WO2013119122A1

WO2013119122A1 - Microbial growth chamber

Info

Publication number: WO2013119122A1
Application number: PCT/NL2013/050079
Authority: WO
Inventors: Martin Hessing; Colin John Ingham
Original assignee: The Microdish Company B.V.
Priority date: 2012-02-10
Filing date: 2013-02-11
Publication date: 2013-08-15
Also published as: WO2013119104A1

Abstract

Described is a device (A) for submersing in a liquid medium, comprising a first section of porous material (20) and a second section of porous material (30) and a first chamber (40), defining a first chamber space (41) within the first chamber (40) and at least one outer space (421, 422) outside the first chamber (40), the first section of porous material (20) having an outer surface (201) being in contact with outer space (421), and an inner surface (202) being in contact with the first chamber space (41), allowing liquid communication between the first chamber space (41) and the outer space (421) through the said first section of porous material (20), the second section of porous material (30) having at least an outer surface (301) being in contact with outer space (422), the second section of porous material (30) being free of contact with the first chamber space (41), as well as a method for assessment of the effect of medium on microbial growth using the said device.

Description

MICROBIAL GROWTH CHAMBER

The present invention relates to a device for submersing in a liquid medium, comprising a first section of porous material and a second section of porous material and a first chamber, defining a first chamber space within the first chamber and at least one outer space outside the first chamber, the first section of porous material having an outer surface being in contact with the outer space, and an inner surface being in contact with the first chamber space, allowing liquid communication between the first chamber space and the outer space through the said first section of porous material, the second section of porous material having at least an outer surface being in contact with outer space, and to a method for assessment of the effect of medium on microbial growth using the said device. Herein, the term 'microbes' is deemed to encompass any microorganism, such as bacteria, viruses and bacteriophages, yeast, fungi, archaea, protists, algae, phyto and zooplankton, planarian etc. Furthermore, the term microbes is also deemed to include genetically modified and otherwise constructed self-replicating entities designed to the basic properties of microbes from the environment. The term 'microbial' is to be understood accordingly.

Such devices are known in the art, e.g. from US7, 01 1 ,957, disclosing a growth chamber, comprising an O-ring, whereto on both sides a porous membrane is attached, therewith forming an incubation chamber in the O-ring. Such chambers are used to allow microbial growth in the natural environment of the microbes. Thereto, a membrane is attached to one side of the O-ring, and the space within the ring is filled with a liquid microbial sample, which sample may also contain added agar. Once filled, the second membrane is attached to the other side of the O-ring and the thus closed chamber is then incubated in the natural environment of the microbes in the sample. Both membranes are identical and have a pore size of 0.03um, therewith being impermeable for microbes, but permeable for at least non-polymeric compounds. Such a chamber is used to grow microbes under natural conditions, which is necessary for microbes that cannot be grown under laboratory conditions. Nutrients, present in the said natural environment can enter the chamber and the microbes present therein can grow. After a certain incubation time, the chamber is taken from the natural liquid habitat and analysed in the laboratory. Similar devices were described by Gavrish et al. , (J. Micr. Meth. 72 (2008) 257-262), where one of the membranes had a pore size of 0.2-0.6 urn, i.e. permeable for both nutrients and microbes, whereas the other membrane having a pore size of 0.03 urn was impermeable for microbes. Such devices were used as microbial traps. To this end, the devices were laid with the large porosity membrane side downward in contact with soil. Micro-organisms could enter the chamber from the soil, and could be further analysed after a certain incubation time at natural conditions.

The devices of the art have however several disadvantages. Importantly, in case a microbe is to be entrapped in the chamber, such as described in US7, 01 1 ,957, the microbial sample has to be taken from the natural environment, and the growth chamber has to be loaded with the sample away from the said natural environments in order to get the sample in the chamber and to seal the said chamber. This may cause stress or death to at least some of the microbes in the sample, so that the sample does not reflect the situation as it was in the natural environment. Further, it is not possible to use such a chamber for microbes, that cannot be sampled, e.g. from difficultly accessible habitats, such as the deep sea. Also, the known devices are not suitable to assess the influence of the natural environment on the entrapped sample, or vice versa, as a proper control is missing, or must be done in a separate chamber, which impairs the reliability of the assessment.

The present invention intends to overcome one or more of the above problems, and is thereto characterized in that the second section of porous material is free of contact with the first chamber space, i.e. that no liquid communication can take place through the said second section of porous material between the chamber space and outer space. In contrast with the devices known in the art, the device of the invention comprises a first section of porous material allowing liquid contact between the chamber and the environment outside the device (herein also Outer space') through the said first section of porous material, but a second section of porous material is present, being in contact with the environment outside the device, but free of contact with the space inside the chamber. This allows a convenient assessment of the effect of the contents of the chamber to a particular natural environment, by comparing the microbial colonisation of both first and second sections of porous material of the device, said colonisation taking place on the outer surfaces of the first and second sections. The chamber can be loaded with a particular medium, optionally comprising microbes, or can be a complete sample, taken from a natural habitat. The device can subsequently be incubated in an environment, such as a natural environment, or a controlled environment such as a growth medium of known composition, optionally comprising microbes.

The pore sizes of the first and second section of porous material can be chosen according to the desired aim. The porosity is chosen such that liquid, preferably water or an aqueous solution can pass through the porous material. So is it possible to choose a porous material such that (non-polymeric) biomolecules and other compounds are capable to move from the environment outside the chamber into the chamber and vice versa, while microbes cannot pass the said sections. In such a case, a pore size of below 0.1 urn would render the porous material impermeable for bacteria, whereas macromolecules and nutrients can still pass. If micro-organisms are intended to pass the porous material, the pore size of e.g. 50 urn. Also, a cut off can be chosen such that only nutrients should pass the porous material, whereas macromolecules and micro-organisms should not pass the porous material. In that case, a pore size of e.g. 10 nm can be chosen. Optionally, the selectivity of the porous membrane may be altered by the chemical nature of the surface as well as pore size. For example, alterations in surface charge or hydrophobicity or the attachment of specific molecules (e.g. antibodies) within the pores of the porous material may be used to alter what molecules or microorganisms can pass through. The sample entrapped in the chamber can e.g. contain microbes, either grown in the laboratory, or derived from a sample taken from a natural habitat. With the device, the effect of the presence of such microbes on the environment can conveniently be assessed by incubation of the device in the environment. As the second section is not in contact with the contents of the chamber, the second section serves as a control, that will be colonised by microbes present in the environment, without subjected to the effect of the contents of the chamber. On the first section however, microbial colonisation is influenced by the contents of the chamber, as liquid exchange between the chamber and the environment is possible over the first section of porous material. To this end, the porous material is preferably suitable for microbes to attach and grown thereon. The device according to the invention for the first time makes it possible to assess the effect of the contents of a particular liquid medium, or the presence of a particular microbe or combination of microbes to the microbial activity in the liquid environment.

The second section of porous material can be integral with the first section of porous material, the second section not being in contact with the chamber space, e.g. comprising an additional impermeable layer between the porous material and the chamber. Also, the first section of porous material can be separate from and not continuous with the second section of porous material, i.e. in that both sections are not from the same piece of material, but are on different pieces. Said pieces may however abut one another if desired.

In a preferred embodiment, the second section of porous material has a planer shape wherein any line, departing perpendicular from the said second section of porous material goes through the chamber space. This means that not only the first section, but also the second section of porous material preferably faces towards the chamber space.

In a particular embodiment, the first section of porous material is on an opposite site of the chamber as compared to the second section of porous material, e.g. both first and second sections of porous material running in parallel planes, the chamber being located between the said planes. Said sections and can e.g. be arranged as the devices of the prior art, wherein the liquid contact between the chamber and the second section porous material is now made impossible e.g. by arrangement of an impermeable layer or wall between the chamber space and the second section of porous material.

In an attractive embodiment, the surface areas of the inner and outer surfaces of the first section of porous material correspond to those of the second section of porous material, respectively, enabling a reliable comparison of the events occurring on both sections of porous materials within the same device.

The skilled person is aware of suitable materials for the first and second sections of porous material. Membranes of the envisaged porosity are available, e.g. from Osmonics Inc. , USA, GE Osmonics Labstore, USA, Millipore, USA. Additionally, suitable membranes may be custom fabricated or commercially manufactured from a variety of materials such as glass, silicon nitride, fibrous materials including cellulose nanofibres, plastics and other polymers, ceramics (including porous metal oxides), metal foams and membranes derived from photoresists via photolithography. Attractively, the chamber is closed, so that only liquid exchange through the first section of the porous material can take place between the chamber space and the environment, wherein the device is submersed. The chamber can be temporarily open, e.g. to allow liquid or a biological sample to enter the chamber space, but it is intended that during use, the chamber is closed, so that liquid exchange will take place between the environment, i.e. the environment, and the chamber space.

