WO2024100076A1

WO2024100076A1 - Method and product for selective growth of microorganisms

Info

Publication number: WO2024100076A1
Application number: PCT/EP2023/081060
Authority: WO
Inventors: Knut Rudi; Inga Leena ANGELL; Ida ORMAASEN; Astrid Randem LUNDE
Original assignee: Norwegian University Of Life Sciences
Priority date: 2022-11-07
Filing date: 2023-11-07
Publication date: 2024-05-16
Also published as: GB202216552D0

Abstract

The invention provides a method for the selective capture and/or growth of one or more microorganisms at a test location or in a liquid sample by placing a solid support with a growth medium which is selective for the one or more microorganisms into a liquid at the location or in the sample, allowing growth and then removing the solid support. The method may be used to detect the presence and/or amount of one or more microorganisms, or a target molecule from the one or more microorganisms, at the test location or sample by analysis of the captured microorganisms. Products for this purpose are also provided.

Description

Method and product

The present invention relates to a method for the selective capture and/or growth of microorganisms in a test location or a liquid sample by use of a solid support with a selective growth medium. Once captured the microorganisms are removed for further analysis. This allows the detection of the presence and/or amount of selected microorganisms at that location or in the liquid sample. Conveniently, at least one means for this detection is used in the method and may be provided by the solid support. A product with a solid support and selective growth medium contained in one or more porous containers, and a product with a solid support comprising a particle which has an affinity binding surface for DNA or RNA and a removable coating with a selective growth medium, are also provided. Background

Microorganisms are ubiquitous in all kinds of micro- and macro-ecology environments on earth. Whilst the presence or absence of particular microorganisms occurs naturally, environmental pressures may influence the presence or amount of those microorganisms. For example, the level of particular microorganisms, such as bacteria, may increase due to the changes in the local environment. The presence and/or amount of different types of microorganisms may be used to determine the status of those sites. For example, certain microorganisms may flourish in the presence of particular contaminants and therefore act as a marker of the environmental status of a particular site. Aquaculture, run-off from agriculture, sewage and wastewater are pollutants that increase the amount of organic carbon, nitrogen, and phosphorus in water. Such pollutants may affect the growth of particular microorganisms in the environment, e.g. providing anoxic conditions or culture conditions which favour particular microorganisms and alter levels of particular microorganisms, e.g. altering the microorganisms (e.g. bacteria) make-up of the benthic community particularly in marine sediments.

There are strict regulations for environmental monitoring in the aquaculture industry to control their impact on the surrounding ecosystems. Identifying and monitoring the presence of microorganisms in non-laboratory sites is time consuming and technically difficult. For example, the current standards for monitoring the state of marine benthic ecosystems are time-consuming, expensive, and unable to test seafloor topography consisting of bedrock free of sediment. By way of example, current monitoring assays for the Norwegian aquaculture industry are expensive and take about 6 months.

Monitoring of the areas close to a facility in production is performed with tendency monitoring. Three parameters are tested: the presence or absence of infauna, a quantitative chemical test, and a qualitative sensory test. The test specimen is obtained using a mechanical grab that brings up a volume of sediment. Areas around an aquaculture facility where particles may sediment are also tendency monitored, but performed more rarely. Tests are focused on a quantitative infauna study, with support from hydrographical, geological, and chemical support parameters, and are only performed on soft sea beads. The studies include one bacterial sensory test by identifying the presence of the white hairy biofilm created by Beggiatoa sp. The presence of Beggiatoa sp. is recorded because of the rapid occurrence around several aquaculture facilities (Norsk-Standard, 2016 “Environmental monitoring of benthic impact from marine fish farms”, Norsk- Standard, 2013, “Water quality, Guidelines for quantitative sampling and sample processing of marine soft-bottom macrofauna”).

Current testing methods are not suitable for detection of all types of locations (e.g. current marine monitoring tests analyse sediment and sediment free, hard bottom surfaces cannot be tested). Whilst some environmental damage may be detected by analysis of other parameters, e.g. hydrogen sulfide, other environmental damage may be initially undetectable, e.g. nitrification. However, this may be assessed through analysis of microorganisms, such as bacteria, present at the testing site. Most current testing does not include microorganism analysis. Even if such analysis were conducted, samples often have microorganisms, such as bacteria, at low abundance making testing and interpretation of the results challenging.

The present invention overcomes these problems and provides a versatile and simple method that may be used at any site and in which even low abundance microorganisms may be detected. The test is rapid and may be completed in as little as 48 hours. This allows regular monitoring.

One example of the inventive method is shown in Figure 1 A in which a porous container is used which contains a solid support with a selective medium for microorganisms such as bacteria (in this case shown as a selective gel-bead complex). The microorganisms of interest are selectively grown and captured by the gel-bead complex and may then be removed for further analysis, e.g. by extraction of their DNA for PCR analysis or sequencing. Conveniently, the solid support has at least one means for detection, e.g. an affinity binding surface for adherence of DNA. Such a method is shown in Figure 1C. For example, paramagnetic alginate beads may be used, the microorganisms selected, the DNA extracted and bound to the beads which may then be analysed. Figure 1 B illustrates an alternative form of a porous container with selective solid supports for microorganism capture and culture.

The method has general applicability and may be used in a variety of different fields. As discussed above it may be used for analysis of the environmental status of a location, e.g. marine waters. Changes in such waters may occur through aquaculture but also as a by-product of other industries such as oil and gas mining and processing. Such a method should replace sediment counting and manual counting of macroorganisms. It may also be used to analyse and monitor wastewater, bacterial contamination in food production settings, or antibiotic resistance in hospitals, to name but a few. In light of the speed, simplicity, and cost of the method, regular rapid monitoring may be conducted, which would allow harmful changes to be rapidly identified before significant harm occurs.

Thus, in a first aspect the present invention provides a method for the selective capture and/or growth of one or more microorganisms at a test location or in a liquid sample, comprising: a) placing a solid support in a liquid at said test location or in said sample, wherein the solid support comprises a growth medium which is selective for said one or more microorganisms, b) allowing the one or more microorganisms to grow on said solid support; c) removing said solid support from the test location or sample.

As used herein, references to entities in the singular includes reference to entities in the plural and vice versa. Reference to a list of alternatives which are conjugated by the term “and/or” indicates that any, all or any combination of those alternatives may be used or present.

Selective capture and growth

As used herein, reference to “selective” refers to the capture and/or growth of said one or more microorganisms at the expense of, or at enhanced rates or levels relative to, other microorganisms that are present in the test location or liquid sample. In particular, the proportion of the one or more microorganisms in the total microorganisms’ amount is higher in the microorganism community on the solid support at the end of the growth period than in the microorganism community in the test location or liquid sample. Preferably, the proportion is increased by at least one fold, e.g. at least 2, 5 or 10 fold relative to the microorganism community. Selection may occur in the capture step, i.e. only certain microorganisms contact and are associated with the solid support. This may occur, for example, by use of physical means, such as a porous container (as described hereinafter) which may limit access to the solid support based on size.

In the alternative, or additionally, selection may occur during growth, i.e. the one or more microorganisms of interest grow to a higher multiple than other microorganisms during the growth period.

Microorganisms which contact and are associated with the solid support are those which are able to grow on or in the solid support, e.g. in the selective growth medium. The “growth period” is the period in which the solid support is in contact with the test location or liquid sample under which growth of the one or more microorganisms is possible and is generally coincident with the placement of the solid support in the test location or liquid sample until removal from that location or sample.

“Growth” of the microorganisms refers to multiplication of the microorganisms over time when under conditions conducive to that growth. Such conditions include appropriate pH, temperature, osmolality, gas concentration, and use of a medium containing essential components necessary for growth. “Capture” of the microorganisms refers to association of the microorganisms with the solid support such that growth of the microorganisms is possible, e.g. in the selective growth medium.

Test location

The test location is any location where a liquid is present that could contain microorganisms such as bacteria. Conveniently, the liquid is water. In the alternative, however, the liquid may be another liquid, for example oil. The test location is generally not a laboratory. Preferably, the location is an environmental location. In particular, the test location may be an aquatic environment (i.e. which contains plants and/or animals), particularly, a fresh or salt-water environment, such as a river, pond, spring, lake, sea or ocean. The test location may also be other bodies of water, including those which are not naturally occurring, such as groundwater, agricultural or commercial run-off, wastewater, tanks, public pools, sinks, drums, and reservoirs. The location is conveniently close to sites of potential contamination or pollution, e.g. around oil/gas sites of production, in food production areas or medical facilities. Preferably, the method is used to monitor the location for evidence of environmental pollution or contamination, particularly resulting from aquaculture.

Samples

In one aspect the solid support is to be placed in liquid at a test location. In the alternative, the solid support may be placed in a liquid sample. As before, such a liquid is one that could contain microorganisms. Whilst the value of the method is that microorganisms may be detected in situ in locations of interest, in some cases it may be more appropriate to use a sample for the method. This may be a sample taken from any of the locations described above. Conveniently, samples of likely polluted liquid are used, e.g. wastewater or other water samples from food production or medical facilities.

Target microorganisms

The one or more microorganisms to be captured and/or grown (also referred to herein as the target microorganisms) are not limited to any particular microorganisms. Conveniently, however, microorganisms are selected that provide a useful readout on the status of the test location or sample. For example, the presence or absence of a microorganism may be indicative of adverse or favourable conditions. The microorganisms of interest are captured and/or grown by use of the selective growth medium for those microorganisms.

Microorganisms include any organisms of microscopic size and may be unicellular or formed of more than one cell. In particular, the microorganisms may be unicellular prokaryotes, such as bacteria or archae, or may be eukaryotes such as unicellular protozoa and microalgae, or multicellular such as fungi (e.g. yeast or mold) and algae. For the purposes of this invention, viruses are not included within the definition of microorganisms.

By way of example, in a medical setting, the one or more microorganisms may be bacteria, which are indicative of contamination or the presence of pathogenic bacteria, which could include antibiotic resistant bacteria or bacteria such as Legionella, Staphylococcus aureus, Listeria monocytogenes or Vibrio salmonicida. Microorganisms indicative of a particular environmental status may also be examined. For example, bacteria or archaea such as sulfur oxidizing bacteria or archaea, hydrogen sulfide producing bacteria or archaea (such as sulfate reducing bacteria or archaea), denitrifying bacteria or archaea, or methanogenic bacteria or archaea may be targeted. Algae or bacteria responsible for algal bloom and production of toxins may also be assessed. (Cyanobacteria are referred to as blue-green algae despite being bacterial in nature.) Pathogenic fungi causing disease in marine animal hosts may also be assessed, such as Microsporidia, Ascomycota, Mucoromycota, and Basidiomycota. Such microorganisms may be used to provide an indication of local conditions. For example, pollution can lead to sedimentation onto the ocean floor leading to rapid depletion of oxygen. This results in anoxic communities on the sediment surface and an increase in the depth of sulfate penetration in the sediments. This allows certain microorganisms to flourish and others to decline. For example, increases in denitrifying bacteria or archaea, sulfur oxidizing bacteria or archaea, hydrogen sulfide producing bacteria or archaea and/or methanogenic bacteria or archaea may be used as indicators of such pollution.

The microorganisms that are captured and/or grown provide an indicator of current conditions which may be monitored to assess any changes. A “fingerprint” of local microorganisms may be used to develop a baseline against which changes may be monitored. As shown in the Examples, various sites exhibit distinguishable communities of bacteria, archae, and eukaryotic microorganisms. Furthermore, as shown in the Examples changes in the microorganism community resulting from anoxia or pollution may be identified by the methods of the invention.

The one or more microorganisms to be captured and grown may be selected from within the same group (i.e. bacteria, archaea, protozoa, microalgae, fungi or algae). In the alternative, they may be from different groups, e.g. a selection of bacteria, archaea, and algae. However, collectively these one or more microorganisms are grown on a selective medium for those selected microorganisms, e.g. a medium that selects for microorganisms that grow in a sulfate reducing medium (such as sulfate-reducing bacteria or archae and sulfur oxidizing bacteria or archaea).

Therefore, in one aspect, each of said one or more microorganisms is selected from a bacterium, archaeon, protozoan, fungus, and alga and said growth medium is selective for said microorganisms. In another aspect, the one or more microorganisms may be provided by only a sub-set of those groups, e.g. may each be from bacteria, archaea or algae, or may each be a bacterium. Preferably, the microorganisms are from different genera to allow easy identification, though this is not essential. When one or more of the microorganisms is a bacterium, preferably, it is selected from the list comprising antibiotic resistant bacteria, sulfur oxidizing bacteria, hydrogen sulfide producing bacteria, denitrifying bacteria, methanogenic bacteria, and pathogenic bacteria. In a preferred aspect, the bacterium is a bacterium from the genus Sulfurimonas (a sulfate oxidising bacteria), a bacterium from the genus Desulfotalea (a sulfate reducing bacterium), a bacterium from the genus Sulfurospirillum (a sulfate reducing bacterium) or a bacterium from the genus Sulfurovum (a sulfate oxidising bacterium). Similarly, when one or more of the microorganisms is an archaeon, preferably, it is selected from the list comprising antibiotic resistant archaea, sulfur oxidizing archaea, hydrogen sulfide producing archaea, denitrifying archaea, and methanogenic archaea.

Solid support

The “solid support” is a component that is solid when present in the test location or sample. For example, it may not be solid if heated. The solid support provides the means for capture and/or growth of the one or more microorganisms and is suitable for handling to allow its containment and removal from the systems used in the method.

The solid support comprises the selective growth medium. This may be evenly distributed throughout the support or may be present in a separate portion. A variety of different forms of this component may be contemplated. The Examples describe the use of particles around which an alginate or agarose gel is bound. Thus, in one example the solid support may have a solid core with a gelatinous coating. In this case, the gelatinous coating may contain or provide the selective growth medium. In the alternative, the solid support may consist entirely of a dissolvable material, e.g. an alginate or agarose particle. Alginate may be formed into a hydrogel on contact with divalent metals such as with Ca²⁺ (e.g. using CaCh as in the Examples), which can be used to generate beads.

In instances in which the selective growth medium is contained in a separate portion of the solid support, conveniently, it is readily removed from the remainder of the solid support, e.g. by heating or dissolution, e.g. using enzymatic lysis. Chemical dissolution may also be used. For example, removing metal ions reduces the integrity of alginate gels causing them to collapse. Solid supports of this sort were used in the Examples in which alginate or agarose gel was used to coat paramagnetic particles. Microorganisms were captured by, and grew, in the alginate/agarose gel and that gel was dissolved to release the microorganisms. After lysis of the microorganisms, released DNA was bound to the core particles from which it was eluted for analysis.

