Methods, devices, kits and uses thereof

Abstract

The invention provides a method for obtaining one or more supporting bacteria for a bacterial composition. The invention further provides devices and kits, which may be used in such methods, and uses of such devices and kits. Such supporting bacteria may be suitable for formation of synthetic bacterial consortia.

Description

[0001] METHODS, DEVICES, KITS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] This patent application pertains to the field of microbiology, specifically to synthetic bacterial consortia.

[0004] BACKGROUND

[0005] The human and plant microbiomes are complex communities of microorganisms that play a pivotal role in the health, disease, growth, and development of the host. While single species bacterial inoculation has been used extensively in agriculture, medicine and industry, many promising bacterial strains are unable to perform their function in certain environments or are not culturable when applied alone.

[0006] Synergistic bacterial consortia represent groups of microorganisms that interact to enhance each other's function or promote survival of the consortia. These consortia benefit from collective metabolic activities and interactions and are thus promising candidates to replace single bacterial inoculation.

[0007] Previous strategies for generating synthetic bacterial consortia have relied on combinatorial culture of two or more isolated and characterised bacterial strains. However, screening a large number of possible combinations of isolated bacteria is time consuming and laborious. Recent approaches have used rationally and / or algorithmically guided approaches to combine specific bacterial strains based on taxonomical, genomic or phenotypic information with moderate success. However, obtaining and analysing such information for each bacterial strain is also laborious, and success is largely dependent on the assumptions and data used to select specific combinations of bacterial strains or to train the specific algorithms to determine the selections. As with most biological systems, bacterial consortia are complex, and so predictions based on such data are rarely successful.

[0008] There is thus an unmet need for a high throughput method of identifying suitable candidate bacterial strains which may generate synthetic bacterial consortia, specifically wherein the bacteria interact synergistically. Specifically, a method that does not exclusive rely on combinatorial culture of bacterial strains, does not require selection based on difficult to obtain and analyse data, and can select specific bacterial strains based on naturally occurring interactions that reflect the complexities of bacterial consortia. SUMMARY OF THE INVENTION

[0009] Bacteria are often affected or dependent on other species in their immediate proximity, and different bacterial strains is such proximity which interact synergistically can form synergistic bacterial consortia. Such synergistic bacterial consortia benefit from collective metabolic activities to enhance one or more functions of its constituent bacterial strains, typically to promote survival of the consortia.

[0010] Without wanting to be bound by theory, the present invention is based on the realisation that bacterial strains which migrate towards each other and spatially co-localize are likely to interact, such interactions possibly being synergistic. Accordingly, by identifying bacterial strains which migrate towards a bacterial strain of interest, such as a bioactive bacterial strain, it is possible to obtain supporting bacteria which interact with the bacterial strain, possibly synergistically, and which may improve one or more biological functions of that bacterial strain.

[0011] Without being bound by theory, the presence of the bacterial strain of interest, such as the bioactive bacterial strain, may be used to apply selection pressure to other bacterial strains. Bacterial strains which overcome such selection pressure and migrate towards the bacterial strain of interest, are thus promising candidates for supporting bacterial which may interact with the bacterial strain of interest, possible synergistically.

[0012] An objective of the invention is thus to provide a method for obtaining supporting bacteria, and thus identifying suitable candidate bacterial strains which may generate synergistic bacterial consortia, possibly together with one or more bacterial strains of interest, such as bioactive bacterial strains.

[0013] Against this background, the inventors have developed a method for obtaining one or more supporting bacteria for a bacterial composition, wherein said supporting bacteria migrate towards a bacterial strain of interest, such as a bioactive bacterial strain.

[0014] A first aspect of the invention relates to a method for obtaining one or more supporting bacteria for a bacterial composition, the method comprising the following steps:

[0015] (i) providing a bacterial composition comprising at least one bioactive bacterial strain in a first position;

[0016] (ii) providing a sample comprising a plurality of different bacteria in a second position; wherein said first and said second positions are separated by a space that is essentially free from bacteria; wherein said first and said second positions are separated by at least one semi- permeable membrane;

[0017] (iii) sampling secondary from said plurality of different bacteria that migrate from said second position towards said first position, thereby obtaining one or more supporting bacteria.

[0018] A second aspect of the invention relates to a device which may be used as part of the method of the first aspect of the invention. Specifically, the second aspect of the invention provides a device for obtaining one or more supporting bacteria for a bacterial composition, the device comprising:

[0019] (i) a first compartment comprising at least one entry opening, said at least one entry opening being exposed on the surface of the device;

[0020] (ii) a second compartment within said first compartment; and

[0021] (iii) at least one inner opening connecting said first compartment with said second compartment; wherein said inner opening comprises a first semi-permeable membrane separating said first compartment and said second compartment.

[0022] A third aspect of the invention provides the use of a device according to the second aspect of the invention for obtaining one or more supporting bacteria for a bacterial composition, the use comprising:

[0023] (i) providing a bacterial composition comprising at least one bioactive bacterial strain in the second compartment of said device;

[0024] (ii) providing sterile medium in the first compartment;

[0025] (iii) placing said device in a sample comprising a plurality of different bacteria, such that the sample contacts the surface of the device; and

[0026] (iv) sampling secondary from said plurality of different bacteria that migrate into said first compartment from said sample comprising a plurality of different bacteria, thereby obtaining one or more supporting bacteria.

[0027] A fourth aspect of the invention provides a kit comprising the device according to the third aspect of the invention and instructions for performing the method according to the first aspect of the invention or the use according to the third aspect of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] Definitions

[0029] As used herein the term "isolating" refers to the separation of one or more bacterial strains from a mixed bacterial population or from a sample comprising said one or more bacterial strains and other components. Accordingly, the term "co-isolation", as used herein, refers to the separation of two or more bacterial strains, where the two or more bacterial strains are separated concomitantly.

[0030] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%.

[0031] The term "bioactive bacterial strain" or "bioactive strain" is to be understood as a bacterial strain with a known biological property (i.e., bioactivity). Such property may include any property inherent to the bacterial strain (such as production of metabolites, antagonism against other organisms, synergism with other organisms, fixation or solubilization of nutrients) or direct and indirect effects on other organisms, such as a host organism.

[0032] As used herein, the term "secondary bacteria" refers to any bacteria or bacterial strain originating or obtained from the plurality of bacteria that migrates towards a bacterial strain or other agent providing certain selection pressure. Accordingly, "secondary bacteria" are bacteria of interest, which as part of the method of the invention are sampled and, optionally, tested for synergism with, for example, the bioactive bacterial strain.

[0033] As used herein, the term "sampled bacteria", "sampled secondary bacteria" or "sampled migrating bacteria", refers to any bacteria or bacterial train that is picked, isolated or otherwise selected from the sample comprising a plurality of different bacteria.

[0034] As used herein, the term "supporting bacteria", "support bacteria" or "supporting bacterial strains" is to be understood as referring to a bacterial strain which is capable of amplifying, stabilizing, prolonging, increasing, maintaining, or otherwise improving a biological property of another bacterial strain (for example the bioactive bacterial strain) when in the presence of said bacterial strain. Additionally or alternatively, the bacterial strain may also be capable of adding one or more biological properties to a co-culture comprising said bacterial strain and another bacterial strain. The biological property may be inherent or brought about by external factors, it may be a biological property exerted on the bacterial strain itself (such as survival, fitness, metabolism, defence, colonization of a host, tolerance and the like) or exerted on another organism, for example a host organism. Alternatively, the supporting bacteria may be compatible with the bioactive bacterial strain. By compatible it should be understood that the bioactive bacterial strain and the supporting bacteria do not decrease, inhibit or otherwise negatively interfere with growth and / or at least one biological property of the other bacterial strain. It should be understood that mention of "supporting bacteria" herein also refers generally to any bacteria which is obtained by any of the methods described herein. Accordingly, supporting bacteria may also be referred to as "obtained bacteria" or "sampled bacteria".

[0035] "Supporting bacteria" may improve the performance of the bioactive bacterial strain and / or themselves when in close proximity to the bioactive bacterial strain. "Supporting bacteria" may also provide desired complementary characteristics to the characteristics of the bioactive bacterial strain. Accordingly, the term "supporting bacteria" may be used interchangeably with "beneficial bacteria", "complementary bacteria", "commensal bacteria" or "mutualistic bacteria".

[0036] As used herein, "free from bacteria" or "essentially free from bacteria" is to be understood as a surface, volume, area, space or object that does not comprise any living bacterial cells or that comprises an abundance of bacterial cells which is below that which can be measured by conventional techniques and / or those techniques currently known in the art.

[0037] The term "semi-permeable membrane" as used herein should be understood as encompassing any surface displaying any level of selective permeability. This selective permeability may largely be determined by the size and / or diameter of the pores in the membrane. It will be appreciated that the pore size dictates the size of the molecules or ions that can pass through, with smaller, molecules typically able to pass through smaller pores.

[0038] The term "migrate" or "migration" is used herein to encompass the direction of growth of a bacterial culture or a particular bacterial strain, which may be part of a culture. As such, the term is not limited to the strict literal definition of movement of a single bacterial cell. Instead, it encompasses the direction in which a bacterial strain grows during multiplication and colony formation on a surface or volume. By way of example, such direction may be determined by favourable conditions at a position (position A), such as improved nutrient availability, allowing enhanced multiplication on a specific surface at position A and / or poor nutrient availability or presence of unfavourable conditions at a second position (position B), leading to decreased multiplication or bacterial death at that second position. In such an example, the bacteria would be understood as migrating towards position A, but not towards position B.

[0039] As used herein the term "synergistic bacterial consortia" or "synergistic bacterial communities", refer to a bacterial composition comprising two or more bacterial species or bacterial strains, wherein at least one biological function or biological effect of at least one bacterial species or strain is improved compared to that of the bacterial species or strain in isolation.

[0040] As used herein, the term "biological function" or "biological effect" are to be understood as any characteristic, function, activity, action, effect or the like of an organism. Such characteristic, function, activity, action, effect or the like may be exerted on the organism itself (for example growth, survival, resistance etc. of the organism) or may be exerted on a separate organism (for example by direct or indirect contact with said organism; such defence or immune induction, growth or survival of the separate organism).

[0041] As used herein, "volume" should be understood as any three-dimensional space. In the context of the methods of the invention, the first and / or second volume may be liquid volumes, gaseous volumes or solid volumes. As an example, the first and / or second volumes may be liquid volumes held in one or more containers or distinguishably placed on a surface, such as liquid droplets. Such containers may for example be, any such used in experimental work, such as reagent tubes, wells in a microtiter plate.

[0042] As used herein, the term "natural microbial sample(s)" is to be understood as a sample obtained directly from a natural environment. The term thus encompasses all types and samples, which are found in nature, including samples of natural origin which have been processed or altered by human action, such as processing of foodstuffs, or production of goods.

[0043] The present specification encompasses various descriptions pertaining to the dimensions of entities such as particles, bacterial cells, aggregates, or other objects. These dimensions, unless explicitly stated otherwise, should be interpreted as the length at the point of maximum extension. This point of maximum extension is typically along the longest axis of the entity in question. Accordingly, it should be noted that the term "size", as used herein, refers to the greatest linear distance between any two points on the surface of the entity, measured along a straight line passing through the centre of the entity, and extending from one surface point to another.

[0044] In the description of the invention various embodiments and / or individual components are disclosed. As will be apparent to the ordinarily skilled practitioner, all combinations of such embodiments and components taught in the disclosure are possible and can result in preferred embodiments of the present invention.

[0045] Any percentages and ratios are calculated by weight unless otherwise indicated. All percentages, parts and ratios are calculated based on the total composition unless otherwise indicated.

[0046] Detailed description of the invention

[0047] Bacteria are often affected or dependent on other species in their immediate proximity, and different bacterial strains is such proximity which interact synergistically can form synergistic bacterial consortia. Without wanting to be bound by theory, the present invention is based on the realisation that bacterial strains which migrate towards each other and spatially co-localize are likely to interact, such interactions possibly being synergistic. Accordingly, by identifying bacterial strains which migrate towards a bacterial strain of interest, such as a bioactive bacterial strain, it is possible to obtain supporting bacteria which interact with the bacterial strain, possibly synergistically, and which may improve one or more biological functions of that bacterial strain. The presence of the bacterial strain of interest, such as the bioactive bacterial strain, may thus be used to apply selection pressure to other bacterial strains. Bacterial strains which overcome such selection pressure and migrate towards the bacterial strain of interest, are thus promising candidates for supporting bacterial which may interact with the bacterial strain of interest, possible synergistically.

[0048] The present invention provides a method for obtaining one or more supporting bacteria for a bacterial composition, wherein said supporting bacteria migrate towards a bacterial strain of interest, such as a bioactive bacterial strain. By obtaining supporting bacteria according to the method of the invention the chance of obtaining a supporting bacterial strain which interacts synergistically with the one or more bacterial strains of interest (such as the bioactive bacterial strains) is increased. Previous strategies for generating synergistic bacterial consortia have relied on combinatorial culture of two or more isolated and characterised bacterial strains, which is time consuming and laborious. Recent approaches have also used rationally and / or algorithmically guided approaches to combine specific bacterial strains based on taxonomical, genomic or phenotypic information with moderate success. However, obtaining and analysing such information for each bacterial strain is also laborious, and success is largely dependent on the assumptions and data used to select specific combinations of bacterial strains or to train the specific algorithms to determine the selections. As with most biological systems, bacterial consortia are complex, and so predictions based on such data are rarely successful.

[0049] The method of the invention thus represents a fast, high throughput workflow to for obtaining one or more supporting bacteria for a bacterial composition compared to single species isolation and subsequent combinatorial testing. Additionally, the method of the invention does not rely on taxonomical, genomic or phenotypic data, and rational or algorithmic selection of bacterial strains for testing but uses the bacterial strain of interest (such as the bioactive strain) as selection pressure on a plurality of bacterial strains to obtain bacterial strains that overcome such pressure to migrate towards the bacterial strain of interest. Such selection is thus governed by natural selection and more closely resembles microbial interactions in natural environments.

[0050] A first aspect of the invention provides a method for obtaining one or more supporting bacteria for a bacterial composition, the method comprising the following steps:

[0051] (i) providing a bacterial composition comprising at least one bioactive bacterial strain in a first position;

[0052] (ii) providing a sample comprising a plurality of different bacteria in a second position; wherein said first and said second positions are separated by a space that is essentially free from bacteria; wherein said first and said second positions are separated by at least one semi-permeable membrane;

[0053] (iii) sampling secondary bacteria from said plurality of different bacteria that migrate from said second position towards said first position, thereby obtaining one or more supporting bacteria.

[0054] In some embodiments, step (iii) comprises sampling secondary bacteria from said plurality of different bacteria that migrate from said second position to said first position. Another aspect of the invention provides a method for obtaining one or more supporting bacteria for a bacterial composition, the method comprising the following steps:

[0055] (i) providing a bacterial composition comprising at least one bioactive bacterial strain in a first position;

[0056] (ii) providing a sample comprising a plurality of different bacteria in a second position; wherein said first and said second positions are separated by a space that is essentially free from bacteria; wherein said first and said second positions are separated by at least one semi-permeable membrane;

[0057] (iii) sampling secondary bacteria from said plurality of different bacteria that migrate from said second position to said first position, thereby obtaining one or more supporting bacteria.