In a preferred embodiment, the first chamber of the device according to the invention comprises at least a first chamber opening, allowing liquid to enter the chamber. The presence of an opening allows easy loading of the chamber with a sample, without the need to load the sample during assembly of the device. Further, this embodiment enables loading of a sample in situ, i.e. at the location of the envisaged liquid environment, in particular a natural environment, when the device is submersed therein. There is no need to take an environmental sample and to take it away from the environment for loading the chamber of the device. To this end, the device can also comprise a second opening to allow any air to escape when the chamber space is filled with the liquid medium.

The diameter of the said first opening is preferably chosen such, that upon immersion of the device, the liquid medium from the environment enters the chamber space without hindrance within e.g. 30 minutes, preferably within 15 minutes, more preferably within 10 minutes, even more preferably within 5 minutes, most preferably within 1 minute.

Preferably, the device comprises means to close the chamber opening, such as a valve or a plug, or is sealable in a different manner. In a very attractive embodiment, the chamber of the device according to the invention is closed when the chamber is immersed in the liquid medium, allowing liquid exchange between the liquid medium and the chamber space through the first section of porous material. The chamber may therefore have an opening to allow the medium from the environment to enter the chamber space upon immersion of the device, however, said opening is closed after immersion to allow the above-mentioned liquid exchange without any additional liquid exchange not through the said first section of porous material.

Once the chamber opening is closed after loading, incubation is allowed to take place without material flow though the chamber opening, but over the sections of porous material. Once the opening is closed after loading of the sample, the device can also be removed from the environment where the sample was taken, and be moved to a second environment, to assess the effect of the sample of the first environment, entrapped in the chamber, to the second environment.

In an attractive embodiment, the first chamber, at the chamber opening, comprises a polymer, that swells and/or otherwise becomes a more significant barrier upon contact with the liquid entering the chamber, the swelling resulting in sealing of the chamber. In this embodiment, the chamber can be loaded with a sample by e.g. submersing the device in a first liquid or humid environment, allow the liquid medium of the first environment to flow into the chamber, where after the chamber opening is sealed by swelling of the polymer e.g. as an effect of contact of the polymer with the environment, or as a result of passing of time. Examples of suitable polymers or materials include gums, gels and natural or synthetic sponges which grow in volume upon hydration whilst being sufficiently impermeable to seal the first chamber. Preferably, the polymer swells upon contact with water, as the samples to be entrapped will usually comprise aqueous media. To this end, the wall of the chamber at the location of the opening can be coated with such a polymer, allowing closure of the opening upon swelling of the polymer.

Such swelling may involve a volume increase of about 4 fold or more of the polymer. So a polymer ring having a diameter of 100 urn, applied to an annular opening of the chamber will be sufficient to close an aperture of 0.8 mm. To this end, the diameter of the opening of the chamber is preferably 5 mm or less, preferably 1 mm or less, more preferably 0.6 mm or less. The opening is preferably more than 0.1 mm, more preferably more than 0.3 mm to allow the liquid medium to enter the chamber within 30 minutes or less after immersion of the device in a liquid medium.

In an attractive embodiment, the first chamber comprises at least one impermeable wall, having an inner surface facing towards the chamber space and an outer surface, the second section of porous material being attached to the said outer surface of the impermeable wall. In this arrangement, the chamber is separated from the second section of porous material by a wall, therewith making it impossible for any liquid medium or compound present therein to move from the chamber over the second section of porous material to the outer surface thereof to the outer environment or vice versa. In another attractive embodiment, the first chamber of the device comprises a second chamber opening, allowing liquid to flow though the chamber. In this embodiment, the device can be designed as a flow cell, wherein the effect of the liquid medium flowing through the chamber on any second liquid medium can be assessed by contacting the outside of the flow chamber with the said second medium. The first medium flowing through the flow cell may comprise a compound attracting microbial growth. Accordingly, the first and second sections of porous material will be susceptible to microbial colonisation from microbes from the second medium, and the effect of the first medium can be conveniently assessed by comparing the colonisation by microbes from the second medium of the first section with the colonisation of the second section. To this end, the device preferably comprises flow enhancing means, such as e.g. venturi elements or other methods of converting liquid flow present into the natural environment into directed flow or active liquid transport including pumps and turbines.

In another embodiment, the device according to the invention further comprises a second chamber having a second chamber space, the second chamber space being in contact with the outer surface of the first section of porous material, allowing liquid communication between the first chamber and the second chamber space through the first section of porous material, the second chamber comprising a third section of porous material, having an outer surface being in contact with outer space, and an inner surface being in contact with the second chamber space, allowing liquid communication between the second chamber space and the outer space through the said third section of porous material; and a third chamber having a third chamber space being in contact with the outer surface of the second section of porous material, the third chamber comprising a fourth section of porous material, having an outer surface being in contact with outer space, and an inner surface being in contact with the third chamber space, allowing liquid communication between the third chamber space and the outer space, allowing liquid communication through the said fourth section of porous material.

In this arrangement, the first chamber is now flanked by two additional chambers, one of which, here the second chamber, is in contact with the first chamber through the first section of porous material, allowing liquid exchange between the first and second chamber through the first section of porous material, whereas contact, and therewith liquid exchange between the third and first chambers is avoided. Both second and third chambers comprise a section of porous material allowing liquid contact with the respective chamber and the outer environment.

With this device it is possible to load a first liquid medium or sample in the first chamber, and allow the second and third chamber to be loaded with a liquid second sample or medium. Said medium can e.g. comprise plankton. The device can then be incubated in an envisaged environment (outer space), to check the effect of the first medium on the plankton in the first and second chamber, to be assessed by microbial colonisation of the third and fourth sections of porous material. The third section allows liquid exchange between the environment and the second chamber, which second chamber is in liquid contact with the first chamber, whereas the fourth section allows liquid exchange between the environment and the third chamber, but there is no liquid exchange between the third chamber and the first chamber. In case of plankton being entrapped in the second and third chamber, the plankton in the second chamber is affected by the first medium in the first chamber, whereas the plankton in the third chamber is not. This difference can be used to deduce which microorganisms (present in the second chamber but absent or at least less numerous in the third chamber) are influenced by metabolites or other compounds (produced directly or indirectly) by microorganisms in the first chamber.

In the above-described multiple chamber device, the first, second and third chambers preferably each comprise a first chamber opening, allowing liquid to enter the respective chamber. Again, the openings in the chambers allow convenient loading of the chambers

One or more of said first chamber openings are preferably sealable in order to avoid exchange between the environment and the content of the chamber through the said chamber openings.

In an attractive embodiment of the above-described multiple chamber device, more than one of the first chamber openings are differentially sealable. This allows sealing of the chambers at different time points and at different location of the device, i.e. loading of the first chamber can take place when the device is held in a first environment, whereas the second and third chambers are sealed at a later stage, e.g. in another environment. Differential loading can be achieved by having a slower rate of sealing the second and third chambers after immersion of the device than the first. Alternatively, differences in the two environments (e.g. salinity, temperature) can be used to seal the first chamber in the first environment and the subsequent chambers in a subsequent environment. This could be achieved, for example, using polymers (such as hydrogels) with different sensitivity to salinity in terms of swelling.

In order to be able to make a sound assessment of the microbial growth, the porous materials of the first and second sections of porous material in the device according to the invention are preferably the same.

As discussed above, the porous materials of at least the first and second sections of porous material are preferably impermeable for microbes. Preferably, the porous materials of at least the first section, but preferably of both the first and second section of porous material (and if present of the third and fourth section as well) is/are at least permeable for non-polymeric molecules. Polymeric macromolecules such as DNA, glycoproteins, proteins and may be too large to pass the porous sections, whereas smaller biomolecules and other chemical compounds, such as nutrients can freely pass the porous materials. In other versions, using larger pores, most macromolecules will pass through with only specific groups of organisms restricted. The choice of porosity of the porous materials, as well as the charge and/or hydrophobicity and/or presence of binding agents such as antibodies determines what molecules can still pass the porous materials and what molecules cannot, as discussed above.

The pore size of the porous materials of at least the first and second sections

(and if present of the third and fourth section as well) of porous material is preferably from 0.025nm to 50um. A pore size of below 0.1 micron makes the material impermeable for microbes, but renders it permeable for many macromolecules. A pore size of 0.03 urn allows nutrients and smaller macromolecules such as proteins to pass. Examples of suitable materials include polycarbonate, 0.22 micron diameter pore size, filters which exclude all except the smallest microorganisms but permit many macromolecules but not necessarily polymers to pass. Other examples are 2 to 5 micron diameter polycarbonate or nylon membranes, which allow the passage of most bacteria and archaea but exclude most protozoa and many other eukaryotic microorganisms.