Thus, in preferred aspects, the solid support is a particle. This particulate form of the solid support may include a solid magnetic core (e.g. a paramagnetic particle) and a coating, for example. Such a coating (e.g. a gel) may be removable by methods described hereinabove and is referred to herein as a “removable coating”. Such a particle is considered a magnetic particle and is a preferred solid support.

The part of the solid support which is not the selective growth medium is preferably inert and non-consumable by micro- or macro-organisms in the test sample or location. Thus, for example, the solid support may be provided by a matrix or carrier (e.g. made of metal, stone, ceramic, plastic, cellulose, nylon, polyethylene, polyester, Teflon, epoxy or rubber), which carries the selective medium.

In the alternative, the solid support may be provided in a non-spherical form, e.g. as a plate, sheet, rod, fibre or molded form of any shape, including shapes with an internal space such as hollow tubes or spheres. To provide ready access to microorganisms, the solid support is in a shape that provides a high surface area and easy access to the microorganisms.

In the method of the invention, a single solid support may be used, if it is of sufficient size, but generally a plurality of essentially identical solid supports is used.

Conveniently, the solid support is of sufficient size to allow easy processing and manual manipulation. For example, the solid support may have a size of from 0.1 to 5 mm in its largest dimension (e.g. diameter for spheres). However, much larger or smaller solid supports may be used with a lower or higher plurality, respectively, when in use in methods of the invention. Conveniently, in each method of the invention, between 1 and 100 solid supports are used, though more may be used when smaller supports are employed.

Selective growth medium

The growth medium that is used is selective for the one or more microorganisms. The medium may be solid and/or liquid and selected as appropriate depending on the solid support that is provided. As referred to hereinbefore, selection refers to enhanced capture and/or growth relative to the capture and/or growth of other microorganisms (i.e. extent of multiplication). This may be achieved by suppressing the growth of microorganisms which are not the target microorganisms or enhancing the growth of the target microorganisms. For example, suppression may be achieved by using an antibiotic if the target microorganisms are antibiotic-resistant microorganisms, such as bacteria (e.g. in a hospital setting). Enhanced growth may be achieved using media which support the growth of particular microorganisms. For example, media which promote growth of sulfate reducing bacteria or archaea may be used, e.g. sulfate reducing medium as used in the Examples. Media are conveniently selected depending on the location and/or sample under investigation. Standard media are known for this purpose, for example as set forth in Standard Methods for the Examination of Water and Wastewater (Clesceri et al., 1999, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, American Water Works Assoc., Water Environ. Fed.). Preferably, the selective medium increases the capture and/or growth of one or more target microorganisms by at least 1-fold (e.g. at least 2, 5 or 10 fold) relative to the proportion in the microorganism community in the test location or liquid sample.

The medium to be used contains essential components for growth that are not found elsewhere in the sample or test location. Typically, this includes: an energy/carbon source, usually in the form of a saccharide such as glucose, all essential amino acids, vitamins, free fatty acids, inorganic salts, and trace elements (usually in the micromolar range), for example, which are necessary for growth and/or survival. The solution is preferably optimized to an appropriate pH and salt concentration suitable for cell survival and proliferation and a buffer may be used to maintain pH (e.g. HEPES). Additional components may be necessary depending on the target microorganisms. For example, for growth of denitrifying bacteria and archaea an electron donor and electron acceptor are also required. Preferably, the starting growth medium (i.e. before placement in the test location or liquid sample) does not contain any microorganisms. Preferably, the solid support is similarly devoid of any microorganisms until in use. The microorganisms that are to be captured and/or grown are sourced from the sample/location and remain with the solid support when it is removed from the sample/source.

Conveniently, the method may comprise the use of more than one different solid supports, e.g. with different selective media. In this way, different sets of microorganisms may be captured, grown, and detected. For example selective media directed to sulfur oxidizing bacteria, hydrogen sulfide producing bacteria, denitrifying bacteria, and methanogenic bacteria may be provided on different solid supports, respectively. These solid supports may be used in separate methods or in the same method.

Incubation

In the method of the invention, the one or more microorganisms are allowed to grow on the solid support. This period of growth is considered the incubation period in which the target microorganisms grow in the selective growth medium. The growth medium is chosen such that when present in the test location (i.e. at the pH, temperature etc.) at that test location, growth of the target microorganisms is possible. When growth in a sample is to be ensured, the conditions of the sample are adjusted to maximize growth of the target microorganisms, e.g. by selection of the pH, temperature, and other incubation conditions. Incubation is conducted until a) selection of the target microorganisms is achieved (when present) and b) measurable amounts of the target microorganisms are obtained (when present). In the latter case, this is dependent on the detection technique to be used. However, routinely, incubation is performed for 48 hours to 100 days, preferably from 7 to 30 days, e.g. from 10 to 20 days.

At the end of the incubation period, conveniently, a total of at least 1x10⁴ target microorganisms are obtained. Removal of the solid support

Following the incubation period, the solid support is removed (spatially separated) from the test location or sample. The mechanism by which the solid support is removed is dependent on the form in which the solid support was provided. In some instances, the solid support may be provided in naked form, i.e. without a covering, coating or container. This may occur in instances in which the test location has an arrangement or structure that would protect the solid support from damage (e.g. a cage or container). In samples in which no such protection is required, naked solid supports may be used. In such cases, the solid support is removed by direct removal of the naked supports. Porous container

In the alternative, the solid support may be provided in a porous container. This allows the solid supports to be easily transported as well as convenient addition and removal from the test location or sample. It also provides protection to the solid supports. This may be required in locations where adverse conditions are expected, e.g. from wildlife or debris. Furthermore, the container may exclude larger organisms from growth on the solid support. The container is necessarily porous to allow the target microorganisms access to the solid support.

Thus, in a preferred aspect, the solid support is enclosed by one or more porous containers which allow liquid and microorganisms to access the solid support, wherein at least one container excludes entities that are 5 mm or larger (e.g. 1 mm or larger), wherein preferably at least one porous container has a pore size of less than 1 mm, preferably less than 10 pm or less than 1 pm.

The “porous” container is porous by virtue of pores which allow liquid and microorganisms to access the solid support(s)/growth medium from the test sample or location. As discussed hereinafter, one or more porous containers may be used. Preferably, at least one of those containers has pores with a size of less than 1 mm. Preferably, when more than one container is used, at least the container immediately surrounding the solid support(s) has that pore size.

Such a pore is not necessarily circular and its shape is dependent on the container that is used. The pore size of less than 1 mm refers to the maximum size of the pore in one dimension on the surface of the container. In the case of circular pores, the size refers to the diameter of the circle. If instead a fibrous container with holes is used, the pores could be rectangular in shape and the size of the pore in that case is considered the longest axis of that rectangle.

The pores in at least one of the porous containers exclude entities that are 5 mm or larger, preferably 1 mm or larger. This pore size specifically excludes debris and wild-life. Furthermore, depending on the microorganisms of interest, at least one porous container may exclude non-target microorganisms, particularly if they may be discriminated based on size. For example, bacteria of the genus Mycoplasma are smaller than most other bacteria and if these are the target bacteria at least one porous container may be used to exclude larger bacteria and other microorganisms to assist selectivity. Most bacteria have a length of 1-10 pm (diameter of 0.2 - 2 pm) whereas Mycoplasma bacteria have a diameter of 0.2 - 0.3 pm (of various shapes from round to oblong). Conveniently, the pore size of at least one porous container is less than 1 mm, preferably less than 10 pm, less than 1 pm or less than 0.45 pm. The latter two pore sizes may be used to exclude larger microorganisms if they are not the target microorganisms.

The container(s) may take any appropriate form providing it is porous, as described above, can contain the solid support(s) and is suitable for the test sample or location environment and incubation step. The container has an exterior material which encloses a void into which an object may be placed. A means by which the solid support(s) (or inner container) can be introduced or removed is also required, which is generally an opening in one area of the material of the container. Conveniently, the container is a lightweight, durable but inert container. The container is conveniently comprised of a natural or synthetic polymer, such as nylon, polyethylene, polyester, Teflon, epoxy, rubber, plastic, silk, cotton, wool, cellulose or protein. The material making up the container may be provided in the form of a solid sheet (with pores) or may not be in a solid form, e.g. comprised of fibres, cloth or layers, which may provide the pores by virtue of their proximity to one another.

Conveniently, the container is flexible, i.e. the material making up the container is flexible. Such a container is conveniently a bag with a single opening.

More than one porous container may be used. When multiple containers are used they may be inserted inside one another and may have the same or different properties. This provides a set of containers surrounding the solid support(s). This is particularly useful in harsh test environments to offer additional protection. In such scenarios, for example, a bag may be used as the first (inner) container containing the solid support(s). The second (outer) container encompasses the first container and can be provided as a more robust, less flexible container. Additional outer containers may also be used. The inner container (closest to the solid support(s)) may comprise pore sizes as discussed above, but the subsequent containers may have larger pores to allow ready flow of liquid (e.g. pores of 0.5- 2cm, for example). Conveniently, 1 , 2, 3 or 4 containers are used. In the examples described hereinafter, rigid cylindrical outer containers are used with pores in their walls and form preferred aspects of the invention.

The size of the containers may be varied taking into account the size of the solid support(s) and the intended use and test sample or location. As noted above, in methods of the invention, a plurality of solid support(s) may be used. Conveniently, all of the solid supports may be provided in a single porous container (or set of containers). In the alternative, multiple, separate porous containers (or multiple sets of containers) may be used in each method, each containing one or more solid supports. By way of example, each separate porous container (or set of containers) may contain from 1 to 100, e.g. 1 to 10, solid supports. Depending on the number of solid supports and the size of each solid support the size of the container may be selected accordingly. Conveniently, the inner container is from 5- 20 cm in size and the outer containers may be from 10-50 cm in size by way of example. The size refers to the maximum size in any direction if fully expanded. Flexible containers may in practice exhibit a smaller size.

Additional component of solid support - means for detection

As described hereinbefore the solid support comprises the selective growth medium. In addition, the solid support may comprise at least one means for detecting the presence and/or determining the amount of the one or more microorganisms, or a target molecule from the one or more microorganisms, or a part of said means for detection. Such means, or parts thereof, are described hereinafter in relation to detection methods of the invention. Sample/location component assessment

Assessment of the test location or sample onsite may be performed to provide information on the status of the sample or test location. This may be used to assess local conditions prior to the incubation step, during the incubation step or after the incubation step has been completed. This information may be used for a variety of purposes. For example, it may provide additional information for categorisation of the test sample or location, e.g. the levels of contaminants or pollution. It may also be used to provide direct information on changes during or after the incubation step, e.g. if particular molecules are released during that step. This may be used to provide a general indicator of which microorganisms are growing during that step. This may provide a useful first indicator of the community of microorganisms present in the test sample or location, or may be used, for example, to determine when incubation should be concluded (e.g. by a colour change on the solid support).

Conveniently, this may be achieved by providing the solid support and/or the porous container, when present, with a test location or sample assessment component which allows assessment of the test location or sample. Such a test location or sample assessment component provides all or part of a detection method allowing assessment of the test location or sample.

In the assessment, conveniently, the presence, absence or level of one or more molecules or entities is determined. By way of example, the levels of hydrogen peroxide, hydrogen sulfide, ammonium, metals, nitrite or nitrate in the test sample or location may be assessed. The entities or molecules may be, but are generally not, derived from microorganisms in the test location or sample, but may affect growth of those microorganisms. They may also be indicative of particular environmental conditions. In some cases, they may be derived from microorganisms and indicative of the growth of particular microorganisms, e.g. denitrifying bacteria based on the products produced by those bacteria during growth.

As noted above, when used, the test location or sample assessment component provides all or part of the assessment method. By way of example, it may provide a reagent that reacts with the entity or molecule of interest to produce a detectable signal. If a test location or sample assessment component is not provided by the solid support/porous container, all components necessary for the analysis may be added separately.

As referred to herein the “signal” is a physical signal which may be detected, e.g. magnetism, optical activity, fluorescence, colour, and so on. The absence, presence or level of the signal may be determined. The detectable signal may result from a reaction (e.g. from an enzymatic reaction) or, by way of example, from binding a label to the entity or molecule of interest or a reaction product derived therefrom. The “label” is any molecule or group of molecules which is detectable and/or generates a signal directly or indirectly. Convenient labels include colorimetric, chemiluminescent, chromogenic, and fluorescent labels. The sample/location assessment component may provide all of the components for the assessment method, i.e. the signal may be generated when the target entity or molecule is present when that sample/location assessment component is present. In the alternative, additional components may be necessary, e.g. the addition of a further reagent to allow the signal to be generated. Conveniently, this may be added by the user and the signal generated assessed. Where no sample/location assessment component is provided by the solid support/porous container, all necessary components for signal generation are added separately.

The signal generated may be assessed quantitatively or qualitatively. In the latter, a threshold analysis may be made to determine whether the test sample/location reaches a particular level for a target entity or molecule.

Where the target entity or molecule derives from microorganism growth, the test location or sample assessment may be used for an initial assessment of whether target microorganisms are present before further analysis of the microorganism community that has been collected. This may provide a crude initial yes:no response, e.g. identifying samples/locations warranting further investigation. Ideally, in light of the intended locations and use of the method, the signal that is generated is readily and simply detectable onsite without further equipment. Thus, in a preferred aspect a colour signal or light is generated. Conveniently, this signal is generated in response to an additional reagent that is added to the test sample or location at the time of assessment. By way of example, an MTT method may be used in which tetrazolium salt is used as the reagent which is reduced to formazan products in the presence of NADH/NADPH, which acts as an indicator of metabolic activity. In an alternative, ATP may be used as a marker of the presence of microorganisms and this may be detected by any appropriate assay, e.g. detecting bioluminescence resulting from interaction of the ATP with added luciferin in the presence of added luciferase.

The sample/location assessment (or testing) may be conducted any time prior to removal of the solid support from the test location or sample. For example, it may be performed prior to the incubation/growth step b) or may be performed during or after that step, e.g. prior to removal of the solid support in step c). The assessment may be used to assess the location or sample as a pre-testing step to assess whether the location is appropriate for the testing to be conducted, e.g. if contamination or pollution is likely that might affect microorganism growth. Alternatively, it may be used to provide additional information on the environment, which can be collated with information derived from the microorganism community analysis. As noted above, it may also be used to provide initial information on the type of microorganism community that is present based on generation of particular molecules by the microorganisms that are present. In a preferred aspect, prior to capture and/or growth of said one or more microorganisms the test location or sample is tested, preferably using the test location or sample assessment component.

