WO2021180941A1 - Dispositif de capture de micro-organismes de l'environnement - Google Patents

Dispositif de capture de micro-organismes de l'environnement Download PDF

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Publication number
WO2021180941A1
WO2021180941A1 PCT/EP2021/056366 EP2021056366W WO2021180941A1 WO 2021180941 A1 WO2021180941 A1 WO 2021180941A1 EP 2021056366 W EP2021056366 W EP 2021056366W WO 2021180941 A1 WO2021180941 A1 WO 2021180941A1
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WO
WIPO (PCT)
Prior art keywords
microorganisms
compartment
nutrient
growth
bulk
Prior art date
Application number
PCT/EP2021/056366
Other languages
English (en)
Inventor
Morten Østergaard ANDERSEN
Magdalena Julia Østergaard ANDERSEN
Andrew Whiteley
Benjamín MOREIRA-GREZ
Original Assignee
Syddansk Universitet
The University Of Western Australia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Syddansk Universitet, The University Of Western Australia filed Critical Syddansk Universitet
Priority to CA3171122A priority Critical patent/CA3171122A1/fr
Priority to EP21710518.8A priority patent/EP4118182A1/fr
Priority to AU2021235144A priority patent/AU2021235144A1/en
Publication of WO2021180941A1 publication Critical patent/WO2021180941A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts

Definitions

  • the present invention relates to a device for capturing microorganisms from the environment and a method for capturing said microorganisms.
  • the present invention relates to a device and a method for capturing microorganisms from soil, water, degrading organic matter and larger living organisms.
  • Microorganisms represent a large diversity of organisms, several of which are likely to be yet unknown. Nevertheless, microorganisms are recognised as being important in many aspects such as human and animal health, agricultural and aquacultural output, food and beverage production, wastewater treatment as well as remediation and restoration of damaged or polluted environments and for several industrial processes such as biological production of chemicals, biomining and leaching processes. New advantages and properties of microorganisms are still being discovered but so far, microorganisms are found to be usable as probiotics, plant growth promotors, biopesticides, biofertilizers and for bioremediation as well as for environmental restoration and biomining. Also, the culture of different microorganisms has led to the discovery of many new biochemicals and pharmaceuticals.
  • microorganisms Despite the still more and more recognised importance of the microorganisms, it is known from sequencing studies that most microorganisms remain uncultured, uncharacterized and non-utilized. It is estimated that only a minority of the diversity of microorganisms has been successfully cultured. This is likely due to the difficulty in replicating the environmental conditions needed for growth by most microbial organisms and the reliance on standard techniques such as culturing and plating of microorganisms on agar. However, provided the beneficial characteristics of the already known microorganisms it would be advantageous to identify further microorganisms for use as e.g. probiotics, biofertilizers, biopesticides or for bioremediation and other processes.
  • an object of the present invention relates to capturing microorganisms with specific characteristics advantageous for use as probiotics, biofertilizers, biopesticides, bioremediation and other microbial processes or for the identification and isolation of new compounds from these microorganisms.
  • This object is solved by providing a device according to the present invention.
  • one aspect of the invention relates to a device for capturing microorganisms from an environment in which the device is placed during use, the device comprising
  • At least one growth compartment comprising at least one entry opening, said at least one entry opening being exposed at a surface of the device for microorganisms to enter the growth compartment from the environment;
  • At least one nutrition compartment separate from said at least one growth compartment; and - at least one release opening fluidly connecting said at least one growth compartment with said at least one nutrition compartment.
  • Another aspect of the present invention relates to a method for capturing microorganisms from an environment; said method comprising the following steps a) providing a device as described herein; b) arranging said device in an environment for entry of microorganisms through the entry opening into said at least one growth compartment, preferably being attracted by chemoattractants from a nutrient bulk being arranged in said at least one nutrient compartment; c) allowing said device to be placed in said environment for a pre-determined period of time; and d) optionally, purifying microorganisms from said device.
  • kits comprising a device as described herein, a nutrient bulk as described herein and/or a solid composition as described herein.
  • Figures 1A-E show the design, construction, assembly and use of an embodiment of a 3D printed Device (Capture Array).
  • Capture Array shows the CAD design of the Capture Array
  • (B) demonstrates an example of a Capture Array loaded with porous ceramics
  • (C) demonstrates the example of a Capture Array from (B) also loaded with Nutrient Capsules
  • (D) describes the mechanism of action of the Capture Array
  • (E) shows four Capture Arrays arranged for use in soil.
  • Figures 2A-D show SEM and EDX images of sintered microporous ceramics.
  • A SEM and
  • B-D SEM/EDX.
  • Figures 3A-B show the relative frequency of Genus Achromobacter (A) and Genus Cupriavidus (B) in samples obtained from soil.
  • Figure 4 shows the relative frequency of Genus Arthrobacter in samples obtained from soil.
  • Figure 5 shows the relative frequency of Family Enterobacteriaceae in samples obtained from soil.
  • Figure 6 shows the relative frequency of Genus Geobacillus in samples obtained from soil.
  • Figure 7A-B shows species that are unassignable species beyond Kingdom Bacteria (A) and Phylum Proteobacteria (B) in samples obtained from soil.
  • Figure 8 shows the relative frequency of Family Enterobacteriaceae in samples obtained from a pond.
  • Figure 9 shows the relative frequency of Family Pseudomonadaceae in samples obtained from a pond.
  • Figure 10 shows the relative frequency of Genus Telmatospirillum in samples obtained from a pond.
  • Figure 11A-D shows the abundance of unclassified microorganisms.
  • A Unclassified bacteria in samples obtained from soil.
  • B Unclassified fungi in samples obtained from soil.
  • C Unclassified bacteria in samples obtained from a pond.
  • D Unclassified fungi in samples obtained from a pond.
  • Figure 12A-C shows the design, construction, assembly and use of a drilled Device.
  • (A) shows the unassembled Device
  • (B) shows an unassembled Device comprising nutrient and capture batches
  • (C) shows an assembled Device arranged in the soil.
  • Figure 13A-H shows the device after use and microorganisms sampled using the device.
  • A shows the device after use in soil for five days.
  • B shows transfer of microorganisms from one porous ceramics to a new porous ceramics.
  • C shows transfer of microorganisms from one porous ceramics to a new porous ceramics.
  • C and (D) show bacteria and fungi spreading from the porous ceramics.
  • E shows microorganisms appearing from a vortex extract of the porous ceramics after 5 days on agar.
  • F shows colonies after secondary plating.
  • G shows growth in LB medium.
  • H shows a still picture from a microscopy video demonstrating motile microorganisms released from the side of the porous ceramics by scratching their surface.
  • Figure 14 shows a device filled with different solid compositions composed of iron, cellulose, tricalcium phosphate and silica.
  • Figure 15A-B shows devices without solid compositions in their growth compartments, one has a nutrient bulk based on glucose.
  • A shows the devices to be arranged in the habitat.
  • B shows the devices retrieved from the habitat.
  • Figure 16A-G shows a microtiter plate sized device and its use for capturing microorganisms.
  • A shows the microtiter plate-sized device;
  • B shows the microtiter plate-sized device including nutrient bulks and solid compositions;
  • C shows the placement of this device in soil;
  • D shows the culture of retrieved solid compositions;
  • E shows the culture of liquid vortex extracts of retrieved solid compositions;
  • F shows a close-up of a single isolate from the vortex extracts for phosphate solubilization on Pikovskaya medium.
  • (G) shows a bar graph having the grow rate of our Genera of microorganisms isolated from the solid compositions from the retrieved device, dark grey is growth on Pikovskaya medium, light grey is growth on phosphate solubilization halo on Pikovskaya medium and white is growth on Jensens medium.
  • Figure 17A-B shows an example of captured strains growing on top of vancomycin resistant Enterococcus faecium (VRE).
  • VRE vancomycin resistant Enterococcus faecium
  • A shows solid compositions retrieved from the device that have been placed on top of vancomycin resistant Enterococcus faecium (VRE) to assess their ability to inhibit VRE. Successful inhibition can be seen around the lower-left solid composition.
  • (B) shows in upper A-D identical cultures of a strain that exhibits inhibition of the surrounding VRE and in lower A-D identical cultures of a strain that does not exhibit inhibition of the surrounding VRE.
  • Figure 18A-C shows the design of a device in the shape of a pill according to the invention and the use hereof in a pig.