The invention further relates to a method for assessment of the effect of medium on microbial growth using the device of the invention, comprising the steps of:

a) allowing a first liquid medium to enter the first chamber space b) sealing the first chamber,

c) incubating the sealed chamber in a second liquid medium,

d) allowing microbes from the second liquid medium to settle and grow on the outer surfaces of the first and second sections of porous material, e) analyse growth of microbes on the first and second sections of porous material, and assess the difference in growth on the first and second sections of porous material.

This method enables a reliable assessment of microbial growth in e.g. a natural environment, but in fact in any envisaged medium, as a function of another medium or contents therein, entrapped in the chamber of the device.

The analysis of microorganisms can be via sequencing representative nucleic acids (DNA or RNA) such as the genes encoding ribosomal RNA, other specific genes or intergenic regions or other non-coding sequences. Random sequencing or total genome sequencing (genomics) or the sequencing may also be used to assess microbial diversity. Analysis of other macromolecules such as proteins (proteomics), lipids, cell wall components, carbohydrates, metabolites (metabolomics). Further, microorganisms may be identified by cellular characteristics (phenotyping) including growth and the catalysis of biochemical reactions.

In a preferred embodiment, the composition of the first medium is known, so that the effect of a known composition can be assessed.

The method encompasses assessment of the effect of one or more one or more factors or a mixture thereof, present in a first liquid medium, on growth of microbes present in a second liquid medium, wherein the first medium is allowed to enter the first chamber space of the device of the invention, and the chamber after sealing is incubated in the second medium. Both the first and second medium can be different natural environments as discussed above.

The factors are preferably chosen from chemical compounds, microbes or a mixture of two or more thereof.

In a particular embodiment of the method of the invention, the factor(s) is/are hindered from equilibration over the first section of porous material with the second medium during the incubation step. This can be effected by limiting the incubation time of the device in the second medium, not allowing an equilibrium to settle. As soon as equilibrium is arrived, the effect of the respective compound is minimised, and colonisation by microbes will not anymore be effected by the said compound. The invention also relates to an assembly comprising a plurality of devices as described above. Such an assembly can be designed by arranging multiple chambers next to one another, sharing wall elements. Such assembly can conveniently be used when different assessments have to be made under the same conditions of incubation time and contact with outer environment.

In an attractive embodiment, at least one chamber of an assembly of the invention is free of a second section of porous material. Such a chamber of the assembly does not necessarily contain a second section of porous material. Such a section can be present in another chamber of the assembly, so that assessment of microbial growth on the second section of porous material can be assessed on the said other chamber. The results of this assessment can also be valid for the chamber without the second section of porous material, as both chambers are in close vicinity to one another and incubated in the same outer environment for the same incubation time. This is particularly true if both chambers comprise the same medium and other ingredients such as microbes. Correspondingly, the assembly may contain at least one chamber being free of a first section of porous material. Such a chamber would function as a control chamber, as it cooperates with the second section of porous material not allowing liquid communication between the said chamber and the outer space. In fact, in the assembly of the invention, the function of the first and second sections of porous material are now divided over different chambers.

In a very attractive embodiment, the assembly of the invention comprises a liquid passage having a liquid inlet and a liquid outlet, the outer surfaces of the first sections of the porous material of the plurality of chambers being in contact with the liquid passage, allowing liquid communication (i.e. liquid exchange) between the plurality of chambers and the liquid passage. This design is very useful as flow cell device. The multiple chambers are in contact with the passage through the first sections of the porous material. A test liquid, such as a polluted liquid can be passed through the passage, and the microbial growth can be assessed for each chamber. Each chamber can comprise a different medium or can be of different microbial content or both. For each chamber, the microbial growth can be assessed, either within the chamber, or on the outer surface of the first section of porous material that is in contact with the liquid in the passage. One or more of the chambers can, instead of a first porous section, cooperate with a second section of porous material, not allowing liquid communication (i.e. liquid exchange) between said chamber and the passage. Such chamber would function as a control for those chambers, allowing liquid communication between the passage and the said chambers. In another attractive embodiment, the assembly does not contain a second section of porous material, in case such controls are not necessary to be made.

Attractively, the liquid passage comprises a passage chamber. In another embodiment, the inner surfaces of the first sections of the porous material of the plurality of chambers are arranged in a single plane. The said plane can define a portion of the said passage chamber.

Attractively, the assembly according to the invention comprises a sheet of impermeable material comprising perforations of equal size, which sheet is mounted on an inner surface of a sheet of porous material having an inner surface and an outer surface and a porosity allowing liquids passage over the said sheet of porous material, the outer surface of the said sheet of porous material being in contact with the passage, wherein the perforations define the plurality of chambers. Chambers are defined by the perforations of the impermeable sheet and the inner surface of the porous material mounted to the said impermeable sheet. The chambers can be open to one side, allowing easy access for loading a medium in the chambers. One or more of the chambers can also, instead, or in addition to, the first section of porous material, be limited by a second section of porous material, not allowing liquid communication between the chamber and the passage there through. This assembly enables a convenient high throughput assessment of microbial growth or any other effect imposed by the liquid flowing through the passage on the content of the chambers and/or vice versa.

In use, the chambers are loaded with a liquid medium of interest, and a liquid of interest is passed through the passage, allowing liquid communication (i.e. exchange) between the passing liquid and the liquid present in the chambers. The liquid in the chambers can vary from one to another, to allow a convenient high throughput assessment. If desired, the passage of the assembly of the invention can also be shut off to allow a static situation within the passage for liquid communication purposes.

The invention also relates to a method for assessment of the effect of medium on microbial growth using the flow chamber device of the invention as described above, comprising the steps of: a) allowing a first liquid medium to flow through the first chamber space, the outer surfaces of the first and second sections of porous material being in contact with a second liquid medium,

b) allowing microbes from the second medium to settle and grow on the outer surfaces of the first and second sections of porous material,

c) analyse growth of microbes on the first and second sections of porous material, and assess the difference in growth on the first and second sections of porous material.

The invention also relates to a method for assessment of the effect of medium on microbial growth using the multi-chamber device of the invention as described above, comprising the steps of:

a) allowing a first liquid medium to enter the first chamber space,

b) sealing the first chamber,

c) allowing a second liquid medium to enter the second and third chamber spaces,

d) sealing the second and third chambers,

e) incubating the device in a third liquid medium,

f) allowing microbes from the third liquid medium to settle and grow on the outer surfaces of the third and fourth sections of porous material, g) analyse growth of microbes on the third and fourth sections of porous

material, and assess the difference in growth on the third and fourth sections of porous material.

The second medium preferably comprises microbes, such as plankton, to check the effect of the medium entrapped in the first chamber on growth of such microbes, and the effect thereof on the effect of microbial growth in a particular environment.

The invention is now further exemplified by the appended figures and examples which are by no means limiting the invention.

Figure 1 shows schematic diagram of a single chamber embodiment of a device according to the invention,

Figure 2 shows a schematic diagram of a flow cell embodiment of a device according to the invention, Figure 3 shows a schematical diagram of a multi chamber embodiment of a device according to the invention, and

Figures 4-6 show schematic diagrams of different embodiments of an assembly comprising a plurality of chambers used as flow cell.

Figures 7A-D show views of an embodiment of a single chamber device of the invention in disassembled state (7A, B) in assembled state (7C, D),

Figure 8 shows a partially assembled device of the invention, having a chamber (25 mm diameter) as explained in example 9 with one piece of PAO at base, the latter is blocked with graphite adhesive (hence black colour). Right: Second piece of PAO without adhesive that will be glued to the upper surface of the rubber ring to assemble the chamber.

Figure 9. shows the device of figure 8, now assembled.

Figure 10. Imaging surface of culture chamber after 8 days, see example 9. Figure 1 1 : Left: Imaging the edge of Surface A in the same experiment as in figure 10; X marks the imaging point. Right: Microbial growth on PAO membrane, see example 9.

Figure 12: Imaging the edge of PAO surface A with two images taken 0.2 sec apart, see example 9.

Figure 13: Diagram of experimental set up of see example 10.

Figure 14 and 15: Microscopy images of test results example 10.