Detection method

As described hereinbefore the method may be used for detecting the presence and/or determining the amount of one or more microorganisms, or a target molecule from one or more microorganisms. Thus, in a further embodiment the invention provides a method of detecting the presence and/or determining the amount of one or more microorganisms, or a target molecule from one or more microorganisms, at a test location or sample by a method comprising: a) selectively capturing and/or growing one or more microorganisms at a test location or in a sample by a method as described hereinbefore, and b) analysing the one or more microorganisms present on the removed solid support to determine whether said one or more microorganisms, or a target molecule from said one or more microorganisms, is present in the test location or sample and/or the amount of said one or more microorganisms or a target molecule from said one or more microorganisms in the test location or sample.

The definitions provided hereinbefore similarly apply. As discussed hereinbelow, the same or different detection methods may be used to detect each of the one or more microorganisms. Conveniently, however, all of the microorganisms, or at least a group of the one or more microorganisms, are detected using the same method. Furthermore, as discussed hereinafter, detection may allow discrimination between different individual microorganisms or may instead identify a group of microorganisms based on common functionality, structure or taxonomy.

The determination that is made in step b) may be qualitative (i.e. identifying the presence or absence of a target microorganism/molecule) and/or quantitative (i.e. determining the amount of a target microorganism/molecule). This may be expressed as an absolute value, a percentage, a ratio or similar indicator. As discussed above, specific microorganisms may be identified, or they may be identified more generally by their species, genus or other taxonomic grouping, or by their functionality, e.g. denitrifying bacteria or archaea. In this regard, reference to a microorganism in the singular may also be considered to encompass microorganisms in the plural, and vice versa.

In determining the presence or absence of a microorganism (or microorganisms) a threshold may be set above which the microorganism (or microorganisms) may be considered to be present. In the alternative, the presence or absence of a microorganism (or microorganisms) may be set simply by the sensitivity of the detection method.

When the presence/amount of a target molecule from one or more microorganisms is detected/determined, similar considerations apply. This information may be used directly to complete the determination or may be used to determine the amount of the one or more microorganisms from which the target molecule is derived. Depending on the specificity of the target molecule, this may provide information on specific microorganisms or taxonomic or functionally related groups of microorganisms. The detection method detects the amount or presence of one or more microorganisms (or target molecules). The method may detect a single type of microorganism or a group of related microorganisms based on common properties without distinction between the different microorganisms in that group. However, alternatively, the one or more microorganisms may be detected as individuals or as a collection of groups of microorganisms. For example, different microorganism species in the same genus may be identified, i.e. the output of the detection method is the presence or amount of one or more genera of microorganisms. When more than one microorganism or group of microorganisms is to be determined those microorganisms are necessarily distinguished in the detection method, e.g. by the selection of the method of detection and/or the specific means for detection used in that method. Furthermore, and as discussed above, the same or different detection methods may be used for each of, or groups of, the one or more microorganisms. Target molecule from microorganisms

As referred to herein a target molecule from the one or more microorganisms may be present on said one or more microorganisms or in the alternative released from said one or more microorganisms either during incubation or post-incubation following further processing. The target molecules from different microorganisms or groups of microorganisms, e.g. DNA or RNA, may be distinguishable or may be the same from all target microorganisms. Reference to determining whether a target molecule from said one or more microorganisms is present (or its levels) refers to the option of determining the presence/amount of a target molecule from each microorganism, each group of microorganisms, or all of said one or more target microorganisms. Target molecules may also be considered markers of one or more of the target microorganisms.

Such a target molecule may be, for example, a molecule expressed on the surface of the one or more microorganisms (e.g. outer membrane protein). Alternatively, the target molecule may be a cytosolic or secreted molecule. Such molecules include virulence factors. For more complex cells, the target molecule may be from an inclusion body, organelle, and/or the nucleus.

Conveniently, the target molecule is a polynucleotide, i.e. RNA or DNA from the microorganisms, and it may be necessary to lyse the microorganisms to release such molecules. As referred to herein, DNA or RNA includes all forms of those molecules, particularly double or single stranded molecules thereof. As described hereinafter, in the analysis step, the target molecule may be assessed directly. In the alternative, related products (e.g. amplification products) or the results of a reaction using that target molecule may be assessed, i.e. the target molecule may be indirectly assessed. However, this indirect assessment provides information on the presence, absence or level of that target molecule and hence the source microorganisms. Whilst the target molecule may be RNA or DNA, equally, when the presence or amount of the one or more microorganisms is to be determined, this may be achieved by analysis of the RNA or DNA of those microorganisms.

First step of analysis

In instances in which the selective growth medium is contained in a separate portion of the solid support, conveniently, it is readily removed from the remainder of the solid support in analysis step b). Preferably, this is performed as the first step in the analysis. Removal may be achieved, for example, by heating or dissolution, e.g. using enzymatic lysis. For example, alginate lyase may be used to dissolve an alginate gel. Microorganism lysis

In order to release target molecules of interest, such as DNA, in a preferred aspect said analysis in step b) comprises the step of lysing said one or more microorganisms. Conveniently, this is achieved by freeze-thawing, sonication, homogenization, heating, or by chemical lysis, by way of example. In another alternative lysis by osmosis may be used. Lysis buffers for these various methods of lysis are well known. Purification before analysis

Conveniently, the molecule (or marker) to be determined (or on which the determination is based) in the analysis step is collected and preferably purified, or at least enriched to remove contaminants. In particular, the DNA or RNA is collected and preferably enriched or purified before said determination. As referred to herein collection refers to separation from other components of the source microorganism, e.g. bacterial, cells. Collection does not necessarily entail purification though the process of collection may increase the purity of the molecule.

Similarly, if a protein is to be used for the analysis, conveniently, that protein would be collected and/or enriched or purified before determination. Purification refers to isolation of the molecule such that it is the predominant (majority) molecule in the solution or composition in which it is contained. In a preferred feature, the molecule is present in the solution or composition at a purity of at least 60, 70, 80, 90, 95 or 99 % w/w when assessed relative to the presence of other components, in the solution or composition.

Any appropriate means for collection/purification may be used depending on the molecule to be determined.

Components involved in the collection and purification of molecules of interest which by so doing contribute to the detection step may be considered a “means for detection or a part thereof”. In the case of polynucleotides and proteins an affinity binding surface may be used (as described hereinafter), which is considered a means for detection. This allows selective binding of the desired molecules, which may be eluted from the affinity binding surface for further analysis.

Appropriate methods and kits for collection and purification of various molecules are known. By way of example, extraction of RNA or genomic DNA may be performed by any convenient method, many of which are known in the art and include, for example, solution-based, silica membrane-based, resin-based and magnetic options. The MagMidi LGC kit (LGC Biosearch™ Technologies) as used in the Example provides an example of the latter. For RNA extraction, appropriate methods are used to ensure retention of the integrity of the RNA, typically by using RNase inhibitory agents.

Analysis of DNA/RNA

To allow accuracy in identification of microorganisms, conveniently, in analysis step b) the DNA or RNA of said microorganisms is identified. This allows the identification of different and specific microorganisms. The analysis of environmental DNA (eDNA) has been obtained directly from environmental samples such as soil, sediment or water (Thomsen and Willerslev, 2015, Biol. Conserv., 183, p4-18) and does not differentiate between free or bound DNA. In eDNA analysis, ancient or present organisms are detected based on their DNA, not their physical presence. eDNA studies allow for taxonomic classification of all organisms present and are commonly performed with general genetic markers (e.g. 16S rRNA or 18S rRNA genes). eDNA can monitor spatiotemporal variations in an ecosystem or the impact external stress factors can cause on an ecosystem (Mathieu et al., 2020, Front. Ecol. Evol., 8, p135; Shade et al., 2018, Trends Ecol. Evol., 33, p731-744). One method for taxonomic classification of eDNA with genetic markers is metabarcoding of eDNA. Metabarcoding of eDNA is a rapid large-scale taxonomic identification that allows for simultaneous identification of many taxa in a complex environmental sample. Metabarcoding uses short regions of the DNA in high- throughput sequencing (HTS), generally called general primers or DNA barcodes. Thus, the analysis of microorganisms’ DNA is particularly advantageous in methods of the invention.

The advantage of using DNA is that the presence of specific microorganisms may be identified based on unique polynucleotide sequences. However, as mentioned hereinbefore the identity of specific microorganisms does not need to be determined but instead specific taxonomic groups or functionally related microorganisms may be identified.

Depending on the information to be obtained, the method of analysis may be selected accordingly. Conveniently, a target polynucleotide sequence specific for one or more of the one or more microorganisms is used for detection. The “specific target polynucleotide sequence” is a sequence of interest, which appears in the genome of at least one of the target microorganisms to be detected, but not in other target microorganisms, which are to be separately identified or in non-target organisms. Preferably, the target polynucleotide sequence is from 6 to 30 nucleotides in length.

RNA may optionally be reverse transcribed to cDNA in an appropriate reaction mix using an appropriate primer (e.g. an oligo(dT) primer), dNTPs, and a reverse transcriptase (e.g. Invitrogen’s SuperScript IV Reverse Transcriptase).

Optionally, relevant sequences from the native genomic DNA obtained from a microorganism (or cDNA reverse transcribed from RNA from a sample) may be amplified to provide amplicons, which may contain the target polynucleotide sequence specific to one or more of the target microorganisms, when that microorganism is present. The amplicons provide a test polynucleotide, which may be double or single stranded, which may contain a target polynucleotide sequence. Amplification may be performed by known amplification techniques such as the polymerase chain reaction (PCR) by the use of appropriate primers (e.g. forward and reverse), NASBA or ligase chain reaction. This amplification may provide amplicons containing only the target polynucleotide sequence (where present) or various amplicons only some of which contain the target polynucleotide sequence of interest (e.g. all 16S rRNA and/or 18S rRNA gene sequences may be amplified, but only some of those are the specific sequences for the one or more target microorganisms to be detected). Thus, in a preferred aspect, genomic DNA is amplified to provide a test polynucleotide, which corresponds to a portion of the genomic DNA, or RNA is reverse transcribed to cDNA, and the cDNA is amplified to provide a test polynucleotide, which corresponds to a portion of the RNA. As referred to herein, a test polynucleotide, which “corresponds” to a genomic DNA or RNA sequence has the same or a complementary sequence thereto, except in the case of a DNA sequence corresponding to an RNA sequence, in which case the DNA sequence contains thymine instead of uracil nucleotides where they appear.

Many suitable techniques for the detection of a target polynucleotide sequence in a test polynucleotide are well known and may be used in this method. Example of such techniques include methods based on PCR amplification with the products monitored (e.g. with fluorogenic probes such as Taqman probes, scorpion probes or Molecular Beacons), non-PCR amplification methods (e.g. stranddisplacement amplification and ligase chain reaction), and non-amplification methods involving signal amplification (e.g. hybridization methods, which attach appropriate labels such as chemiluminescent labels or nanoparticles).

Conveniently, real time PCR methods may be used, such as quantitative PCR. Such qPCR methods have been used in the Examples. Probe-based methods for detecting target nucleotide sequences may also be used. Appropriate primers and probes may be selected to detect a specific microorganism or specific taxonomic groups or functionally related microorganisms. When probe-based methods are used they are based on conserved or known target polynucleotide sequences. Conveniently, probes carry a label as described hereinbefore.

To obtain yet further information, particularly for the identification of unknown microorganisms, DNA or RNA may be sequenced. The target polynucleotide sequence may have known flanking sequences which allow the generation of appropriate primers, which bind to regions which flank the target sequence. However, such flanking sequences are not necessary and sequencing that is not reliant on such sequences may be used, e.g. fragmentation followed by ligation of adapters for sequencing.

Any appropriate target polynucleotide sequence may be used based on knowledge of the genome/RNA of the microorganism to be detected. Ideally, and particularly for multiplexing, a sequence is selected that has a conserved segment that is common to a group of, or all, microorganisms, flanked by a variable region. The conserved region can be used e.g. for primer binding for amplification, and the variable region provides the specific target polynucleotide sequence, which acts as a marker or signature of the target microorganism. Conveniently, for prokaryotes, the target polynucleotide sequence is the 16S rRNA gene, which is an approximately 1500 base pair gene that codes for a portion of the 30S ribosome, or rRNA transcribed from said gene. For eukaryotes, conveniently, the target polynucleotide sequence is the 16S rRNA gene homologue, the 18S rRNA gene. In limited cases where the 16S rRNA (or 18S rRNA) sequence cannot be used to discriminate between different prokaryotes (or eukaryotes), an alternative target polynucleotide sequence should be selected. In an alternative embodiment the target polynucleotide sequence may be an antibiotic resistance gene, i.e. a gene which encodes a protein or peptide that confers partial or complete resistance to an antibiotic to the host microorganism. In the case of bacteria or archaea, a variety of different peptides/proteins may offer antibiotic resistance and include those that decrease affinity of the antibiotic for the bacterium or archaeon, decrease the antibiotic uptake, and improve its removal or modify the antibiotic.

In a preferred aspect, the target polynucleotide sequence is a 16S rRNA gene or an antibiotic resistance gene (prokaryotes) or an 18S rRNA gene (eukaryotes).

The presence or absence of a particular target polynucleotide sequence provides a qualitative assessment of the presence/absence of a target microorganism (or group of microorganisms). Quantitative assessment may be provided by quantifying the signal generated during detection, e.g. to determine the copy number of the target polynucleotide sequence, for example. Analysis of non-polynucleotide molecules

Whilst DNA/RNA provides a convenient molecule for analysis and can provide detailed information on the microorganism community, other molecules may be used as the target molecule or as an indicator of the presence or amount of the one or more microorganisms. In this embodiment, the presence and/or amount of said one or more microorganisms is determined by identifying a protein of said microorganism(s). Depending on the protein selected, e.g. a virulence factor, this may provide information on a particular taxonomic group or functionally related family of microorganisms. As used herein, a “protein” is a sequence of 2 or more amino acids joined by one or more peptide bonds. Detection principles

If the “amount” of a microorganism is to be reliably quantified and the amount of that microorganism is not assessed directly, the amount of a target molecule from that microorganism may be used to provide that information indirectly, if it is present in an amount that directly correlates to the amount of that microorganism. However, precise quantification is not necessary if instead trends or monitoring is to be performed.