  • A shows the separate parts of a pill forming a growth compartment and a nutrition compartment with a release opening connecting the two compartments as well as the inclusion of a nutrient bulk and a solid composition.
  • B shows the principle of the pill with the interaction between the pill and the microorganisms of the gut.
  • C shows how the pill may be fed to a mini-pig for testing of the compatibility of the pill with a living animal.
  • a nutrition compartment is to be understood as a separate compartment in the device in which nutrition such as a nutrient bulk may be included.
  • a nutrient bulk is to be understood as any type of growth medium either in liquid form or in a solid form, preferably in solid form, from which soluble or suspended nutrients may be derived for promoting the attraction and/or growth of the microorganisms.
  • Solid or liquid nutrient bulks may also yield nutrient gasses, aluminium carbide may for example be used as part of a nutrient bulk to generate methane gas as a nutrient for methanotrophic organisms.
  • the nutrients may also function as chemoattractants.
  • the nutrient bulk may provide the nutrient in a slow-release (or controlled release) manner that allows a long-term release of nutrients from the nutrient bulk.
  • the nutrients may be provided in the nutrient bulk as nutrient salts.
  • solid lipids are to be understood as hydrophobic molecules (solubility in water less than lg/L at 20°C) that are solid at the temperature wherein the device is used. It is to be understood that this temperature may vary, a solid lipid suitable for use in a device that is used in a cold environment may not be suitable as a solid lipid if the device is used in a warm environment.
  • the solid lipid composition may be tailored to the intended use of the device.
  • the solid lipids have a melting point above 0°C.
  • lipids include linear and/or circular and/or branched and/or aliphatic organic molecules including alkanes, paraffin, triglycerides, diglycerides, monoglycerides, free fatty acids, fatty alcohols, fatty aldehydes, fatty esters, fatty amines, fatty acid conjugates, phospholipids, cholesterol, other sterols, components of the cell membrane of cells and/or intracellular storage vesicles and salts thereof.
  • the solid lipids are solid fatty acids such as Caprylic acid, Pelargonic acid, Capric acid, Undecylic, Why acid, Tridecylic acid, Myristic acid, Pentadecylic acid, Palmitic acid, Margaric acid, Stearic acid, Nonadecylic acid and Arachidic acid.
  • Solid fatty acids such as Caprylic acid, Pelargonic acid, Capric acid, Undecylic, Why acid, Tridecylic acid, Myristic acid, Pentadecylic acid, Palmitic acid, Margaric acid, Stearic acid, Nonadecylic acid and Arachidic acid.
  • slow-release is to be understood as the nutrients being released from the nutrient bulk over a longer period of time such as 1 day, 2 days, 3 days, 1 week, 1 month, 1 year.
  • Slow-release and long-term release are here used as synonyms that mean that at least some of the initial nutrient bulk is left after the release period (e.g. 5 wt%, such as 3 wt%, like 1 wt%), thus ensuring that nutrients have been released during the entire release period.
  • the wt% left may be more than e.g. 5 wt%.
  • the amount of nutrient bulk left and the time period where release is taking place will depend on the design of the nutrient bulk.
  • essential nutrients are to be understood as known by the skilled person in the art i.e. nutrients required for the survival of a particular microorganism but which cannot be synthesized by the microorganism. Different microorganisms will have different essential nutrients.
  • Essential nutrients may be specific chemicals such as a fatty acid or an amino acid, or it may be an element, such as carbon, nitrogen, oxygen, phosphate or sulphur.
  • lack of essential nutrients is to be understood as the nutrient bulk to be devoid of one or more of nutrients known to be essential for the survival of microorganisms.
  • the lack may be of one or more particular chemicals such as fatty acids or amino acids or it may be the lack of an element, such as carbon, nitrogen, oxygen, phosphate or sulphur.
  • the nutrient bulk may contain the essential nutrient as an insoluble compound that does not transfer to the neighbouring growth compartment.
  • growth compartment is to be understood as a separate compartment in the device in which the microorganisms settle and grow if the growth conditions provided by the nutrient bulk and/or the environment and the conditions of the growth compartment allow survival and growth of the microorganisms.
  • entry opening is to be understood as an opening from the growth compartment to the outer surface of the device allowing microorganisms to enter into the growth compartment from the surrounding environment.
  • the size of the entry opening allows microorganisms to enter into the growth compartment, where they may settle and grow if the growth conditions are suitable for the microorganism.
  • a release opening is to be understood as an opening connecting the growth compartment and the nutrition compartment allowing nutrients e.g. from the nutrient bulk to flow from the nutrition compartment to the growth compartment.
  • microorganisms are to be understood as single or multicellular organisms with at least one dimension being less than 1 mm. This may be Bacteria such as eubacteria and archaebacteria but also fungi, protozoa, algae, arthropods, nematodes, rotifers and other organisms.
  • environment is to be broadly understood as the surroundings of the device from where it is placed in use i.e. it could be terrestrial (e.g. soil, rock, earth, pasture, field), aquatic (e.g. sea, lake, stream, fish pond, sewer, wastewater, groundwater, underground aquifers or reservoirs of e.g. oil or gas), artificial (e.g. in structures, vehicles, buildings or infrastructure possibly within their materials e.g. cement, brick or mortar), within production or distribution systems (e.g. oil, gas or water pipelines and tanks or in fermentation tanks) and within or on living organisms (e.g. within or on a plant, animal or human such as on the surface (e.g. on or in skin, hair, bark, roots) or in the interior (e.g. in the gastrointestinal/reproductive/urinary system, ears, eyes, nose, lungs of an animal or a human or under the bark of a plant).
  • terrestrial e.g. soil, rock, earth, pasture, field
  • aquatic e.g. sea,
  • the present invention relates to the surprising fact that a device according to the present invention advantageously can be used for capturing microorganisms from the environment.
  • a device according to the present invention advantageously can be used for capturing microorganisms having specific characteristics due to a selection based on the availability of essential nutrients either as soluble, insoluble or gas compositions.
  • One aspect of the invention relates to a device for capturing microorganisms from an environment in which the device is placed during use, the device comprising
  • At least one growth compartment comprising at least one entry opening, said at least one entry opening being exposed at a surface of the device for microorganisms to enter the growth compartment from the environment;
  • the device comprises at least one growth compartment capable of maintaining and growing the microorganism and at least one nutrition compartment capable of holding nutrition such as a nutrient bulk used both in the process of capturing the microorganisms as well as in the process of selecting and maintaining the microorganisms of interest.
  • the device also comprises an entry opening for allowing the microorganisms to enter into the growth compartment from the surrounding environment.
  • the size of the entry opening allows at least one microorganism to enter the growth compartment, preferably being of the size of less than 5 mm, such as less than 4.5 mm, like less than 4 mm, such as less than 3.5 mm, like less than 3 mm, such as less than 2.5 mm, like less than 2 mm, such as less than 1.5 mm, like less than 1 mm, such as less than 0.9 mm, like less than 0.8 mm, such as less than 0.7 mm, such as less than 0.6 mm, like less than 0.5 mm in at least one dimension.
  • the at least one dimension is the diameter of the entry opening.
  • the entry opening may also comprise a valve to control when the device opens or closes for microbial entry/exit.
  • the device might comprise one growth compartment, two growth compartments, three growth compartments, four growth compartments, five growth compartments, six growth compartments, seven growth compartments etc.
  • the device might comprise one nutrition compartment, two nutrition compartments, three nutrition compartments, four nutrition compartments, five nutrition compartments, six nutrition compartments, seven nutrition compartments etc.
  • the number of nutrition compartments resembles the number of growth compartments. In a further embodiment, the number of nutrition compartments are higher than the number of growth compartments. In a still further embodiment, the number of nutrition compartments are lower than the number of growth compartments.
  • a growth compartment may be connected to one or more nutrition compartments.
  • a nutrition compartment may be connected to one or more growth compartments.
  • one nutrition compartment is connected with several growth compartments such as eight growth compartments.
  • several growth compartments would be affected by the content of the nutrition compartment and the influence of the content of the growth compartment may be investigated. This may minimize the size of the devices and the use of the number of nutrient bulks.
  • each nutrition compartment of the device is connected to one growth compartment enabling just one nutrient bulk to influence the selection and maintaining of the selected microorganisms. In a further embodiment, several nutrient bulks may influence the selection and growth to the selected microorganisms.
  • the number of release openings connecting a growth compartment and a nutrition compartment can be one or more.