In figure 1 , a device (1 ) according to the invention is shown having a single chamber 40, defining chamber space 41 . The top of the chamber is limited by a first section of porous material 20, having an outer surface 201 , and an inner surface 202. This first section of porous material 20 allows liquid communication between the chamber space 41 , over the first section of porous material 20, with an outer space 421 . The porous material can be chosen such, that microorganisms, in particular bacteria and other single or cell organisms cannot pass, but nutrients and other compound present in the chamber 40 and or the outer space 421 can pass. The chamber is further limited by a continuous vertical wall 401 and a bottom wall 402, impermeable for both microorganism and nutrients or other compounds. Bottom wall 402 has an inner surface 4021 , facing towards the chamber space 41 , and an outer surface 4022 to which a second section of porous material 30 is attached. Said second section of porous material 30 has an outer surface 301 , being in contact with outer space 422. Outer space 421 and outer space 422 can be the same space. There is no liquid communication possible between the chamber space 41 and the outer space 422 over the second section of porous material 30, as the impermeable nature of the bottom wall 30 does not allow liquid contact of the outer space with the chamber space or vice versa.

Vertical wall 401 comprises an opening 431 , allowing medium entry from the outer space 441 , 422 into the chamber space 41 . Around opening 44, a swellable polymers is arranged, rendering the chamber sealable upon contact of the device with a medium, such as an aqueous medium.

In a use of device 1 , a first medium is allowed to enter the first chamber space 41 . This can be achieved e.g. by removing the first section of porous material and allowing the medium to enter the chamber, and closing the chamber 40 by placing the first section of porous material on the vertical wall 401 . If opening 431 is present, the medium can enter via the said opening 431 , where after the opening is preferably sealed. No displacement of the first section of porous material is needed in that case.

The sealed chamber can be relocated and be brought into contact with a second medium in the outer space 421 and 422, allowing microbes from the second medium to settle and grow on the outer surfaces 201 , 301 of the first and second sections of porous material 20, 30 respectively. The growth of microbes on the first and second sections of porous material can be analysed, and the difference in growth on the first and second sections of porous material can be assessed.

In figure 2, a similar device as depicted in figure 1 is shown. Only differing features are indicated. The same features as present in the device of figure 1 are indicated by the same reference numbers. By the presence of a second opening 432 in the vertical wall 401 , opposite to the opening 431 , a flow cell arrangement is obtained. Flow enhancing means 45, such as venturi elements, are present in the chamber 40.

In a use of the device of figure 2, a first liquid medium is allowed to flow through the first chamber space 41 , entering the said chamber space through the first opening 431 and leaving the chamber by the second opening 432, or vice versa. The outer surfaces 201 , 301 of the first and second sections 20, 30, respectively, of porous material are in contact with a second liquid medium, constituting outer space 421 , 422. Microbes from the second medium are allowed to settle and grow on the outer surfaces 201 , 301 of the first and second sections 20, 30 of porous material, and the growth of microbes on the first and second sections (20, 30) of porous material is analysed, and the difference in growth on the first and second sections (20, 30) of porous material are assessed.

In figure 3, a multi chamber device 10 is shown, comprising a first central chamber 40, having the same configuration as the device 1 of figure 1 . The same features as present in the device of figure 1 are indicated by the same reference numbers. Above the said chamber 40, a second chamber 50 is arranged defining a second chamber space 51 , and liquid communication, i.e. liquid exchange, between the first chamber space 41 and second chamber space 51 is possible through the first section 20 of porous material. The second chamber has an optionally sealable opening 531 . The second chamber comprises a third section 70 of porous material, having an inner surface 702 facing to second chamber space 51 , and an outer surface 701 , facing outer space 423. The third section of porous material allows liquid exchange between the second chamber space 51 and the outer space 423.

Below chamber 40, a third chamber 60 is arranged defining a third chamber space 61 , and liquid communication between the first chamber space 41 and third chamber space 61 via a second section 30 of porous material is not possible by the presence of the bottom wall 402 of the first chamber. The third chamber has an optionally sealable opening 631 . The third chamber comprises a fourth section 80 of porous material, having an inner surface 802 facing to third chamber space 61 , and an outer surface 801 , facing outer space 424. The fourth section of porous material allows liquid exchange between the third chamber space 61 and the outer space 424.

In a use of the device 10, a first liquid medium is allowed to enter the first chamber space 41 , where after the first chamber can be sealed. A second medium is allowed to enter the second and third chamber spaces 51 , 61 , where after the second and third chambers 50, 60 are sealed. The device 10 is incubated in a third medium, which can be identical to the second medium. Microbes from the third medium are allowed to settle and grow on the outer surfaces 701 , 801 of the third and fourth sections 70, 80 of porous material, and the growth of microbes on the third and fourth sections 70, 80 of porous material are analysed, and the difference in growth on the third and fourth sections (70, 80) of porous material is assessed. In figure 4 an assembly according to the invention comprises a housing 7, comprising inlet channel 732 with an inlet 731 and an outlet channel 733 with an outlet 735. Inlet 731 is connected to a feed tube 734 via connector 736, whereas outlet 735 is connected with outlet tube 738 via connector 737. A liquid passage, in particular a passage chamber, is denoted by 721 . Passage chamber 721 is connected to inlet channel 732 and outlet channel 735 and made liquid-tight by the use of rubber washers 739. The passage chamber 721 is in contact a sheet 800 of porous material, such as porous aluminium oxide (20 to 200 nm diameter pores), facing with an outer surface 801 thereof towards the passage chamber 721 . The outer surface 801 is in contact with the passage chamber 721 , and defines the upper boundary thereof allowing liquid exchange between the chambers and the liquid passage. At the opposite surface, i.e. inner surface 802, porous sheet 800 is mounted on a perforated sheet 71 of impermeable material such as glass. With 'impermeable' is meant that no passage of contents of liquid through the said material is possible. Each perforation 710 defines a chamber, also being denoted by reference number 710, the bottom thereof being defined by the inner surface 802 of the sheet 800 of porous material. The inner surface 802 is therefore in contact with the spaces of chambers 710 at the location where the said inner surface 802 defines the respective bottoms of the said chambers 710 with dimensions of e.g. a few mm across. This allows liquid communication between the passage chamber 721 and the chambers 710. So the sheet 800 constitutes a first section of porous material for each chamber as defined herein. Passage chamber 721 may be regarded as an equivalent of the outer space as defined herein and as shown in the previous figures. The perforations 710 are separated from one another by the sheet 71 of impermeable material, shown by position 72. Below this position 72 between the chambers 710, the porous material does not allow liquid communication between the chambers 710 and the passage chamber 721 . This portion of the porous material can be regarded as a second section of porous material as defined herein, as this portion is free of liquid contact with the chambers. However, the assembly can be free of a second section. On the other hand, some of the perforations can be made in the impermeable sheet 800 such, that only an intrusion is formed in the said sheet, and not a hole. Such intrusion would have a bottom section of the said impermeable material, and therewith, no contact with the porous material mounted thereon is possible. Again, the porous material at the location of such bottom section is free of liquid contact with such a chamber and can therefore be regarded as second section of porous material as defined herein. It is also possible to arrange a chamber such, that this chamber is in liquid contact with the inner surface 802 of the porous material 800, but not allowing liquid communication with the passing chamber 721 , e.g. as is the case for chamber 71 1 . The presence of such chambers 71 1 is however optionally. The chambers 710 of the assembly are closed by a cap element 780, fixed to the housing but removable if desired. The chambers can be protected by an additional impermeable sheet or layer 73, such as glass. The said additional impermeable layer 73 may be transparent or otherwise permit imaging through the material to the chambers 710 beneath.

A source of liquid, such as water from a natural source or a polluted environment, is piped from a distant location through the device from feed tube 734 via inlet channel 732 into the flow chamber 721 and then out via outlet channel 733 and outlet tube 738 to a second distant location.

In use, a liquid is passed from inlet tube 734 via inlet 731 and inlet channel

732 to passage chamber 721 . The said passing liquid is in liquid communication with any liquid or medium present in chambers 710 via the sheet 800 of porous material. During passage through the passage chamber 721 , liquid communication, e.g. interaction or exchange can take place between the passing liquid and the liquid medium in the chambers 710. The pore size can be chosen such, that passage of nutrients and other small molecules are allowed, but that passage of microbes is not possible. Such liquid communication is not possible between the passage chamber 721 and chamber 71 1 . Each of the chambers 710 can be loaded with different medium or having different contents, and the influence of the passing liquid on the composition of the media or vice versa can be assessed by allowing microbes to grow on either side of the porous material, i.e. on the inner surface 201 or the outer surface 202, whatever is applicable. The liquid leaves the passage chamber 721 via outlet channel 733 and outlet tube 738.

The device as indicated in Figure 4 can be used to culture and isolate new microorganisms sensitive to a liquid source passing through the device including such factors as nutrients, gases, pH or temperature of this liquid.