The step of detection may be performed in one or more separate assays to determine the presence or amount of each of the one or more target microorganisms or groups of the target microorganisms, using the same or different detection methods. This is particularly appropriate when different means of detection are to be used for the different target microorganisms. Conveniently, the assay may be “multiplexed” (i.e. detection of multiple different microorganisms or multiple groups of microorganisms in a single assay) by use of means of detection, which can discriminate between different microorganisms (or groups of microorganisms), e.g. primers or probes to specific target sequences.

Multiplexing, in which multiple analytes (in this case multiple microorganisms or their components) are measured in a single assay, is achieved by discrimination between markers of different target microorganisms (or groups of microorganisms) within a single sample. Conveniently, this is achieved by attaching different labels to those different markers. Most conveniently the marker of a specific microorganism (or group of microorganisms) is a specific target polynucleotide sequence, and the presence of that target polynucleotide sequence is identified by the generation of a signal, which is different to the signal generated by a specific target polynucleotide sequence to a different target microorganism.

The detection methods described hereinbefore or hereinafter may be performed entirely independently of the solid support used in methods of the invention, or, as described hereinafter, may in part use components of the solid support, namely a means for detection (or part thereof) which is provided by the solid support.

Means for detection

In a preferred aspect, therefore, the present invention provides a detection method of the invention, wherein at least one means for detecting the presence and/or determining the amount of said one or more microorganisms, or a target molecule from said one or more microorganisms, or a part of said means for detection, is used in the analysis step b) and preferably said solid support additionally comprises said means or part thereof.

As referred to herein, a “means” for this detection is a component of a detection method for the one or more microorganisms (or one or a group thereof) or the target molecule. More than one means for detection may be used in the methods described herein. One or more of the required means may be provided by the solid support.

Various detection methods may be used are described hereinafter. This component may, for example, form a reagent in a detection method or may aid performance of the detection method or allow for collection and/or purification of the microorganisms or their target molecules. As such a means for detection may be a component for capture such as an affinity binding surface or a reagent involved in the generation of a signal, e.g. an antibody or enzyme that may be used for detection, as described in more detail hereinafter. A part of said means for detection refers to a sub-portion of the relevant component, e.g. which by itself does not aid performance of the detection method but provides part of a component that has that effect. More than one means for detection may be used (and may be provided on the solid support) when the detection method requires a multiple component detection system and/or different detection means are required for different microorganisms.

A means that may be used is entirely dependent on the detection method to be used. However, conveniently, a means is used, which is sufficiently robust to survive potentially harsh incubation conditions, particularly, when that means is comprised in the solid support. In one example, a means for detection is an affinity binding surface for said one or more microorganisms (or one or a group thereof), or a target molecule from said one or more microorganisms (or one or a group thereof), preferably an affinity binding surface for DNA, RNA or a protein from said one or more microorganisms. When used in a detection method of the invention, the step of analysis in the detection method comprises releasing the microorganisms from the solid support and/or lysing said microorganisms, and binding the one or more microorganisms or target molecules to that means for detection or part thereof.

Such an affinity binding surface binds its target microorganism or target molecule selectively, i.e. binds that entity or molecule to the exclusion of at least some other entities or molecules. However, it does not necessarily bind that entity or molecule to the exclusion of all other entities or molecules. Thus, for example, the affinity binding surface may bind all bacteria non-selectively, but not other microorganisms, or may bind all polynucleotides non-selectively, but not nonpolynucleotide molecules. Later steps in the determination method may allow discrimination between the microorganisms/polynucleotides to allow determination of the presence or amount of the target microorganisms/molecules. In the examples described herein, beads which bind DNA were used to capture DNA after lysis of the microorganisms. The DNA was eluted for further analysis.

As discussed above, a means for detection may be a reagent in the detection method. (Optionally this may be a second means for detection, which is used in the detection method.) In one example in this connection a means for detection may be a molecule that is capable of interacting directly or indirectly with one or more of the one or more microorganisms, or a target molecule from said one or more microorganisms, to allow the generation of a detectable signal. In the method of detection of the invention, the step of analysis comprises releasing the microorganisms from the solid support and/or lysing the microorganisms, and binding one or more of the one or more microorganisms or target molecules therefrom to a means for detection or part thereof thereby generating a detectable signal. To distinguish between different microorganisms, a means for detection must be provided in a method which allows discrimination between the microorganisms if more than one microorganism is to be detected by a single detection method (in the alternative, different detection methods may be used for discriminating between microorganisms or groups of microorganisms). This may be achieved, for example, by using a molecule that is capable of interacting directly or indirectly with one or more of the microorganisms, but not with other target microorganisms, e.g. by selection of a specific binding molecule or enzyme etc.

Direct interaction refers to the molecule, which is a means for detection, interacting with the microorganism/target molecule without the intermediacy of other molecules. Indirect interaction refers to the use of an intermediary molecule or entity facilitating interaction with the target microorganism/molecule. A means for detection in this regard, together with one or more ancillary molecules generates a signal. The “signal” is as described hereinbefore. The absence, presence or level of the signal may be determined. Conveniently, the signal is generated by a process as described for the sample/location component assessment as described hereinbefore. Preferably the signal that is generated is colour or light. However, since the analysis step of the method of detection is most likely to be conducted in a laboratory (unlike the local component assessment, which is carried out onsite), alternative signals may also be used, which require additional equipment for assessment, e.g. fluorescent analysis of DNA.

Various different systems may be used for detection of the microorganisms or the target molecule (which include use of a means for detection). This will depend partially on the microorganism or target molecule to be detected. Furthermore, the detection method necessarily must identify the target microorganisms or target molecules directly or indirectly. Target molecules may be used to identify the target microorganisms. As mentioned previously, identification may not be of a specific microorganism, but could be of a taxonomic or functional group of microorganisms. Nevertheless, the signal generated should indicate the presence (or amount) of the target microorganism(s)/molecule(s).

In one example, the step of analysis involves detection of the presence of a molecule produced by one or more of the microorganisms. This molecule may act as a substrate for a chemical reaction, e.g. an enzymatic reaction. The substrate is consumed or modified during that reaction to produce a resultant product. That product may itself produce a detectable signal or indirectly produce a detectable signal through the intermediacy of other molecules. The detectable signal may be a colour change. This signal would then indicate the presence of a target molecule, which may be used to determine the presence or extent of the target one or more microorganisms. Thus, in a preferred aspect the molecule which is a means of detection is an enzyme, which preferably interacts with a substrate from one or more of the one or more microorganisms, preferably, to produce a product, which directly or indirectly produces a detectable signal, preferably, a colour change.

By way of example, the substrate that is generated could be hydrogen peroxide, e.g. generated by enzymes such as superoxide dismutase. This may be used in colour reactions (e.g. using a catalase to produce a coloured product). In the alternative, the molecule produced may be an enzyme, which is detected by analysis of its products. For example, catalases which are produced by aerobic bacteria, decompose hydrogen peroxide. Hydrogen peroxide may be added for detection of the catalases. Known colour reactions may be used in which the catalases mediate the oxidation of certain organic compounds by hydrogen peroxide to produce a coloured product. Thus, in a preferred aspect, a means of detection is an enzyme that interacts with a substrate from said one or more microorganisms (or the target molecule) to produce a product, which directly or indirectly produces a detectable colour change, pH change, change in redox potential or change in glucuronidase activity.

An alternative means for detection may be an antibody or another specific binding molecule (e.g. an aptamer, RNA/DNA probe or a binding molecule generated or modified by in vitro evolution). Antibodies include monoclonal or polyclonal antibodies, chimeric and humanised antibodies, and include antibody fragments such as, for example, Fab, F(ab’)2, Fab’, and Fv fragments. Such antibodies and their fragments may be used to bind directly to a molecule on the one or more microorganisms or the target molecule and may be detected by any appropriate means. For example, the antibody may carry a detectable label (as described hereinbefore) or may be identified using routine assays such ELISA assays. The specific binding molecule may be any molecule, which selectively and specifically binds to a particular molecular partner, in this case to at least one of the one or more microorganisms or a target molecule thereof. A molecule which binds specifically to such microorganisms or molecules binds with a greater affinity than that with which it binds to other molecules (e.g. other microorganisms or target molecules), or at least most other molecules.

To allow separate identification of one or more microorganisms from the target microorganisms, different means for detection should be used. This may involve the use of different antibodies or other reagents specific for one or more of the target microorganisms. Conveniently, when polynucleotides are analysed, e.g. by PCR, the different one or more microorganisms may be discriminated. Determination of presence/amount of microorganisms

The signal that is generated in the detection step is used to determine the amount or presence of said one or more microorganisms or target molecules as described hereinbefore. In particular, a quantitative or qualitative result is obtained, which defines that amount or presence. This result can be used for classification purposes. By way of example, the presence and/or amount of said one or more microorganisms is used to generate a quantitative or qualitative value indicative of the status of the test location or sample. Conveniently, the status may be a binary determination, e.g. indicative of anaerobic or aerobic conditions (by virtue of the microorganisms captured and grown at that site). Thus, the method of detection allows the determination of aerobic or anaerobic conditions in the test location or sample. As referred to herein, anaerobic conditions refer to anoxic conditions in which insufficient oxygen is present to allow aerobic growth of microorganisms, such as bacteria. In contrast under aerobic conditions oxygen is sufficient to allow aerobic growth of microorganisms, such as bacteria.

In another example, it may identify contaminated or non-contaminated locations or samples, again based on the microorganisms which are present. In the alternative a non-binary status may be provided, e.g. an indication of environmental health or pollution, i.e. the environmental status may be determined. This would allow monitoring over a period of time by monitoring changes to that health or pollution status.

The quantitative or qualitative value indicative of the status of the test location or sample may be determined by reference to calibration curves or by comparison to a set of controls. Algorithms may be used to generate the status value based on such calibration data.

Products

The invention also relates to products that may be used in methods of the invention. Thus, in a further aspect, the present invention provides a product for selective capture and/or growth and optionally detection of one or more microorganisms in a liquid at a test location or in a liquid sample, said product comprising: a) a solid support comprising a growth medium which is selective for said one or more microorganisms; b) one or more porous containers enclosing said solid support, wherein said one or more containers allow liquid and microorganisms to access the solid support and at least one porous container excludes entities that are 5 mm or larger, wherein preferably at least one porous container has a pore size of less than 1 mm, preferably less than 10 pm or less than 1 pm.

The components of the product have the definitions as described hereinbefore, in particular the solid support and/or said one or more microorganisms and/or said one or more containers are as defined hereinbefore.

The present invention also provides in a further embodiment, a product for selective capture and/or growth and optionally detection of one or more microorganisms in a liquid at a test location or in a liquid sample, said product comprising a solid support comprising a particle and a portion which is a removable coating which contains a growth medium which is selective for said one or more microorganisms and said particle has an affinity binding surface for DNA or RNA from said one or more microorganisms. The components of the product have the definitions as described hereinbefore, in particular, the solid support and/or said one or more microorganisms are as defined hereinbefore, but in which the solid support has the specific features described above. The removable coating is as defined hereinbefore. The solid support may be enclosed by one or more porous containers which allow liquid and microorganisms to access the solid support, wherein preferably said at least one porous container excludes entities that are 5 mm or larger, as defined hereinbefore.

The products described herein may be used to put the methods of the invention into effect.

The products described above may also be provided in kit form, with the same components, which may also be provided with additional components for use, e.g. standardizing materials, e.g. microorganisms for comparative purposes, reagents for microorganism isolation and lysis, reagents for DNA isolation and elution, primers for amplification and/or appropriate enzymes, buffers and solutions. Optionally, said kit may also contain a package insert describing how the method of the invention should be performed, optionally providing standard graphs, data or software for interpretation of results obtained when performing the invention.

The invention also extends to kits or products of the invention for use in methods of the invention as well as use of kits or products of the invention in methods of the invention.

By way of illustration, methods of putting the invention are described in more detail in the Examples. Whilst the invention is not limited to such methods, preferred aspects described herein are preferred aspects applicable to all methods described herein.

In a particularly preferred aspect, the method of the invention is a method of detecting the presence and/or determining the amount of one or more microorganisms, or a target molecule from one or more microorganisms, at a test location or sample by a method comprising: a) selectively capturing and/or growing one or more microorganisms at said test location or in said sample by: i) placing a solid support in a liquid at said test location or in said sample, wherein the solid support:

1) is a particle, 2) comprises a removable coating which contains a growth medium which is selective for said one or more microorganisms, and

3) additionally comprises at least one means for detecting the presence and/or determining the amount of said one or more microorganisms, or a target molecule from said one or more microorganisms, or a part of said means for detection as described hereinbefore, and

4) is enclosed by one or more porous containers as described hereinbefore; ii) allowing the one or more microorganisms to grow on said solid support; iii) removing said solid support from the test location or sample; and b) analysing the one or more microorganisms present on the removed solid support to determine whether said one or more microorganisms, or a target molecule from said one or more microorganisms, is present in the test location or sample and/or the amount of said one or more microorganisms in the test location or sample, by: i) removing the removable coating from the remainder of the solid support, ii) releasing said one or more microorganisms from said coating, iii) lysing said one or more microorganisms, iv) binding DNA or RNA from said microorganisms to one of said means for detection, preferably an affinity binding surface for DNA or RNA, and v) identifying the DNA or RNA of said microorganisms, preferably by qPCR or sequencing.

The definitions and preferred aspects of all of these components is as described hereinbefore.

The methods described in the Examples form further preferred aspects of the invention. All combinations of the preferred features described above are contemplated, particularly as described in the Examples. The invention will now be described by way of non-limiting Examples with reference to the drawings in which:

Figure 1 shows a representative example of a method and product of the invention. A) Shows the use of a porous container (a bag) in which a solid support with a selective medium (in this case a selective gel-bead complex) is placed. Bacteria of interest grow selectively in the medium and are therefore associated with the gelbead complex. B) Shows an alternative form of the product that may be used for analyzing test samples or locations. C) Shows an example of the process of preparation to analysis. The solid support (e.g. a gel-bead complex) is prepared then placed in a porous container, which is incubated in a liquid sample or environment of interest. The solid support is then collected and the DNA extracted. In this example, the beads are able to bind DNA and in this way the DNA is collected and may be eluted for further analysis such as by qPCR or sequencing.