[0058] Said bioactive bacterial strain and said sample comprising a plurality of different bacteria are separated by at least one semi-permeable membrane. The semi-permeable membrane may avoid mixing of the bioactive bacterial strain and the migrating secondary bacteria, which may facilitate further downstream isolating, characterization and maintenance of the sampled secondary bacteria. Additionally or alternatively, the semi-permeable membrane may facilitate maintaining the bioactive strain in said first position, in order to have a fixed reference point to be used when sampling said secondary bacteria. Additionally or alternatively, said semi-permeable membrane may also function as a scaffold to which said migrating secondary bacteria may attach or be contacted to, and which facilitates sampling said secondary bacteria by sampling directly from said semi-permeable membrane. Accordingly, it may be advantageous for said at least one semi-permeable membrane to be configured to exclude passage of bacterial cells. Thus, in some embodiments, at least one semi-permeable membrane is configured to exclude passage of bacterial cells.

[0059] One skilled in the art will appreciate that, the selection pressure produced by said bioactive strain may encompass, for example, bacterial metabolites, including proteins, nucleic acids, or viral particles. Such bacterial metabolites may only produce such selection pressure by being diffused out of said first position and towards said second position, where they may contact the sample comprising a plurality of different bacterial cells. Accordingly, to reach said second position and produce said selection pressure, such bacterial metabolites must be able to cross said semi-permeable membrane. Accordingly, in some embodiments said at least one semi-permeable membrane is configured to allow for passage of bacterial metabolites. In a particular embodiment, said at least one semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

[0060] It will be appreciated that in order to exclude passage of bacterial cells and allow for passage of bacterial metabolites, the semi-permeable membrane must have a pore diameter larger than said metabolites and smaller than said bacterial cells.

[0061] Accordingly, in some embodiments, at least one semi-permeable membrane has a pore diameter of about 0.1 pm to about 1 pm, such as about 0.1 pm to about 1 pm, about 0.15 pm to about 0.9 pm, about 0.15 pm to about 0.8 pm, about 0.15 pm to about 0.7 pm, about 0.15 pm to about 0.6 pm, about 0.15 pm to about 0.5 pm, about 0.15 pm to about 0.45 pm, about 0.2 pm to about 0.45 pm, about 0.2 pm to about 0.4 pm, or about 0.2 pm to about 0.3 pm, or about 0.15 pm to about 0.25 pm. In a preferred embodiment, at least one semi-permeable membrane has a pore diameter of about 0.2 pm to about 0.45 pm. In a further preferred embodiment, at least one semi-permeable membrane has a pore diameter of about 0.4 pm to about 0.5 pm.

[0062] In some additional or alternative embodiments, at least one semi-permeable membrane has a pore diameter of less than about 2um, such as less than about 2 pm, less than about 1.95 pm, less than about 1.9 pm, less than about 1.85 pm, less than about 1.8 pm, less than about 1.75 pm, less than about 1.7 pm, less than about 1.65 pm, less than about 1.6 pm, less than about 1.55 pm, less than about 1.5 pm, less than about 1.45 pm, less than about 1.4 pm, less than about 1.35 pm, less than about 1.3 pm, less than about 1.25 pm, less than about 1.2 pm, less than about 1.15 pm, less than about 1.1 pm, less than about 1.05 pm, less than about 1 pm, less than about 0.95 pm, less than about 0.9 pm, less than about 0.85 pm, less than about 0.8 pm, less than about 0.75 pm, less than about 0.7 pm, less than about 0.65 pm, less than about 0.6 pm, less than about 0.55 pm, less than about 0.5 pm, less than about 0.45 pm, less than about 0.4 pm, less than about 0.35 pm, less than about 0.3 pm, less than about 0.25 pm, less than about 0.2 pm, less than about 0.15 pm, or less than about 0.1 pm. In a preferred embodiment, at least one semi- permeable membrane has a pore diameter of less than about 0.45 pm. In a further preferred embodiment, at least one semi-permeable membrane has a pore diameter of less than about 0.5 pm.

[0063] As previously described, the semi-permeable membrane may facilitate maintaining the bioactive strain in said first position, in order to have a fixed reference point to be used when sampling said migrating secondary bacteria. Additionally or alternatively, said semi-permeable membrane may also function as a scaffold to which said migrating secondary bacteria may attach or be contacted to. One skilled in the art will appreciate, that to the abovementioned end, it may be advantageous for at least one semi- permeable membrane to be in contact or contiguous to said first position. Accordingly, in a particular embodiment, at least one semi-permeable membrane is contiguous to said first position.

[0064] It may be advantageous, but not necessary, to separate said first position and said second position by at least two semi-permeable membranes. One skilled in the art will appreciate that an additional semi-permeable membrane may be used, as described previously as a scaffold to which said migrating secondary bacteria may attach or be contacted to. Additionally or alternatively, said additional semi-permeable membrane may be used as an obstacle to said migrating secondary bacteria, for example by impeding, but not excluding, passage of bacterial cells. Such a use of an additional semi-permeable membrane may be advantageous to increase the likelihood that the migrating secondary bacteria that migrate past said additional semi-permeable membrane, and which are sampled in step (iii) are subject to selection pressure by the bioactive strain. By way of example, bacteria may migrate from said second position towards said first position without being subjected to selection pressure by the bioactive strain, for example due to nutrient availability, predation or parasitism of the bioactive bacterial strain, and / or lack of competition in said space between said first position and second position, or otherwise due to the aleatory and / or stochastic nature of bacterial multiplication and colony formation. Accordingly, if said migrating secondary bacteria are subjected to strong selection pressure in favour of migrating towards said first position, they will be able to migrate through said additional filter, but bacteria which are not subject to said selectin pressure in favour of migrating towards said position are less likely to migrate through said additional filter. It will further be appreciated that an additional semi-permeable membrane may be advantageous when the method is carried out in a liquid medium or a minimally viscous medium, as the additional semi-permeable membrane may prevent excessive non-selection pressure driven migration of secondary bacteria towards the first position (i.e., by stochastic migration due to Brownian motion or dilution). Accordingly, addition of the additional semi-permeable membrane increases the likelihood of the secondary bacteria migrating due to selection pressure.

[0065] Accordingly, in some embodiments said first and second positions are separated by at least a first semi-permeable membrane and a second semi-permeable membrane, wherein the distance between said first position and said first semi-permeable membrane is less than the distance between said first position and said second semi-permeable membrane. In a particular embodiment, said first semi-permeable membrane is contiguous to said first position and / or said second semi-permeable membrane is contiguous to said second position. In a preferred embodiment, said first semi-permeable membrane is contiguous to said first position and said second semi-permeable membrane is contiguous to said second position.

[0066] As detailed previously, it may be advantageous for at least one semi-permeable membrane to allow for passage of bacterial metabolites and exclude passage of bacterial cells. It will be appreciated that if two or more semi-permeable membranes are present, it will be advantageous for said first semi-permeable membrane that is closest to said first position, to allow for passage of bacterial metabolites and exclude passage of bacterial cells. Accordingly, in some embodiments, said first semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

[0067] It will be appreciated that in order to exclude passage of bacterial cells and allow for passage of bacterial metabolites, said first semi-permeable membrane must have a pore diameter larger than said metabolites and smaller than said bacterial cells.

[0068] In some embodiments, said first semi-permeable membrane has a pore diameter of about 0.1 pm to about 1 pm, such as about 0.1 pm to about 1 pm, about 0.15 pm to about 0.9 pm, about 0.15 pm to about 0.8 pm, about 0.15 pm to about 0.7 pm, about 0.15 pm to about 0.6 pm, about 0.15 pm to about 0.5 pm, about 0.15 pm to about 0.45 pm, about 0.2 pm to about 0.45 pm, about 0.2 pm to about 0.4 pm, or about 0.2 pm to about 0.3 pm, or about 0.15 pm to about 0.25 pm. In a preferred embodiment, said first semi- permeable membrane has a pore diameter of 0.2 pm to about 0.45 pm. In a further preferred embodiment, said first semi-permeable membrane has a pore diameter of about 0.4 pm to about 0.5 pm.

[0069] In an additional or alternative embodiment, said first semi-permeable membrane has a pore diameter of less than about 2um, such as less than about 2 pm, less than about 1.95 pm, less than about 1.9 pm, less than about 1.85 pm, less than about 1.8 pm, less than about 1.75 pm, less than about 1.7 pm, less than about 1.65 pm, less than about 1.6 pm, less than about 1.55 pm, less than about 1.5 pm, less than about 1.45 pm, less than about 1.4 pm, less than about 1.35 pm, less than about 1.3 pm, less than about 1.25 pm, less than about 1.2 pm, less than about 1.15 pm, less than about 1.1 pm, less than about 1.05 pm, less than about 1 pm, less than about 0.95 pm, less than about 0.9 pm, less than about 0.85 pm, less than about 0.8 pm, less than about 0.75 pm, less than about 0.7 pm, less than about 0.65 pm, less than about 0.6 pm, less than about 0.55 pm, less than about 0.5 pm, less than about 0.45 pm, less than about 0.4 pm, less than about 0.35 pm, less than about 0.3 pm, less than about 0.25 pm, less than about 0.2 pm, less than about 0.15 pm, or less than about 0.1 pm. In a preferred embodiment, at least one semi-permeable membrane has a pore diameter of less than about 0.45 pm. In a further preferred embodiment, said first semi-permeable membrane has a pore diameter of less than about 0.5 pm.

[0070] As detailed previously, said second semi-permeable membrane may be used as an obstacle to said migrating secondary bacteria, for example by impeding, but not excluding, passage of bacterial cells. Such second semi-permeable membrane may be advantageous to increase the likelihood that the migrating secondary bacteria that migrate past said second semi-permeable membrane, and which are sampled in step (iii) are subject to selection pressure by the bioactive strain. For example, if said migrating secondary bacteria are subjected to strong selection pressure in favour of migrating towards said first position, they will be able to migrate through said additional filter, but bacteria which are not subject to said selectin pressure in favour of migrating towards said position are less likely to migrate through said additional filter.

[0071] Accordingly, in some embodiments, said second semi-permeable membrane is configured to impede passage of bacterial cells.

[0072] It will be appreciated that in order to impede passage of bacterial cells, said second semi-permeable membrane must have a pore diameter larger than at least the smallest dimension of said bacterial cells, but less than a pore diameter that allows bacterial strains to migrate without being impeded.

[0073] Accordingly, in some embodiments, said second semi-permeable membrane has a pore diameter of about 5 pm to about 200 pm, such as about 5 pm to about 200 pm, about 5 pm to about 150 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, about 20 pm to about 100 pm, about 30 pm to about 100 pm, about 50 pm to about 100 pm, about 10 pm to about 40 pm, or about 10 pm to about 30 pm. In some embodiments, said second semi-permeable membrane has a pore diameter of about 10 pm to about 20 pm.

[0074] In some additional or alternative embodiments, said second semi-permeable membrane has a pore diameter greater than 50 pm, such as greater than about 50 pm, greater than about 60 pm, greater than about 70 pm, greater than about 80 pm, greater than about 90 pm, greater than about 100 m, greater than about 110 pm, greater than about 120 pm, greater than about 130 pm, greater than about 140 pm, greater than about 150 pm, greater than about 160 pm, greater than about 170 pm, greater than about 180 pm, greater than about 190 pm, or greater than about 200 pm. In some embodiments, said second semi-permeable membrane has pore diameter greater than 10 pm, such as greater than 10 pm, greater than 15 pm, greater than 20 pm, greater than 30 pm, greater than 40 pm or greater than 50 pm.

[0075] In an alternative embodiment, said second semi-permeable membrane is configured to allow passage of bacterial cells.

[0076] It will be appreciated that, as described above, the preferred pore size of the first and / or second semi-permeable membrane may vary according to the conditions in which the method is carried out and the nature of the one or more bioactive bacterial strains and / or of the sample comprising a plurality of bacterial strains. By way of example, it may be advantageous when the method is carried out in a liquid medium or a minimally viscous medium, for said second semi-permeable membrane separating said sample comprising a plurality of bacterial strains and said space essentially free from bacteria to have a pore size which impedes excessive non-selection pressure driven migration of secondary bacteria towards the first position (i.e., by stochastic migration due to Brownian motion or dilution). Alternatively, in particulate matter samples (e.g., soil samples) it may in turn be advantageous for said semi-permeable membrane to have a larger pore-size to allow for increased migration of said migrating secondary bacteria.

[0077] Accordingly, in some embodiments wherein the method is carried out in liquid or minimally viscous liquid, said second semi-permeable membrane has a pore size of less than 100 pm, such as less than 100 pm, less than 90 pm, less than 80 pm, less than 70 pm, less than 60 pm, less than 50 pm, less than 40 pm, less than 40 pm, less than 30 pm, less than 20 pm, less than 15 pm or less than 10 pm. In a preferred embodiment, less than 15 pm. In some alternative embodiments wherein the method is carried out in particular matter, solid matter and / or in soil, said second semi-permeable membrane has a pore diameter greater than 50 pm, such as greater than about 50 pm, greater than about 60 pm, greater than about 70 pm, greater than about 80 pm, greater than about 90 pm, greater than about 100 pm, greater than about 110 pm, greater than about 120 pm, greater than about 130 pm, greater than about 140 pm, greater than about 150 pm, greater than about 160 pm, greater than about 170 pm, greater than about 180 pm, greater than about 190 pm, or greater than about 200 pm. It will be appreciated that the distance between said first and second semi-permeable membranes may determine the available distance that said migrating secondary bacteria can migrate, particularly in certain embodiments wherein said first and second semi- permeable membranes are contiguous to said first and second positions. It may thus be advantageous for said first and second semi-permeable membrane to be at a distance that allows for selection pressure from said bioactive bacterial strain to effect said migrating secondary bacteria. This may, for example, be the distance to which a sufficient concentration of bacterial metabolites can diffuse to. One skilled in the art will appreciate that such distance that allows for selection pressure from said bioactive strain will also be dependent on other factors, such as the nature of the bioactive bacterial strain, the amount of time said bioactive bacterial strain is cultured in said first position and the nature of the environment in and around said first and second positions.

[0078] In some embodiments, the distance between said first and said second semi-permeable membrane is less than 500 mm, such as about 500 mm, about 475 mm, about 450 mm, about 425 mm, about 400 mm, about 375 mm, about 350 mm, about 325 mm, about 300 mm, about 275 mm, about 250 mm, about 225 mm, about 200 mm, about 175 mm, about 150 mm, about 125 mm, about 100 mm, about 75 mm, about 50 mm, about 25 mm, about 15 mm, about 10 mm, about 5 mm, about 2 mm or about 1 mm.

[0079] Similarly, one skilled in the art will appreciate that the distance between said first and second position may determine the distance said migrating secondary bacteria can migrate and the distance said selection pressure from said bioactive bacterial strain may affect said secondary bacteria.

[0080] One skilled in the art will appreciate that the method of the invention may be carried out in a variety of set-ups, having different distances between said first and second position, different distances between said first and second semi-permeable membrane and different time said secondary bacteria migrate from said second to said first position before being sampled.

[0081] Accordingly, in some embodiments said first position and said second position are at a distance of about 100 mm to about 1 mm, such as about 100 mm, about 80 mm, about 60 mm, about 50 mm, about 45 mm, about 40 mm, about 35 mm, about 30 mm, about 25 mm, about 20 mm, about 15mm, about 10 mm, about 5 mm, about 2 mm or about 1 mm. It will be appreciated that the methods of the first aspect of the invention may be carried out using standard laboratory equipment commonly used in the field of microbiology. For example, said first position, comprising the bioactive bacterial strain, may be situated in a 15 ml Falcon tube and said second position may be situated outside a 50 ml Falcon tube. Placing said 15 ml Falcon tube inside said 50 ml Falcon tube thus leads to the distance between said first and said second position to be equal to the difference between the radius of said 15 ml Falcon tube and said 50 ml Falcon tube. Accordingly, in a preferred embodiment said first position and said second position are at a distance of about 12 mm. A similar or corresponding arrangement and / or experimental set-up may be prepared using any other standard laboratory equipment, such as centrifugation-, culture- or conical centrifugation tubes, tissue culture plates, or petri dishes.