In figure 5 an assembly according to the invention comprises is as described for Figure 4 with the following changes. The same reference numbers are used for the same features, but not always indicated. As second sheet 900 of impermeable material, such as glass, may line the opposite surface of the passage chamber 721 with an upper surface 901 facing the flow chamber and a lower surface 902 facing the housing 7. However, this second impermeable material may also be part of the housing, if the housing is made of material, suitable to be impermeable. The passage chamber 721 is, via sheet 800 of porous material, in liquid communication with an outer environment 1000, for example a location in the sea in which the device is immersed. A portion 803 of sheet 800 of porous material may be masked by an additional sheet of impermeable material 73, so that this portion 83 cannot be in contact with the outer environment 1000. A second source of liquid, such as water from another source is piped from a distant location 1001 through the device from inlet tube 734 via inlet channel 732 into the flow chamber 721 and then out via outlet channel 733 and outlet tube 738 to a second distant location 1002. A distant location means for example from 5 cm to 10 metres from the main body of the device.

Isolation of microorganisms sensitive to two types of environment can be performed using the version of the device of figure 5, as will be exemplified in example 7.

In another version of the device (Figure 6) most aspects are the same as described in figures 4 and 5. The same reference numbers are used for the same features, but not always indicated. However, there is a second sheet 1 100 of porous material, such as porous aluminium oxide with an upper surface1 101 facing the passage chamber 721 , and a second surface 1 102 facing the housing 7. This feature replaces the sheet 900 of impermeable material illustrated in Figure 5, and is to be regarded as the second section of porous material as defined herein, as it is not in contact with an outer environment, whereas sheet 800 is in contact with outer environment via the outer surface 802 thereof, i.e. constituting a first section of porous material as defined herein.

In figure 7, a device of the invention is shown comprising a cylindrical main housing 1 , comprising two handles 2 for attachment to any desired structure, a cylindrical chamber receiving area 14 and a female screw thread 15, axially positioned for receiving closure cap 12, having male screw thread 13. Both main housing 1 and cap 12 have an axial opening 15 and 1 1 , respectively, of the same size. A different size is also possible. The receiving area 14 of the main housing receives, in the following order: 1 ) a disc shaped porous material 5, providing the second section of porous material, in analogy with reference number 30 in figure 1 ; 2) a liquid impermeable washer 6 (analogous to bottom wall 402 in figure 1 ), e.g. of metal, optionally followed by a second washer 7 for spacing purposes of the chamber. Thereto, washers 6 and 7 can be of different thickness;3) a cylindrical chamber wall element 8, analogous to number 401 in figure 1 , defining a chamber 9; 4) a disc shaped porous material 10, being of different or the same material as porous material 5, forming the first section of porous material. Assembled, washers 6 and 7 prevent contact of the porous disc 5 with the chamber, avoiding liquid exchange through porous disc 5 between the environment and the chamber via opening 15. Liquid exchange between chamber 9 and the environment is however possible through porous disc 10, via opening 1 1 of cap 12. Preferably the surface area of the surfaces of both porous discs 5 and 9 facing outwards, i.e. towards the outer space correspond to one another, as well as the surfaces facing inward, i.e. towards the chamber 9, in order to provide equal circumstances for both first and second porous sections.

The device of figure 7 can conveniently be adapted to provide a multiple chamber as depicted in figure 3 (not shown), by assembling, in the main housing, in the following order, 1 ) a porous disc 5,9; 2) a cylindrical chamber wall 8; 3) a porous disc 5,9; 4) one or more washers 6,7; 5) a cylindrical chamber wall 8; 6) a porous disc 5,9; 7) a cylindrical chamber wall 8; 8) a porous disc 5,9, followed by the cap 12. The receiving area 14 of the main housing 1 can be designed of accordingly longer, to accommodate all necessary elements.

Nota that the main housing may also comprise one or more radially arranged apertures, creating an opening between the outside of the housing and receiving area 14, to provide an opening to allow liquid to enter the chamber upon immersion of the assembled device. To this end, wall element 8 also comprises an aperture, aligned with that of the housing once assembled.

Figure 8 shows a partially assembled culture chamber (25 mm diameter) with one piece of PAO at base, the pores in the latter are blocked with graphite adhesive (hence black colour). Right: Second piece of PAO without adhesive that will be glued to the upper surface of the rubber ring to assemble the chamber. See also example 9. Figure 9 shows the assembled culture chamber of figure 8 with unblocked face (i.e. PAO with carbon graphite adhesive only around the periphery) upwards and blocked PAO downwards (out of sight).

Figure 10 shows Imaging surface of culture chamber after 8 days. Imaging was of 4 x 2.8 mm areas of the PAO surfaces using an Olympus BX41 microscope equipped with a x4 objective lens. Syto 9 and hexidium iodide staining (showing stained bacteria as an intense white) both indicate that there is substantially more growth on the unblocked PAO surface (A) than the blocked (B), see also example 9. Therefore, most of the microorganisms visualized on surface A are dependent on the contents of the central chamber.

Figure 1 1 shows imaging the edge of Surface A in the same experiment as Figure 3. Left: X marks the imaging point, on the interface between the inner section of the PAO that has unblocked pores that communicate with the inner chamber and the outer section of PAO that is blocked by the carbon graphite adhesive bonding the material to the rubber ring. Right: The only significant growth (as revealed by hexidium iodide staining) was on the inner, unlocked section of the PAO.

Figure 12 shows imaging the edge of PAO surface A with two images taken 0.2 sec apart using hexidium iodide stained samples then imaged by fluorescence microscopy. Two objects show rapid changes in position (1 and 2). Object 1 is not present in the left hand frame but is in the right. Object 2 is visible in the left hand frame but not the right. The reason for this observation is both objects are rapidly moving microorganisms (10 to 30 microns long). These were not found on surface B (blocked) indicating a difference in the microbial population.

Figure 13 shows a diagram of an experimental set up as used in example 10, viewed from above. Upper section: 1 : water. 2: microscope slides above and below agar film (3). 4: channel formed by agar and microscope slides. Lower section: 5: channel closed.

Figure 14 shows results Trial 1 of experiment 10 viewed by microscopy. (A) Starting situation with 0.5 mm width channel open. 3: agar, 4: channel open, 5: channel closed. (B) After 2 h showing nearly complete closure. C: Closure after 4 h now excludes particles the size of microorganisms.

Figure 15 shows results of trial 4(A) and trial 2(B) of experiment 10 viewed by microscopy after 4 hours. The presence of sea salts in the agar allows closure of the 0.6 mm width channel. Where the agar was formulated without salts closure does not occur.

EXAM PLE 1

Isolation of previously uncultivated microorganisms that grow on compounds liberated from asphalt or ones, which are dependent on microorganisms that grow on asphalt.

This experiment describes a method for isolating microorganisms that are dependent (directly or indirectly) on the degradation of asphalt. Such microorganisms are likely to be valuable in industrial bioconversions and in the degradation (removal) unwanted materials.

The chamber of a single chamber device according to the invention, e.g. as described in figure 1 , is loaded with the poorly soluble material asphalt as growth substrate. The main body of the chamber is fabricated from stainless steel, with the dimensions of the device being 2 cm across. The porous membranes are clamped into the device using silica seals allowing them to be removed later for microbial analysis. The porous membrane chosen is porous aluminium oxide with a pore size of 0.2 microns (as available from companies General Electric, Synkera, SmartMembranes, and SPI Supplies) allowing nutrients including proteins to pass the membrane but retaining microorganisms including bacteria. The stainless steel device with silicon rubber and porous aluminium oxide is designed to be highly resistant to corrosion in salt water. The device is lowered into the sea using a fine cable and is protected by an open cage of stainless steel (mesh diameter 2 mm) to protect from fish and other macroscopic sea life. The device is implanted in the sea attached to a marker buoy. The chamber fills with seawater and the chamber opening seals within 2 h. by using a swellable hydrogel ring (e.g. polyvinyl copolymers), attached around the chamber opening. The device is then incubated in situ for from 1 to 60 days. Some of the microorganisms (total) trapped within the growth chamber grow - some directly on the substrate (group A), some growing dependent on the growth of group A (group B), and some independently of group A or the asphalt. However, groups A, B and C are currently indistinguishable from each other as a mixture so will be referred to as group A/B/C. Further, the two outer surfaces of the first and second sections of the porous material will also support growth of microorganisms. The surface of the second section, i.e. the outer surface with no direct connection with the central chamber (301 ) will accumulate microorganisms from the environment (group D) most of which cannot degrade or grow on asphalt. The surface of the first section, i.e. the outer porous surface with connection with the chamber will accumulate not only group D but also microorganisms that are directly or indirectly influenced by asphalt degradation (group E). Sequencing whole genomes, specific genes (such as the rRNA encoding genes), proteomics or other analytical method is used to identify groups A/B/C, D and E. In the subsequent analysis, by subtracting group D from group E (group F) it is possible to increase the chance that microorganisms within group F are identified which are interacting with groups A and B, i.e. those unique to group E. For example, group D (determined by sequencing subcloned fragments of DNA encoding 16S rRNA genes) comprises primarily of SAR bacteria, Ralstonia spp. , Rhodospirillum spp. and Sinorhizobium spp. Group E comprises SAR bacteria, Ralstonia spp. , Rhodospirillum spp. , Chromatobacterium spp and Burkholderia spp. The organisms present in E not D (group F) are Chromatobacterium spp and Burkholderia spp. and these therefore are candidate species for growth on asphalt. The group of bacteria Sinorhizobium spp. are present in D not E and are candidates for microorganisms that have their growth inhibited by asphalt. EXAM PLE 2

Screening for microorganisms from deep sea water that are stimulated in growth by microorganisms from shallow sea water.