Figure 2 shows bacterial abundance in samples incubated anaerobically (right hand bar for each time point) or aerobically (left hand bar for each time point) after 10 or 17 days of culture, as assessed by average 16S rRNA gene copy number, in experiments 1 (A), 2 (B), and 3 (C).

Figure 3 shows eukaryotic microorganism abundance in samples incubated anaerobically (right hand bar for each time point) or aerobically (left hand bar for each time point) after 10 or 17 days of culture, as assessed by average 18S rRNA gene copy number, in experiments 1 (A), 2 (B), and 3 (C).

Figure 4 shows the appearance of the samples and agarose beads after 10 or 17 days of culture. (A) After 10 days, the colour of the water in the aerobe and anaerobe beakers differed. (B) All beads after 10 days were intact. (C) The water in the beakers was filtered after 17 days, and the colour difference was very clear. (D) The agarose beads had been distributed to each corner of the tea bag in close proximity to the sand. The black colour may indicate sulfur oxidation.

Figure 5 shows the experimental set-up for the sea experiment. 12 boxes, each containing a tea bag with three agarose beads, were attached to a board. Big holes were drilled in half of the boxes to provide an aerobe milieu. The other half only had a small hole, facilitating an anaerobe milieu. Fish feed was used to imitate pollution.

Figure 6 shows the prokaryotic and eukaryotic abundance in samples from the sea experiment. Average 16S rRNA gene copy number (A) and 18S rRNA gene copy number (B) in samples from aerobe/anaerobe (left/right hand bar for each test pair) conditions with or without fish feed at the sea bottom are shown. Figure 7 shows principal component (PCA) analyses of the microbiota composition (16S rRNA) in the agarose beads after one week at sea. Each spot represents one sample. The numbering indicates if the conditions were aerobic (1) or anaerobic (0), while the shape indicates if salmon feed was added (square), or if no feed was added (diamonds). The space between each spot indicates the difference in the microbiota composition.

Figure 8 shows principal component (PCA) analyses of the microbiota composition (18S rRNA) in the agarose beads after one week at sea. Each spot represents one sample. The numbering indicates if the conditions were aerobic (1) or anaerobic (0), while the shape indicates if salmon feed was added (square), or if no feed was added (diamonds). The space between each spot indicates the difference in the microbiota composition.

Figure 9 shows the significant OTUs with an a = 0.05 for the interaction between sediment and growth condition. The difference in abundance between the significant OTUs and the four factors is shown, where OTUs with a positive value for the Iog2 Fold-Change were significantly abundant in anaerobic D sediment. In contrast, OTUs with a negative value were significantly abundant in aerobic and B sediment. OTUs with an abundance > 1% were included, and the data was based on results from 16S rRNA sequencing. The figure was created with DESeq2 and phyloseq. The adjusted p-values were used to find the significant OTUs. The star signifies that Acidobacteria is not a genus, but the lowest classified level for this OTU and is therefore included.

Figure 10 shows the abundance plot for the genera present at > 1% in the samples rarefied at 60 000 sequences per sample. The different genera are presented in the same order in the bar plot as in the legend, and the black lines show the separation between the different OTUs in each sample. The reduced bars for B and D illustrate that few of their genera had an abundance > 1%. Some genera are marked with a number to ease visualisation. The figure was created with phyloseq.

Figure 11 shows a PCoA plot with the Bray Curtis dissimilarity for the clustering of alginate beads in the experiment based on the total genome of each bead. The different sediment types are marked with colours, and the various growth conditions are marked with shapes. The anaerobe clusters are placed on top of each other, illustrated with a circle (top left). The B1- samples are marked with a circle (bottom middle), and the B2- are marked with a circle (second circle from top, left). The squares (top right) are the original sediment samples. The plot was created based on the results from 16S rRNA sequencing and the OTUs present in > 1% of the samples. The plot was made with phyloseq ordination and explained 72% of the variance.

Figure 12 shows the PCoA plot with Bray Curtis dissimilarity for the clustering of alginate beads from each environmental site based on the total genome of each bead. The test runs were performed at different depths and included all the beads, shown with shape from each site. The original sediment samples were collected from Bunnefjorden. The figure shows the clustering of alginate beads within each ball in the prototype. The prototype consists of 4 balls with a total of 9 alginate beads. Balls 1, 2, 3, and 4 were from Fagerstrand (5 m), balls 5, 6, 7, and 8 were from Bunnefjorden (30 m), and balls 13, 14, 15, and 16 were from Alta (76 m). The figure was created based on ordination in phyloseq.

Figure 13 shows significant OTUs for the three sites in the environmental study compared two by two with an a = 0.05. a) Significant OTUs between 5 m and 30 m. The OTUs that were significant for 5 m have a positive log2-FC value, and the OTUs significant for 30 m have a negative value, b) Significant OTUs between 76 m and 30 m. The OTUs significant for 76 m have a positive log2-FC value, and the OTUs significant for 30 m have a negative value, c) Significant OTUs between 76 m and 5 m. The OTUs significant for 76 m have a positive log2-FC value, and the OTUs significant for 5 m have a negative value.

Figure 14 shows abundance plots for the 30 most abundant bacterial orders at fish farm 1 (A) and fish farm 2 (B) and the 10 most abundant bacterial phyla at fish farm 1 (C) and 30 most abundant genera at fish farm 1 (D). In A and B, the dominant orders at site C1 are Campylobacterales and Enterobacterales and at site C2, Enterobacterales. In C, the dominant phyla at site C1 are Campylobacterota and Proteobacteria and at site C2, Proteobacteria. In D, the dominant genera at site C1 are Sulfurovum and Litorilituus and at site C2, Litorilituus. Figure 15 shows PCoA plots with Bray-Curtis dissimilarity for the order (A) and genus (B) rank. The numbers in the legend indicate the fish farm (either fish farm 1 or 2), and the two sites C1 and C2.

Figure 16 shows boxplots of A) the results of Figure 15A, PCo1 (order rank) and B) nEQR results at the same sites. The numbers in the legend indicate the fish farm (either fish farm 1 or 2), and the two sites C1 and C2.

EXAMPLES

Experiment 1 : In vitro analysis of aerobic versus anaerobic environments using a solid support of agarose with a selective growth medium

In this experiment, two environments were fabricated, one aerobe and one anaerobe. The growth conditions in polluted areas at the sea bottom are anaerobic. Sulfur oxidizing and denitrifying bacteria thrive in these areas. Unaffected areas at the sea bottom are aerobic. The objective of the experiment was to discriminate between the two environments based on the presence of sulfur oxidizing and denitrifying bacteria and other microorganisms, using the method of the invention.

Materials and Methods

Agarose beads: The agarose beads used in the experiment consisted of low melting agarose, paramagnetic beads, and a selective growth medium. The agarose beads contained 3% agarose in enrichment culture (Cardoso et al., 2006, Biotech. Bioeng., 95, p1148-1157); 0.8 g/L K₂HPO₄, 0.3 g/L KH₂PO₄, 0.4 g/L NH₄CI, 0.01 g/L MgCI₂, and 2g/L NaHCCh, to which 29.8 mM thiosulfate and 52.6 mM nitrate had been added. The low melting point agarose gave shape to the beads and at the same time, it could easily be melted at a suitable temperature for downstream analysis. The paramagnetic beads enabled DNA extraction. The growth medium used was selective towards environmental bacteria, especially sulfur oxidizing and denitrifying bacteria and other related microorganisms, facilitating growth on the agarose beads. Test system: A simple system, simulating the sea bottom, was set up using marine sand and seawater collected from the Oslo fjord. Three agarose beads were put directly onto marine sand in a beaker, and covered with sterile seawater. As negative controls, autoclaved sand was used. All samples were incubated at 10°C with discreet shaking to imitate the conditions at the sea bottom. Half of the samples were incubated aerobically, the other half anaerobically. Samples were collected after 10 and 17 days.

Analysis: DNA was extracted from the beads. Quantification of the extracted DNA was performed by qPCR amplification of the 16S rRNA (bacterial abundance) or 18S rRNA (eukaryotic abundance) genes. The 16S rRNA genes were then sequenced to classify the bacterial and eukaryotic communities growing on the beads.

In this and subsequent examples, reference to bacteria or bacterial communities includes also archaea and archaeal communities.

Results

Figure 2A shows the results from this experiment. There was a slight difference in average 16S rRNA copy numbers in the aerobe sample compared to the anaerobe sample after 10 days of incubation. After 17 days of incubation, the copy number was higher for the aerobe sample, and lower for the anaerobe sample.

Quantification of eukaryotic DNA using the 18S rRNA gene revealed higher amounts of eukaryotic DNA in the aerobe sample compared to the anaerobe sample (Figure 3A).

A Bray Curtis PCoA plot based on the microbial compositions as assessed by 16S rRNA gene sequencing showed that the aerobic samples clustered away for the anaerobic samples indicating that the microbial communities in the samples within each milieu are similar to each other, but deviate between each condition (data not shown).

Noticeable changes in colour occurred around the agarose beads after 10 days (data not shown). The sand around the beads in the anaerobe milieu had turned grey, implying that sulfur oxidation had occurred. The difference between the samples in each milieu was even clearer after 17 days. The beads in the anaerobe system had turned black (data not shown).

A PLS discriminant analysis on the 16S data separated the samples with high accuracy, even after cross evaluation. The only sample that could not be separated was aerobe at 17 days. This might imply that it had started to become anaerobic. Of all the bacteria present in the samples, sulfur oxidizing and denitrifying bacteria were crucial in the separation of anaerobe at 17 days from the other samples.

Experiment 2: In vitro analysis of aerobic versus anaerobic environments using a solid support of agarose with a selective growth medium contained in a bag

In contrast to the first experiment, in which agarose beads were placed on sand directly, in this experiment, the beads were placed in a bag and then placed on the sand to assess the effect of using the bag.

Materials and Methods

The agarose bead, test system, and analysis were set up as in experiment 1 except that sterile tea bags were used to contain the 3 agarose beads and placed on top of the sand and covered with sterile seawater.

Results gPCR guantification of DNA by 16S rRNA gene amplification revealed that the DNA concentration in the aerobe samples was slightly higher than in the anaerobe samples after 10 days (Figure 2B). The copy numbers in the samples incubated for 17 days were similar to the 10 days samples. The 18S rRNA copy number was higher in the aerobe samples than the anaerobe samples (Figure 3B). A Bray Curtis PCoA plot illustrated that the bacterial composition in the aerobe differed from the composition in the anaerobe samples (data not shown). The clustering of aerobe and anaerobe was more distinct in this experiment compared to experiment 1 without a tea bag. In experiment 2, no distinct colour differences were observed between the sand incubated aerobically and anaerobically, presumably due to the separation of the sand and agarose beads.

Experiment 3: In vitro analysis of aerobic versus anaerobic environments using a solid support of agarose with a selective growth medium contained in a bag

Experiment 3 is a repeat of experiment 2 but using a different marine sand.

Results

The 16S rRNA gene copy numbers for the samples were in concordance with experiment 2 (Figure 2C). The 18S rRNA copy numbers of the samples (eukaryotic DNA) were lower than the 16S rRNA copy numbers (Figure 3C), suggesting that the eukaryotic abundance is lower than the prokaryotic abundance. A Bray Curtis PCoA showed that the 10 days samples both from the aerobe and anaerobe condition clustered well together (data not shown). Both after 10 and 17 days, a colour difference of the water in the aerobe and anaerobe beakers was noticeable (Figure 4C). The agarose beads were intact (Figure 4B). After 17 days, the tea bag in the anaerobe beaker had turned black in the places the agarose beads had been close to the sand (Figure 4D).

Experiment 4: Verification of testing method at sea

In this experiment 12 boxes, each containing a tea bag with 3 agarose beads, were attached to a board and placed at the sea bottom. Fish feed was used to imitate pollution.

Materials and Methods

The experimental setup included an aerobe and an anaerobe condition. A total of 12 boxes containing a tea bag with three agarose beads were used in the experiment (Figure 5). Big holes were drilled in half of the boxes to ensure good water flow, promoting aerobic conditions. In comparison, the anaerobe boxes only had a small hole.

In addition to the size of the holes, fish feed was used to create an anaerobe environment. The feed was put in tubes to prevent it from disappearing from the box. A hole in the end of the tube enabled leakage of some of the food to the box. Half of the anaerobe boxes contained fish feed, as well as half of the aerobe boxes, enabling investigation of the effect of fish feed. The boxes were attached to a board and placed at the sea bottom. After 7 days, the boxes were recovered, DNA was extracted and quantified with qPCR using 16S and 18S covering primers. Sequencing of the 16S and 18S rRNA gene was performed as in previous experiments.

Results

The average 16S rRNA copy numbers of the samples from the boxes with fish feed were higher than the samples without fish feed (Figure 6A), indicating that the copy numbers are more dependent on the presence of fish food than aerobe/anaerobe conditions. A quantification of eukaryotic organisms was performed as well, using the 18S rRNA gene. The 18S rRNA copy number of the aerobe samples was higher both with and without fish feed compared to the anaerobe samples (Figure 6B), indicating that the presence of oxygen is the limiting factor of eukaryotic abundance.

The microbiota composition in the sea experiment showed a clear distinction between the environmental conditions tested, with a very clear distinction between the aerobic and anaerobic conditions, in addition to conditions with and without salmon feed (Figure 7, based on 16S rRNA gene sequencing; Figure 8, based on 18S rRNA gene sequencing). Interestingly, the porous bag in the anaerobic environment without feed contained bacteria that can form nanowires, suggesting biogeochemical processes in the beads.

These results show that the agarose beads are able to capture and culture bacteria/archaea and eukaryotic microorganisms from the local environment and the population that is collected is indicative of the local environment, in this case showing that different microbiota compositions were present in aerobic or anaerobic, or polluted or non-polluted, environments.

Experiment 5: Use of alginate beads for analysis of microbial communities in laboratory samples and at environmental sites

Materials and Methods

Development and optimisation of the protocol

Several parameters were tested to create alginate beads equal in size with good integrity that could easily be dissolved. 50 pl large alginate beads were created with concentrations from 1-6% sodium alginate together with 1-2% of CaCh and paramagnetic beads. Droplet creation was tested with different pipette sizes and various gelatinisation times from 5-30 minutes.