[0082] It will be appreciated by one skilled in the art, that in order to promote migration and survival of said migrating secondary bacteria, it may be advantageous for said space essentially free from bacteria to comprise components and / or conditions which allow for the growth of bacteria. Accordingly, in some embodiments, said space essentially free from bacteria further comprises bacterial nutrients.

[0083] It will moreover be appreciated that it may be advantageous for such space essentially free from bacteria to comprise, additionally or alternatively, compounds and / or conditions which affect growth, survival and / or migration of certain bacteria differently than other bacteria. This may, for example, be advantageous to apply a secondary selection pressure (in addition to that exerted by the bioactive bacterial strain). Accordingly, it may be possible to select from said migrating secondary bacteria only bacterial strains which can metabolise a specific compound or survive and grow in specific conditions. By way of example, such condition may be the humidity and / or water content of said space essentially free from bacteria. Low humidity and / or water content may select for migrating secondary bacteria, which are tolerant to such conditions (i.e., are drought resistant). Such secondary bacteria may in turn, form synergistic bacterial consortia with said bioactive strain, possibly also having increased drought resistance.

[0084] Accordingly, in some embodiments, said space essentially free from bacteria further comprises one or more compounds which affect growth, survival and / or migration of certain bacteria differently than other bacteria. In one embodiment, said space essentially free from bacteria further comprises one or more compounds selected from the list consisting of an antimicrobial, a host signalling compound, a heavy metal and a contaminant. It will be appreciated that, it may be further advantageous to increase, decrease or otherwise change the aforementioned conditions and / or the concentration of the aforementioned compounds throughout said space essentially free from bacteria. Such difference in conditions and / or concentration may, for example, be advantageous in order to allow for certain growth of migrating secondary bacteria but exclude growth of other bacteria.

[0085] Accordingly, in some embodiments, the concentration of the bacterial nutrients and / or compounds at or contiguous to said first position is different to the concentration at or contiguous to said second position. In a preferred embodiment, the concentration of the bacterial nutrients and / or compounds gradually increases and / or decreases from said first position towards said second position.

[0086] In an additional or alternative embodiment, the pH and / or salinity at or contiguous to said first position is different to the pH and / or salinity at or contiguous to said second position. In a preferred embodiment, the pH and / or salinity gradually increases and / or decreases from said first position towards said second position.

[0087] As previously described, it may be advantageous for said space essentially free from bacteria to allow for growth of bacteria, in order to allow for bacterial migration. It will further be appreciated that, it may also be advantageous for said first and / or said second positions to allow for growth of bacteria, in order to maintain said bioactive bacterial strains and / or said secondary bacteria. Accordingly, in some embodiments, said first position, said second position and / or said space essentially free from bacteria comprise a bacterial growth medium. One skilled in the art will appreciate that bacterial growth media may comprise any substance or matter on or in which bacteria can grow and multiply. Such bacterial growth media may thus be in any state of matter or shape. In some embodiments, the bacterial growth medium is solid, gelatinous, or liquid.

[0088] While it will be appreciated that different bacterial strains may grow on a wide variety of bacterial growth medias, certain medias are commonly used in the field of microbiology. Certain ingredients, conditions, nutrients and the like, which are useful in the preparation of bacterial growth media is detailed in M. Bonnet, J.C. Lagier, D. Raoult, S. Khelaifia. Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology, New Microbes and New Infections, Volume 34, 2020. Specific bacterial growth media are further known in the art and disclosed and catalogued in specific databases, such as the Deutsche Sammlung von Mikroorganismen un Zellkulturen (DSMZ) MediaDive database. In a preferred embodiment, the bacterial growth medium is selected from the group consisting of agar-based growth media, liquid growth media, animal or plant tissue homogenate media, a foodstuff and soil. In a preferred embodiment, the bacterial growth medium is soil.

[0089] In an alternative preferred embodiment, the bacterial growth medium is a foodstuff. In some embodiments, the foodstuff is selected from the group consisting of a dairy product, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume. In a preferred embodiment, the foodstuff is a dairy product. In some embodiments, the foodstuff is selected from the group consisting of milk, whey butter, cheese, yogurt, kefir, cream, buttermilk, sour cream, and quark.

[0090] One skilled in the art will appreciate that the first aspect of the invention provides a method of obtaining supporting bacteria. Without wanting to be bound by theory, such supporting bacteria may be able to form synergistic bacterial consortia with said bioactive bacterial strain. Members of such synergistic bacterial consortia interact to promote survival of the consortia, generally by enhancing biological effects or functions of each member of the consortia. It will thus be appreciated that, at least one biological effect or biological function of the bioactive strain may be increased, decreased, or otherwise changed when said bioactive bacterial strain is in the presence of one or more said supporting bacteria.

[0091] One skilled in the art will appreciate that improving and / or increasing certain biological effects and / or biological functions may be advantageous for the further use and utility of a bacterial consortium comprising said bioactive bacterial strain and said supporting bacteria. By way of example, increasing and / or improving the stability and / or adaptability of the bioactive bacterial strain may be advantageous for the further use of the bacterial consortium, as it would be biologically active for longer and / or biologically active in a broader range of conditions. Additionally or alternatively, improving and / or increasing host colonization success of the bioactive strain may be advantageous to exert one or more functions of the bioactive strain more efficiently, rapidly, successfully or potently in a host organism.

[0092] Accordingly, in one embodiment, the bioactive strain has increased or improved stability in the presence of said one or more supporting bacteria. In an additional or alternative embodiment, the bioactive strain has increased or improved adaptability in the presence of said one or more supporting bacteria. In a further additional or alternative embodiment, the bioactive strain has increased or improved host colonization success in the presence of said one or more supporting bacteria. In a further embodiment, the bioactive bacterial strain has increased or improved nutrient availability, drought tolerance, temperature tolerance, and / or overall fitness in the presence of said one or more supporting bacteria. In an alternative embodiment, the bioactive bacterial strain has increased or improved fermentation capabilities, acidification capabilities, and / or production of one or more aroma and / or flavour compounds in the presence of said one or more supporting bacteria.

[0093] One skilled in the art will further appreciate that the one or more supporting bacteria may have additional and / or different biological effects, functions and / or properties that the bioactive strain, and that these may also be advantageous and / or useful for the use and utility of the bacterial consortium. By way of example, the supporting bacteria may have a fungicidal effect not present in the bioactive bacterial strain, which may be advantageous for use of the consortia comprising said supporting bacteria and said bioactive bacterial strain in agriculture. Accordingly, in some embodiments, the supporting bacteria has one or more biological effects, functions and / or properties not present in the bioactive bacterial strain. In a preferred embodiment, the biological effect, function or property is selected from the group consisting of a biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, pathogen antagonism, plant growth promotion, nitrogen fixation, phosphorous solubilization, plant induced systemic resistance (ISR), plant defence priming, enhanced nutrient cycling, rhizosphere modification, drought tolerance, salinity tolerance, and pathogen exclusion. In some embodiments, the supporting bacteria has one or more biological effects, functions or properties selected from the group consisting of fermentation, acidification, production of one or more aroma compounds, production of one or more flavour compounds, production of one or more nutrients or minerals.

[0094] It will be appreciated that biofilm formation is a known marker of synergy within multistrain bacterial consortia and synergistic effects within a bacterial community have been shown to promote biofilm biomass, resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms. High prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat have been strongly suggested to represent interspecific cooperation in soil bacterial consortium. Moreover, improved or increased biofilm formation of the bacterial consortia, may protect and promote growth of the individual members of the consortia. Accordingly, in a preferred embodiment, the bioactive bacterial strain and said one or more supporting bacteria have increased biofilm formation capabilities when cultured or applied together, compared to the biofilm formation capability of the bioactive bacterial strain and the one or more supporting bacteria cultured or applied separately. One skilled in the art will appreciate that the bioactive bacterial strain may have or produce a biological property, such as a biological effect or function, that is exerted on a separate organism, for example by direct or indirect contact with said organism. Such biological property may be characteristic and / or inherent of said bioactive strain or may be obtained, for example through interaction with other members of a bacterial consortia. In some embodiments, at least one biological property of the bioactive strain is increased or improved in the presence of said one or more supporting bacteria. In one embodiment, the at least one biological property is selected from the group consisting of improved growth, yield, defence, robustness and / or tolerance of a plant that is contacted with said first strain. In an additional or alternative embodiment, the at least one biological property is selected from the group consisting of improved growth, yield, defence, robustness and / or tolerance of a crop that is contacted with said first strain.

[0095] In an additional or alternative embodiment, the at least one biological property is selected from the list consisting of a biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, pathogen antagonism, plant growth promotion, nitrogen fixation, phosphorous solubilization, plant induced systemic resistance (ISR), plant defence priming, enhanced nutrient cycling, rhizosphere modification, drought tolerance, salinity tolerance, and pathogen exclusion.

[0096] In a further additional or alternative embodiment, the at least one biological property selected from the list consisting of bioremediation, biodegradation, fermentation, biocatalysis, acidification, a probiotic effect, aroma development, flavour development, food texturization, bioprotection effect and stability.

[0097] One skilled in the art will further appreciate that the bioactive bacterial strain may have or produce a biological property, such as a biological effect or function, that is exerted on a foodstuff composition, for example by direct or indirect contact with said foodstuff. Such biological property may be characteristic and / or inherent of said bioactive strain or may be obtained, for example through interaction with other members of a bacterial consortia. In some embodiments, at least one biological property of the bioactive strain is increased or improved in the presence of said one or more supporting bacteria. In one embodiment, the at least one biological property is selected from the group consisting of improved or increased fermentation, acidification, production of one or more aroma compounds, production of one or more flavour compounds, production of one or more nutrients or minerals. In some embodiments, the foodstuff is a dairy product.

[0098] One skilled in the art will further appreciate that such biological properties of a bacterial strain may be increased, decreased or otherwise changed in the presence of one or more different bacteria directly (i.e., through contacting) or indirectly (i.e., through release biological agents or changes in the environmental conditions). In one embodiment, the at least one biological property of the bioactive bacterial strain is increased or improved when contacted with said one or more supporting bacteria. In an additional or alternative embodiment, the at least one biological property of the bioactive bacterial strain is increased or improved when contacted with a biological agent secreted and / or produced by said one or more supporting bacteria. In a preferred embodiment, the biological agent is a metabolite, a protein, a nucleic acid, or a viral particle.

[0099] It will be appreciated by one skilled in the art that the bioactive bacterial strain may comprise any bacterial strain. It will further be appreciated that, it may be advantageous for said bioactive bacterial strain to have and / or be capable of one or more specific biological effects or properties, such as those described above. In performing the method of the first aspect of the invention, it may thus be advantageous to select a bacterial strain with known or theorised specific biological effects or properties. By way of example, the bioactive bacterial strain may be known to be particularly useful in some process, such as an industrial, manufacturing, agricultural or medicinal process, or to treat or prevent one or more specific diseases in a subject. The bioactive strain may thus, be a well characterised bacterial strain, and may also have been isolated. Accordingly, in one embodiment, said bioactive bacterial strain is a bacterial isolate.

[0100] It will further be appreciated that certain applications or uses of a bacterial consortium comprising said bioactive strain may preferably not include bacteria that are or are suspected to be pathogenic. For example, uses in medicinal and / or food and / or feed related processes may preferably avoid use of pathogenic bacterial. Accordingly, in one embodiment, said bioactive bacterial strain is a non-pathogenic bacterial strain.

[0101] It will be appreciated that any sample which comprises a plurality of bacterial strains may be provided in step (ii) of the method of the first aspect of the invention. Without being bound by theory, it is thought that natural microbial samples comprise variety and abundance of bacterial strains, and thus are appropriate samples for use in the method of the invention. Accordingly, in some embodiments, the sample comprising a plurality of different bacteria is a natural microbial sample. In some embodiments, the sample comprising a plurality of different bacteria is selected from the group consisting of a soil sample, a water sample, a sediments sample, a faecal sample, a plant tissue sample, an animal tissue sample, a human tissue sample, a foodstuff sample, and a wastewater sample. In a preferred embodiment, the sample comprising a plurality of different bacteria is a soil sample. In an alternatively preferred embodiment, the sample comprising a plurality of different bacteria is a foodstuff sample.

[0102] One skilled in the art will appreciate that the sample provided in step (ii) of the method of the first aspect of the invention may also comprise isolated, and optionally previously characterised, bacteria. This may be advantageous, for example, to select supporting bacteria from a pre-defined or known library of isolated bacteria having a specific characteristic (for example, being safe for human consumption or being able to grow in certain conditions). Accordingly, in some embodiments, the sample comprising a plurality of different bacteria is a bacterial library, comprising two or more isolated bacterial strains.

[0103] One skilled in the art will appreciate that step (iii) of sampling secondary bacteria that migrate from said second position towards said first position may be carried out by any means that separates, isolates or otherwise obtains said secondary bacteria. It may be advantageous to separate only a representative fraction of the space between said first and second position (i.e., a volume), which comprises said migrating secondary bacteria. By way of example, doing so may allow for further sampling to occur at different times. Accordingly, in some embodiments, sampling said secondary bacteria that migrate from said second position towards said first position comprises excising one or more volumes of said space between said first and second positions. It may further be advantageous to sample the secondary bacteria that migrate furthest towards said first position (i.e., towards said bioactive bacterial strain), as these secondary bacteria are most likely subjected to increased selection pressure by said bioactive bacterial strain. Accordingly, in a preferred embodiment, said one or more volumes are excised contiguous to said first position.

[0104] Additionally or alternatively, the migrating secondary bacteria may contact the at least one and / or the first semi-permeable membrane. It will be appreciated that, such secondary bacteria contacting a semi-permeable membrane may attach to said semi-permeable membrane on the side facing said space between said first and second positions. It may therefore be advantageous and convenient, to simple sample said bacteria from said side facing the space between said first and second positions of said semi-permeable membrane. Accordingly, in some embodiments, sampling said secondary bacteria that migrate from said second position towards said first position comprises eluting bacteria attached to said semi-permeable membrane and / or said first semi-permeable membrane. In a preferred embodiment, said secondary bacteria are eluted from the side facing said space between said first and second positions of said semi-permeable membrane. It will be appreciated that repeated sampling of said migrating secondary bacteria may be advantageous, for example, to obtain migrating secondary bacteria at different time points during migration, or to obtain migrating secondary bacteria at different distances from said first and / or secondary positions. Accordingly, in some embodiments, step (iii) is repeated one or more times.

[0105] Following step (iii) of sampling secondary bacteria that migrate from said second position towards said first position, it may be advantageous to multiply and / or culture the bacterial strains sampled. Such bacterial culture derived from the secondary bacteria or from the supporting bacteria may for example, be suitable for further characterization, maintenance and / or other use of said bacterial strains. Accordingly, in some embodiments, the method of the invention further comprises comprising a step of isolating said secondary bacteria.

[0106] In some embodiments, one or more bacterial strains are isolated from said sampled secondary bacteria by inoculating said sample in bacterial culture media.