Microorganisms grow at all depths in the sea. Due to motility, transport by larger organisms and currents microorganisms from different depths can recirculate and interact. However, no good method exists for determining such interactions.

The chamber of a single chamber device according to the invention, e.g. as described in figure 1 , is loaded with microorganisms. To do this, the device is implanted in the sea attached to a marker buoy at a depth of 10 m for 2 hours. The chamber fills with seawater and the chamber opening seals trapping microorganisms (group G) from 10 m depth due to the swelling of the hydrogel (e.g. polyvinyl copolymers) to block the 2 mm diameter aperture within 2h. The device is then lowered to a depth of 50 m and incubated for 1 to 60 days. The two outer surfaces of the first and second section of porous material device will also support growth of microorganisms. The outer surface with no connection with the central chamber, i.e. of the second section of porous material, will accumulate (by growth and attachment) microorganisms from the environment (100 m depth, group H). The outer porous surface with connection with the central compartment, i.e. of the first section of porous material, will accumulate group H but also microorganisms that are directly or indirectly influenced by group G (group I). Sequencing whole genomes, specific genes (such as the rRNA encoding genes), proteomics or other analytical method is used to identify groups G, H and I . In the subsequent analysis, by subtracting group G from group H (group J) it is possible to increase the chance that microorganisms within group J are identified which are from 100 m depth and are likely interacting with the microorganisms from 10 m depth.

EXAM PLE 3

Screening for microorganisms sensitive to liquid flow in a natural environment. In this example, it is shown that using a device according to the invention, designed as flow chamber such as shown in figure 2, the effect of liquid flow on microorganisms growing on a surface can be measured, for example in studying fouling. In nature, it is difficult to manipulate or assess the effect of liquid flow and also difficult to separate the physical effects of liquid flow (e.g. shearing) on the provision of nutrients. The device of the invention offers novel solutions to this problem.

The central chamber of the device of the invention has two apertures, such as shown in figure 2. The device is placed in a liquid environment so that liquid flows through the central chamber. By manipulating the dimensions of the central chamber or adding a flow enhancing element a liquid flow is created that is more rapid than that flowing over the outside of the device. As discussed above, one of the surfaces allows liquid communication between the chamber and the outer environment (corresponding to surface 201 shown in Figure 1 ), whereas the other surface is only in contact with the outer environment (corresponding to surface 301 shown in Figure 1 ). Microorganisms growing as a biofilm or microcolonies on the outer surface that is not in liquid contact with the chamber, (group K) will only experience liquid flow and nutrients delivered from the environment (422 in Figure 1 ). Microorganisms growing on the surface allowing liquid communication between the chamber and the outer environment (group L) will experience both the environment and the accelerated flow through the central chamber. By comparing the populations of cells, proteins, nucleic acids or other parameters on the two surfaces (groups K and L) the effect of the flow of liquid through chamber 41 can be assessed.

EXAM PLE 4

Screening for microorganisms, which generate compounds or extracellular enzymes that can be used to prevent fouling of ships.

There is a need to isolate microorganisms that reduce the fouling of ships or other surfaces. However, such organisms are likely not to be isolated by adhering to the surfaces they are designed to protect. Furthermore, it is not easy to screen for such microorganisms under the conditions under which fouling occurs. The device of the invention offers a more effective route to this goal.

The central chamber of a single chamber device according to the invention, e.g. as described in figure 1 , is loaded with microorganisms into the central chamber (group M) and attached to the hull of the ship with the outer porous surfaces being equally exposed to the outer environment, with the chamber opening of the chamber sealing after 2 h due to the swelling of the hydrogel to block the aperture, while the microorganisms being unable to penetrate the porous material. After a cruise of 60 days the two surfaces (+/- communication with central chamber) are assessed by degree of growth (groups N and O). Devices for which the surface communicating with the chamber show less microbial growth (less fouling or a more limited biofilm) compared to the porous surface not communicating with the chamber contain samples within the central chamber that may be of value in preventing fouling. Identification of the microbial contents of the central chamber (group M) provides leads as to microbes that prevent fouling. Dilution of such mixtures of microorganisms into new chambers and repeating the process for one or more cycles (incubation attached to the ship, identification of the contents of chamber at the end of this process) may be used to purify, isolate and/or identify the desired microorganisms further. This approach can also be used to assess the antimicrobial activity of compounds under such circumstances, by introducing in the chamber the antimicrobial compound to be tested, and to check, after the incubation, the difference in microbial growth on both outer porous surfaces of the device. To this end, the compound to be tested can be provided in a slow release vehicle to avoid premature leakage of the compound from the chamber. EXAM PLE 5

Isolation of new groups of microorganisms growing in liquid culture, that are dependent on a particular compound or another group of microorganisms for growth.

Previous versions of the device have been orientated towards isolating microorganisms growing on surfaces (e.g. as biofilms). It is also desirable to look at microorganism in suspension (such as planktonic growth). The device for this purpose is shown in Figure 3; it comprises a central chamber (40), and two outer chambers (50 and 60) rather than a single chamber of the foregoing examples.

The central chamber (40) is loaded with microorganisms (group P) or with a growth substrate such as a slowly degraded polymer such as cellulose. To load with microorganisms, the device is implanted in the sea attached to a marker buoy at a depth of 10 m for 2 hours. The chamber fills with seawater and the chamber opening seals trapping microorganisms (group P) from 10 m depth due to the swelling of the hydrogel to block the aperture, while the microorganisms being unable to penetrate the porous material. The outer chambers (50 and 60) will also be filled with water and microorganisms from the same environment; however, these will not be sealed in this step as the material intended to seal apertures 531 and 631 takes longer to seal the outer chambers. This can be achieved by choosing another sealing polymer, or by a larger aperture for the outer chambers (e.g. 4 mm diameter) so that the apertures in the outer chamber take 10 hours to seal. The entire device is then lowered to 100 m. During this time microorganisms from 100 m replace those from 10 m in the outer chambers (group Q) but not those from 10 m (central chamber) as the latter is already sealed.

The device is incubated for e.g. 20 days. The microorganisms in the central chamber influence the growth of the microorganisms in chamber 50 (via nutrients, signalling molecules, antibiotics and other molecules that can penetrate the porous material 20 allowing group Q to develop into group R) but not chamber 60 (group Q developing into group S), as between outer chamber 60 and the central chamber, no liquid communication is possible. By comparing groups R and S the influence of group P on group R can be deduced.

EXAM PLE 6

Isolation of new microorganisms The device as indicated in Figure 4 can be used to culture and isolate new microorganisms sensitive to a liquid source including such factors as nutrients, gases, pH or temperature of this liquid. Reference numerals refer to those as used in figure 4. Microorganisms are inoculated into one or more identical chambers 710 onto surface 802 of porous material 800 and, if present, also in chamber 71 1 . Chamber(s) 710 allow the microorganisms to receive nutrients and other low molecular weight materials through the porous membrane (800) via the flow chamber (721 ). Chamber 71 1 is not connected with flow chamber 721 via a porous surface. Microorganisms growing in chamber(s) 722 are responsive to the liquid from flow chamber (721 ) and comprise group T. Microorganisms isolated from chamber 71 1 can be expected not to respond to the liquid in the flow chamber and comprise group U. Analysis can be via DNA sequencing (individual genes including those coding for rRNA or whole genomes or cloned fragments) or other analysis including proteomics and lipid analysis to give an indication of the microbial diversity within each group of microorganisms. By subtracting group U from group T gives group V, microorganisms that respond or are likely to respond to the water or other liquid from chamber 721 .