  • said at least one nutrition compartment is arranged next to said at least one growth compartment. As this would minimize the distance between the two thereby reducing the size and material needed for producing the device.
  • the rate of release of nutrients from the nutrition compartment to the growth compartment can be regulated by varying the size of the release opening connecting the two.
  • a slow rate of release would enable a long-term supply of nutrients for extended growth periods, it would also enable growth of microorganisms that are inhibited by too-high concentrations of nutrients, it would also prevent the formation of hypertonic environments detrimental to microorganisms.
  • the design of the release opening may influence the flow between the growth compartment and the nutrition compartment and hereby influence the selection and growth of the selected microorganisms. Flow may be reduced by reducing the dimensions of the release opening and/or by increasing its length i.e. the distance between the growth compartment and the nutrition compartment.
  • the release opening could be an opening as such or a pore or duct. In one embodiment, the release opening is a pore or a duct.
  • the design of the release opening would depend on the overall design of the capture device.
  • the release opening is a pore connecting the nutrition compartment and the growth compartment being arranged next to one another.
  • the pore may be covered by a membrane or filter that allows the exit of nutrients from the nutrient compartment while preventing the entry of microorganisms into the nutrient compartment.
  • a growth compartment may be connected to two or more nutrient compartments and where these nutrient compartments may be connected to two or more growth compartments.
  • This design could minimize the size of the device and/or the nutrients that need to be added by allowing fewer nutrient compartments than growth compartments.
  • 3 nutrients (A, B, C) placed in 3 nutrient compartments could be released through multiple release openings to growth compartments to give them 8 different combinations of nutrients (A, B, C, A+B, A+C, B+C, A+B+C, and none of A/B/C).
  • the size of the release opening prevents microorganisms from entering the nutrient compartment.
  • the size of the release opening is preferably below 500 nm in at least one dimension, such as below 450 nm, like below 400 nm, such as below 350 nm, like below 300 nm, such as blow 250 nm, like below 200 nm, such as below 150 nm, like below 100 nm.
  • the size of the release opening is below 200 nm. In one embodiment, the size is measured as the diameter of the release opening. This prevents the microorganisms from entering the nutrition compartment, maintaining them in the growth compartment.
  • the opening may be larger than 200 nm but covered by a membrane or filter with pores below 200nm.
  • the release opening comprises a valve.
  • the flow from the nutrient compartment to the growth compartment may be controlled. It is to be understood that in case the device comprises more than one release opening, not all of the release openings need comprise a valve.
  • the valves may be mechanically and/or electronically controlled and enable the user to control the specific nutrition provided to the growth chamber, in particular if the growth chamber is connected to several nutrition compartments.
  • the device there are no movable, mechanical or electronic parts in the device to simplify its design, exclude a power source and to lower its production cost and time.
  • designing the diameter and length of the entry e.g. below 1 mm and release openings e.g. below 200 nm and/or by using a slow release nutrient bulk, long term release of nutrients can be provided to both the growth compartment and/or to the external environment without any power source or external manipulation.
  • the entry opening is sufficiently large to allow the entry of at least two different microorganisms i.e. by allowing the entry opening to be larger than the size of the microorganisms allowing the microorganisms to enter the growth compartment and not only block the entry opening.
  • This may enable the establishment/ capture of multi-species consortia in the growth compartments and could be beneficial as it is recognized that many microorganisms are only capable of growing in such consortia and that such consortia may have properties that no single microorganisms has (such as their ability to degrade/produce different chemicals/compounds).
  • the entry opening is above or equal to 1 mm.
  • the entry opening is the only opening exposed to the surface of the device i.e. chemicals, liquids or gasses will only be exchanged between the external environment and the growth compartment via the entry opening.
  • this embodiment would not have larger areas of membranes or filters that allow exchange of chemicals, gasses and liquids between the growth compartment and the external compartment.
  • the influence of the external environment could be minimised allowing chemicals to accumulate to higher concentrations within the growth compartment, such chemicals may be produced by the growing/capture microorganisms (e.g. antibiotics or antifungals) or be chemicals that are already present in the growth compartment or released to it from the nutrient compartments.
  • the device may be of any shape advantageous for the particular use of the device.
  • the device comprises at least one planar or substantially planar outer surface. This is particularly beneficial in cases, where the device is to be arranged on a substantially planar surface enabling the device to maintain in a given position.
  • the shape of the device advantageously reflect the use of the device.
  • the device is a tablet, pill or capsule that may be swallowed by or inserted into a bodily cavity of an individual such as an animal or a human.
  • the device is a patch for being arranged on the dermis on an individual such as an animal or a human.
  • the device may also consist of just one growth compartment and one nutrient compartment to minimize its size.
  • the device is a pill comprising at least one growth compartment having an entry opening, and at least one nutrient compartment, wherein the at least one growth compartment and the at least one nutrient compartment are separated by a spacer having a release opening.
  • the device is a pill comprising a growth compartment having an entry opening, and a nutrient compartment, wherein the growth compartment and the nutrient compartment are separated by a spacer having a release opening.
  • the pill may be assembled from two outer parts, each comprising a compartment, which are separated by the spacer when the two parts are assembled.
  • a solid nutrition bulk is arranged in one compartment (nutrition compartment) and a solid composition is arranged in the other compartment (growth compartment).
  • the capture device is made of plastic.
  • other materials may also be used such as other polymers, metals, alloys, glasses, silicones and ceramics or combinations thereof as well as combinations thereof with plastic.
  • the device is transparent. This allows visual inspection or measurement of its content possibly aided with a microscope, spectroscope, spectrometer, fluorimeter, luminometer and/or light source or light detector.
  • a transparent device may also allow the growth or manipulation of photosynthetic, phototrophic or photoresponsive microorganisms.
  • the nutrition compartment, nutrient bulk, growth compartment and/or solid composition within the growth compartment may be added a chemical such as a dye or fluorophore that aides or enables to study/visualization of the growth or nutrient compartment.
  • The may be a pH sensitive indicator dye or fluorophore, or a compound that indicates whether a chemical is being produced, degraded or solubilized.
  • the device comprises a base part and a lid, said base part comprising insertion openings exposed to a surface of said base part; said insertion openings allowing insertion and retrieval of said nutrient bulk into/from said at least one nutrition compartment and optionally said solid composition into/from said at least one growth compartment; said lid, in use, being arranged on said surface of said base part, where said insertion openings are exposed, covering said insertion openings.
  • This embodiment allows easy insertion of the nutrient bulk into the nutrition compartment(s) of the device and of potential material such as porous materials into the growth compartment(s) and following removal hereof after capturing of microorganisms.
  • the lid prevents that the nutrient bulk and/or solid compositions fall out during use. It may also prevent the contamination of the nutrition and growth compartments during use of the device and ensures that the only entry of microorganisms into the device is through the entry opening.
  • the lid may be made of a material as the base part of the device as illustrated in fig. 11. However, the lid may be any kind of material or design, which cover potential insertion openings of the base part of the device when in use. In one embodiment, the entry opening is arranged in said lid. In another embodiment, the lid is transparent.
  • the lid comprises entry openings connecting the growth compartments in the base part to the surface of the device.
  • the lid comprises insertion openings being connected with the insertion openings in the base part. This may allow easier retrieval of nutrient bulk and/or solid compositions after use.
  • the device may be tailored to attract microorganism with desirable properties by tailoring the nutrient bulk and/or material in the growth compartment.
  • oil and nitrate may be combined as the electron donor and acceptor in the nutrient as a bait for oil oxidizing/nitrate reducing bacteria (other pollutants may be used instead of oil e.g. pesticides).
  • phosphate-solubilizing bacteria for agricultural biofertilizer use or for use in biomining/ore leaching, all nutrient elements could be provided except phosphate in the nutrient bulk while the growth compartment could comprise phosphate e.g. as a solid substrate.
  • microorganisms may be selected that produce anti-microbial compounds, which could have a value as antibiotics, antifungals, anti-cancer drugs or other pharmaceuticals, pesticides or biocides while the microorganism itself may be potential probiotic candidates e.g. for treatment/prevention of human or animal gastro-intestinal illnesses or as biopesticides for plant illnesses. Another application would be to test which microorganisms would amplify upon the addition of nutrients to an environment.
  • Such data could be used to guide, set or control nutrient emissions to the environment and/or be used to predict whether toxic or otherwise problematic microorganisms (e.g. cyanobacteria or algae) may be amplified in an environment.