[0107] It will be appreciated that the one or more of the sampled secondary bacteria may be characterised using a variety of methods known in the art and commonly used in the field of microbiology. Characterising bacteria may, for example, be suitable to identify taxonomical information of such bacteria. Such bacteria may be characterized morphologically, based on the size, shape and colour of a bacterial colony thereof, biochemically, based on the nature of the metabolites produced by such bacteria, or by sequencing, based on the nature and sequence of the nucleic acid component of the bacteria. Sequencing is generally considered the optimal method of identifying the nature and taxonomical information of a bacteria.

[0108] Accordingly, in some embodiments, the method of the invention further comprises a step of isolating nucleic acid from said secondary bacteria and sequencing said nucleic acids. One skilled in the art will appreciate that different sequencing methods are known in the art and may be used interchangeably dependent on which information is required. By way of example, sequencing of the 16S ribosomal RIMA (rRNA) allows for genus level identification, while metagenome sequencing allows for strain level identification. In some embodiments, sequencing the nucleic acid comprises sequencing the 16S ribosomal RNA, shotgun metagenome sequencing or whole genome sequencing.

[0109] As previously described and without wishing to be bound by theory, it is thought that the sampled migrating secondary bacteria and the bioactive bacterial strain have increased probability of interacting, such as by interacting synergistically. Accordingly, the sampled secondary bacteria and the bioactive bacterial strain are candidates for forming a synergistic bacterial consortium. It will be appreciated that supporting bacteria, which may be able to form synergistic bacterial consortia with said bioactive bacterial strain, may be obtained by selecting from sampled secondary bacteria, those which interact synergistically with the bioactive bacterial strain.

[0110] Specifically, one or more isolated bacterial strains isolated from said sampled secondary bacteria may be co-cultured with the bioactive bacterial strain and screened for synergism and synergistic bacterial consortia may be selected from said co-cultures. For example, such co-cultures may comprise only two or more, three or more, four or more, five or more or six or more bacterial strains from said sampled secondary bacteria, and the bioactive bacterial strain. Accordingly, combinatorial co-culture of one or more isolated bacterial strains isolated from said sampled secondary bacteria and the bioactive bacterial strain may be screened to select synergistic bacterial consortia.

[0111] Accordingly, in some embodiments, the method further comprises the steps of:

[0112] (iv) co-culturing said one or more secondary bacteria with said bioactive bacterial strain; and

[0113] (v) selecting one or more supporting bacteria that cause a bioactive bacterial strain to perform a function differently than in absence of the one or more secondary bacteria.

[0114] As previously described, and as will be appreciated, biofilm formation is a known marker of synergy within multi-strain bacterial consortia and synergistic effects within a bacterial community have been shown to promote biofilm biomass, resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms. Accordingly, coculture of one or more isolated bacterial strains isolated from said sampled secondary bacteria and the bioactive bacterial strain may be screened to select synergistic bacterial consortia by screening for co-cultures having increased biofilm formation capabilities.

[0115] Accordingly, in some embodiments, said function performed differently is biofilm formation. In a preferred embodiment, said selection comprises co-culturing said secondary bacteria with said bioactive bacterial strain and measuring biofilm formation capability of the co-culture. In an additional embodiment, the secondary bacteria are selected if the biofilm formation capability of the co-culture is greater than the biofilm formation capabilities of a reference. It will be appreciated that the reference may be any biofilm formation capability value, which has been determined to be an effective cut-off value for determining bacterial synergism of two or more bacterial strains based on the biofilm formation capabilities. In some embodiments, the reference is determined as part of the method of the invention. In an alternative embodiment, the reference may be a known or calculated value. In some embodiments, the reference is the bioactive bacterial strain and / or the one or more secondary bacteria cultured separately. In an alternative embodiment, the reference is the average biofilm formation capability of the individual bacteria in said co-culture, when each bacterial strain is cultured separately.

[0116] Specific bacterial cultures comprising a known composition of bacterial strains are known to have specific biofilm formation capabilities. For example, a bacterial culture comprising Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus has previously been shown to produce high levels of biofilm (Yang, N., Nesme, J., Roder, H.L. et al. Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis. npj Biofilms Microbiomes 7, 82 (2021)). Accordingly, in some embodiments, the reference is one or more bacterial culture with known biofilm formation capabilities. In a preferred embodiment, the reference bacterial culture with known biofilm formation capability comprises Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus.

[0117] Methods of measuring biofilm formation of a bacterial culture are known in the art. For example, a list of suitable methods is disclosed in Wilson C et al. Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review. Res Rev J Eng Technol. 2017. In some embodiments, said biofilm formation is determined using a method selected from the group consisting of viable cell counting, flow cytometry-based counting, light and / or fluorescence microscopy, dry mass assessment, total organic carbon assessment, crystal violet assay, an ATP bioluminescence assay, and total protein determination. In a preferred embodiment, said biofilm formation capability is determined using a Crystal Violet assay.

[0118] As previously described, the first aspect of the invention provides a method for obtaining one or more supporting bacteria, which have increased likelihood of interacting with the bioactive strain, such as by interacting synergistically. Accordingly, the sampled secondary bacteria and the bioactive bacterial strain are candidates for forming a synergistic bacterial consortium. Without wanting to be bound by theory, it is thought that bacterial strains forming a synergistic bacterial consortium have improved and / or increased biological properties, compared to each bacterial strain by itself. As previously described such biological properties may be exerted on a separate organism, such as a host organism to which the bacterial consortium is contacted or exposed to.

[0119] One skilled in the art will appreciate that it may be advantageous to identify supporting bacteria, which have increased likelihood of forming a synergistic bacterial consortium with said bioactive strain, by screening the sampled secondary bacteria to identify those that can increase, decrease or otherwise change at least one biological property of the bioactive strain. Additionally or alternatively, the sampled secondary bacteria may be screened to identify those that can increase, decrease or otherwise change a particular biological property of the bioactive strain. By way of example, sampled secondary bacteria may be screened for their capability to increase growth promotion of a plant (i.e., a host), which is known to be mediated by the bioactive bacterial strain.

[0120] Accordingly, in some embodiments, the method of the first aspect of the invention, further comprises the steps of:

[0121] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a target host organism; and

[0122] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied on the target host organism.

[0123] In one embodiment, the target host organism is a plant or a crop. In a preferred embodiment, the target host organism is a plant.

[0124] It will be appreciated that the biological property of the bioactive bacterial strain may be any direct or indirect effect, action, or result exerted on the host organism by the bioactive bacterial strain. In some embodiments, the biological property of the bioactive strain exerted on the target host organism is selected from the group consisting of improved growth, yield, defence, robustness, tolerance, a biopesticide biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, Pathogen antagonism, Plant Growth Promotion, Nitrogen Fixation, Phosphorous Solubilization, Pant Induced Systemic Resistance (ISR), Plant Defence Priming, Enhanced Nutrient Cycling, Rhizosphere Modification, Drought Tolerance, Salinity Tolerance, and Pathogen Exclusion and combinations thereof. In a preferred embodiment, the biological property of the bioactive strain is improved growth, yield, or defence of a plant or a crop, preferably a plant.

[0125] Alternatively, sampled secondary bacteria may be screened for their capability to increase or improve at least one biological property of the bioactive bacterial when applied to a composition on which the bioactive strain is known to exert one or more biological functions. In some embodiments, the composition is a foodstuff composition.

[0126] Accordingly, in some embodiments, the method of the first aspect of the invention, further comprises the steps of:

[0127] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a foodstuff composition; and

[0128] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied to the foodstuff composition.

[0129] In one embodiment, the foodstuff composition is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume. In a preferred embodiment, the foodstuff composition is a dairy product or a precursor thereof.

[0130] It will be appreciated that the biological property of the bioactive bacterial strain may be any direct or indirect effect, action, or result exerted on the composition (e.g., the foodstuff composition) by the bioactive bacterial strain. In some embodiments, the biological property of the bioactive strain exerted on the foodstuff composition is selected from the group consisting of improved and / or increased fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in the foodstuff composition, and combinations thereof. In a preferred embodiment, the biological property is acidification and / or fermentation of the foodstuff composition.

[0131] For some applications, it may be advantageous to have reduced production of unwanted compounds, e.g. to prevent malodours or unpleasant taste. Thus, in an embodiment, the biological property is decreased production of one or more aroma and / or flavour compounds produced in the foodstuff composition.

[0132] It will be appreciated by one skilled in the art that step (iv), step (v), step (vi) and step (vii) may each be carried out as part of the invention or not carried out. Similarly, each of the abovementioned steps may be carried out in any order, unless dictated otherwise by context. Additionally or alternatively, each step of isolating the bacterial strains, characterising the bacterial strains, isolating nucleic acids or sequencing said nucleic acids, may also be carried out or not carried out, and may be carried out before or after any other steps described, unless dictated otherwise by context. By way of example, isolating and sequencing nucleic acids from the migrating secondary bacteria may be carried out regardless of whether all or any of steps (iv), (v), (vi) and / or (viii) were carried out. Moreover, steps (vi) and (vii) of applying secondary bacteria and the bioactive strain to a host and selecting supporting bacteria may be carried out regardless of whether steps (iv) and (v) were carried out.

[0133] One skilled in the art will appreciate that the method of the first aspect of the invention may be carried out in several distinct variants and using a variety of equipment, devices, or set-ups. In performing the method of the first aspect of the invention, it may be advantageous to use a device comprising the features necessary for performing the method of the invention. Such devices may vary depending on specific factors and intended use of the method of the first aspect of the invention, such as the nature of the bioactive bacterial strain and the sample comprising a plurality of different bacteria.

[0134] Accordingly, a second aspect of the invention provides a device for obtaining one or more supporting bacteria for a bacterial composition, the device comprising:

[0135] (i) a first compartment comprising at least one entry opening, said at least one entry opening being exposed on the surface of the device;

[0136] (ii) a second compartment within said first compartment; and

[0137] (iii) at least one inner opening connecting said first compartment with said second compartment; wherein said inner opening comprises a first semi-permeable membrane separating said first compartment and said at least one second compartment.

[0138] One skilled in the art will appreciate that said second compartment may be used to hold or contain a bacterial strain, such as the bioactive bacterial strain used in the method of the first aspect of the invention. Accordingly, said first semi-permeable membrane may separate said bacterial strain in said second compartment from the exterior of said second compartment. In some embodiments, the device comprises two or more, three or more, four or more, five or more, six or more, ten or more, twenty or more, fifty or more or hundred or more second compartments, each connected with said first compartment by at least one inner opening.

[0139] As will be appreciated and as previously described in regard to the first aspect of the invention, said first semi-permeable membrane may avoid mixing of the bacterial strain in said second compartment and other bacteria on the exterior of said second compartment, such as the migrating secondary bacteria referred to in the first aspect of the invention. This may facilitate further downstream isolating, characterization, and maintenance of the sampled secondary bacteria. Additionally or alternatively, said first semi-permeable membrane may facilitate maintaining the bacterial strain inside said second compartment. Additionally or alternatively, said first semi-permeable membrane may also function as a scaffold to which said bacteria on the exterior of said second compartment may attach or be contacted to, and which facilitates sampling such bacteria by sampling directly from said first semi-permeable membrane. Accordingly, it may be advantageous for said first semi-permeable membrane to be configured to exclude passage of bacterial cells. Accordingly, in some embodiments, said first semi- permeable membrane is configured to exclude passage of bacterial cells.

[0140] As will be appreciated and as previously described, a bacterial strain provided inside said second compartment may produce selection pressure on bacteria outside said second compartment. Such selection pressure may encompass, for example, bacterial metabolites, including proteins, nucleic acids, or viral particles. Such bacterial metabolites may only produce such selection pressure by being diffused out of said second compartment, where they may contact the bacteria on the exterior of said second compartment. Accordingly, to diffuse outside said second compartment and produce said selection pressure, such bacterial metabolites must be able to cross said first semi- permeable membrane. Accordingly, in some embodiments said first semi-permeable membrane is configured to allow for passage of bacterial metabolites.

[0141] In a particular embodiment, the first semi-permeable membrane is configured to allow for passage of bacterial metabolites, and / or exclude passage of bacterial cells.

[0142] In some embodiments, the first semi-permeable membrane has a pore diameter of about 0.1 pm to about 1 pm, such as about 0.1 pm to about 1 pm, about 0.15 pm to about 0.9 pm, about 0.15 pm to about 0.8 pm, about 0.15 pm to about 0.7 pm, about 0.15 pm to about 0.6 pm, about 0.15 pm to about 0.5 pm, about 0.15 pm to about 0.45 pm, about 0.2 pm to about 0.45 pm, about 0.2 pm to about 0.4 pm, or about 0.2 pm to about 0.3 pm, or about 0.15 pm to about 0.25 pm. In a preferred embodiment, said first semi- permeable membrane has a pore diameter of 0.2 pm to about 0.45 pm. In a further preferred embodiment, said first semi-permeable membrane has a pore diameter of about 0.4 pm to about 0.5 pm.

[0143] In an additional or alternative embodiment, the first semi-permeable membrane has a pore diameter of less than about 2um, such as less than about 2 pm, less than about 1.95 pm, less than about 1.9 pm, less than about 1.85 pm, less than about 1.8 pm, less than about 1.75 pm, less than about 1.7 pm, less than about 1.65 pm, less than about 1.6 pm, less than about 1.55 pm, less than about 1.5 pm, less than about 1.45 pm, less than about 1.4 pm, less than about 1.35 m, less than about 1.3 pm, less than about 1.25 pm, less than about 1.2 pirn, less than about 1.15 pm, less than about 1.1 pm, less than about 1.05 pm, less than about 1 pm, less than about 0.95 pm, less than about 0.9 pm, less than about 0.85 pm, less than about 0.8 pm, less than about 0.75 pm, less than about 0.7 pm, less than about 0.65 pm, less than about 0.6 pm, less than about 0.55 pm, less than about 0.5 pm, less than about 0.45 pm, less than about 0.4 pm, less than about 0.35 pm, less than about 0.3 pm, less than about 0.25 pm, less than about 0.2 pm, less than about 0.15 pm, or less than about 0.1 pm. In a preferred embodiment, at least one semi-permeable membrane has a pore diameter of less than about 0.45 pm. In a further preferred embodiment, said first semi-permeable membrane has a pore diameter of less than about 0.5 pm.

[0144] In some embodiments, the first compartment is essentially free from bacteria. As will be appreciated, it may be advantageous for said first compartment to be essentially free from bacteria to facilitate and improve sampling of secondary bacteria migrating into said compartment from outside the device. Without wanting to be bound by theory, it is thought that a space essentially free from bacteria (i.e., the first compartment) will promote migration and survival of said migrating secondary bacteria due to decreased competition and will improve sampling of said bacteria due to lack of non-migrating bacteria in said space.

[0145] One skilled in the art will thus appreciate that the space inside said first compartment, but excluding said second compartment, may be essentially free from bacteria, and may thus be the space essentially free from bacteria referred to in the first aspect of the invention. Accordingly, in some embodiments, said first compartment is essentially free from bacteria, excluding said second compartment.

[0146] It will be appreciated that said entry opening may comprise a second semi-permeable membrane. It may be advantageous in, but not necessary, to separate the interior of said first compartment and the exterior of the device by a second semi-permeable membrane. One skilled in the art will appreciate that a second semi-permeable membrane may be used, as described previously as a scaffold to which said migrating secondary bacteria may attach or be contacted to. Additionally or alternatively, said second semi-permeable membrane may be used as an obstacle to bacteria on the outside of the device, for example by impeding, but not excluding, passage of bacterial cells from outside the device to inside said first compartment. Such a use of a second semi-permeable membrane may be advantageous to increase the likelihood that bacteria that migrate said second semi-permeable membrane and into the device are subject to selection pressure by a bacterial strain, which may be provided in said second compartment.