EXAM PLE 7

Isolation of new microorganisms

Isolation of microorganisms sensitive to two types of environment can be performed using the version of the device described in Figure 5. Reference numerals refer to those as used in figures 4 and 5. The device is submerged in the sea to a depth of 1 metre so that the immediate outer aqueous environment 1000 contacts the outer surface 802 of sheet 800 of porous material where it is exposed to the environment but not where it is masked by an impermeable surface (surface 803 marked by impermeable material 72). Water from another distant or separate source, for example 10 metres away (10001 ) and piped to this location via an impermeable tubing, flows through the flow chamber (721 ) from source 1001 in a path from 734 via 731 , 732, 721 , 733, 735 then out at 738 to location 1002. Microorganisms growing on surface 802 of the porous material are responsive to nutrients and other small molecules (e.g. antibiotics) from both water sources (group W). In contrast, those microorganisms growing on surface 901 of an impermeable material such as glass bordering the flow chamber 721 only experience water from one source (group X). After 10 days operation of the device in this environment the microorganisms are analysed by proteomic analysis, nucleic acid sequencing or culture. By subtracting sequences or the identity or abundance of the microorganisms comprising group X from group W (to give group Y). Group Y microorganisms are likely to include microorganism dependent on both water sources for growth.

EXAM PLE 8

Isolation of new microorganisms

This example has a similar purpose to the situation described in example 7, however, a device as described in figure 6 is used. Reference numerals refer to those as used in figures 4, 5 and 6. In this device there are two porous materials bordering the flow chamber (sheet 800 with surface 801 and sheet 1 100 with surface 1 10, respectively, therewith bordering the flow chamber 721 . These surfaces can be porous aluminium oxide. The experimental set up is otherwise the same as example 6 except that microorganisms growing on surface 801 (exposed to both environments 1000 and 1001 , group Z) are compared to microorganisms growing on surface 1 101 (exposed only to water source 1001 ; group AA). Microorganisms growing on surface 801 are in liquid contact with both the liquid from source 1001 passing through the passage chamber 721 and with the outer environment 1000, whereas the microorganisms growing on surface 1 101 are only in liquid contact with the liquid from environment 1001 passing through passage chamber 721 . Environment 1000 can e.g. be sea or lake water at the location where the device is submersed, whereas environment 1001 can be any liquid, such as another water source from elsewhere and passed through the passage chamber 721 . Deducting group AA from group Z yields group BB, microorganisms sensitive to, or requiring, both water sources.

EXAMPLE 9

Validation experiments

Materials:

Rubber rings (from Gamma, NL with 25 mm outer diameter, 18 mm inner diameter, 2 mm height), carbon graphite adhesive (Graphite Conductive Adhesive 502, catalogue no. 12684-30 from Electron Microscopy Sciences, USA), 25 mm diameter PAO membrane (Anodisc 25, Whatman NL via SPI supplies USA, catalogue no. 6809-6002 with 20 nm pore), artificial sea salts (catalogue no: S9883 from Sigma Chemicals, NL), Syto 9 DNA staining dye (Invitrogen, NL), hexidium iodide DNA staining dye (Invitrogen, N L), agar (Eiken, Japan). Biological Samples:

Seaweed (Fucus guiryi) and water from beach/sea (intertidal region) on the North Sea near Leiden, NL. Google map location: (https://maps. google. nl/maps?daddr=52.162455,4.349205&hl=nl&sll=52.162798,4.35 1 101 &sspn=0.020165,0.038581 &t=h&mra=mift&mrsp=1 &sz=15&z=15)

Chamber assembly:

One disc of PAO is coated with graphite adhesive both to block the pores (but leaving an outer face of PAO to interact with environmental bacteria) and to glue it to the rubber ring (Fig 8). This assembly was then washed in ethanol and water to remove any toxic compounds from the adhesive. The interior of the chamber was loaded with homogenized seaweed (Fucus guiryi) in sea water (2 g sea weed per ml) then the chamber sealed with the second PAO section using carbon graphite adhesive around the periphery but (unlike the first section of PAO) the pores remained unblocked (Fig. 9). The outer surface of the PAO connected by pores to the central chamber is surface A. The outer surface of the blocked PAO is surface B.

EXAM PLE 9A

Initial Control Experiment to Determine if Sea Weed is a Suitable Growth Substrate.

The intention of the following experiment 9B is that nutrients in the culture chamber support the growth of microorganisms on the chamber surface A. The microorganisms from the sea water should only grow, or at least grow better or differently, on the outer surface of the culture chamber if they can access the nutrients or other compounds derived from the sea weed (i.e. it is expected that surfaces A and B will show differences in the associated microorganisms. Therefore, it was tested that the sea weed acts as an appropriate source of nutrients or other growth factors.

1 % w/v Eiken agar was supplemented with 3% (w/v) sea salts and shredded sea weed at the same density (2 g/ml), this was autoclaved and poured as an agar plate (1 ). A control plate was poured except for the absence of the sea weed (plate 2). Only plate 1 supported the visible growth of microorganisms derived from sea water after 2 days at 20 °C. Further, this differential growth (on plate 1 not 2) could be detected also by placing sterile PAO strips on the agar plates and inoculating these with sea samples and observing microbial colonies. This showed that at least some of the nutrients released from the seaweed penetrated the PAO. This control experiment suggested that the sea weed could support microbial growth in the presence of suitable salts and therefore was suitable for example 9B.

EXAM PLE 9B:

Culture Chamber Testing

The entire culture chamber assembly was incubated in sterile artificial sea water (3% w/v sea salts) spiked with 1 ml of sea water from the same environment as the sea weed to provide microorganisms. After 8 days the entire culture chamber was stained with hexidium iodide and Syto 9 in artificial sea water and imaged by fluorescence microscopy (Fig. 10). The with areas correspond to microorganisms indicating that the growth of microorganisms was far greater (> 100 fold) on the unblocked PAO surface (A) relative to the blocked surface (B). Further, imaging the region of the unblocked PAO near the area where it becomes blocked (due to bonding to the rubber ring) supported the observation that the unblocked area of the PAO (supplied by the sea weed in the inner chamber) supported growth (figure 1 1 ). Further, quantifying the data using the software programme ImageJ to analyse TI FF files indicated that for the unblocked PAO the hexidium iodide staining population was from 3 to 6 fold more abundant than the Syto 9 staining population. In contrast, growth on the blocked side showed similar staining for both dyes (1 : 1 ratio). This suggests that the 2 populations differed in composition as well as abundance. This latter conclusion is also supported by the observation that motile (moving) microorganisms were observed on the unblocked but not blocked PAO (figure 12).

From the validation experiments it can be concluded that a cultivation chamber can be constructed with one porous surface connected to a central chamber and one, otherwise identical surface, blocked. If the central chamber was filled with a complex nutrient source then this enriches microorganisms when used for in situ cultivation (increases numbers, allows microorganism to grow that could not grow before) that are exposed to this source. Therefore the device can be used to isolate specific subpopulations of microorganisms from the environment. EXAM PLE 10

Channel closure

Materials:

Agar (Eiken, Japan), Salt Solution (Sea Salts, Sigma NL, dissolved as a 3 percent w/v solution in water), insoluble blue dye (Ultramarine Blue), three micron diameter polystyrene spheres (Polysciences), microscope slides (Thermo, Germany, 76 x 26 mm). Experiment:

2% w/v agar was resuspended in 3% salt solution with 0.02% blue pigment added was liquefied by heating to 90 degrees centigrade poured on an agar slide to create a 1 mm thick layer and air dried until both solidified. The blue dye was present to allow the channel to be visualized by transmission microscopy (walls are blue, channel is transparent). A channel (0.5 mm diameter) was cut in the dried agar (solidified and reduced in depth from 1 mm to 0.5 mm) and a second slide was placed on top of the agar to create a channel open at both ends. This assembly was placed in 50 ml water (Trial 1 , containing 3% w/v sea salts. Trial 2, distilled water) and the channels imaged ever hour using an Olympus BX41 microscope using white light to assess the channel closure. In a second set of experiments the gel was formulated as before, but using distilled water rather than salt solution. The channel was cut as before. These assemblies were placed in 50 ml water (Trial 3, containing 3% w/v sea salts. Trial 4, distilled water) and, again, imaged ever hour using an Olympus BX41 microscope to assess the channel closure.

Results and conclusion:

The channels closed over a period of 2 to 4 hours immersion. After closure it was not possible to passage 3 micron diameter beads through the channels (simulating microorganisms). Surprisingly, channel closure was achieved with both high and low external salt concentrations, which suggests that the method is widely usable in the environment (from fresh to sea water). Closure was maintained for > 48 h. I n trials 3 and 4, when salt was not present in the agar, closure was not achieved. Therefore, using an agar polymer containing sea salts is a viable way of sealing a culture chamber device after immersion in an aqueous environment after filling the chamber. Repeating these experiments without the blue dye gave the same result - i.e. this component was not essential.