  • the device comprises a nutrition compartment with a mix of nutrients (nutrient bulk) that are selected so that attraction and growth of microorganisms with desirable properties are encouraged.
  • the nutrient compartment is connected to a growth compartment preferably comprising a solid substrate, preferably a porous solid substrate, for entrapping the attracted microorganisms and/or providing them with a large surface area to which they can adhere/adsorb and grow on. Only the growth compartment is connected to the outside environment when in use. When placed in the environment, the nutrient compartment starts to release nutrients through the growth compartment to the environment, attracting microorganisms through chemotaxis. Once the microorganisms enter the growth compartment, they get trapped in the solid substrate wherein they grow.
  • the device offers a slow release of nutrients, which enables long-term attraction and culture.
  • At least one of said at least one nutrition compartments comprises a nutrient bulk.
  • the nutrient bulk is designed specifically for the purpose of attracting and selecting specific microorganisms of interest based on its nutrient composition.
  • the nutrient bulk is a solid nutrient bulk.
  • the solid nutrient bulk is a pellet, a pill or a powder.
  • said nutrient bulk comprises soluble nutrients such as water soluble nutrients and/or suspendable nutrients such as water-suspendable nutrients.
  • Soluble nutrients may be compounds such as organic acids, carbohydrates, lipids, amino acids and inorganic ions like sulphate, phosphate, ammonia and/or nitrate.
  • Suspendable nutrients may be compounds such as microplastic, certain carbohydrates, oil, alkanes and other hydrophobic chemicals, metals, ores and insoluble salts. This enables the nutrient bulk to release such nutrients once the nutrient bulk comes into contact with a solvent such as water.
  • the device comprises an internal or is connected to an external water or liquid reservoir that is fluidly linked to the nutrient compartment and/or the growth compartment, preferably the nutrient compartment.
  • the device does not comprise water but will be activated once placed in a water-containing environment. This would allow the device to be stored and distributed; it would also allow the device to be placed in a dry environment for activation by the later addition of water to that environment, e.g. triggered by rainfall.
  • said one or more essential nutrients is selected from the group consisting of: a vitamin, carbohydrate, protein co-factor, fatty acid, amino acid, nucleotide, peptide, protein, another organic molecule, a gas or where it is selected from the group of chemicals comprising e.g. C, N, O, S, P, Fe, Ca and Mg. These latter groups are commonly known as "carbon sources", “nitrogen sources” etc.
  • said nutrient bulk comprises one or more of the following components: A fatty acid, a carbohydrate, an amino acid, a nitrate, a sulfate, ammonia, sulphur or iron, preferably Myristic Acid, Glucose, Glycine, NaN03, Na2S04, NH4CI, Sulphur (S°), S1O2 and/or FeCh.
  • said nutrient bulk comprises chemoattractants.
  • the chemoattractants diffuse via the release opening into the growth compartment and further via the entry opening into the environment to attract specific microorganisms. It is known that specific microorganisms are attracted by different chemoattractants e.g. carbohydrates, amino acids, peptides and proteins and inorganic ions.
  • the nutrient bulk is a slow-release (or controlled release) matrix providing a long-term release of the soluble and/or suspended nutrients and/or chemoattractants.
  • the slow-release matrix providing a long-term release can e.g. be obtained by amending the size of the release opening or by amending the composition and/or design of the slow-release matrix.
  • said nutrient bulk is made from nutrient salts embedded in solid lipids, preferably solid fatty acids.
  • the amount of nutrient salts may be 50-95 wt% together with at least 5 wt% solid lipids, such as 75 wt% nutrient salts and 25 wt% solid lipids, like 67 wt% nutrient salts and 33 wt% solid lipids.
  • the nutrient bulk being a slow-release matrix may be prepared according to the methods as known to the persons skilled in the art and described in WO2017/059866 A2 as well as being exemplified herein in example 1 for enabling a slow-release of the nutrients in a long-term fashion.
  • said nutrient bulk lacks one or more essential nutrients.
  • the nutrient bulk is devoid of one or more essential nutrients, which are necessary for the proper growth of the microorganisms, the nutrient bulk is not able to support the growth of the microorganisms in a sufficient manner by itself but the microorganism needs to derive the lacking essential nutrients from alternative sources in order to survive in the device.
  • the nutrient bulk is not able to support the growth of the microorganisms in a sufficient manner by itself but the microorganism needs to derive the lacking essential nutrients from alternative sources in order to survive in the device.
  • only microorganisms capable of deriving the lacking essential nutrients from alternative sources will grow in the device leaving other microorganisms either to die or to migrate to other places optimal for their growth.
  • said nutrient bulk lacks one or more essential nutrients, said essential nutrients are present within the growth compartment either as gasses such as l ⁇ and/or ChU or as part of said solid composition.
  • the microorganisms may be able to derive the lacking essential nutrients from the content of the growth compartment e.g. by fixating gasses such as nitrogen e.g. from the air or gases dissolved in liquid present in this compartment.
  • the microorganisms would be able to derive the lacking essential nutrients from solid sources present in the growth compartment.
  • at least one of said at least one growth compartment comprises one or more solid compositions.
  • said one or more solid compositions comprises one or more essential nutrients as insoluble nutrients.
  • the insoluble nutrients may be solubilized, metabolized or degraded by microorganisms.
  • the insoluble nutrients may be attached to the internal surfaces of the growth compartment by covalently or non-covalently bindings between the material of the growth compartment and the insoluble nutrients.
  • the insoluble nutrients may be part of a solid composition, which can be placed into the growth compartment i.e. the solid composition is separate from the growth compartment.
  • the solid composition may easily be arranged in the growth compartment and/or retrieved from the growth compartment as e.g. demonstrated in the examples.
  • the solid composition may be a pellet.
  • the device comprises more solid compositions, which comprises different essential nutrients for capturing different types of microorganisms.
  • a device may comprise a solid composition comprising iron in one growth compartment, a solid composition comprising cellulose in another growth compartment and a solid composition comprising tricalcium phosphate and silica.
  • the solid composition in the growth compartment does not comprise nutrients. In a further embodiment, the solid composition in the growth compartment is impermeable i.e. it is not porous.
  • said solid composition is a primarily inorganic material such as a natural or synthetic rock, clay, sand, mineral, ore, sediment, soil, a glass, a ceramic or mine tailings.
  • the solid composition comprises at least 80 wt% inorganic material, such as at least 85 wt% inorganic material, like at least 90 wt%, such as 95 wt% inorganic material, like 98 wt% inorganic material, such as 99 wt% inorganic material.
  • said solid composition comprises a water insoluble phosphate salt, preferably Ca3(P04)2, Cas(P04)3(0H), Mg3(P04)2 or other calcium or magnesium salts.
  • said solid composition comprises S1O2 and a phosphate salt, such as 80-20 wt% S1O2 and 20-80 wt% Ca3(P04)2, such as 30-70 wt% S1O2 and 70-30 wt% Ca3(P04)2, such as 40-60 wt% S1O2 and 60-40 wt% Ca3(P04)2, such as 50 wt% S1O2 and 50 wt% Ca3(P04)2.
  • a phosphate salt such as 80-20 wt% S1O2 and 20-80 wt% Ca3(P04)2, such as 30-70 wt% S1O2 and 70-30 wt% Ca3(P04)2, such as 40-60 wt% S1O2 and 60-40 wt% Ca3(P04)2, such as 50 wt% S1O2 and 50 wt% Ca3(P04)2.
  • said solid composition is porous.
  • said porous solid composition is a porous ceramic such as a phosphate (e.g. Ca3(P04)2), sulfate (e.g. CaSC ), oxide (e.g. S1O2, AI2O3, Fe304, FeO, Fe203) or a halogen (e.g.
  • LiCI, NaCI, KCI organic such as cellulose, lignin, lignocellulose, alginate, collagen, chitin, polyethylene, polystyrene, polylactic acid, polycaprolactone and other natural and synthetic polymers; metallic such as iron, uranium, copper and alloys like steel, bronze and brass; glassy such as silicate glass and borosilicate glass; a construction material such as concrete and cement; a natural inorganic material like igneous, metamorphic and sedimentary rocks, sediments and soils; or organic material such as wet or dehydrated parts of food, plants and animals.
  • the porous composition could also be a porous hydrogel.
  • said porous composition has pores with dimensions that are sufficiently large to allow bacteria and other microorganisms to penetrate into the habitat but sufficiently small to trap them and/or to exclude larger organisms.