[0147] Accordingly, in some embodiments the entry opening comprises a second semi- permeable membrane. In a preferred embodiment, said second semi-permeable membrane is configured to allow passage of bacterial cells.

[0148] In some embodiments, said second semi-permeable membrane has a pore diameter of about 5 pm to about 200 pm, such as about 5 pm to about 200 pm, about 5 pm to about 150 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, about 20 pm to about 100 pm, about 30 pm to about 100 pm, about 50 pm to about 100 pm, about 10 pm to about 40 pm, or about 10 pm to about 30 pm. In some embodiments, said second semi-permeable membrane has a pore diameter of about 10 pm to about 20 pm.

[0149] In an additional or alternative embodiment, said second semi-permeable membrane has a pore diameter greater than 50 pm, such as such as greater than about 50 pm, greater than about 60 pm, greater than about 70 pm, greater than about 80 pm, greater than about 90 pm, greater than about 100 pm, greater than about 110 pm, greater than about 120 pm, greater than about 130 pm, greater than about 140 pm, greater than about 150 pm, greater than about 160 pm, greater than about 170 pm, greater than about 180 pm, greater than about 190 pm, or greater than about 200 pm. In some embodiments, said second semi-permeable membrane has pore diameter greater than 10 pm, such as greater than 10 pm, greater than 15 pm, greater than 20 pm, greater than 30 pm, greater than 40 pm or greater than 50 pm.

[0150] One skilled in the art will appreciate that the device may be of any shape advantageous for the particular use of the device. Use of the device may for example entail placing the device in a sample comprising bacteria, in order to allow for such bacteria to migrate into the device. The shape, size, and material of the device may therefore be dependent on the sample on or in which the device is to be placed. Accordingly, in some embodiments the device is configured to be inserted into a sample comprising a plurality of different bacteria. As will be appreciated and as previously described, soil may be an advantageous sample to for use of the device, as it comprises a variety of different bacteria, many of which may be useful for agricultural, industrial, or medicinal purposes. Accordingly, in a preferred embodiment, the device is configured to be inserted into a soil sample.

[0151] Alternatively, the device may be configured to be inserted in a liquid or minimally viscous liquid. As noted previously, foodstuff samples may be an advantageous sample for use of the device, as it comprises a variety of different bacteria, many of which may be useful in food manufacturing. Certain foodstuff samples may particularly be liquid and or minimally viscous. Accordingly, the device may be configured to be inserted in a foodstuff sample. In some embodiments, the foodstuff sample is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume. In some embodiments, the foodstuff sample is a dairy product or a precursor thereof.

[0152] It will be appreciated that, as described above, the preferred pore size of the first and / or second semi-permeable membrane may be adapted such that the device is configured for use in different samples and / or on the nature of the one or more bioactive bacterial strains to be used. By way of example, a device configured to be used in a liquid or a minimally viscous liquid may advantageously comprise a second semi-permeable membrane which impedes excessive non-selection pressure driven migration of secondary bacteria into the first compartment. Alternatively, in particulate matter samples (e.g., soil samples) it may in turn be advantageous for said semi-permeable membrane to have a larger pore-size to allow for increased migration of the migrating secondary bacteria into the first compartment.

[0153] Accordingly, in some embodiments wherein the device is configures to be used and / or inserted in a liquid or minimally viscous liquid, said second semi-permeable membrane has a pore size of less than 100 pm, such as less than 100 pm, less than 90 pm, less than 80 pm, less than 70 pm, less than 60 pm, less than 50 pm, less than 40 pm, less than 40 pm, less than 30 pm, less than 20 pm, less than 15 pm or less than 10 pm. In a preferred embodiment, less than 15 pm. In some alternative embodiments wherein the device is configures to be used and / or inserted in a particular matter sample, solid matter and / or in a soil sample, said second semi-permeable membrane has a pore diameter greater than 50 pm, such as greater than about 50 pm, greater than about 60 pm, greater than about 70 pm, greater than about 80 pm, greater than about 90 pm, greater than about 100 pm, greater than about 110 pm, greater than about 120 pm, greater than about 130 pm, greater than about 140 pm, greater than about 150 pm, greater than about 160 pm, greater than about 170 pm, greater than about 180 pm, greater than about 190 pm, or greater than about 200 pm.

[0154] A third aspect of the invention provides the use of a device according to the second aspect of the invention to obtain one or more supporting bacteria for a bacterial composition.

[0155] In some embodiments, the use comprises: (i) providing a bacterial composition comprising at least one bioactive bacterial strain in the second compartment of said device;

[0156] (ii) providing a medium in the first compartment;

[0157] (iii) placing said device in a sample comprising a plurality of different bacteria, such that the sample contacts the surface of the device; and

[0158] (iv) sampling secondary from said plurality of different bacteria that migrate into said first compartment from said sample comprising a plurality of different bacteria, thereby obtaining one or more supporting bacteria.

[0159] As described previously, it may be advantageous for said first compartment to be essentially free from bacteria. For the same reason it is thus also advantageous for said medium which is provided in said first compartment to be essentially free from bacteria. In some embodiments, the medium is essentially free from bacteria. In some embodiments, the medium is sterile.

[0160] As previously described, it be advantageous to select a bacterial strain with known or theorised specific biological effects or properties. By way of example, the bioactive bacterial strain may be known to be particularly useful in some process, such as an industrial, manufacturing, agricultural or medicinal process, or to treat or prevent one or more specific diseases in a subject. The bioactive strain may thus, be a well characterised bacterial strain, and may also have been isolated. Accordingly, in one embodiment, said bioactive bacterial strain is a bacterial isolate. In an alternative or additional embodiment, said bioactive bacterial strain is a non-pathogenic bacterial strain.

[0161] It will be appreciated that the device may be placed in or on any sample which comprises a plurality of bacterial strains. In some embodiments, the sample comprising a plurality of different secondary bacteria is a natural microbial sample. In some embodiments, the sample comprising a plurality of different secondary bacteria is selected from the group consisting of a soil sample, a water sample, a sediments sample, a faecal sample, a plant tissue sample, an animal tissue sample, a human tissue sample, a foodstuff sample, and a wastewater sample.

[0162] One skilled in the art will appreciate that step (iv) of sampling secondary bacteria that migrate into said first compartment may be carried out by any means that separates, isolates or otherwise obtains said secondary bacteria. It may be advantageous to separate only a representative fraction of said medium in said first compartment. By way of example, doing so may allow for further sampling to occur at different times. Accordingly, in some embodiments, sampling said secondary bacteria that migrate into said first compartment comprises excising one or more volumes of medium inside said first compartment. In some embodiments, the excised volumes do not comprise said second compartment. It may further be advantageous to sample the secondary bacteria that migrate. In a preferred embodiment, said one or more volumes are excised contiguous to said second compartment.

[0163] Additionally or alternatively, the migrating secondary bacteria may contact the first semi- permeable membrane. It will be appreciated that, such secondary bacteria contacting a semi-permeable membrane may attach to said semi-permeable membrane on the side facing away from said second compartment. It may therefore be advantageous and convenient, to simply sample said bacteria from said side facing away from said second compartment of said semi-permeable membrane. Accordingly, in some embodiments, sampling said secondary bacteria that migrate towards said second compartment comprises eluting bacteria attached to said semi-permeable membrane. In a preferred embodiment, said secondary bacteria are eluted from the side facing away from said second compartment.

[0164] It will be appreciated that repeated sampling of said migrating secondary bacteria may be advantageous, for example, to obtain migrating secondary bacteria at different time points during migration, or to obtain migrating secondary bacteria at different distances from said second compartment. Accordingly, in some embodiments, step (iv) is repeated one or more times.

[0165] In some embodiments, the use further comprises comprising a step of isolating said secondary bacteria. In some embodiments, one or more bacterial strains are isolated from said sampled secondary bacteria by inoculating said sample in bacterial culture media.

[0166] It will be appreciated that the one or more of the sampled secondary bacteria may be characterised using a variety of methods known in the art and commonly used in the field of microbiology. Such bacteria may be characterized morphologically, based on the size, shape and colour of a bacterial colony thereof, biochemically, based on the nature of the metabolites produced by such bacteria, or by sequencing, based on the nature and sequence of the nucleic acid component of the bacteria. Sequencing is generally considered the optimal method of identifying the nature and taxonomical information of a bacteria.

[0167] Accordingly, in some embodiments, the use further comprises a step of isolating nucleic acid from said secondary bacteria and sequencing said nucleic acids. One skilled in the art will appreciate that different sequencing methods are known in the art and may be used interchangeably dependent on which information is required. In some embodiments, sequencing the nucleic acid comprises sequencing the 16S ribosomal RIMA, shotgun metagenome sequencing or whole genome sequencing.

[0168] It will be appreciated that supporting bacteria, which may be able to form synergistic bacterial consortia with said bioactive bacterial strain, may be obtained by selecting from sampled secondary bacteria, those which interact synergistically with the bioactive bacterial strain. Specifically, one or more isolated bacterial strains isolated from said sampled secondary bacteria may be co-cultured with the bioactive bacterial strain and screened for synergism and synergistic bacterial consortia may be selected from said cocultures. For example, such co-cultures may comprise only two or more, three or more, four or more, five or more or six or more bacterial strains from said sampled secondary bacteria, and the bioactive bacterial strain. Accordingly, combinatorial co-culture of one or more isolated bacterial strains isolated from said sampled secondary bacteria and the bioactive bacterial strain may be screened to select synergistic bacterial consortia.

[0169] Accordingly, in some embodiments, the use further comprises the steps of:

[0170] (iv) co-culturing said one or more secondary bacteria with said bioactive bacterial strain; and

[0171] (v) selecting one or more supporting bacteria that cause a bioactive bacterial strain to perform a function differently than in absence of the one or more secondary bacteria.

[0172] As previously described, biofilm formation is a known indicator of bacterial synergism in multi-species bacterial consortia. Accordingly, in some embodiments, said function performed differently is biofilm formation. In a preferred embodiment, said selection comprises co-culturing said secondary bacteria with said bioactive bacterial strain and measuring biofilm formation capability of the co-culture. In an additional embodiment, the secondary bacteria are selected if the biofilm formation capability of the co-culture is greater than the biofilm formation capabilities of a reference.

[0173] It will be appreciated that the reference may be any biofilm formation capability value, which has been determined to be an effective cut-off value for determining bacterial synergism of two or more bacterial strains based on the biofilm formation capabilities. In some embodiments, the reference is determined as part of the method of the invention. In an alternative embodiment, the reference may be a known or calculated value. In some embodiments, the reference is the bioactive bacterial strain and / or the one or more secondary bacteria cultured separately. In an alternative embodiment, the reference is the average biofilm formation capability of the individual bacteria in said co-culture, when each bacterial strain is cultured separately. In some embodiments, the reference is one or more bacterial cultures with known biofilm formation capabilities. In a preferred embodiment, the reference bacterial culture with known biofilm formation capability comprises Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus.

[0174] In a preferred embodiment, said biofilm formation capability is determined using a Crystal Violet assay.

[0175] One skilled in the art that the sampled secondary bacteria may be screened to identify those that can increase, decrease or otherwise change a particular biological property of the bioactive strain, such as a biological property exerted on a host organism.

[0176] Accordingly, in some embodiments, the use further comprises the steps of:

[0177] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a target host organism; and

[0178] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied on the target host organism.

[0179] In some embodiments, the target host organism is a plant or a crop. In a preferred embodiment, the target host organism is a plant.

[0180] It will be appreciated that the biological property of the bioactive bacterial strain may be any direct or indirect effect, action, or result exerted on the host organism by the bioactive bacterial strain. In some embodiments, the biological property of the bioactive strain exerted on the target host organism is selected from the group consisting of improved growth, yield, shelf-life, defence, nutrition, robustness, tolerance, a biopesticide biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, Pathogen antagonism, Plant Growth Promotion, Nitrogen Fixation, Phosphorous Solubilization, Pant Induced Systemic Resistance (ISR), Plant Defence Priming, Enhanced Nutrient Cycling, Rhizosphere Modification, Drought Tolerance, Salinity Tolerance, and Pathogen Exclusion and combinations thereof. In a preferred embodiment, the biological property of the bioactive strain is improved growth, yield, or defence of a plant or a crop, preferably a plant. In an alternative embodiment, the use further comprising the steps of:

[0181] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a foodstuff composition; and

[0182] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied to the foodstuff composition.

[0183] In some embodiments, the foodstuff composition is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume. In a preferred embodiment, the foodstuff composition is a dairy product or a precursor thereof.

[0184] In some embodiments, the biological property of the bioactive strain exerted on the foodstuff composition is selected from the group consisting of improved and / or increased fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in the foodstuff composition, and combinations thereof.

[0185] A fourth aspect of the invention provides a kit comprising the device of any of the second aspect of the invention and instructions for performing the method according to the first aspect of the invention or the use according to the third aspect of the invention. In some embodiments the kit further comprises one or more bioactive bacterial strains. In an additional or alternative embodiment, the kit comprises a sample comprising a plurality of secondary bacteria. In a further embodiment, the kit comprises means for sampling secondary bacteria that migrate into said first compartment of the device.

[0186] A further aspect of the invention provides one or more supporting bacteria obtained or obtainable by any of the methods according to the first aspect of the invention or any of the uses according to the third aspect of the invention. A further aspect of the invention provides a use of any supporting bacteria according to the previous aspect of the invention to improve or increase at least one biological property of a bioactive bacterial strain.

[0187] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0188] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. All patents, patent applications and / or other disclosures described herein are hereby incorporated by reference in their entirety.

[0189] Preferences, options, and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention.

[0190] Description of the Figures

[0191] Figure 1. - Simplified and schematic representation of one possible spatial set-up that may be used in one non-limiting embodiment of the method of the invention. The first position comprises one bacterial strain and the second position comprises a plurality of bacterial strains. The first and second positions are separated by a space essentially free from bacteria to which certain bacteria from said plurality of bacterial strains in said second position can migrate to. The first and second positions are separated by at least one semi- permeable membrane, which in this example excludes crossing of bacterial cells. An additional semi-permeable membrane which allows or impedes crossing of bacterial cells may be optionally included.

[0192] Figure 2. - Simplified and schematic representations of two possible exemplary and nonlimiting embodiments of the device according to certain aspects of the invention. The first compartment (1) of the device provided in a sample comprising a plurality of bacterial strains (3) (for example, a soil sample (A) or a foodstuff sample (B)). A second compartment (2) is within said first compartment and may comprise a bacterial strain (4) (such as the bioactive bacterial strain) inside said second compartment (2). Figure 3. - Bar diagram shows normalized OD590 (Crystal Violet assay) of bioactive bacterial strain SI-391 co-cultured with obtained supporting bacteria (recruited isolate 53), compared to SI-391 or recruited isolate 53 alone.

[0193] Figure 4. - Bar diagram shows normalized OD590 (Crystal Violet assay) of bioactive bacterial strain SI-930 co-cultured with obtained supporting bacteria (recruited isolates 87, 102 and 112), respectively, compared to SI-930 or each of recruited isolates 87, 102 or 112 alone.

[0194] Figure 5. - Representative images show attraction assay comparing colony size and morphology of culture of bioactive strain SI-391 and obtained supporting bacteria RI-53 or bioactive strain SI-930 and obtained supporting bacteria RI-87, each alone or in proximity of each other.

