensor (LI-190R from Li-Cor) located above the ETFE open window of the chamber (50).

By controlling the temperature of the gas atmosphere within the chamber, the broth temperature was maintained at 28 ℃ during the light cycle (where PID control was used to keep the variation within the + -0.2 ℃ oscillation range) and at 25 ℃ during the dark cycle (again where PID control maintained the variation of + -0.2 ℃). The air atmosphere inside the chamber is heated to the desired temperature by an air heater device installed inside the chamber, which has to intermittently overcome the temperature of the air blown in the chamber (which is 21 ℃) to control the humidity, and the temperature of the ambient air outside the chamber (which is also 21 ℃). The humidity in the gas chamber is also controlled so as to maintain a humidity of 82% or less by pumping a gas mixture having a lower humidity. Liquid media was pumped throughout the system by peristaltic pump "FMP 50" from Boyser. One valve can divert liquid media to an outlet (143) into the vessel for biomass collection and further liquid media sampling when needed, while another valve (78) enables insertion of newly grown media from the auxiliary tank (71) into the system.

During the experiment, the introduction of the gas containing CO was intermittently carried out in the chamber₂So as to make CO₂Is passed through the membrane bioreactor into a liquid medium to sustain the growth of photoautotrophic microorganisms. CO in the chamber₂The concentration can be maintained as desired at a pH in the liquid medium (between 9.8-9.9 pH).

During the experiment, the optical density was seen to increase by about 11OD within 35 days; the optical density corresponds to the growth rate of the microbial culture inside the bioreactor, and it is represented by a solid line in the graph illustrated in fig. 20.

This experiment shows that the technique is also effective for autotrophic and/or photoautotrophic organisms and that it is possible to control the temperature, pH and concentration of gases, nutrients and metabolites in the liquid medium by controlling the gas atmosphere in the chamber.

Furthermore, during the duration of the two runs, the temperature in the liquid medium was successfully maintained at the desired conditions (about 28.0+ -0.2 during the light cycle and about 25.0+ -0.2 during the dark cycle), demonstrating that the system was able to successfully control the liquid temperature by controlling the temperature of the gas atmosphere within the chamber. The liquid temperature during the 10 days of the experiment is shown by the graph illustrated in fig. 23.

These two experiments (described in examples 1 and 2) demonstrate that the technique is applicable to phototrophic, chemotrophic and mixotrophic organisms.

Although specific embodiments of the invention have been disclosed herein in detail, this has been done by way of example only and for purposes of illustration. The foregoing embodiments are not intended to be limiting with respect to the scope of the following appended claims. The inventors contemplate that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.