  • the pores may be larger than the size of the microorganisms to be captured/grown such as around 1 mm, like around 500 m ⁇ h, such as around 200 m ⁇ h, like around 100 m ⁇ h, such as around 50 m ⁇ h, like around 20 m ⁇ h, such as around 10 m ⁇ h, like around 5 m ⁇ h, such as around 2 m ⁇ h, like around 1 m ⁇ h for at least one dimension.
  • the size could be optimized to allow entry of smaller microorganisms while excluding the entry of larger microorganisms. To trap the microorganisms it would be advantageous if the porous geometry does not allow direct access through the habitat.
  • pores in the porous composition may also be advantageous, as this would allow passage of nutrients and gasses through the porous composition.
  • a preferable embodiment would be a porous composition that contains larger pores that allow entry into and entrapment of microorganisms in the porous composition as well as smaller pores that allow passage of chemicals and gasses to these.
  • at least 10% of the pores are of the size as indicated herein, such as at least 20% of the pores, like at least 30% of the pores, such as at least 40% of the pores, like at least 50% of the pores.
  • the size of the pores are within a range of 0.1-1000 m ⁇ h, such as a range of 0.3-800 m ⁇ h, like a range of 0.5-700 m ⁇ h, such as a range of 0.7-500 m ⁇ h, like a range of 0.8-250 m ⁇ h, such as a range of 0.9-100 m ⁇ h, like a range of 1-50 m ⁇ h, such as 1-10 mGh.
  • the void volume of the porous solid composition is at least 10% v/v, such as at least 20% v/v, like at least 30% v/v, such as at least 40% v/v, like at least 50% v/v, such as at least 60% v/v, like at least 70% v/v, such as at least 80% v/v, like at least 90% v/v.
  • the void volume of the porous solid composition is below 90% v/v, such as below 80% v/v, like below 70% v/v, such as below 60% v/v, like below 50% v/v, such as below 40% v/v, like below 30% v/v, such as below 20% v/v, like below 10% v/v.
  • said solid composition is a biomass such as wood, straw, fibre, compost, algae, seaweed, exoskeletons, skeletons, shells, seeds, seed capsules, sludge and/or faeces, and/or a partially or fully isolated or purified component thereof such as lignin, alginate and cellulose.
  • said solid composition is a solid food or feed substance such as a meat, a vegetable, a fruit, a cereal or a food protein and/or carbohydrate, and/or a component thereof such as gluten and/or pectin.
  • At least one of said one or more solid compositions is inoculated with at least one microorganism prior to being arranged in the growth compartment. This would allow moving the microorganisms to a different habitat as well as allow for selection of other microorganisms, which are symbiotic with the inoculated microorganisms or which are able to compete with the inoculated microorganisms e.g. by combating the inoculated microorganisms by producing specific anti-microbials. The latter may serve as a method for discovering microorganisms with inhibitory activity against human, animal or plant pathogens.
  • the solid composition may be prepared by many different means such as casting, molding, sintering, additive manufacturing (e.g. 3D printing) or subtractive manufacturing (e.g. milling or drilling) as known by the persons skilled in the art e.g. as described in WO2017/059866 A2 and as also exemplified herein in examples 1 and 2 for enabling capture of microorganisms in the growth compartment.
  • additive manufacturing e.g. 3D printing
  • subtractive manufacturing e.g. milling or drilling
  • a further aspect of the invention relates to a method for capturing microorganisms from an environment; said method comprising the following steps a) providing a device as described herein; b) arranging said device in an environment for entry of microorganisms through the entry opening into said at least one growth compartment, preferably being attracted by chemoattractants from a nutrient bulk being arranged in said at least one nutrient compartment; c) allowing said device to be placed in said environment for a pre-determined period of time.
  • the pre-determined period of time could be 1 day, 1 week, 1 month etc. this would depend on the environment and the microorganisms to be captured.
  • the pre determined period of time should allow the device to collect and grow the microorganisms in the growth compartment. Thus, it should allow the microorganisms to grow sufficiently in the growth compartment for the amount of microorganisms to be sufficient for subsequent purification.
  • the method further comprises the step of purifying microorganisms from said device.
  • the microorganism may be purified in a manner allowing the microorganisms to be collected for further growth and optimisation outside said device such as on agar or in liquid medium.
  • the purified microorganisms may be further optimised with respect to certain specific characteristics like processing of biomass.
  • said growth compartment comprises a solid composition.
  • said method further comprises the step of removing said solid composition from said growth compartment and arranging said solid composition in a habitat different from the habitat where the device were arranged. Hence, the solid compositions are removed from the device and placed in a new environment to transfer microorganisms from the initial environment to this new environment.
  • said method further comprises the step of removing said solid composition from said growth compartment and arranging said solid composition next to a new solid composition to allow migration of microorganisms from said solid composition to said new solid composition.
  • the solid composition from the growth environment is, after the capture and growth of microorganisms from one environment, used to transfer these microorganisms to a new environment e.g. by placing the solid composition in this new environment.
  • This could be, for example, a transfer of soil microorganisms from one site (say a productive field or a healthy ecosystem) to a new site (say a new field or damaged or degraded ecosystem) or of probiotics from one individual (e.g. a healthy animal or person) to another (e.g. a sick animal or person).
  • the solid compositions may be incubated, cultured or grown and/or propagated to additional solid compositions prior to the placement in the new environment.
  • the method of capturing the microorganisms includes a selection system such as a double-selection system using at first a selection based on the chemoattractants present in the nutrient bulk placed in the nutrient compartment acting as a bait for luring the microorganisms into the growth compartment of the device through the entry opening and a second selection based on the nutrient bulk lacking one or more essential nutrients.
  • a selection system such as a double-selection system using at first a selection based on the chemoattractants present in the nutrient bulk placed in the nutrient compartment acting as a bait for luring the microorganisms into the growth compartment of the device through the entry opening and a second selection based on the nutrient bulk lacking one or more essential nutrients.
  • said nutrient bulk lacks one or more essential nutrients, said one or more essential nutrients are either to be obtained from a gas or from a solid composition present in the growth compartment. If the lacking essential nutrients cannot be derived from the growth compartment such as from potential insoluble nutrients provided e.g. by a porous rock trap habitat. Microorganisms might be able to derive the lacking essential nutrients from gas or dissolved gasses such as a gas selected from the group of H2, N2, NH3, C2H2, C2H4, CO, CPU, H2S, SO3 and/or CO2.
  • said nutrient bulk slowly releases the nutrients from said nutrient bulk such as slow release of glucose, urea, NO3 , PO4 3 and/or NH4T
  • said nutrients such as slow release of glucose, urea, NO3 , PO4 3 and/or NH4T
  • a slow release will only drain the nutrient content of the nutrient bulk to a minor degree enabling the content of nutrients in the nutrient bulk to last for a longer period of time.
  • said environment is soil or water. In an even further embodiment, said environment is soil, water, a plant, plant roots or an individual such as an animal or human.
  • a further aspect of the invention relates to the use of a device as described herein and/or a method as described herein for capturing microorganisms from an environment, such as soil, water, a plant, plant roots, or an individual such as an animal or human.
  • the microorganisms are isolated from the surface, cavity or internal of said individual, such as from the skin or gut.
  • a further aspect of the invention relates to the use of microorganisms captured by a device as described herein, microorganisms purified by a method as described herein or said solid compositions containing microorganisms as described herein as probiotics, anti-microbials, biofertilizers, biopesticides, and/or bioremediation or for use in biomining, bioleaching or for producing/isolating/purifying new compounds such as new pharmaceuticals.
  • the captured anti-microbial microorganisms are used in human or animal health, in crop/plant protection or in food/beverage production and/or preservation.
  • the captured anti-microbial microorganisms is used as sources of new chemical compounds e.g. new antibiotics, anti-fungals, anti-parasite drugs, immunosuppressive drugs, cancer drugs, preservatives, biocides, pesticides and fungicides.
  • the present invention relates to a kit comprising a device as described herein, and nutrient bulk as described herein and/or a solid composition as described herein.
  • the invention further relates to a kit ready for use, where the device is provided together with the nutrient bulk(s) of interest and optionally also a solid composition(s) of interest.
  • the nutritional bulk is arranged in said nutrition compartment and optionally said solid composition is arranged in at least one growth compartment.
  • the device comprises a base part and a lid.
  • the kit is sterile.
  • a sterile, packaged and water-free device can be provided in a kit being ready for use.