[0195] Figure 6. - Green house trial of bioactive bacterial strain SI_391 and supporting bacterial strain RI_53 co-inoculated on tomato plants. Fruit count (A), Ripening score (B) and Fruit weight (C) were compared between the co-culture (SI_391 + RI_53) as compared to the constituent bacteria alone.

[0196] Figure 7. - Violin plots show the average pH obtained in the soy-milk / soy-yogurt after 8 hours of fermentation by specific combination of the bioactive bacterial strains and each of the bacterial strains in the bacterial library tested.

[0197] Certain embodiments and features of the present invention are also outlined in the following numbered items:

[0198] 1. A method for obtaining one or more supporting bacteria for a bacterial composition, the method comprising the following steps:

[0199] (i) providing a bacterial composition comprising at least one bioactive bacterial strain in a first position;

[0200] (ii) providing a sample comprising a plurality of different bacteria in a second position; wherein said first and said second positions are separated by a space that is essentially free from bacteria; wherein said first and said second positions are separated by at least one semi-permeable membrane;

[0201] (iii) sampling secondary bacteria from said plurality of different bacteria that migrate from said second position towards said first position, thereby obtaining one or more supporting bacteria.

[0202] 2. The method of item 1, wherein at least one semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

[0203] 3. The method of any one of the preceding items, wherein at least one semi-permeable membrane has a pore diameter of about 0.1 pm to about 1 pm, such as about 0.1 pm to about 1 pm, about 0.15 pm to about 0.9 pm, about 0.15 pm to about 0.8 pm, about 0.15 pm to about 0.7 pm, about 0.15 pm to about 0.6 pm, about 0.15 pm to about 0.5 pm, about 0.15 pm to about 0.45 pm, about 0.2 pm to about 0.45 pm, about 0.2 pm to about 0.4 pm, or about 0.2 pm to about 0.3 pm, or about 0.15 pm to about 0.25 pm.

[0204] 4. The method of items 1 or 2, wherein at least one semi-permeable membrane has a pore diameter of less than about 2um, such as less than about 2 pm, less than about 1.95 pm, less than about 1.9 pm, less than about 1.85 pm, less than about 1.8 pm, less than about 1.75 pm, less than about 1.7 pm, less than about 1.65 pm, less than about 1.6 pm, less than about 1.55 pm, less than about 1.5 pm, less than about 1.45 pm, less than about 1.4 pm, less than about 1.35 pm, less than about 1.3 pm, less than about 1.25 pm, less than about 1.2 pm, less than about 1.15 pm, less than about 1.1 pm, less than about 1.05 pm, less than about 1 pm, less than about 0.95 pm, less than about 0.9 pm, less than about 0.85 pm, less than about 0.8 pm, less than about 0.75 pm, less than about 0.7 pm, less than about 0.65 pm, less than about 0.6 pm, less than about 0.55 pm, less than about 0.5 pm, less than about 0.45 pm, less than about 0.4 pm, less than about 0.35 pm, less than about 0.3 pm, less than about 0.25 pm, less than about 0.2 pm, less than about 0.15 pm, or less than about 0.1 pm.

[0205] 5. The method of any one of the preceding items, wherein at least one semi-permeable membrane is contiguous to said first position.

[0206] 6. The method of item 1, wherein said first and second positions are separated by at least a first semi-permeable membrane and a second semi-permeable membrane, wherein the distance between said first position and said first semi-permeable membrane is less than the distance between said first position and said second semi-permeable membrane. 7. The method of item 6, wherein said first semi-permeable membrane is contiguous to said first position and / or said second semi-permeable membrane is contiguous to said second position.

[0207] 8. The method of any one of items 6 or 7, wherein said first semi-permeable membrane has a pore diameter of about 0.1 pm to about 1 pm, such as about 0.1 pm to about 1 pm, about 0.15 pm to about 0.9 pm, about 0.15 pm to about 0.8 pm, about 0.15 pm to about 0.7 pm, about 0.15 pm to about 0.6 pm, about 0.15 pm to about 0.5 pm, about 0.15 pm to about 0.45 pm, about 0.2 pm to about 0.45 pm, about 0.2 pm to about 0.4 pm, or about 0.2 pm to about 0.3 pm, or about 0.15 pm to about 0.25 pm.

[0208] 9. The method of any one of items 6 or 7, wherein said first semi-permeable membrane has a pore diameter of less than about 2um, such as less than about 2 pm, less than about 1.95 pm, less than about 1.9 pm, less than about 1.85 pm, less than about 1.8 pm, less than about 1.75 pm, less than about 1.7 pm, less than about 1.65 pm, less than about 1.6 pm, less than about 1.55 pm, less than about 1.5 pm, less than about 1.45 pm, less than about 1.4 pm, less than about 1.35 pm, less than about 1.3 pm, less than about 1.25 pm, less than about 1.2 pm, less than about 1.15 pm, less than about 1.1 pm, less than about 1.05 pm, less than about 1 pm, less than about 0.95 pm, less than about 0.9 pm, less than about 0.85 pm, less than about 0.8 pm, less than about 0.75 pm, less than about 0.7 pm, less than about 0.65 pm, less than about 0.6 pm, less than about 0.55 pm, less than about 0.5 pm, less than about 0.45 pm, less than about 0.4 pm, less than about 0.35 pm, less than about 0.3 pm, less than about 0.25 pm, less than about 0.2 pm, less than about 0.15 pm, or less than about 0.1 pm.

[0209] 10. The method of any one of items 6-9, wherein said first semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

[0210] 11. The method of any one of items 6-10, wherein said second semi-permeable membrane has a pore diameter of about 5 pm to about 200 pm, such as about 5 pm to about 200 pm, about 5 pm to about 150 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, about 20 pm to about 100 pm, about 30 pm to about 100 pm, about 50 pm to about 100 pm, about 10 pm to about 40 pm, or about 10 pm to about 30 pm. The method of any one of items 6-10, wherein said second semi-permeable membrane has a pore diameter greater than 50 pm, such as greater than about 50 pm, greater than about 60 pm, greater than about 70 pm, greater than about 80 pm, greater than about 90 pm, greater than about 100 pm, greater than about 110 pm, greater than about 120 pm, greater than about 130 pm, greater than about 140 pm, greater than about 150 pm, greater than about 160 pm, greater than about 170 pm, greater than about 180 pm, greater than about 190 pm, or greater than about 200 pm. The method of any one of items 6-12, wherein said second semi-permeable membrane is configured to allow passage of bacterial cells. The method of any one of items 6-13, wherein said second semi-permeable membrane is configured to impede passage of bacterial cells. The method of any one of items 6-14, wherein the distance between said first and said second semi-permeable membrane is less than 500 mm, such as about 500 mm, about 475 mm, about 450 mm, about 425 mm, about 400 mm, about 375 mm, about 350 mm, about 325 mm, about 300 mm, about 275 mm, about 250 mm, about 225 mm, about 200 mm, about 175 mm, about 150 mm, about 125 mm, about 100 mm, about 75 mm, about 50 mm, about 25 mm, about 15 mm, about 10 mm, about 5 mm, about 2 mm or about 1 mm. The method of any one of the preceding items, wherein said first position and said second position are at a distance of about 100 mm to about 1 mm, such as about 100 mm, about 80 mm, about 60 mm, about 50 mm, about 45 mm, about 40 mm, about 35 mm, about 30 mm, about 25 mm, about 20 mm, about 15mm, about 10 mm, about 5 mm, about 2 mm or about 1 mm. The method of any one of the preceding items, wherein said space essentially free from bacteria further comprises bacterial nutrients. The method of any one of the preceding items, wherein said space essentially free from bacteria further comprises one or more compounds selected from the list consisting of an antimicrobial, a host signalling compound, a heavy metal and a contaminant. 19. The method of item 17 or 18, wherein the concentration of the bacterial nutrients and / or compounds at or contiguous to said first position is different to the concentration at or contiguous to said second position.

[0211] 20. The method of item 19, wherein the concentration of the bacterial nutrients and / or compounds gradually increases and / or decreases from said first position towards said second position.

[0212] 21. The method of any one of the preceding items, wherein the pH and / or salinity at or contiguous to said first position is different to the pH and / or salinity at or contiguous to said second position.

[0213] 22. The method of item 21, wherein the pH and / or salinity gradually increases and / or decreases from said first position towards said second position.

[0214] 23. The method of any one of the preceding items, wherein said first position, said second position and / or said space essentially free from bacteria comprise a bacterial growth medium.

[0215] 24. The method of item 23, wherein the bacterial growth medium is solid, gelatinous, or liquid.

[0216] 25. The method of item 23, wherein the bacterial growth medium is selected from the group consisting of agar-based growth media, liquid growth media, animal or plant tissue homogenate media, a foodstuff and soil.

[0217] 26. The method of item 23, wherein the bacterial growth medium is soil.

[0218] 27. The method of item 23, wherein the bacterial growth medium is a foodstuff.

[0219] 28. The method of item 23, wherein the foodstuff is selected from the group consisting of a dairy product, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume.

[0220] 29. The method of item 28, wherein the foodstuff is a dairy product. 30. The method of item 29, wherein the foodstuff is selected from the group consisting of milk, whey butter, cheese, yogurt, kefir, cream, buttermilk, sour cream, and quark.

[0221] 31. The method of any one of the preceding items, wherein the bioactive strain has increased or improved stability in the presence of said one or more supporting bacteria.

[0222] 32. The method of any one of the preceding items, wherein the bioactive strain has increased or improved adaptability in the presence of said one or more supporting bacteria.

[0223] 33. The method of any one of the preceding items, wherein the bioactive strain has increased or improved host colonization success in the presence of said one or more supporting bacteria.

[0224] 34. The method of any one of the preceding items, wherein the bioactive bacterial strain has increased or improved nutrient availability, drought tolerance, temperature tolerance, and / or overall fitness in the presence of said one or more supporting bacteria.

[0225] 35. The method of any one of the preceding items, wherein the bioactive bacterial strain has increased or improved fermentation capabilities, acidification capabilities, and / or production of one or more aroma and / or flavour compounds in the presence of said one or more supporting bacteria.

[0226] 36. The method of any one of the preceding items, wherein the bioactive bacterial strain and said one or more supporting bacteria have increased biofilm formation capabilities when cultured or applied together, compared to the biofilm formation capability of the bioactive bacterial strain and the one or more supporting bacteria cultured or applied separately.

[0227] 37. The method of any one of the preceding items, wherein at least one biological property of the bioactive strain is increased or improved in the presence of said one or more supporting bacteria. 38. The method of item 37, wherein the at least one biological property is selected from the group consisting of improved growth, yield, defence, robustness and / or tolerance of a plant that is contacted with said first strain.

[0228] 39. The method of item 37, wherein the at least one biological property is selected from the list consisting of a biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, pathogen antagonism, plant growth promotion, nitrogen fixation, phosphorous solubilization, plant induced systemic resistance (ISR), plant defence priming, enhanced nutrient cycling, rhizosphere modification, drought tolerance, salinity tolerance, and pathogen exclusion.

[0229] 40. The method of any one of items 37, wherein the at least one biological property selected from the list consisting of bioremediation, biodegradation, fermentation, bio-catalysis, acidification, a probiotic effect, aroma development, flavour development, food texturization, bioprotection effect and stability.

[0230] 41. The method of item 37, wherein the at least one biological property is selected from the group consisting of improved fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in a foodstuff incubated in the presence of said first strain.

[0231] 42. The method of any one of items 37-41, wherein the at least one biological property of the bioactive bacterial strain is increased or improved when contacted with said one or more supporting bacteria.

[0232] 43. The method of any one of items 37-41, wherein the at least one biological property of the bioactive bacterial strain is increased or improved when contacted with a biological agent secreted by said one or more supporting bacteria.

[0233] 44. The method of item 43, wherein the biological agent is a metabolite, a protein, a nucleic acid, or a viral particle.

[0234] 45. The method of any one of the preceding items, wherein said bioactive bacterial strain is a bacterial isolate.

[0235] 46. The method of any one of the preceding items, wherein said bioactive bacterial strain is a non-pathogenic bacterial strain. 47. The method of any one of the preceding items, wherein the sample comprising a plurality of different bacteria is a natural microbial sample.

[0236] 48. The method of item 47, wherein the sample comprising a plurality of different bacteria is selected from the group consisting of a soil sample, a water sample, a sediments sample, a faecal sample, a plant tissue sample, an animal tissue sample, a human tissue sample, a foodstuff sample, and a wastewater sample.

[0237] 49. The method of item 48, wherein the sample comprising a plurality of different bacteria is a soil sample.

[0238] 50. The method of item 48, wherein the sample comprising a plurality of different bacteria is a foodstuff sample.

[0239] 51. The method of any one of items 1-46, wherein the sample comprising a plurality of different bacteria is a bacterial library, comprising two or more isolated bacterial strains.

[0240] 52. The method of any one of the preceding items, wherein sampling said secondary bacteria that migrate from said second position towards said first position comprises excising one or more volumes of said space between said first and second positions.

[0241] 53. The method of item 52, wherein said one or more volumes are excised contiguous to said first position.

[0242] 54. The method of any one of the preceding items, wherein sampling said secondary bacteria that migrate from said second position towards said first position comprises eluting bacteria attached to said semi-permeable membrane and / or said first semi- permeable membrane.

[0243] 55. The method of any one of the preceding items, wherein step (iii) is repeated one or more times.

[0244] 56. The method of any one of the preceding items, further comprising a step of isolating said secondary bacteria.

[0245] 57. The method of any one of the preceding items, further comprising a step of isolating nucleic acid from said secondary bacteria and sequencing said nucleic acids. 58. The method of item 57, wherein sequencing said nucleic acid comprises sequencing the 16S ribosomal RIMA, shotgun metagenome sequencing or whole genome sequencing.

[0246] 59. The method of any one of the preceding items, further comprising the steps of:

[0247] (iv) co-culturing said one or more secondary bacteria with said bioactive bacterial strain; and

[0248] (v) selecting one or more supporting bacteria that cause a bioactive bacterial strain to perform a function differently than in absence of the one or more secondary bacteria.

[0249] 60. The method of item 59, wherein said function performed differently is biofilm formation.

[0250] 61. The method of item 60, wherein said selection comprises co-culturing said secondary bacteria with said bioactive bacterial strain and measuring biofilm formation capability of the co-culture.

[0251] 62. The method of item 61, wherein the secondary bacteria are selected if the biofilm formation capability of the co-culture is greater than the biofilm formation capabilities of a reference.

[0252] 63. The method of item 62, wherein the reference is the bioactive bacterial strain and / or the one or more secondary bacteria cultured separately.

[0253] 64. The method of item 62, wherein the reference is one or more bacterial cultures with known biofilm formation capabilities.

[0254] 65. The method of item 64, wherein the bacterial culture with known biofilm formation capability comprises Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus.

[0255] 66. The method of any one of items 62-65, wherein said biofilm formation capabilities are determined using a Crystal Violet assay.

[0256] 67. The method of any one of the preceding items, further comprising the steps of: (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a target host organism; and

[0257] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied on the target host organism.

[0258] 68. The method of item 67, wherein the target host organism is a plant.

[0259] 69. The method of any one of items 67-68, wherein the biological property of the bioactive strain exerted on the target host organism is selected from the group consisting of improved growth, yield, defence, robustness, tolerance, a biopesticide biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, Pathogen antagonism, Plant Growth Promotion, Nitrogen Fixation, Phosphorous Solubilization, Pant Induced Systemic Resistance (ISR), Plant Defence Priming, Enhanced Nutrient Cycling, Rhizosphere Modification, Drought Tolerance, Salinity Tolerance, and Pathogen Exclusion and combinations thereof.