Several solvents were tested to ease the extraction of bacterial cells and DNA from the alginate beads. Different concentrations and ratios of PBS (1x and 10x), EDTA (0.1 M, 0.5 M, and 1 M) and sodium citrate (0.1 M, 0.5 M, and 1 M) were tested as solvents with unequal dissolution times and speed (rpm). The volume of solvents was reduced down to the smallest volume capable of complete dissolution of the alginate bead.

Due to amplicon PCR inhibition, the protocol was optimized based on dissolution time, solvent concentration, and enzymatic addition of alginate lyase.

E. coli were used as the bacteria attached to the beads and optimization evaluated by the yield of DNA, performance of the dissolved alginate bead solution during DNA extraction, and inhibition of qPCR. dsDNA was quantified with the Qubit dsDNA HS and BR Assay Kit (InvitrogenTM, USA). The manufacturer's protocol was followed, and 2 pl of Template DNA was used. The kit contains a reagent that specifically binds to dsDNA and emits fluorescence that can be detected by the Qubit FlurometerTM (Invitrogen, USA). The inhibition evaluation was performed with 16S rRNA qPCR with 0, 5, 25 and 125x dilutions of the DNA eluate. The 16S rRNA qPCR working solution consisted of 1x HOT FIREPol EvaGreen qPCR supermix, 0.2 pM PRK341 F forward primer (5’-CCTACGGGRBGCASCAG- 3’, Invitrogen, USA, SEQ ID NO: 1), 0.2 pM PRK806R reverse primer (5’-GGACTACYVGGTATCTAAT-3’ Invitrogen, USA, SEQ ID NO: 2), and 2 pl DNA template (an overall range of 30 ng/pl - 150 ng/pl). The amplification was performed using the following program: 95°C for 15 minutes followed by a 40-cycle step with 95°C in 30 sec, 55°C in 30 sec, and 72°C for 45 sec with the C1000 Touch Thermal cycler CFX96 Real-Time system (BIO-RAD, USA).

The optimised protocol

Sulfate reducing medium: The sulfate reducing medium was made based on the sulfate reducing medium in Standard Methods for the Examination of Water and Wastewater (Clesceri et al., 1999, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, American Water Works Assoc., Water Environ. Fed.), but with some modifications. 0.031 M L-(+)-Lactic acid sodium salt (Sigma-Aldrich, USA), 3 g Nutrient Broth (5 g peptone and 3 g Beef extract per L) (Merck KGaA, Germany), 0.008M MgSO4 x 7H2O (VWR Chemicals, USA), 0.011 M Na2SO4 (Merck, Germany), 0.0007 M CaCh x 2H2O (Merck KGaA, Germany), 0.003 M K2HPO4 (Merck KGaA, Germany), mixed with 1 L MilliQ water until fully dissolved. The solution was autoclaved before pH was adjusted to 7.5 ± 0.3 using 0.5 M NaOH or HCI. The medium was stored at 2- 4°C after autoclavation and used throughout the period.

The two temperature-sensitive compartments of the medium, 0.001 M Fe(ll) (NH4SO4)2 x 7H2O (KEBO-Lab, Norway) and 0.0057 M L-(+)-ascorbic acid (Merck, Germany), were filter sterilized with a 0.45 pm filter and were freshly made for each experiment due to the short shelf life of the solutions. The Fe(ll)(NH4SO4)2 x 7H2O and L-(+)-ascorbic acid were aseptically added to 0,1 mL of each solution per 10 mL of the basal medium on the day of use.

Creation of paramagnetic alginate beads: Sodium alginate (Sigma-Aldrich, USA) was prepped in a 6% concentration (Soo et al., 2017, Int. J. Polymer Sci. , Article ID 6951212) with the sulfate reducing medium and mixed until even on a magnetic stirring device with occasionally heating in a 40°C water bath. A correct ratio of alginate solution and magnetic particle suspension BLm from the mag midi kit (LGC Genomics, UK) was homogenized to create 50 pl large paramagnetic alginate beads where each bead consisted of 16 pl magnetic particle suspension. The inspiration for the magnetic alginate gel beads was Bee et al. (2011 , J. Colloid Interface Sci., 362, p486-492), Rocher ef al. (2008, Water Res., 42 p1290-1298) and Soo et al. (2017, supra).

A 2% CaCh x 2H2O (Merck KGaA, Germany) (Soo et al., 2017, supra) was placed on a magnetic stirring device with a small magnet. The turn of the magnet created a water vortex that was able to drag the alginate drop under the water surface right after impact.

With a chopped off disposable 1.5 ml Pasteur pipette, the alginate magnetic particle solution was dripped into the centre of the water vortex in the CaCh solution from a height of 2-3 cm. The alginate beads were allowed to gelatinize in the CaCh solution for 30 minutes (Soo et al., 2017, supra) for an even gelatinization throughout the alginate bead. Floating alginate beads were removed because of their assumed lack of integrity.

The alginate beads were washed in Mil liQ water three times to remove excess calcium ions before they were stored at 4°C in a sealed beaker with volume to volume with MilliQ water. The alginate beads were not found viable for more than ten days at 4°C in MilliQ water.

Incubation with samples:

Laboratory test: For the test on growth conditions and the selective properties of the alginate bead, sediment samples were collected from two locations in Emmerstadsbukta. The selectiveness for the alginate beads was tested as a two factorial design observing the effect of selectiveness with different sediments and growth conditions. Four beakers were filled with sediment and autoclaved seawater, and alginate beads in an empty triangular autoclaved teabag were placed on top of the sediment. The teabags were filled with a few acid-washed glass beads (3-5 mm in size, Sigma-Aldrich, USA) to increase the weight of the bag and four alginate beads. The teabags were sealed with a staple, and the system was lowered down on top of the sediment. The four beakers were incubated in the dark at 10°C for four days.

Beakers were placed to grow aerobically and anaerobically. The anaerobic beakers were placed in an incubation box with a suitable amount of AnaeroGenTM 3.5 L anaerobic bags (Thermo Fisher diagnostic, Norway) and two anaerobic indicators (Thermo Fisher, USA) to achieve an environment with 9-13% CO2 and < 1% O2. The aerobic beakers were covered up to avoid contamination.

After the incubation period, the teabags were aseptically removed from the incubation systems, and the beads were carefully collected.

Environmental test: Alginate beads in tea bags were loaded in penetrable balls connected by a rope and separated with small weights. This was anchored and connected allowing the prototype to move with the currents, but the alginate beads remained in contact with the sediment.

Three different testing sites were used. The first was performed at 5 m depth at FI1 at Fagerstrand in the Oslofjord, a location with assumed good conditions based on an oxygen concentration between 20-50% at the seafloor. The second was performed at 30 m depth at Gp2 in Bunnefjorden with an assumable worse condition based on an oxygen concentration between 5-20%. Both locations are from the inner part of the Oslo fjord. The third was conducted in Alta under an active merd in an aquaculture facility at 76 m depth.

Cell extraction after incubation: After incubation in a sample, incubated alginate beads were collected and placed in separate 1.5 ml Eppendorf tubes. To the tubes were added 100 pl of 0.5 M EDTA (Sigma Chemical Company, USA) solution and 100 pl of 0.5 M sodium citrate (Sigma-Aldrich, USA) solution to remove the Ca²⁺ ions. The Eppendorf tubes were incubated at 30 minutes, 1200 rpm at 65°C using Thermomixer C (Eppendorf, Germany). The alginate polymers were enzymatically removed with 20 pl of 2U alginate lyase solution (Sigma-Aldrich, USA) made with 1x PBS (Rabille et al., 2019, Sci. Rep., 9, p1 -17), with an incubation step for 1 hour, 300 rpm at 37°C. DNA extraction and quantification ofdsDNA: DNA extraction was performed using the mag midi kit (LGC Genomics, UK), following the manufacturer's protocol with some modifications. To the dissolved alginate bead solution was added 250 pl Lysis buffer BLm, 25 pl protease solution and 250 pl 97% ethanol (Kemetyl, Norway). The entire volume of 795 pl was placed on a magnet. After discarding the supernatant, the volumes for washing buffers were followed as set out in the manufacturer's protocol. 170 pl of BLm 1 was used to resuspend the pellet, followed by 10 minutes of incubation at room temperature with regular use of the vortex. The samples were placed on a magnet, and the supernatant was discarded. The washing step was repeated twice with 175 pl of BLm 2.

The pellet was air-dried for 6 minutes at 55°C to allow for complete evaporation of the remaining buffers. Next, the pellet was resuspended with 63 pl elution buffer BLm and incubated at 55°C for 10 minutes at 900 rpm. The sample was placed on a magnet. After pellet formation, 50 pl of the eluate was transferred to a new Eppendorf tube. E.coli cells and an unincubated alginate bead were used as positive controls. Two negative controls were included - one had an unincubated alginate bead and 50 pl nuclease-free water, while the other had 50 pl nuclease- free water and 16 pl mag particle suspension BLm. Eluted DNA was quantified with the Qubit Fluorometer, as mentioned above.

DNA sequencing:

Illumina 16S rRNA gene sequencing

Library preparation: The first step in the library preparation was an amplicon PCR with a reaction cocktail made of a 1x HOT FIREPol Blend Master Mix RLT (Solis BioDyne, Estonia), 0.2 pM PRK341 F forward primer (5’- CCTACGGGRBGCASCAG-3’, Invitrogen USA, SEQ ID NO: 1), 0.2 pM PRK806R reverse primer (5’-GGACTACYVGGTATCTAAT-3’, Invitrogen USA, SEQ ID NO: 2), and 2 pl DNA template (a range between 0.05 ng/pl - 18 ng/pl). Nuclease free water was used as negative control and eluted DNA from E.coli as a positive control. Next, the PCR reactions were amplified with the following program starting with 95°C for 15 minutes before 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec, followed by 7 minutes at 72°C with a 2070 Thermocycler (Applied Biosystems, USA).

Gel electrophoresis of amplicons: Amplicons were checked with gel electrophoresis on 1.5% Agarose gel made of UltraPureTM Agarose (Invitrogen, USA) with 1x trisacetate EDTA (TAE) buffer added 2.5 pl/50 pl PeqGreen (Peqlab, Germany). 5 pl of a 100 bp ladder (Solis BioDyne, Estonia) was added to the first well of each gel, and 4 pl of the PCR products were applied. The gel electrophoresis was run at 86V for 30 minutes before visualisation (=466 bp) with a GelDocTMXR (BIO-RAD, USA).

AMPure clean-up of PCR products: The AM Pure clean-up of the PCR products was performed by a Biomek 3000 robot (Beckman Coulter, USA) with an in-house made AMPure solution with 1.0x volume of Sera-Mag Speed Beads (Thermo scientific, UK) to volume of DNA sample. The samples were cleaned in 80% freshly made ethanol and eluted in nuclease-free water.

The attachment of indexes and quantification of products: Index PCR was performed using reactions containing 1x FIREPol Master Mix Ready to Load (Solis BioDyne, Estonia), 0.2 pM of each primer (16S rRNA gene Illumina primers, sequences not provided) and 2 pl DNA template. An epMotion 5070 robot (Eppendorf, Germany) was used to distribute the primers to the reaction mix. Amplification was performed using the following program: 95°C for 5 minutes followed by ten cycles of 95°C for 30 sec, 55°C for 1 minute, and 72°C for 45 sec, followed by 7 minutes on 72°C. As described above, the PCR products were checked with gel electrophoresis (=595 bp).

After Index PCR, DNA quantification was performed by the plate reader Varioscan Lux (Thermo Fisher Scientific, USA) with 70 pl of the Qubit working solution and 2 pl template DNA. The fluorescence values from the plate reader were analysed with a standard curve created with a selected number of representative samples based on fluorescence values. The DNA concentrations were measured with the Qubit dsDNA HS and BR Assay Kit. Library normalization and validation: The samples were normalized to equal concentrations and pooled with a Biomek 3000 robot. After pooling, a manual AMPure clean-up was performed with 0.1% Sera Mag Beads in a 1:1 ratio. 150 pl of the pooled PCR product was transferred to a new Eppendorf tube together with the Sera Mag beads. The homogenous solution was incubated for 5 minutes at room temperature before being brought into contact with the magnet for 2 minutes to create a pellet. The supernatant was discarded when clear, and 200 pl freshly made 80% ethanol was added without resuspending the pellet. After 30 sec incubation, the supernatant was discarded, and the process was repeated twice. The excess ethanol was removed, and the pellet was air-dried for 15 minutes for complete evaporation. Next, 40 pl nuclease-free water was used to resuspend the pellet of the magnet. The dissolved pellet was incubated for 2 minutes on a magnet before 35 pl of the eluate was removed and kept.

The cleaned product was checked on a 1.5% agarose gel, and the band size should be around 550-600 bp. For verifying the library before sequencing, the sample was added 1x Gel loading dye Purple SDS (BioLabs, USA) before application on a gel. The library was quantified with the Qubit Fluorometer as described above.

The sequencing was performed on a MiSeq with 300 bp paired-end sequencing. The sequencing for the selective properties of the alginate beads was sequenced on the in-house MiSeq and had a failed run at the forward read. The sequencing of the test runs from the prototype was performed at the Norwegian Sequencing Centre (Oslo).

Data analysis of 16S rRNA sequencing results

The data was demultiplexed into each original sample, and each paired-end sequence was merged. The primer sequences were removed. A quality filtration was performed on the sequences, subsequently calculations on the unique sequences and their abundance were performed. All sequences with > 97% similarity were clustered into OTUs, and chimaeras were filtered away. The OTUs were gathered in an OTU table, and the taxonomy was added using the Rdp 16S v18 database (Maidak et al., 2000, Nucl. Acids Res., 28, p173-174). The data were rarefied to 60 000 sequences per sample for the test of selective properties and 10 000 sequences per sample for the prototype test runs. The data was loaded into R (version 4.1.2) as a Phyloseq object, and Phyloseq was used to create different plots (McMurdie and Holmes, 2013, Pios One, 8, e61217). The sample complexity was further reduced with a cut of abundance in the samples at > 1%. All plots were created with the Bray Curtis dissimilarity index. Principal coordinate analysis (PCoA) plots were used to study the clustering between the conditions or locations. PCoA is a multivariate analysis that analyses the proximity matrix for dissimilarity or similarity, which allows for cluster recognition and interpretation (Korstanje, 2021 , https://towardsdatascience.com/principal-coordinates-analysis-cc9a572ce6c).