  • Example 1 Baiting, capturing and enriching soil and aquatic microorganisms
  • the device was then designed in Tinkercad and 3D printed using a Moonray DLP 3D printer using grey resin (Sprintray).
  • the device was washed with isopropanol, cured and sterilized using UV light, loaded with the porous ceramics and the nutrient pellets, closed using bolts and nuts. The device was then placed at 5cm depth in leaf covered soil (31°59’01.7"S 115°49'11.9"E) or on the bottom of a 10cm deep pond (31°59O2.1"S 115°49'10.3"E) for 9 and 7 days, respectively.
  • the device was then retrieved along with control samples of soil or sediment, the porous rock trap habitats were removed from the device, and their DNA was isolated using a PowerSoil DNA kit (Qiagen).
  • the DNA was sent for 16S sequencing and the sequences were analyzed in R using the Qiime2 workflow.
  • Figure 1A-E The design of the device 101 is shown in Figure 1A-E.
  • Figure 1A shows the CAD design of the device 101 demonstrating several nutrition compartments 103 having a release opening 105 connecting the nutrition compartments 103 with the growth compartment 109.
  • the growth compartments 109 further comprises entry openings 107 for the microorganisms to enter the growth compartment 109 when the device 101 is in use.
  • Porous ceramics 111 may be inserted in the growth compartments as shown in Figure IB.
  • the nutrient pellets 113 are arranged in the nutrient compartments as shown in Figure 1C separately from the porous ceramics 111.
  • the one or more device 101 are placed e.g. onto soil as shown in Figure IE.
  • Figure ID demonstrates the functioning of the device according to the invention showing the slow-release of the nutrient bait 115 from the nutrition compartment 103 comprising the nutrient pellet via the release opening 105 into the growth compartment 109 with the porous ceramics diffusing further to the outside via the entry opening 107.
  • the diffusing nutrients attracts the microorganisms to enter 117 the growth compartment 109 being trapped herein.
  • the porous ceramics used were investigated using SEM and EDX. Close inspection with SEM ( Figure 2A) reveals that the pores have a non-linear geometry with diameters varying in size but where many are larger than 1 pm and smaller than 10 pm corresponding to the diameter of many microorganisms, which enables the infiltration, and entrapment of these. SEM/EDX ( Figure 2B-2D) reveals that the ceramic components are well distributed in the material.
  • the ability of the different nutrient pellets to attract different types of microorganisms were investigated in triplicates for traps having been placed in soil or in a pond, respectively.
  • the different types of microorganisms were identified using DNA measurements of the 16S RNA and the result is demonstrated in the Figures 3-10 and in Tables 1-2.
  • the relative frequency (the number 16S sequences belonging to a particular species divided by the total number of 16S sequences) of Genus Achromobacter and Genus Cupriavidus from samples obtained from placement of the device in soil is shown in Figure 3A and 3B, respectively. Both are enriched in the 'no nutrients', 'C14' and 'C14+NH4CI' samples, Achromobacter is also enriched in the 'C14+S'.
  • Achromobacter is known to include members that fix nitrogen and solubilize phosphate and that are useful for promoting the growth of vegetables.
  • Cupriavidus are also known to fix nitrogen and are common nodulation bacteria that stimulate plant growth.
  • the soil control samples as such show only low abundance illustrating the ability of the different nutrient pellets to enrich the amount of specific microorganisms.
  • Genus Arthrobacter The relative frequency of Genus Arthrobacter from samples obtained from placement of the device in soil is shown in Figure 4. This Genus is particularly enriched in the 'C14+G+G' samples.
  • the soil samples as such show only low abundance illustrating the ability of the different nutrient pellets to enrich the amount of specific microorganisms.
  • the relative frequency of Family Enterobacteriaceae from samples obtained from placement of the device in soil is shown in Figure 5. This family is particularly enriched the 'C14+G+G+N03' samples. Enterobacteriaceae are known to ferment glucose and reduce nitrate. The soil samples as such show only low abundance illustrating the ability of the different nutrient pellets to enrich the amount of specific microorganisms.
  • Genus Geobacillus from samples obtained from placement of the device in soil is shown in Figure 6. This genus is particularly enriched in the 'C14+G+G+S04' samples. Geobacillus are known to reduce sulfate. The soil samples as such show only low abundance illustrating the ability of the different nutrient pellets to enrich the amount of specific microorganisms.
  • the relative frequency of species that are unassignable species beyond Kingdom Bacteria and Phylum Proteobacteria from samples obtained from placement of the device in soil are shown in Figures 7A and 7B, respectively.
  • the soil samples as such show only low abundance illustrating the ability of the different nutrient pellets to enrich the amount of specific microorganisms.
  • the relative frequency of Family Enterobacteriaceae from samples obtained from placement in a pond is shown in Figure 8. This family is particularly enriched in the 'C14+G+G' and 'C14+G+G+N03' samples. Enterobacteriaceae are known to ferment glucose and reduce nitrate.
  • the pond samples as such show only low abundance illustrating the ability of the different nutrient pellets to enrich the amount of specific microorganisms.
  • Genus Telmatospirillum from samples obtained from placement in a pond is shown in Figure 10. This genus is particularly enriched in the 'C14' samples.
  • Table 1 and Table 2 show the relative frequency data (%) shown for some microorganisms from the MicroCaptureArray samples incubated in soil or in a pond, respectively.
  • the device according to the present invention may be used for targeted capture of microorganisms with desirable metabolic properties.
  • microorganisms may be used in e.g. agriculture, wastewater treatment, environmental remediation, biomining (e.g. ore solubilization) or for pharmaceutical/probiotics discovery.
  • the device may be used to find rare species in an environment that can be activated by the addition of particular nutrients, this may be valuable for knowing how an ecosystem might react to different types of nutrient input e.g. of N, P or K fertilizer input. This could be used to determine what nutrients that triggers eutrophication or the growth of harmful, toxic and/or pathogenic microorganisms.
  • Example 2 Baiting, capturing and enriching microorganisms from compost heap and soil
  • Scope The scope of this example is to demonstrate an alternative design of a Device according to the invention and methods for capturing, growing, isolating, utilizing and further culturing the captured microorganisms.
  • glucose and stearic acid powder was mixed in the weight ratio 97:3, the mixed powder was placed in a steel mold and was then compressed to a cylindrical pellet using a piston with 10 tons of pressure for 5 minutes. After compression the solid pellet was removed.
  • the devices were then placed at 10 cm depth in a leaf covered grass lawn and near the bottom of a ⁇ lm tall enclosed 3 year old compost heap full of degrading organic material.
  • the air temperature was 4°C
  • soil temperature was 4.5°C
  • the compost temperature was 5.0°C.
  • Figure 13A shows the device retrieved from soil.
  • the porous ceramics ("Samples" below) were then taken out for further analysis. Some were frozen for DNA extraction and sequencing; background soil and compost samples were also taken. Samples 1-8 from compost were distributed as following : #1-3 half for sequencing, half for plating, #4 and 8 for ceramic to ceramic transfer, # 5-7 for sequencing. Sample 9-16 from soil were distributed as following : # 9-11 for plating, # 12-13 for ceramic to ceramic transfer, # 14-16 for sequencing.
  • Samples for plating were transferred to a 1.5 mL tube. 500 pL filtrated and autoclaved soil or compost solution was added and samples were vortexed for 30 seconds. 250 pl_ of the solution was transferred to agar plates which were based on compost and soil agar, respectively (lg/L Glucose, 0,5 g/L dipotassium phosphate, 17 g/L soil or compost extract, pH 6.5, 15 g/L Agar). The remaining ceramics was placed in the center of a separate agar plate after the vortexing. The plates were incubated at 25°C under both aerobic and anaerobic conditions.
  • the samples for ceramic to ceramic transfer were, after retrieval from the device, placed in liquid soil or compost medium together with identical but new and sterile ceramics. This was done to propagate and further culture/ expand the microorganisms to multiple ceramics.
  • Figure 12A show the device comprising a base part 219 and a lid 221. Furthermore, screws 223 are shown for attaching the lid 221 to the base part 219.
  • the base part 219 comprises one nutrition compartment 203 and eight growth compartments 209.
  • a nutrient bulk 213 may be inserted into the nutrition compartment and porous ceramics 211 into the eight growth compartments of the base part 219 as shown in figure 12B.
  • Figure 12C shows the device 201 in use placed in the soil having the lid attached to the base part of the device.
  • Figure 13A shows the device with samples after retrieval from soil. A similar results was shown for the device retrieved from compost (data not shown).