[0260] 70. The method of any one of items 1-66, further comprising the steps of:

[0261] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a foodstuff composition; and

[0262] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied to the foodstuff composition.

[0263] 71. The method of item 70, wherein the foodstuff composition is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume.

[0264] 72. The method of item 71, wherein the foodstuff composition is a dairy product or a precursor thereof.

[0265] 73. The method of any one of items 70-72, wherein the biological property of the bioactive strain exerted on the foodstuff composition is selected from the group consisting of improved and / or increased fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in the foodstuff composition, and combinations thereof. A device for obtaining one or more supporting bacteria for a bacterial composition, the device comprising:

[0266] (i) a first compartment comprising at least one entry opening, said at least one entry opening being exposed on the surface of the device;

[0267] (ii) a second compartment within said first compartment; and

[0268] (iii) at least one inner opening connecting said first compartment with said second compartment; wherein said inner opening comprises a first semi-permeable membrane separating said first compartment and said second compartment. The device of item 74, wherein said first compartment is essentially free from bacteria. The device of item 74 or 75, wherein the first semi-permeable membrane has a pore diameter of about 0.1 pm to about 1 pm, such as about 0.1 pm to about 1 pm, about 0.15 pm to about 0.9 pm, about 0.15 pm to about 0.8 pm, about 0.15 pm to about 0.7 pm, about 0.15 pm to about 0.6 pm, about 0.15 pm to about 0.5 pm, about 0.15 pm to about 0.45 pm, about 0.2 pm to about 0.45 pm, about 0.2 pm to about 0.4 pm, or about 0.2 pm to about 0.3 pm, or about 0.15 pm to about 0.25 pm. The device of item 74 or 75, wherein the first semi-permeable membrane has a pore diameter of less than about 2um, such as less than about 2 pm, less than about 1.95 pm, less than about 1.9 pm, less than about 1.85 pm, less than about 1.8 pm, less than about 1.75 pm, less than about 1.7 pm, less than about 1.65 pm, less than about 1.6 pm, less than about 1.55 pm, less than about 1.5 pm, less than about 1.45 pm, less than about 1.4 pm, less than about 1.35 pm, less than about 1.3 pm, less than about 1.25 pm, less than about 1.2 pm, less than about 1.15 pm, less than about 1.1 pm, less than about 1.05 pm, less than about 1 pm, less than about 0.95 pm, less than about 0.9 pm, less than about 0.85 pm, less than about 0.8 pm, less than about 0.75 pm, less than about 0.7 pm, less than about 0.65 pm, less than about 0.6 pm, less than about 0.55 pm, less than about 0.5 pm, less than about 0.45 pm, less than about 0.4 pm, less than about 0.35 pm, less than about 0.3 pm, less than about 0.25 pm, less than about 0.2 pm, less than about 0.15 pm, or less than about 0.1 pm. 78. The device of any one of items 74-77, wherein the first semi-permeable membrane is configured to allow for passage of bacterial metabolites, and / or exclude passage of bacterial cells.

[0269] 79. The device of any one of items 74-78, wherein the entry opening comprises a second semi-permeable membrane.

[0270] 80. The device of item 79, wherein said second semi-permeable membrane has a pore diameter of about 5 pm to about 200 pm, such as about 5 pm to about 200 pm, about 5 pm to about 150 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, about 20 pm to about 100 pm, about 30 pm to about 100 pm, about 50 pm to about 100 pm, about 10 pm to about 40 pm, or about 10 pm to about 30 pm.

[0271] 81. The device of item 79, wherein said second semi-permeable membrane has a pore diameter greater than 50 pm, such as such as greater than about 50 pm, greater than about 60 pm, greater than about 70 pm, greater than about 80 pm, greater than about 90 pm, greater than about 100 pm, greater than about 110 pm, greater than about 120 pm, greater than about 130 pm, greater than about 140 pm, greater than about 150 pm, greater than about 160 pm, greater than about 170 pm, greater than about 180 pm, greater than about 190 pm, or greater than about 200 pm.

[0272] 82. The device of any one of items 79-81, wherein said second semi-permeable membrane is configured to allow passage of bacterial cells.

[0273] 83. The device according to any one of items 74-82, wherein the device is configured to be inserted into a sample comprising a plurality of different bacteria.

[0274] 84. The device according to item 83, wherein the sample is a soil sample.

[0275] 85. The device according to item 83, wherein the sample is a foodstuff sample.

[0276] 86. The device according to item 85, wherein the foodstuff sample is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume.

[0277] 87. The device according to item 85, wherein the foodstuff sample is a dairy product or a precursor thereof. 88. Use of a device according to any one of items 74-87 for obtaining one or more supporting bacteria for a bacterial composition, the use comprising:

[0278] (i) providing a bacterial composition comprising at least one bioactive bacterial strain in the second compartment of said device;

[0279] (ii) providing a medium in said first compartment;

[0280] (iii) placing said device in a sample comprising a plurality of different bacteria, such that the sample contacts the surface of the device; and

[0281] (iv) sampling secondary bacteria from said plurality of different bacteria that migrate into said first compartment from said sample comprising a plurality of different bacteria, thereby obtaining one or more supporting bacteria.

[0282] 89. The use according to item 88, wherein the medium is essentially free from bacteria.

[0283] 90. The use according to item 89, wherein the medium is sterile.

[0284] 91. The use according to any one of items 88-90, further comprising the steps of:

[0285] (iv) co-culturing said one or more secondary bacteria with said bioactive bacterial strain; and

[0286] (v) selecting one or more supporting bacteria that cause a bioactive bacterial strain to perform a function differently than in absence of the one or more secondary bacteria.

[0287] 92. The use of item 91, wherein said function performed differently is biofilm formation.

[0288] 93. The use of item 92, wherein said selection comprises measuring the biofilm formation capability of the co-culture.

[0289] 94. The use of item 94, wherein the secondary bacteria are selected if the biofilm formation capability of the co-culture is greater than the biofilm formation capabilities of a reference.

[0290] 95. The use of item 94, wherein the reference is the bioactive bacterial strain and / or the one or more secondary bacteria cultured separately.

[0291] 96. The use of item 94, wherein the reference is one or more bacterial cultures with known biofilm formation capabilities. 97. The use of item 96, wherein , the reference bacterial culture with known biofilm formation capability comprises Stenotrophomonas rhizophila, Xanthomonas retro flexus, Microbacterium oxydans, and Paenibacillus amylolyticus.

[0292] 98. The use of any one of items 92-97, wherein said biofilm formation capabilities are determined using a Crystal Violet assay.

[0293] 99. The use according to any one of items 88-98, further comprising the steps of:

[0294] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a target host organism; and

[0295] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied on the target host organism.

[0296] 100. The use of item 99, wherein the target host organism is a plant.

[0297] 101. The use of any one of items 99 or 100, wherein the biological property of the bioactive strain expressed on the target host organism is selected from the group consisting of improved growth, yield, defence, robustness, tolerance, a biopesticide biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, Pathogen antagonism, Plant Growth Promotion, Nitrogen Fixation, Phosphorous Solubilization, Pant Induced Systemic Resistance (ISR), Plant Defence Priming, Enhanced Nutrient Cycling, Rhizosphere Modification, Drought Tolerance, Salinity Tolerance, and Pathogen Exclusion and combinations thereof.

[0298] 102. The use according to any one of items 88-98, further comprising the steps of:

[0299] (vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a foodstuff composition; and

[0300] (vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied to the foodstuff composition.

[0301] 103. The use of item 102, wherein the foodstuff composition is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume.

[0302] 104. The method of item 102, wherein the foodstuff composition is a dairy product or a precursor thereof. 105. The use of any one of items 102-104, wherein the biological property of the bioactive strain exerted on the foodstuff composition is selected from the group consisting of improved and / or increased fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in the foodstuff composition, and combinations thereof.

[0303] 106. A kit comprising the device of any one of items 74-87 and instructions for performing the method according to any one of items 1-73 or the use according to any one of items 88-105.

[0304] 107. One or more bacterial strains obtainable by the method according to any one of claims 1-73 or the use according to any one of items 88-105.

[0305] EXAMPLES

[0306] Certain embodiments and features of the present invention are presented in the following non-limiting examples.

[0307] EXAMPLE 1 - Obtainment of supporting bacteria to specific bioactive bacterial strains from a soil sample

[0308] Background and aim

[0309] The method of the invention provides an efficient method of obtaining supporting bacterial strains that may form synergistic bacterial consortia with a bioactive bacterial strain. The method uses the bioactive bacterial strain to produce selection pressure thereby increasing the likelihood that secondary bacteria that migrate towards said bioactive strain can interact with said bioactive strain, possibly synergistically. The present example demonstrates the usefulness of one embodiment of the invention to obtain supporting bacteria using two different bioactive bacterial strains.

[0310] Methods and Results

[0311] Experimental set-up A 15 mL falcon tube was placed inside a 50 mL falcon tube. Holes were made on the side of both tubes and a filter membrane having a pore size of 0.22 pm was fitted on the outside of the 15 ml falcon tube, covering the holes. Both tubes were then filled with sterile soil. A 1 ml culture of the bioactive bacterial strain (SI-391) at ODeoo diluted in sterile PBS was inoculated into the 15 ml falcon tube and both falcon tubes were transferred to a 1 L pot containing non-sterile soil. A simplified schematic of the experimental set-up is presented in Figure 2A.

[0312] Sampling

[0313] After incubation for 7 days in soil the falcon tubes were removed and the 0.22 pm filter was recovered. The side of the filter membrane that had been facing away from the 15 ml falcon tube was then stamped on plates containing R2A bacterial growth medium supplemented with cycloheximide and nystatin to inhibit fungal growth. Bacterial growth was observed on the plates and the bacteria from the plates was obtained, diluted to IO-6to 1015and streaked on new plates containing R2A medium. Single colonies were identified on the new plates and picked based on morphology to obtain isolated bacterial cultures of bacteria that had migrated from the soil and towards the bioactive bacterial strain.

[0314] An identical experimental set-up and sampling was carried out using bioactive bacterial strain SI-930). Bacteria growth was also observed on the R2A plates following stamping, and bacteria could be isolated.

[0315] EXAMPLE 2 - Biofilm formation of co-culture of obtained supporting bacteria and bioactive bacterial strain

[0316] Background and aim

[0317] Example 1 demonstrated that the invention can be used to obtain and isolated bacterial strains that migrate from the soil and towards the bioactive bacterial strain. To further validate that such bacteria may interact with said bioactive strain, possibly synergistically, a co-culture of the obtained bacteria and the bioactive bacterial strain can be screened for synergism.

[0318] Biofilm formation has been extensively used as a marker of synergy within a multi-strain bacterial co-cultures and synergistic effects within a bacterial community have been shown to promote biofilm biomass. Accordingly, co-cultures of the obtained bacteria and the bioactive bacterial strain can be screened for synergism by measuring the biofilm formation capabilities of the co-culture.

[0319] Methods

[0320] Co-cultures consisting of an obtained bacterial isolate and the bioactive bacterial strain or each bacteria separately were normalized based on OD600 to setup a Crystal Violet assay.

[0321] Overnight cultures inoculated in Tryptone soy broth were OD normalized to the same cell density. 2pl of normalized culture was inoculated in 198pl of TSB in a 96-well plate and incubated for 24h at 25°C on agitation (200rpm). After 24h, the wells are washed with lx Phosphate buffer saline (PBS), and then stained with 160 pL of 0,5% CV stained. The stained wells were further washed three times with PBS, and biofilm production was measured spectrophotometrically at OD590 against a blank control. Biofilm production was compared between co-cultures and their individual bacteria using the same assay.

[0322] Results

[0323] Obtained bacterial isolate RI_53 tested positively for synergism with bioactive bacterial strain SI_391. In a co-culture with obtained bacterial isolate RI_53, the biofilm formation of SI_391 increased significantly (p < 0.001) (Figure 3).

[0324] Obtained bacterial isolates RI_87, RI_102, RI_112 tested positively for synergism with bioactive bacterial strain SI_930. In two-strain co-cultures with obtained bacterial isolates RI_87, RI_102, RI_112, the biofilm formation of SI_930 increased significantly (p < 0.001) (Figure 4). A quantitative summary of the biofilm assay can be found in Table 1.

[0325] Table 1 - Biofilm formation (Crystal Violet assay - OD590) of (co-)cultures of obtained bacterial isolates (obtained in Example 1) and the bioactive bacterial strains.

[0326] EXAMPLE 3 - In-vitro interaction assay

[0327] Background and aim

[0328] An alternative approach to biofilm formation as exemplified in Example 2 to assess whether two bacterial strains may interact synergistically is by culturing each bacterial strain separately but in proximity to each other. Bacterial strains that possibly interact synergistically may grow towards each other, grow quicker or change in colony morphology. On the other hand, bacterial strains that interact negatively or antagonistically will grow slower or away from each other. Accordingly, the obtained bacterial isolates obtained in example 1 were tested for synergism with the bioactive bacterial strain in vitro by culturing each bacterial strain in the presence of the other.

[0329] Methods

[0330] Overnight cultures of bioactive bacterial strains SI_391 and SI_930 and obtained bacterial isolates RI_53, RI_87, RI_102 and RI_112 were inoculated in Tryptone soy broth and OD normalized to the same cell density.

[0331] 1 pl droplets of one bioactive bacterial strain and the corresponding obtained isolate were applied on different arms of V-shaped pattern on plates containing R2A medium to generate a spatial gradient between the two strains (Figure 5). Each obtained isolate was paired with the bioactive bacterial strain that had been used to obtain said isolate in example 1.

[0332] The plates were incubated for 13 days at 25°C and colony morphology monitored as a factor of distance between the two strains.

[0333] Results

[0334] Obtained bacterial isolate RI_53 tested positively for synergism with bioactive bacterial strain SI_391. Colonies of SI_391 showed a remarkably larger size when co-inoculated with RI_53. Obtained bacterial isolates RI_87, RI_102, RI_112 also tested positively for synergism with bioactive bacterial strain SI_930. Colonies from SI_930 showed a remarkably larger size when co-inoculated with RI_87 or RI_102 or RI_112, although the obtained bacterial strain remained unaffected.

[0335] Representative images of colony size and morphology following the abovementioned in- vitro interaction assay for each of SI-391 and RI_53, and SI_930 and RI_87 are shown in Figure 5.

[0336] EXAMPLE 4 - Co-culture effect on plant growth promotion.

[0337] Background and aim

[0338] Obtained bacterial isolate RI_53 tested positively for synergism with bioactive bacterial strain SI_391 in biofilm formation assays and in vitro interaction assays (see Examples 1- 3). The co-culture of RI_53 and SI_391 was further tested for its ability to affect growth of a plant being treated with the co-culture, as compared to the constituent strains alone.

[0339] Methods

[0340] In a greenhouse trial the co-culture was tested for its plant growth promotion ability in tomato plants. Co-culture (RI_53 + SI_391) or the constituent strains were cultured to ODeoo=0.1 and coated on the surface of sterilized tomato seeds. Harvest weight, fruit count, and fruit ripening were measured 65 days after transplantation of the seeds into fertilized potting soil.

[0341] Results

[0342] Co-culture of RI_53 and SI_391 showed improved plant growth characteristics as compared to the constituent strains alone. The co-culture showed an increased number of fruits at harvest, earlier ripening of the fruit, as well as higher fruit weight as compared to the constituent strains.