Claims

1 . Device (A) for submersing in a liquid medium, comprising a first section of porous material (20) and a second section of porous material (30) and a first chamber (40), defining a first chamber space (41 ) within the first chamber (40) and at least one outer space (421 , 422) outside the first chamber (40), the first section of porous material (20) having an outer surface (201 ) being in contact with the outer space (421 ), and an inner surface (202) being in contact with the first chamber space (41 ), allowing liquid communication between the first chamber space (41 ) and the outer space (421 ) through the said first section of porous material (20), the second section of porous material (30) having at least an outer surface (301 ) being in contact with outer space (422), the second section of porous material (30) being free of contact with the first chamber space (41 ).

2. Device (A) according to claim 1 , wherein the first chamber is closed.

3. Device (A) according to claim 1 , wherein the first chamber (40) comprises at least a first chamber opening (431 ), allowing liquid to enter the chamber (40).

4. Device (A) according to claim 3, comprising means to close the chamber opening (43).

5. Device (A) according to any of the preceding claims, wherein the chamber is closed when the chamber is immersed in the liquid medium, allowing liquid exchange between the liquid medium and the chamber space (41 ) through the first section of porous material (20).

6. Device (A) according to claim 5 or 6, wherein the first chamber (40) at the chamber opening (43) comprises a polymer (44), swellable upon contact with liquid entering the chamber (40), the swelling resulting in sealing of the chamber (40).

7. Device (A) according to claim 7, wherein the swellable polymer comprises gums, gels, such as agar, and natural or synthetic sponges.

8. Device (A) according to any of the claims 3 - 7, wherein the first chamber opening has a diameter of 0.1 - 5 mm.

9. Device (A) according to any of the preceding claims, wherein the first chamber comprises at least one impermeable wall (402), having an inner surface

(4021) facing towards the chamber space (41 ) and an outer surface (4022), the second section of porous material (30) being attached to the said outer surface

(4022) of the impermeable wall (402).

10. Device (A) according to any of the claims 2 - 9, wherein the first chamber (40) comprises a second chamber opening (432), allowing liquid from the outer space to enter the first chamber space (41 ) through the first chamber opening (431 ) and to leave the chamber space (41 ) through the said second chamber opening (432).

1 1 . Device (A) according to claim 10, wherein the device comprises flow enhancing means (45).

12. Device (A) according to any of the preceding claims, wherein the porous materials of at least the first and second sections of porous material (20, 30) are the same.

13. Device (A) according to any of the preceding claims, wherein the first section of porous material (20) is separate from and not continuous with the second section of porous material (30).

14. Device (A) according to any of the preceding claims, wherein the second section of porous material (30) has a planer shape wherein any line, departing perpendicular from the said second section of porous material (30) goes through the chamber space (41).

15. Device (A) according to any of the preceding claims, wherein the surface area of the first section of porous material (20) is on an opposite side of the chamber (40) as compared to the second section of porous material (30).

16. Device (A) according to any of the preceding claims, wherein porous materials of at least the first and second sections of porous material (20, 30) are impermeable for microbes.

17. Device (A) according to any of the preceding claims, wherein the porous materials of at least the first section of porous material (20) is at least permeable for non-polymeric molecules.

18. Device (A) according to any of the preceding claims, wherein the pore size of the porous materials of at least the first and second sections of porous material (20, 30) is from 0.025 nm to 50 urn.

19. Device (B) according to any of the preceding claims, the device further comprising

- a second chamber (50) having a second chamber space (51 ), the second chamber space (51 ) being in contact with the outer surface (201 ) of the first section of porous material (20), allowing liquid communication between the first chamber (41 ) and the second chamber space (51 ) through the first section of porous material (20), the second chamber (50) comprising a third section of porous material (70), having an outer surface (701 ) being in contact with outer space (423), and an inner surface (702) being in contact with the second chamber space (51 ), allowing liquid communication between the second chamber space (51 ) and the outer space

(423) through the said third section of porous material (70),

- a third chamber (60) having a third chamber space (61 ), the third chamber space (61 ) being in contact with the outer surface (301 ) of the second section of porous material (30), the third chamber (60) comprising a fourth section of porous material (80), having an outer surface (801 ) being in contact with outer space

(424) , and an inner surface (802) being in contact with the third chamber space (61 ), allowing liquid communication between the third chamber space (61 ) and the outer space (424) through the said fourth section of porous material (80).

20. Device (B) according to claim 19, wherein the first, second and third chambers (40, 50, 60) each comprise a first chamber opening (431 , 531 , 631), allowing liquid to enter the respective chamber (40, 50, 60).

21 . Device (B) according to claim 20, wherein one or more of the first chamber openings (40, 50, 60) are sealable.

22. Device (B) according to claim 21 , wherein more than one of the first chamber openings (40, 50, 60) are differentially sealable.

23. Assembly comprising a plurality of devices of any of claims 1 - 18.

24. Assembly according to claim 23, wherein at least one chamber is free of a second section of porous material.

25. Assembly according to claim 23 or 24, comprising a liquid passage(721) having a liquid inlet (731 ) and a liquid outlet (735), the outer surfaces (201 ) of the first sections (200) of the porous material of the plurality of chambers (710) being in contact with the liquid passage (721 ) , allowing liquid communication between the plurality of chambers (710) and the liquid passage (721 ) through the porous material (800).

26. Assembly according to claim 25, wherein the liquid passage (721 ) comprises a passage chamber.

27. Assembly according to any of the claims 23 - 26, wherein the inner surfaces (202) of the first sections of porous material (200) of the plurality of chambers (71 1 ) are arranged in a single plane.

28. Assembly according to any of the claims 23 - 27, comprising a sheet of impermeable material (71 ) comprising perforations (710) of equal size, which sheet is mounted on an inner surface (802) of a sheet (800) of porous material having an inner surface (802) and an outer surface (801 ) and a porosity allowing liquid passage over the said sheet (800) of porous material, the outer surface (801 ) of the said sheet of porous material being in contact with the passage (721 ), wherein the perforations define the plurality of chambers (710).

29. Method for assessment of the effect of medium on microbial growth using the device of any of the preceding claims, comprising the steps of:

a) allowing a first liquid medium to enter the first chamber space (41 ), b) sealing the first chamber (40),

c) incubating the sealed chamber (40) in a second liquid medium, d) allowing microbes from the second liquid medium to settle and grow on the outer surfaces (201 , 301) of the first and second sections (20, 30) of porous material,

e) analyse growth of microbes on the first and second sections (20, 30) of porous material, and assess the difference in growth on the first and second sections (20, 30) of porous material.

30. Method according to claim 29, wherein the composition of the first medium is known.

31 . Method according to claim 29 or 30 for assessment of the effect of one or more one or more factors or a mixture thereof, present in a first liquid medium, on growth of microbes present in a second liquid medium, wherein the first liquid medium is allowed to enter the first chamber space (41 ), and the chamber after sealing is incubated in the second liquid medium.

32. Method according to claim 31 , wherein the factors are chosen from chemical compounds, microbes or a mixture of two or more thereof.

33. Method according to claim 31 or 32, wherein the factor(s) is/are hindered from equilibration over the first section (20) of porous material with the second medium during the incubation step.

34. Method for assessment of the effect of liquid medium on microbial growth using the device (B) of any of the claims 19 - 22, comprising the steps of: a) allowing a first liquid medium to flow through the first chamber space (41 ), the outer surfaces (201 , 301 ) of the first and second sections (20, 30) of porous material being in contact with a second liquid medium,

b) allowing microbes from the second liquid medium to settle and grow on the outer surfaces (201 , 301) of the first and second sections (20, 30) of porous material,

c) analyse growth of microbes on the first and second sections (20, 30) of porous material, and assess the difference in growth on the first and second sections (20, 30) of porous material.

35. Method for assessment of the effect of liquid medium on microbial growth using the device (B) of any of the claims 19 - 22, comprising the steps

a) allowing a first liquid medium to enter the first chamber space (41 ), b) sealing the first chamber,

c) allowing a second liquid medium to enter the second and third chamber spaces (51 , 61),

d) sealing the second and third chambers (50, 60)

e) incubating the device in a third liquid medium,

f) allowing microbes from the third liquid medium to settle and grow on the outer surfaces (701 , 801 ) of the third and fourth sections (70, 80) of porous material, g) analyse growth of microbes on the third and fourth sections (70, 80) of porous material, and assess the difference in growth on the third and fourth sections (70, 80) of porous material.

36. Method according to claim 35, wherein the second liquid medium comprises microbes.