  • Figure 13B shows transfer of microorganisms from incubated porous ceramics to new sterile ceramics by arranging the porous ceramics from the device next to new ceramics.
  • Figure 13C and 13D show bacteria and fungi spreading onto the surrounding agar from incubated porous ceramics inserted into the agar after 5 days of culture in soil. Similar results were achieved from a porous ceramics after 5 days of culture in compost.
  • Figure 13E shows many different bacterial and fungal colonies appearing after the first plating of the vortexed ceramics extract on agar after 5 days.
  • Figure 13F shows colonies appearing 3 days after secondary plating of picked colonies from the first plating on agar.
  • Figure 13G shows an example of a microbial culture growing in LB medium that were picked from the vortexed ceramics extract that was plated on agar.
  • Figure 13H shows still pictures from a microscopy video demonstrating motile microorganisms released from the side of the porous ceramics by scratching their surface.
  • the device may be designed by drilling instead of 3D printing.
  • the device may have one nutrient compartment that is connected to multiple growth compartments.
  • the device traps microorganism, some of these are motile. Some of the trapped species are bacterial, some are fungal, the growth compartment and solid compositions placed therein may capture different species that form different types of colonies. The trapped species may be released from the traps and be cultured subsequently in liquid medium or on agar wherefrom pure cultures may be isolated. The inoculated solid compositions (here ceramics) retrieved from the device may also be propagated directly to other sterile solid compositions (here ceramics). When placed in new environments, inoculated solid compositions (here ceramics) release microorganisms to this environment thus inoculating it, here this was done with agar but it could have been the inoculation of soil, water, animals or humans or other environments.
  • the inoculated ceramics may be studied to characterize the interaction between the habitats and trapped microorganisms.
  • the nutrients in the nutrient bulk does not have to be encapsulated in fatty acids in order to functionally attract microorganisms into the ceramic rock traps.
  • the ceramics may be made of pure tricalciumphosphate and still be able to capture microorganism.
  • the size and length of the entry and release openings dictate the solubilization and release rate of the nutrients from the nutrient bulk, a completely and rapidly water soluble nutrient bulk (here glucose, the pellets used here dissolve in 1-2 hours in water) can remain after 3 weeks of in situ culture if the openings are small/long enough thus providing a slow release of nutrients over this period.
  • Solid compositions for placement in the growth compartments composed of iron, cellulose, tricalcium phosphate and silica were created.
  • the tricalcium phosphate, iron and silica cylinders were created by mixing 6g myristic acid with 25g Ca3(P04)2, Fe304 or S1O2 powder, respectively, and then molding and sintering the samples as in example 2.
  • the cellulose samples were created by dissolving/suspending cellulose in water and molding the mixture in the silicone mold from examples 1+2. The silicone mold was then dehydrated at 60°C creating dry cellulose cylinders that could then be removed from the mold.
  • the solid compositions were placed in the growth compartments in a device loaded with glucose/stearic acid (97:3, as in example 2) in its nutrition compartment. The devices were then placed in similar locations to those used in example 2.
  • the loaded device 301 is shown in figure 14 having the cellulose nutrient bulk 313, 2x solid compositions comprising tricalcium phosphate 326, 2x solid compositions comprising silica 325, 2x solid compositions comprising cellulose 327 and 2x solid compositions comprising iron 329.
  • the different solid compositions will promote the capture of different microorganisms such as attraction of iron oxidizing bacteria for the iron containing solid compositions, cellulose degrading bacteria and fungi for the cellulose containing solid compositions, phosphate solubilizing microorganisms for the tricalciumphosphate containing solid compositions while the silica containing solid compositions may act as a porous no-nutrient control composition.
  • the devices' growth compartments were left empty i.e. without a solid composition. The devices were then placed in a water stream. After 24 hours they were retrieved, the water in the growth compartments was removed to a microplate using a pipette. A drop of water from the growth compartments and from a control sample from the stream was then studied using a microscope. DNA was isolated from the content of the microplate and analysed as described in example 1. Results
  • the placed and retrieved devices 401 are shown in figure 15A and 15B, respectively.
  • the highly soluble nutrient bulk 413 did not dissolve despite being submerged in running water for 24 hours.
  • the two-compartment system limits the dissolution of soluble nutrients provided in the nutrient bulk and can therefore provide slow-release of nutrients to the growth compartment and surrounding environment. This appears to attract and concentrate microorganisms within the growth compartment.
  • Example 5 Culture of trapped microorganisms that can solubilize phosphate and/or fix nitrogen
  • a device was used with the overall size and shape of a microtiter plate (figure 16A).
  • the device contained 16 nutrition compartments for nutrient bulks (four compartments in four columns; noted 1,2,2, 1 in figure 16A) and 64 growth compartments (8 columns each with 8 compartments).
  • the device was 3D printed by Materialise (Leuven, Belgium) using Multi Jet Fusion and PA12 material.
  • the nutrition compartments were partly filled with nutrient bulks (illustrated as white fillings in figure 16B).
  • myristic acid and either Pikovskaya's medium broth powder, Jensen's medium broth powder, LB broth powder or glucose powder were mixed in a glass petri-dish on a hot-plate at 80°C in a 1:2 mass ratio.
  • these pellets could be inserted as nutrient bulks in the nutrition compartments.
  • Pikovskaya's medium is a medium that enriches for phosphate solubilizing microorganisms as its only phosphate source is insoluble calcium phosphate. It is also enables a quantitative measurement of the ability of the microorganisms to solubilize phosphate in the form of a solubilization halo surrounding a colony wherein insoluble calcium phosphate has been solubilized.
  • Jensens's medium is a medium that enriches for nitrogen fixing microorganisms as it does not contain any sources of nitrogen.
  • Each nutrition compartment was connected to four growth compartments.
  • Solid compositions were inserted into a subset of growth compartments (illustrated as white fillings in figure 16B).
  • the solid compositions were solid Tricalcium phosphate compositions made as described in example 2, except for a mass ratio 7:25 myristic acid:tricalcium phosphate.
  • the device was sealed with transparent microtiter plate sealing tape and was buried in root-rich forest soil (55°21’57.6"N 10°25’56.4"E).
  • the device was retrieved after 24 days and the tricalcium phosphate compositions were either placed directly on selective media (figure 16D) or vortexed with water to release trapped microorganisms with subsequent plating of the water (figure 16E).
  • Figure 16G shows the grow rate of the Genera of microorganisms isolated from the solid compositions from the retrieved device, dark grey is growth on Pikovskaya medium, light grey is growth of phosphate solubilization halo on Pikovskaya medium and white is growth on Jensens medium.
  • Table 3 lists the identity, growth rate and phosphate solubilization rate on Pikovskaya medium (PVK) and growth rate Jensen's medium (J). Numbers refer to daily increase in colony/halo size (mm 2 ).
  • Table 3 The microbial culture that was best at solubilizing phosphate, number 18 in the table, was identified using NCBTs blast database and its 16S sequence to be a new species in the Serratia Genus.
  • the device may be used to capture phosphate solubilizing and nitrogen fixing microorganisms. These can be transferred to a new environment directly while still on the solid composition or they can be retrieved from these and brought into culture, enabling isolation and characterization. Such species may hold value in areas like agriculture, forestry, horticulture, environment restoration and mining.
  • the device enables the capture and isolation of new species such as the species present in culture 18 and enables capture of microorganisms that can fix nitrogen (I ) from the environment as well as capture of phosphate solubilizing microorganisms.
  • Example 6 Culture of trapped microorganisms that can kill or inhibit pathogens
  • a device was used with the overall size and shape of a microtiter plate, identical to the one used in example 5. It contained 16 nutrition compartments for nutrient bulks, these were filled with nutrient bulks consisting of myristic acid and LB broth powder (prepared as described in example 5). Each nutrition compartment was connected to four growth compartments comprising solid compositions being solid Tricalcium phosphate compositions. These were made as in example 5. Half of the solid compositions had been seeded with either Extended spectrum beta -lactamase (ESBL) producing Escherichia coli (ESBLpE) or vancomycin resistant Enterococcus faecium (VRE). These pathogens were obtained from the Department of Clinical Microbiology at Odense University Hospital.
  • ESBL Extended spectrum beta -lactamase
  • EBLpE Escherichia coli
  • VRE vancomycin resistant Enterococcus faecium
  • the device was sealed with transparent microtiter plate sealing tape and was buried in root-rich soil at 37°C.