[0343] A quantitative summary is shown in Table 2 and corresponding bar graphs are shown in Figure 6.

[0344] Table 2 - Tomato plant growth characteristics of plants treated with co-culture of RI_53 and SI_391 as compared to constituent strains alone.

[0345] EXAMPLE 5 - Obtainment of supporting bacteria to specific bioactive bacterial strains from a bacterial library for use in improving food characteristics during fermentation

[0346] Background and aim

[0347] Having shown the applicability of the method and device of the invention in soil samples and the use of the obtained co-cultures in plant growth promotion ((see Examples 1-4), it was then sought to show that the method and device of the invention could be used to obtain supporting bacteria from a bacterial library to improve food characteristics during fermentation by two bioactive bacterial strains.

[0348] Several strains have been identified that can lower the pH and coagulate soy milk into soyyoghurt under 20hrs at 37°C, also over seven successive cycles. For health benefits, it can be of interest to fortify this soy-yoghurt with vitamin B12. For flavour benefits, it may also be of interest to produce soy-yoghurt with a more pleasant aroma similar to that associated with dairy yoghurt and reduce or eliminate the planty, beany or earthy aroma and / or flavour.

[0349] Propionibacterium freudenreichii (Pf) is known to produce vitamin B12 in fermented products and Lactobacillus are known to produce a more pleasant aroma in fermented products. It has previously been found that Lactobacillus sanfrancinensis (Ls) produce yogurt with less off-flavour compounds.

[0350] Here we aimed at obtaining a supporting bacterial strain to be combined with the bioactive bacterial strains Pf and Ls to form a three-member consortium that is efficient at fermentation (reduce pH to below 5 in under 8h) and produces soy-yoghurts that are fortified with B12 and have better aromas.

[0351] Methods

[0352] Experimental set-up in sov-voohurt:

[0353] Holes were made on the bottom of a 24-well microtiter plate, and a 15pm filter membrane was fitted on the outside of the 24-well microtiter plate covering the holes. To create a three-zone setup, cell-culture inserts with a 0.22pm membrane were placed inside the wells of the modified plate and subsequently placing the plate inside an omniplate (Figure 2B).

[0354] The omni plate (also referred to as "reservoir zone" herein) was filled with soymilk and inoculated with 11 strains (108cells / ml) of interest selected for fermentation or acidification potential. This was incubated for 8h at 37°C, to allow the liquid soymilk to ferment into a semi-solid soy-yoghurt. The wells in the modified plate were then filled with sterile soymilk (i.e., to create a compartment essentially free from bacteria to which the bacterial cells could migrate into, also referred to as "recruitment zone" herein).

[0355] Cell culture inserts were placed inside the well and filled with soymilk (also referred to as "bait zone" herein), which was inoculated with (i) Ls, (ii) Pf or (iii) Ls and Pf at 109cells / ml (also referred to as "the bait strain(s)" herein). As cell density in the bait zone is 10X higher than in the reservoir zone, a selection gradient is created. The dual plate setup was further incubated for 8h at 37°C.

[0356] The 11 strains of interest inoculated in the reservoir zone comprised Lactobacillus (6), Corynebacterium (1), Enterococcus (1), Leuconostoc (1) and Lactoplantibacillus (1) that were selected for their ability to ferment soy-milk into soy-yoghurt.

[0357] Sampling procedure

[0358] 8 h after incubation, the cell culture inserts were removed from the wells, and the outside of the 0.22pm membrane was sampled by washing it with PBS (Phosphate buffer saline). DNA was extracted from the membrane and reservoir using the NucleoSpin DNA Extraction Kit. 16 V3-V4 amplicon sequencing was performed on the DNA on an Illumina MiSeq to identify the strains that migrated towards the bioactive bacterial strain.

[0359] Distinct bacterial strains from the bacterial library which were sampled and identified as migrating towards the bioactive strains are labelled as La, Lf, Em, Cc and Lm. Bacterial strains from the bacterial library which were not sampled are labelled as Lp, Ljo, Lj, Lh.

[0360] 6 - Combinatorial fermentation

[0361] Background and aim The bacterial strains sampled in Example 5 as migrating towards the bioactive strains are thought to have higher likelihood of interacting synergistically or otherwise complementarily with the bioactive bacterial strains. Accordingly, it was tested whether consortia of the bioactive bacterial strains (Ls, Pf or Ls+Pf) in combination with each of these bacterial strains showed increased fermentation capabilities.

[0362] As a control, non-migrating bacterial strains (not sampled) were also tested for their fermentation capabilities.

[0363] Method

[0364] Overnight cultures of each bacterial strain in the bacterial library and the bioactive strains were grown in MRS and ODeoo normalized to setup a soymilk fermentation assay.

[0365] Each of the 11 bacterial strains in the bacterial library were tested for fermentation of soymilk to soy-yoghurt over 8h at 37°C either as single strains, or in two-strain or three- strain combinations with the bioactive bacterial strains Pf, Ls or Pf+Ls. pH was measured after 8 hours of incubation.

[0366] Results

[0367] The bioactive bacterial strains Pf and Ls were shown to reduce pH to 6.3 and 5.1, respectively. The co-culture of Pf and Ls reduced pH to 5.1.

[0368] When the sampled strains were co-inoculated with Pf+Ls as three-strain-consortia, four out of the five sampled strains (La, Lf, Em and Cc) showed enhanced fermentation of soymilk to soy-yoghurt (P<0.05) in the range of 4.89>pH>4.71. Lm was the only recruited strain that did not reduce pH below 5 (Figure 7 and Table 3).

[0369] In contrast none of the combination between non-sampled strains (Lp, Ljo, Lj or Lh) and the bioactive bacterial strains showed a pH decrease below 5.

[0370] Table 3 - pH values obtained by fermentation with co-culture or single strains.

[0371]

[0372] The results clearly demonstrate that consortia comprising bioactive bacterial strains and supporting bacteria as obtained by the method of the invention were more likely to lead to improved biological efficacy (i.e., increased fermentation) as compared to consortia comprising the bioactive bacterial strains and other bacteria.

[0373] REFERENCES

[0374] M. Bonnet, J.C. Lagier, D. Raoult, S. Khelaifia. Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology, New Microbes and New Infections, Volume 34, 2020

[0375] 2. Wilson C etal. Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review. Res Rev J Eng Technol. 2017.

[0376] 3. Yang, N., Nesme, J., Roder, H.L. et al. Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis. npj Biofilms Microbiomes 7, 82 (2021)

Claims

CLAIMS1. A method for obtaining one or more supporting bacteria for a bacterial composition, the method comprising the following steps:(i) providing a bacterial composition comprising at least one bioactive bacterial strain in a first position;(ii) providing a sample comprising a plurality of different bacteria in a second position; wherein said first and said second positions are separated by a space that is essentially free from bacteria; wherein said first and said second positions are separated by at least one semi-permeable membrane;(iii) sampling secondary bacteria from said plurality of different bacteria that migrate from said second position towards said first position, thereby obtaining one or more supporting bacteria.

2. The method of claim 1, wherein at least one semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

3. The method of any one of the preceding claims, wherein at least one semi- permeable membrane has a pore diameter of about 0.2 pm to about 0.45 pm.

4. The method of any one of the preceding claims, wherein at least one semi- permeable membrane is contiguous to said first position.

5. The method of claim 1, wherein said first and second positions are separated by at least a first semi-permeable membrane and a second semi-permeable membrane, wherein the distance between said first position and said first semi-permeable membrane is less than the distance between said first position and said second semi-permeable membrane.

6. The method of claim 5, wherein said first semi-permeable membrane is contiguous to said first position and / or said second semi-permeable membrane is contiguous to said second position.

7. The method of any one of claims 5 or 6, wherein said first semi-permeable membrane has a pore diameter of about 0.2 pm to about 0.45 pm.

8. The method of any one of claims 5-7, wherein said first semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

9. The method of any one of claims 5-8, wherein said second semi-permeable membrane has a pore diameter of about 10 pm to about 30 pm.

10. The method of any one of claims 5-8, wherein said second semi-permeable membrane has a pore diameter greater than 50 pm.

11. The method of any one of claims 5-10, wherein said second semi-permeable membrane is configured to allow passage of bacterial cells.

12. The method of any one of claims 5-10, wherein said second semi-permeable membrane is configured to impede passage of bacterial cells.

13. The method of any one of the preceding claims, wherein said space essentially free from bacteria further comprises bacterial nutrients.

14. The method of any one of the preceding claims, wherein said first position, said second position and / or said space essentially free from bacteria comprise a bacterial growth medium.

15. The method of claim 14, wherein the bacterial growth medium is selected from the group consisting of agar-based growth media, liquid growth media, animal or plant tissue homogenate media, a foodstuff and soil.

16. The method of claim 15, wherein the bacterial growth medium is soil.

17. The method of claim 15, wherein the bacterial growth medium is a foodstuff.

18. The method of claim 17, wherein the foodstuff is a dairy product.

19. The method of any one of the preceding claims, wherein the bioactive bacterial strain has increased or improved nutrient availability, drought tolerance, temperature tolerance, and / or overall fitness in the presence of said one or more supporting bacteria.

20. The method of any one of the preceding claims, wherein the bioactive bacterial strain has increased or improved fermentation capabilities, acidification capabilities, and / or production of one or more aroma and / or flavour compounds in the presence of said one or more supporting bacteria.

21. The method of any one of the preceding claims, wherein the bioactive bacterial strain and said one or more supporting bacteria have increased biofilm formation capabilities when cultured or applied together, compared to the biofilm formation capability of the bioactive bacterial strain and the one or more supporting bacteria cultured or applied separately.

22. The method of any one of the preceding claims, wherein at least one biological property of the bioactive strain is increased or improved in the presence of said one or more supporting bacteria.

23. The method of claim 22, wherein the at least one biological property is selected from the group consisting of improved growth, yield, defence, robustness and / or tolerance of a plant that is contacted with said first strain.

24. The method of claim 22, wherein the at least one biological property is selected from the list consisting of a biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, pathogen antagonism, plant growth promotion, nitrogen fixation, phosphorous solubilization, plant induced systemic resistance (ISR), plant defence priming, enhanced nutrient cycling, rhizosphere modification, drought tolerance, salinity tolerance, and pathogen exclusion.

25. The method of claim 22, wherein the at least one biological property selected from the list consisting of bioremediation, biodegradation, fermentation, bio-catalysis, acidification, a probiotic effect, aroma development, flavour development, food texturization, bioprotection effect and stability.

26. The method of claim 22, wherein the at least one biological property is selected from the group consisting of improved fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in a foodstuff incubated in the presence of said first strain.

27. The method of any one of the preceding claims, wherein said bioactive bacterial strain is a bacterial isolate.

28. The method of any one of the preceding claims, wherein the sample comprising a plurality of different bacteria is a natural microbial sample.

29. The method of claim 28, wherein the sample comprising a plurality of different bacteria is selected from the group consisting of a soil sample, a water sample, a sediments sample, a faecal sample, a plant tissue sample, an animal tissue sample, a human tissue sample, a foodstuff sample, and a wastewater sample.

30. The method of claim 29, wherein the sample comprising a plurality of different bacteria is a soil sample.

31. The method of claim 29, wherein the sample comprising a plurality of different bacteria is a foodstuff sample, preferably a dairy sample.

32. The method of any one of claims 1-31, wherein the sample comprising a plurality of different bacteria is a bacterial library, comprising two or more isolated bacterial strains.

33. The method of any one of the preceding claims, wherein sampling said secondary bacteria that migrate from said second position towards said first position comprises excising one or more volumes of said space between said first and second positions.

34. The method of any one of the preceding claims, wherein sampling said secondary bacteria that migrate from said second position towards said first position comprises eluting bacteria attached to said semi-permeable membrane and / or said first semi- permeable membrane.

35. The method of any one of the preceding claims, further comprising a step of isolating said secondary bacteria.

36. The method of any one of the preceding claims, further comprising the steps of:(iv) co-culturing said one or more secondary bacteria with said bioactive bacterial strain; and(v) selecting one or more supporting bacteria that cause a bioactive bacterial strain to perform a function differently than in absence of the one or more secondary bacteria.

37. The method of claim 36, wherein said function performed differently is biofilm formation.

38. The method of any one of the preceding claims, further comprising the steps of:(vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a target host organism; and(vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied on the target host organism.

39. The method of claim 38, wherein the target host organism is a plant.

40. The method of any one of claims 38 or 39, wherein the biological property of the bioactive strain exerted on the target host organism is selected from the group consisting of improved growth, yield, defence, robustness, tolerance, a biopesticide biofungicide effect, bioherbicide effect, bioinsecticide effect, bio-stimulation, Pathogen antagonism, Plant Growth Promotion, Nitrogen Fixation, Phosphorous Solubilization, Pant Induced Systemic Resistance (ISR), Plant Defence Priming, Enhanced Nutrient Cycling, Rhizosphere Modification, Drought Tolerance, Salinity Tolerance, and Pathogen Exclusion and combinations thereof.

41. The method of any one of claims 1-37, further comprising the steps of:(vi) applying said one or more secondary bacteria and said bioactive bacterial strain to a foodstuff composition; and(vii) selecting one or more supporting bacteria that increase or improve at least one biological property of the bioactive bacterial when applied to the foodstuff composition.

42. The method of claim 41, wherein the foodstuff composition is selected from the group consisting of a dairy product or precursor thereof, an alcoholic beverage or precursor thereof, a vegetable, a grain product, and a legume.

43. The method of any one of claims 41 or 42, wherein the biological property of the bioactive strain exerted on the foodstuff composition is selected from the groupconsisting of improved and / or increased fermentation, acidification, and / or production of one or more aroma and / or flavour compounds produced in the foodstuff composition, and combinations thereof.

44. A device for obtaining one or more supporting bacteria for a bacterial composition, the device comprising:(i) a first compartment comprising at least one entry opening, said at least one entry opening being exposed on the surface of the device;(ii) a second compartment within said first compartment; and(iii) at least one inner opening connecting said first compartment with said second compartment; wherein said inner opening comprises a first semi-permeable membrane separating said first compartment and said second compartment, and wherein said first compartment is essentially free from bacteria.

45. The device of claim 44, wherein the first semi-permeable membrane has a pore diameter of about 0.2 pm to about 0.45 pm.

46. The device of any one of claims 44 or 45, wherein the first semi-permeable membrane is configured to allow for passage of bacterial metabolites and exclude passage of bacterial cells.

47. The device of any one of claims 44-46, wherein the entry opening comprises a second semi-permeable membrane.

48. The device of claim 47, wherein said second semi-permeable membrane is configured to allow passage of bacterial cells.

49. Use of a device according to any one of claims 44-48 for obtaining one or more supporting bacteria for a bacterial composition, the use comprising:(i) providing a bacterial composition comprising at least one bioactive bacterial strain in the second compartment of said device;(ii) providing medium in said first compartment;(iii) placing said device in a sample comprising a plurality of different bacteria, such that the sample contacts the surface of the device; and(iv) sampling secondary bacteria from said plurality of different bacteria that migrate into said first compartment from said sample comprising a plurality of different bacteria,thereby obtaining one or more supporting bacteria.

50. The use according to claim 49, wherein the medium is essentially free from bacteria.

51. A kit comprising the device of any one of claims 44-48 and instructions for performing the method according to any one of claims 1-43 or the use according to any one of claims 49 or 50.

52. One or more bacterial strains obtainable by the method according to any one of claims 1-43 or the use according to any one of claims 49 or 50.

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