Statistical differences between the factors in the experimental designs were calculated with DESeq2 (Love et al., 2014, Genome Biol., 15, p1 -21), where all samples not directly included in the design were removed. DESeq2 calculated the significant OTUs with a log2-FC value as the response variable. The null hypothesis was a log2-FC = 0 and signified no difference between the groups. The alternative hypothesis was that the log2-FC 0.

Shotgun metagenome sequencing

The Shotgun metagenome sequencing library preparations were performed based on the Nextera DNA Flex Library Prep Protocol (Illumina, USA). The manufacturer’s protocol was followed with some exceptions, and the protocols for DNA inputs <100 ng were followed throughout the procedure. The number of PCR cycles was chosen according to the different groupings of DNA input from the table in the manufacturing protocol. The index primers were provided by Nextera.

The PCR products were verified with 1x Gel loading dye Purple SDS (BioLabs, USA) on a 2% agarose gel with 5 pl PCR product for 45 minutes on 86V. The cleaned product and the pooled library were checked on a 2% agarose gel run for 55 minutes at 86V. The finished library was sent to Novogene UK and sequenced on a NovaSeq 6000 with 150 bp paired-end reads.

The raw data was processed through FastQC (Andrews, 2010, FastQC, Babraham Bioinformatics, Babraham Institute, Cambridge, UK) to quality check the sequences before trimming with Trimmomatic (Bolger et al., 2014, Bioinf. , 30, p2114-2120) and a new quality check with FastQC (Andrews, 2010, supra). metaSPAdes (Nurk et al., 2017, Genome Res., 27, p824-834) was used to assemble, and Maxbin2 (Wu et al., 2016, Bioinf. , 32, p605-607) for binning. The bins were de-replicated with dRep (Olm et al., 2017, ISME J., 11 , p2864-2868). The dRep step includes Prodigal, a fast gene-prediction package that generates an amino acid and nucleic acid FASTA file. The amino acid FASTA file from Prodigal was entered into GhostKoala (Kanehisa et al., 2016, J. Mol. Biol., 428, p726-731) to identify the metabolic pathways and for taxonomic classification.

The metabolic pathways were extracted from GhostKoala, and genes from nitrogen metabolism, sulfur metabolism, and alginate lyase were searched for. Manual searches in the GhostKoala database studied the metabolic pathways belonging to the genes of interest. The pathways were sorted into complete and incomplete pathways to identify the most probable metabolic pathway for the genus in each bin. Taxonomic classifications were performed with GhostKoala, which classifies based on an amino acid sequence. The classification was performed by sub-setting the raw data based on a cut-of-score on values > 400 for the GHOSTX score. The most abundant taxonomic classification was found by further reduction of the data frame to unique genera and the occurrence of each genus. The most prevalent genus was used for taxonomic classification, and the percentage occurrence was calculated.

Results

Optimisation

Alginate beads were created that were equal in size, had good integrity, and were easily dissolved after incubation. Based on the experiences of Soo et al. (2017, supra), the 6% sodium alginate solution and 2% CaCh solution and a gelatinization step of 30 minutes were selected to give stable alginate beads with good size and integrity.

Optimisation of the protocol was performed due to inhibition of the amplicon PCR reaction. The evaluation was based on the best performing DNA extraction, the yield of eluted DNA, and the qPCR results. The parameters tested were the dissolution time, the solvents concentration, and the addition of an enzymatic step. The optimised protocol consisted of 100 pl 0.5 M EDTA and 100 pl 0.5 M sodium citrate for 30 minutes at 1200 rpm and 65°C together with 20 pl 2U alginate lyase for 1 hour at 300 rpm and 37°C.

Bacterial abundance in incubated beads - laboratory test

The test of selective properties was set up with four categories as a two-factorial design to test in which environment the selective properties of the alginate bead performed best. The categories were: assumable good sediment with aerobic conditions (B1-), assumable good condition with anaerobic conditions (B2-), assumable bad conditions with aerobic conditions (D1-), and assumable bad conditions with anaerobic conditions (D2-).

The results were based on 15 samples from the 16S rRNA sequencing. There were 5003 OTUs, where 41 OTUs > 1%. The samples were rarefied at 60 000 sequences. The average DNA concentrations were 1.74 ng/pl ± 1.14, and the DNA concentrations for the categories were 3.34 ng/pl ± 0.27 for B1-, 1.12 ng/pl ± 0.39 for D1-, 0.64 ng/pl ± 0.06 for B2- and 0.85 ng/pl ± 0.30 for D2-.

Selective properties were tested and resulted in 28 significant OTUs out of 41 for the interaction between sediment type and growth conditions based on a log2-FC value. The OTU with the highest log2-FC value was Petrocella, and the OTU with the lowest log2-FC value was Thalassocella. There were 20 significantly abundant OTUs with a positive log2-FC value and eight with a negative value.

Desulfobacterium, Desulfosarcina, Desulforhopalus, Desulfobulbus, and Desulfosalsimonas are all members of the order Desulfobacterales that reduce sulfate to sulfides under strictly anaerobic conditions (Zhou et al., 2021 , Sci Total Environ., 772, p145464). They were all significant for anaerobic and D sediment but had low abundance in all samples (Figures 9 and 10). The members of the Desulfobacterales order were mainly found in the D21 sample. Sulfurovum and Thioprofundum are sulfate oxidizing bacteria significant for anaerobic and D sediment and observed in the D sediment samples with low abundance (data not shown). All mentioned genera in this paragraph have a log2-FC value above five, indicating a large difference between the groups. The significant OTUs in the main experiment were significant based on one out of two factors or interactions between both. The samples from each group were placed together to better visualise the differences in abundance between the environmental factors in the test for selective properties (Figure 10). B1- share distinct similarities based on the abundance of genera Colwellia, Pseudoalteromonas, Thalassocella, and Psychromonas that have a high abundance in the four samples. All four genera were significant, and Colwellia, Pseudoalteromonas, and Thalassocella have a negative log2-FC value (Figure 9). Pseudoalteromonas and Colwellia have a high abundance for all aerobic samples and Thalassotaela for 50% of the aerobic samples. D1- shares several genera with B1- but has an overall higher diversity and abundance of Psychromonas with 63.6% compared to 19.5%. Psychromonas were one of the most abundant genera in some samples and provide 94.77% of the sample B22 and 91.05% of sample B24. B2- and D2- have a high diversity with abundant genera but a lower abundance for the genera shared with B1-.

The original sediment samples contained fewer OTUs with an abundance >1%, with 23% for the B sediment sample and 18.17% for the D sediment sample, compared to 94.57% for the alginate beads. The dominant genera from the alginate beads were not abundant in the original sediment samples. The B sediment sample had Desulfosalsimonas, a sulfate-reducing bacteria, as the most abundant genus at 17.25%. The D sediment sample had Granulosicoccus, a nitrate-reducing bacteria, as the most abundant genus at 31.7% (Baek et al., 2014, Int. J. System. Evol. Micrbiol., 64, p4103-4108).

The variance between the four categories in the test for selective properties was calculated with the Bray Curtis dissimilarity index to include the relative abundance of each genus in the sample. The results are presented as a PCoA plot (Figure 11). The PCoA plot shows the similarity or dissimilarity between samples based on their distance and the percentage variation between the categories. The aerobe samples were separated into two clusters in the PC1 and PC2 direction, while the anaerobe samples were clustered together. Samples belonging to B sediment were divided into two clusters in the PC1 and PC2 directions. Samples belonging to D sediment were clustered based on PC2 direction. All the samples from the test in selective properties were clustered away from the original sediment samples. Based on the 72% variance explained by the plot, the anaerobe samples appear most similar. This assumption correlates well with the abundance plot in Figure 10.

Shotgun metagenome sequencing was performed on the same DNA extracted for the 16S rRNA sequencing, resulting in nine metagenome-assembled genomes (MAGs) for all the samples. The nine MAGs represented all four growth categories, and several specific samples had more than one MAG designated to them. The MAGs were classified through GhostKoala. The classification in GhostKoala was performed based on the amino acid sequence from prodigal. The output from GhostKoala was loaded into R-studio. The sequences with a GHOSTX score > 400 were selected, and the number of occurrences of each unique genus was calculated. The genus with the highest occurrence was used for taxonomic classification, as none of the MAGs consisted of just one genus.

The nine MAGs contain Psychromonas, Pseudoalteromonas, Colwellia and Psychrosphaera, which were all genera equal to the significant OTUs in the test for selective properties. Two of the B1 samples were Colwellia, which correlates well with the results from 16S rRNA sequencing. The bin from the D2- sample was classified as Psychromonas, which correlates well with earlier findings. The Arcobacter in the D1- sample was not a significant OTU but had an average of 3.79%.

Environmental test

Testing at three sites resulted in a collective 2502 OTUs where 140 had an abundance >1%. Thirty-six samples were retrieved and analysed with 16S rRNA sequencing at each test site. The average DNA concentrations for each test run were 0.101 ng/pl ± 0.07 for Fagerstrand (5 m depth), 0.35 ng/pl ± 0.19 for Bunnefjorden (30 m depth), and 0.6 ng/pl ± 0.41 for Alta (76 m depth).

Figure 12 illustrates the three test runs separated in clusters based on the depth (equal to location) in a PCoA plot measured with Bray Curtis dissimilarity. The figure shows a distinct clustering between the three test runs and a difference in the bacterial composition in the three locations. The three original sediment samples collected from Bunnefjorden were clustered together away from the prototype samples. Figure 12 also illustrates the separation between the beads in each ball in the different prototype test runs. The alginate beads from the test run at Fagerstrand were primarily separated in the PC1 direction, stretching from -0.01 to 0.25, as shown by the short arrows in the bottom right quadrant. The same situation was observed in the cluster belonging to the alginate beads from Bunnefjorden. The cluster was entirely separated from the other clusters and had a separation along PC1 from -0.25 to -0.55. The short arrows in the bottom left quadrant illustrate the cluster's endpoint and the difference in the distribution of the samples from the different balls within the cluster. The variance in the Alta cluster was mainly described in the PC2 direction, spreading from -0.2 to 0.55. The lower part of the cluster was closer to the cluster from Fagerstrand than Bunnefjorden. The top and bottom circles (right hand of the figure) show the separation in the distance between alginate beads from ball 13 and ball 16. Ball 16 was the ball placed furthest from the anchor, and the ball with the greatest potential for movement.

The significant associations between the OTUs and the three locations were calculated with DESeq2, and plots with log2-FC values were created to compare the three locations (Figure 13A-C). In Figure 13A, the differences between the samples from Fagerstrand and Bunnefjorden were compared. This comparison resulted in 72/140 significant OTUs. In this comparison, only one bacterium with the possibility for sulfate metabolism was found based on the test of selective properties. Sulfurimonas was significant for Bunnefjorden and is a known sulfate oxidising and nitrogen reducing marine bacteria (Han and Perner, 2015, Front. Microbiol., 6, p989). The second comparison in Figure 13B shows the differences between the samples from Alta and Bunnefjorden. The comparison resulted in 87/140 significant OTUs. The OTU with the highest log2-FC value classified down to the genus level was Sulfurimonas, with five significant OTUs. Sulfurimonas is a sulfate oxidising bacterium. Desulfotalea is an obligate anaerobic sulfate reducing bacterium and was found significant for Alta with one OTU (Rabus et al., 2004, Environ. Microbiol., 6, p887-902). Sulfurospirillum was significant for Alta with two OTUs. The last bacterium of interest was Sulfurovum, a sulfate oxidizing bacterium significant for Alta with one OTU. All mentioned genera have a log2-FC value between 5 and 10, which indicates a large difference between the groups. The last comparison in Figure 13C illustrates the difference between the samples from Alta and Fagerstrand. The comparison resulted in 84/140 significant OTUs. Equal to the last comparison, Sulfurimonas was the genus with the highest log2-FC value classified down to the genus level with five significant OTUs. This comparison has the same number of significant OTUs as the comparison for Bunnefjorden for Desulfotalea, Sulfurospirillum, and Sulfurovum.

The abundance of genera present at >1% in the samples based on the 16s rRNA sequencing was determined (data not shown). The abundance of each genus in the alginate beads showed a visually large difference between the three locations (data not shown). There was an evenness in the distribution of the abundance of genera between the alginate beads within one test run. Alginate beads from Fagerstrand have abundant Pseudoalteromonas and Psychromonas with some Colwellia. OTUs belonging to both Pseudoalteromonas and Psychromonas were significant towards the location at Fagerstrand. Alginate beads collected from Bunnefjorden had abundant Colwellia and Thalassotalea.

Conclusions

Alginate beads were significantly enriched with sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB), reflective of the intended outcome of using a sulfate reducing medium with the alginate beads. Furthermore, bacteria associated with the bead were distinctive for different environmental conditions.

Experiment 6: Use of alginate beads for assessment of environmental status of the seafloor

The method of the invention was used to evaluate the environmental status of the seafloor by selecting microorganisms indicative of that status. In overview, bags containing selective beads (as discussed hereinafter) were placed on the seafloor. After approximately a week they were collected. DNA from the collected microorganisms was extracted and sequenced to identify the microorganisms and thereby determine the status of the site. Two sites at two different fish farms were investigated, one close to the farm (C1) and one at a distant site (C2). Materials and Methods

Bead preparation

A solution of sulfate reducing medium and sodium alginate was used to create the beads, using the process described in Example 5. To prepare the sulfate reducing medium, 3.5 g sodium lactate, 1.0 g beef extract, 2.0 g peptone, 2.0 g MgSCU, 1.5 g Na2SC>4, 0.5 g K2HPO4, and 0.1 g CaCh were added to 1 L reagent grade water and stirred on a magnetic stirrer with heating until dissolved. The solution was autoclaved and stored in a refrigerator until use.

3 g sodium alginate was added to 50 mL sulfate reducing medium and stirred on a magnetic stirrer with heating until dissolved. The solution was autoclaved and stored in a refrigerator until use. 20 g CaCh was added to 1 L reagent-grade water, generating a 2 % CaCh solution. The solution was autoclaved and stored in a refrigerator until use.

The beads were prepared as described in Example 5 by adding sodium alginate/sulfate reduced medium solution dropwise to the CaCh solution. The beads were kept on the stirrer for 30 minutes to allow for complete hardening of the sodium alginate and then rinsed as described in Example 5 before storage in a refrigerator.

Bead bags were created as in Example 5 comprising an autoclaved teabag with one sterile glass marble and four alginate beads. The bags were stapled closed and added to 40 mL sterile water and frozen in a 50 ml falcon tube.