  • the soil had been collected in an old birch forest (55°21'57.6"N 10°25'56.4"E).
  • the device was retrieved after 1 week and solid compositions were removed and placed directly on semi-solid Mueller Hinton medium inoculated with ESBLpE or VRE as seen on figure 17A.
  • bacterial isolates could be retrieved from the clearing zones. At least three different strains could be isolated. These were plated onto blood agar inoculated with ESBLpE, VRE, methicillin resistant Staphylococci aureus (MRSA) or Candida albicans. All three strains inhibited ESBL, VRE and MRSA whereas two of them could inhibit Candida albicans.
  • Figure 17B shows the inhibition of VRE by one of these strains. Upper A-D reflects identical colonies of this strain and a zone of VRE inhibition can be seen surrounding the colonies. Lower A-D are identical colonies that do not exhibit any inhibition of VRE and thus, no zone of VRE inhibition is observed.
  • the solid compositions can be inoculated with microorganisms prior to being placed in the environment. This inoculation may promote the capture of species from the environment that are capable of killing the microorganisms that were seeded onto the solid compositions in the first place.
  • the device can be used to capture anti-microbial microorganisms, these may find use in human or animal health, in crop/plant protection or in food/beverage production and in many other areas. They may also serve as sources of new chemical compounds e.g. new antibiotics, anti-fungals, anti-parasite drugs, immunosuppressive drugs, cancer drugs, biocides, pesticides and fungicides.
  • Example 7 Use of a pill-sized device that can pass through the gastrointestinal tract
  • a device was used with the overall size and shape of a pill (length 2 cm, width 1cm).
  • the pill was 3D printed by Materialise (Leuven, Belgium) using Multi Jet Fusion and PA12 material.
  • FIG 18A it contained two separate outer parts 531a, b; each providing a compartment.
  • One compartment (nutrition compartment) that could be filled with a nutrient bulk 513 and one (growth compartment) that could be filled with a solid composition 511.
  • the nutrient bulk consisted of myristic acid and LB medium and the solid composition consisted of tricalcium phosphate, both were made as described in example 5.
  • the part 531a providing the growth compartment also comprised an entry opening 507 to the outside environment.
  • a spacer 537 with a release opening was arranged in the middle to separate the two compartments.
  • the two ends 531a, b were then attached to each other forming a pill with separate nutrient and growth compartments.
  • the pill was fed to mini-pigs by placing it at the end of a steel rod where it could be ejected from to be swallowed by the pigs (Figure 18C).
  • the pigs were stabled a week before the experiment. After the intake of the pill, the pigs were monitored daily for welfare and health and their faeces was examined for the presence of the pill. The study was done at University of Southern Denmark's animal facility (The Biomedical Laboratory).
  • the device may consist of several small parts that when assembled create a nutrient and growth compartment, which during passage in the gut is able to interact with the microenvironment of the animal as illustrated in Figure 18B.
  • Figure 18B illustrates the pill 501 as imagined in the large intestine interacting with the microorganisms 539 of the gut.
  • the pill releases nutrients from the nutrient bulk 513 through the release opening and entry opening (upward pointing arrow) and the migration of microorganisms through the entry opening to the solid composition (downward pointing arrow).
  • the device may have the size and shape of a pill that can be swallowed when fed to e.g. a pig.
  • the pill passed through the mini-pig without incidence and could be collected after passage.
  • the passage time was 1 week.
  • the pig did now show any signs of any adverse effects to the treatment.
  • the device may be used to capture microorganisms from humans or animals for use as e.g. probiotics or to transfer microorganisms from a healthy animal/human to a sick animal/human in a manner similar to a faecal transplant.
  • a device for capturing microorganisms from an environment in which the device is placed during use comprising
  • At least one growth compartment comprising at least one entry opening, said at least one entry opening being exposed at a surface of the device for microorganisms to enter the growth compartment from the environment;
  • At least one nutrition compartment separate from said at least one growth compartment; and - at least one release opening fluidly connecting said at least one growth compartment with said at least one nutrition compartment.
  • At least one of said at least one nutrition compartments comprises a nutrient bulk, optionally said nutrient bulk comprises chemoattractants.
  • At least one of said at least one growth compartment comprises one or more solid compositions, such as one or more porous solid compositions.
  • the size of the entry opening allows at least one microorganism to enter the growth compartment.
  • the size of the release opening prevents microorganisms from entering the nutrient compartment.
  • the device comprises a base part and a lid, said base part comprising insertion openings exposed to a surface of said base part; said insertion openings allowing insertion and retrieval of said nutrient bulk into/from said at least one nutrition compartment and optionally said solid composition into/from said at least one growth compartment; said lid, in use, being arranged on said surface of said base part, where said insertion openings are exposed, covering said insertion openings.
  • a method for capturing microorganisms from an environment comprising the following steps a) providing a device according to any of the items 1-10; b) arranging said device in an environment for entry of microorganisms through the entry opening into said at least one growth compartment, preferably being attracted by chemoattractants from a nutrient bulk being arranged in said at least one nutrient compartment; c) allowing said device to be placed in said environment for a pre-determined period of time; and d) optionally, purifying microorganisms from said device.
  • a kit comprising a device as described by any of the items 1-10, and a nutrient bulk as described by any of the items 2-10 and/or a solid composition as described by any one of the items 5-10.

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  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
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  • Biomedical Technology (AREA)
  • Microbiology (AREA)
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  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un dispositif de capture de micro-organismes à partir d'un environnement dans lequel le dispositif est placé pendant l'utilisation, le dispositif comprenant au moins un compartiment de croissance comprenant au moins une ouverture d'entrée, ladite au moins une ouverture d'entrée étant exposée à une surface du dispositif pour que des micro-organismes pénètrent dans le compartiment de croissance à partir de l'environnement ; au moins un compartiment de nutrition séparé dudit au moins un compartiment de croissance ; et au moins une ouverture de libération reliant de manière fluidique ledit au moins un compartiment de croissance audit au moins un compartiment de nutrition. L'invention concerne également un procédé de capture des micro-organismes à l'aide du dispositif.
PCT/EP2021/056366 2020-03-13 2021-03-12 Dispositif de capture de micro-organismes de l'environnement WO2021180941A1 (fr)

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CA3171122A CA3171122A1 (fr) 2020-03-13 2021-03-12 Dispositif de capture de micro-organismes de l'environnement
EP21710518.8A EP4118182A1 (fr) 2020-03-13 2021-03-12 Dispositif de capture de micro-organismes de l'environnement
AU2021235144A AU2021235144A1 (en) 2020-03-13 2021-03-12 Device for capturing microorganisms from the environment

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EP20163020.9 2020-03-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4317401A1 (fr) * 2022-08-02 2024-02-07 Karlsruher Institut für Technologie Dispositif contenant un matériau élastomère macroporeux pour l'enrichissement et la culture de micro-organismes in situ
WO2024104961A1 (fr) 2022-11-18 2024-05-23 Bioomix Aps Nouvelle souche de pseudomonas

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150166954A1 (en) * 2012-08-27 2015-06-18 Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet Cell selection method and cells obtained therefrom
US20150167043A1 (en) * 2012-03-28 2015-06-18 Northeastern University Nanofluidic device for isolating, growing, and characterizing microbial cells
WO2017059866A2 (fr) 2015-10-09 2017-04-13 Syddansk Universitet Matière première pour impression 3d, et utilisations de cette matière première
US20170247652A1 (en) * 2014-10-20 2017-08-31 Northeastern University Device and Method for High Throughput Bacterial Isolation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150167043A1 (en) * 2012-03-28 2015-06-18 Northeastern University Nanofluidic device for isolating, growing, and characterizing microbial cells
US20150166954A1 (en) * 2012-08-27 2015-06-18 Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet Cell selection method and cells obtained therefrom
US20170247652A1 (en) * 2014-10-20 2017-08-31 Northeastern University Device and Method for High Throughput Bacterial Isolation
WO2017059866A2 (fr) 2015-10-09 2017-04-13 Syddansk Universitet Matière première pour impression 3d, et utilisations de cette matière première

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4317401A1 (fr) * 2022-08-02 2024-02-07 Karlsruher Institut für Technologie Dispositif contenant un matériau élastomère macroporeux pour l'enrichissement et la culture de micro-organismes in situ
WO2024104961A1 (fr) 2022-11-18 2024-05-23 Bioomix Aps Nouvelle souche de pseudomonas

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