Placement of beads at the site

Bead bags were thawed one day before use. On the day of use, a bead bag was placed into each of three chambers generated by cross-sectional division of an end sealed cylinder, which was provided in two longitudinal parts hinged together to allow opening and closing and with large pores in the body of the cylinder walls to allow influx of seawater. These devices were sealed and submerged on the seafloor with weights. Two separate experiments were conducted at distinct aquaculture farms in Finnmark, Norway. The bead-containing devices were deployed at two different sites at each fish farm, with one site, C1 , located near the sea cages, and the other, C2, positioned farther away from the sea cages.

In the first experiment, conducted in March 2023, the devices were left on the seafloor for seven days, with depths at C1 and C2 measuring 130 m and 60 m, respectively. After sampling, portions were promptly stored at -20°C within two hours post-sampling and remained frozen for three weeks before being sent to the laboratory for DNA analysis.

In the second experiment, conducted in June 2023, the devices were left on the seafloor for eight days, with depths at C1 and C2 measuring 62 m and 38 m, respectively. Following sampling, the samples were promptly refrigerated and sent to the laboratory on the day following collection.

DNA extraction and 16S rRNA sequencing

The beads were treated with EDTA, sodium citrate and alginate lyase using an inhouse protocol before DNA was extracted using the Quick-DNA Fecal/Soil Microbe 96 Magbead Kit (Zymo Research, USA) in a KingFisherFlex instrument (Thermo Fisher Scientific, USA).

Construction of a 16S rRNA library was performed by a two-step PCR. The first PCR was performed with a mix containing 1x HOT FIREPol® Blend Master Mix Ready to Load (SolisBiodyne, Estonia), 0.2 uM of each of nanopore modified 16S rRNA long range primers and 2 pl template DNA. The PCR cycling conditions consisted of 95°C for 15 min followed by 30 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 80 sec. The PCR products were cleaned using an in-house made sera mag speed beads solution before a second PCR was performed with a mix containing 1x HOT FIREPol® Blend Master Mix Ready to Load, primers from PCR Barcoding Expansion 1-96 kit (ONT, UK) and purified PCR product from the first PCR. The PCR cycling conditions consisted of 95°C for 15 min, followed by 10 cycles of 95°C for 30 sec, 62°C for 15 sec, 65°C for 120 sec before a final elongation step at 65°C for 10 min. The PCR products were then quantified and pooled together with equal amounts of DNA from each sample before purification using a sera mag speed beads solution.

The remaining steps involving DNA end preparation, DNA repair and adapter ligation were performed using the Ligation Sequencing Kit V14 (ONT, UK) and the recommended third-party reagents following ONT’s recommended protocols. Sequencing was performed using an R10.4.1 Flow cell (ONT, UK) on a MinlON Mk1C device (ONT, UK) installed with MinKNOW22.12.5 and basecaller Guppy 6.4.6.

Bioinformatics and statistics

The SituSeq workflow was used to analyze the Nanopore-generated 16S rRNA amplicon data (Zorz et al., 2023, ISME Communications, 3(1), 33).

The first step of the workflow was a preprocessing step in which FASTQ files were concatenated, primers were removed, and sequences were filtered by length. In the available pre-made Preprocessing script, the following parameters were used: minLength = 600, maxLength = 1500, trimLeft = 100, and trimRight = 100.

Next, the taxonomy was assigned using the script Stream 1A using the parameters as set in the original script. The silva data base (silva_nr99_v138.1_train_set.fa.gz) was used as taxonomic database, and the output .csv files contained taxonomic information for the Nanopore sequences (Gldckner et al., 2017, J. Biotechnol., 261, P169-176; Quast et al., 2012, Nucl. Acids Res., 41 (D1), D590-D596; Yilmaz et al., 2013, Nucl. Acids Res., 42(D1), D643-D648).

To summarize and visualize the data, the script Stream 1B was used and modified. The output .csv files Phylum_summary, Order_summary and Genus_summary from the two aquaculture sites were used to create bar plots showing the relative abundance of the most abundant orders, as well as phyla and genera.

Macrofauna using standard analysis to determine environmental status Sediment samples were collected according to ISO 5667-19:2004: Guidance on sampling of marine sediments using a 1000 cm³ Van Veen grab. The macrofauna was analyzed according to ISO 16665:2014. Water quality - Guidelines for quantitative sampling and sample processing of marine soft-bottom macro fauna.

The environmental status for the macrofauna was based on the nEQR values, which represent a summary of the environmental status.

Results

Figure 14 shows the bar plots depicting the relative abundance of the most abundant orders. Figures 14A and B show the order bar plots for fish farms 1 and 2, respectively. Figures 14C and D show the phyla and genera output for fish farm 1 (by way of example), respectively.

The PCoA plots with Bray-Curtis dissimilarity created from the Order_summary and Genus_summary files are shown in Figures 15A and B, respectively.

At the order level, excluding the control samples, the first PCo axis explained 85.10 % of the total variance, and the second explained 11.69 %. Overlap between the different fish farms and sites is shown in the figure. A PERMANOVA with Bray- Curtis dissimilarity and 1000 permutations revealed significant difference in terms of bacterial composition between the two sites (C1 and C2) (p = 0.000999, F-statistic = 155.57, R² = 70.85 %).

The results obtained were compared to known techniques to establish the environmental status of the site by sampling marine sediments. As noted in the material and methods section, the environmental status for the macrofauna was based on the nEQR values, which represent a summary of the environmental status. Values may range from 0 to 1. Low values indicate a poor environmental status, while high values indicate a good state. For fish farm 1, the values for CI and C2 were 0.46 and 0.88, respectively. The corresponding values for farm 2 were 0.35 and 0.90. The variance between these values for the different locations on each fish farm reflected the variance observed at the different sites in the PCo1 axis thus reflecting the utility of the method in assessing environmental status. This is shown in Figure 16 in which boxplots for the PCo1 axis of the order rank (as shown in Figure 15A) are shown in A) and the boxplots generated for the same locations using the standard environmental test are shown in B). Conclusions

Nanopore sequencing was used to clearly distinguish the microbiota at the C1 and C2 sites. At the genus level, a strong association of Sulfurovum was observed with C1 and Litorilituus with C2. We have previously identified Sulfurovum as being closely linked to sediment samples near aquaculture sites, while Litorilituus remains less well-understood.

Sulfurovum is likely favored in sulfide-rich environments, but the criteria for selecting Litorilituus are still unclear.

There was a strong association between the macrofauna indexes and the score for PCo1. The method may therefore be used for analysis of environmental conditions on the seafloor connected to aquaculture, as an alternative to traditional macrofauna analyses.

Claims

Claims:

1. A method for the selective capture and/or growth of one or more microorganisms at a test location or in a liquid sample, comprising: a) placing a solid support in a liquid at said test location or in said sample, wherein the solid support comprises a growth medium which is selective for said one or more microorganisms, b) allowing the one or more microorganisms to grow on said solid support; c) removing said solid support from the test location or sample.

2. The method as claimed in claim 1 , wherein the solid support is enclosed by one or more porous containers which allow liquid and microorganisms to access the solid support, wherein at least one porous container excludes entities that are 5 mm or larger, wherein preferably at least one porous container has a pore size of less than 1 mm, preferably less than 10 pm or less than 1 pm.

3. The method as claimed in claim 1 or 2, wherein the solid support is a particle, preferably a magnetic particle.

4. The method as claimed in any one of claims 1 to 3, wherein said solid support comprises a portion which contains the selective growth medium.

5. The method as claimed in claim 4, wherein said portion containing the selective growth medium is readily removable from the remainder of the solid support.

6. The method as claimed in claim 5, wherein said portion comprises agarose or alginate and/or said portion is removable by heating or dissolution.

7. The method as claimed in any one of claims 1 to 6, wherein said test location is an aquatic environment (e.g. around oil/gas sites of production), a food production area or a medical facility.

8. The method as claimed in any one of claims 1 to 6, wherein said sample is wastewater.

9. The method as claimed in any one of claims 1 to 8, wherein each of said one or more microorganisms is selected from a bacterium, archaeon, protozoan, fungus and alga and said growth medium is selective for said microorganisms, wherein preferably a) said bacterium is selected from the list comprising antibiotic resistant bacteria, sulfur oxidizing bacteria, hydrogen sulfide producing bacteria, denitrifying bacteria, methanogenic bacteria and pathogenic bacteria (e.g. selected from Legionella, Staphylococcus aureus or Listeria monocytogenes and Vibrio salmonicida), or b) said archaeon is selected from the list comprising sulfur oxidizing archaea, hydrogen sulfide producing archaea, denitrifying archaea and methanogenic archaea.

10. The method as claimed in any one of claims 1 to 9, wherein said solid support additionally comprises at least one means for detecting the presence and/or determining the amount of said one or more microorganisms, or a target molecule from said one or more microorganisms, or a part of said means for detection.

11. The method as claimed in claim 10, wherein one of said means for detection is an affinity binding surface for said one or more microorganisms, or a target molecule from said one or more microorganisms, preferably an affinity binding surface for DNA, RNA or a protein from said one or more microorganisms.

12. The method as claimed in claim 10, wherein one of said means for detection is a molecule that is capable of interacting directly or indirectly with said one or more microorganisms, or a target molecule from said one or more microorganisms, to allow the generation of a detectable signal.

13. The method as claimed in claim 12, wherein said molecule is an antibody or other specific binding molecule, or an enzyme which preferably acts on a substrate from said one or more microorganisms.

14. The method as claimed in any one of claims 1 to 13, wherein said solid support, and/or said porous container when present, contains a test location or sample assessment component which allows assessment of the test location or sample.

15. The method as claimed in any one of claims 1 to 14, wherein prior to capture and/or growth of said one or more microorganisms the test location or sample is tested, wherein preferably said testing uses the test location or sample assessment component as defined in claim 14.

16. The method as claimed in claim 14 or 15, wherein the levels of hydrogen peroxide, hydrogen sulfide, ammonium, metals, nitrite or nitrate are tested.

17. A method of detecting the presence and/or determining the amount of one or more microorganisms, or a target molecule from said one or more microorganisms, at a test location or sample by a method comprising: a) selectively capturing and/or growing one or more microorganisms at a test location or in a sample by a method as claimed in any one of claims 1 to 16, and b) analysing the one or more microorganisms present on the removed solid support to determine whether said one or more microorganisms, or a target molecule from said one or more microorganisms, is present in the test location or sample and/or the amount of said one or more microorganisms or target molecule from said one or more microorganisms in the test location or sample.

18. The method as claimed in claim 17, wherein said analysis in step b) comprises the step of lysing said one or more microorganisms.

19. The method as claimed in claim 17 or 18, wherein the presence and/or amount of said one or more microorganisms is determined in said analysis step b) by identifying the DNA or RNA of said microorganisms, preferably by qPCR or sequencing and/or wherein the target polynucleotide sequence in the DNA or RNA is the 16S rRNA gene, an antibiotic resistance gene or the 18S rRNA gene.

20. The method as claimed in claim 19, wherein the DNA or RNA is collected and/or purified before said determination.

21. The method as claimed in claim 17 or 18, wherein the presence and/or amount of said one or more microorganisms is determined in said analysis step b) by identifying a protein of said microorganisms.

22. The method as claimed in any one of claims 17 to 21, wherein at least one means for detecting the presence and/or determining the amount of said one or more microorganisms, or a target molecule from said one or more microorganisms, or a part of said means for detection, is used in the analysis step b) and preferably said solid support additionally comprises said means or part thereof.

23. The method as claimed in claim 22, wherein one of said means for detection is an affinity binding surface for said one or more microorganisms, or a target molecule from said one or more microorganisms, preferably an affinity binding surface for DNA, RNA or a protein from said one or more microorganisms, and said step of analysis comprises releasing said microorganisms from said solid support and/or lysing said microorganisms, and binding said one or more microorganisms or target molecules therefrom to said means for detection or part thereof.

24. The method as claimed in claim 22, wherein one of said means for detection is a molecule that is capable of interacting directly or indirectly with said one or more microorganisms, or a target molecule from said one or more microorganisms, to allow the generation of a detectable signal, and said step of analysis comprises releasing said microorganisms from said solid support and/or lysing said microorganisms, and binding said one or more microorganisms or target molecules therefrom to said means for detection or part thereof thereby generating a detectable signal.

25. The method as claimed in claim 24, wherein said signal is a change in colour or the generation of light.

26. The method as claimed in claim 24 or 25, wherein the molecule which is one of said means of detection is a) an enzyme, which preferably interacts with a substrate from said one or more microorganisms, preferably to produce a product which directly or indirectly produces a detectable signal, preferably a colour change or b) an antibody or other specific binding molecule, which directly or indirectly produces a detectable signal, preferably a colour change.

27. The method as claimed in any one of claims 17 to 26, wherein said solid support comprises a portion which contains the selective growth medium and said portion is removed from the remainder of the solid support in step b), preferably by heating, preferably as the first step in said analysis.

28. The method as claimed in any one of claims 17 to 27, wherein the presence and/or amount of said one or more microorganisms is used to generate a quantitative or qualitative value indicative of the status of the test location or sample.

29. A product for selective capture and/or growth and optionally detection of one or more microorganisms in a liquid at a test location or in a liquid sample, said product comprising: a) a solid support comprising a growth medium which is selective for said one or more microorganisms; b) one or more porous containers enclosing said solid support, wherein said one or more containers allow liquid and microorganisms to access the solid support and at least one porous container excludes entities that are 5 mm or larger, wherein preferably at least one porous container has a pore size of less than 1 mm, preferably less than 10 pm or less than 1 pm.

30. The product as claimed in claim 29, wherein the solid support is as defined in any one of claims 3-6 or 9-14 and/or said one or more microorganisms are as defined in claim 9 and/or said one or more containers are as defined in claim 14.

31. A product for selective capture and/or growth and optionally detection of one or more microorganisms in a liquid at a test location or in a liquid sample, said product comprising a solid support comprising a particle and a portion which is a removable coating which contains a growth medium which is selective for said one or more microorganisms and said particle has an affinity binding surface for DNA or RNA from said one or more microorganisms.

32. The product as claimed in claim 31 , wherein the solid support is as defined in any one of claims 3-6 or 9-14 and/or wherein the solid support is enclosed by one or more porous containers which allow liquid and microorganisms to access the solid support, wherein at least one porous container excludes entities that are 5 mm or larger, preferably as defined in claim 2 or 14 and/or said one or more microorganisms are as defined in claim 9.