The present invention relates to a method of manufacturing a microbial starter culture, the microbial starter culture itself, and a method of microbial inoculating an apparatus for treating raw water by use of the microbial starter culture.

Background of the invention

Raw water, such as groundwater typical undergoes a two-step treatment comprising aeration and passage through granular media filters to remove e.g. suspended matter, iron, manganese, ammonium and nitrite before distribution as drinking water. Purification of groundwater for ammonium is traditionally performed either with biological oxidation to nitrite followed to nitrate by natural microorganisms (nitrification) or by chemical oxidation to free nitrogen with chlorine.

The granular media filters become biologically active during the commissioning time by colonisation with indigenous microorganisms from the source water. However, typically it takes several months for the natural microbial processes to be efficient during commissioning of new filters. The alternative chemical purification with chlorine works immediately after the start, but leads to a large and unwanted continuous chemical consumption, as approximately ten times the amount of chlorine is needed relative to the ammonium amount.

A main problem with new granular media filters for purifying groundwater by e.g. nitrification is the unpredictable manner by which the required microbial processes naturally is starting up. The typical timeframe to establish an effective microbial process in new drinking water filter materials is two to three months. During this period, the produced drinking water is discharged to sewer or recipient and the raw water, such as abstracted groundwater, used energy and manpower are wasted. Prior art document JP H09 22596 discloses method of manufacturing a nitrification filter material for oxidising ammonia in waste water to nitrate. To shorten the time required water is circulated through a filter. By this method robust and slow-growing bacteria will out- compete new and fast-growing bacteria. Such slow-growing bacteria are undesirable when starting up new granular media filters. Description of the invention

It is an object of embodiments of the invention to provide a method of manufacturing a microbial starter culture.

It is a further object of embodiments of the invention to provide a microbial starter culture for fast and predictable initiation of the microbial process in new granular media filters.

It is an even further object of embodiments of the invention to provide a microbial starter culture comprising fast-growing microorganisms.

According to a first aspect, the invention provides a method of manufacturing a microbial starter culture for a water treatment filter, the method comprising the step of: - filling a plurality of first containers with water, a filter material, and nitrifying

microorganisms and/or Mn-oxidising microorganisms;

- adding a mineral medium to the first containers;

- filling a plurality of second containers with a filter material and water;

- adding a mineral medium to the second containers; and - executing a selection process which comprises a step of defining a growth rate for each first container, the growth rate expressing the growth of microorganisms taking place in the first containers, and a step of defining a first group of microorganisms such that the

microorganisms in the first group have a higher growth rate than microorganisms not being in the first group, and transferring the microorganisms from the first group to the second containers by transferring a part of the first group of microorganisms to each of the second containers.

In one embodiment, the invention provides a method of manufacturing a microbial starter culture for a water treatment filter, the method comprising the step of:

- filling a plurality of first containers with water, a filter material, and nitrifying

microorganisms;

- adding a mineral medium to the first containers; - filling a plurality of second containers with a filter material and water;

- adding a mineral medium to the second containers; and

- executing a selection process which comprises a step of defining a nitrification rate for each first container, the nitrification rate expressing the nitrification taking place in the first containers by the microorganisms, and a step of defining a first group of microorganisms such that the microorganisms in the first group have a higher nitrification rate than microorganisms not being in the first group, and transferring the microorganisms from the first group to the second containers by transferring a part of the first group of

microorganisms to each of the second containers. The method comprises a step of filling a plurality of first containers with water, a filter material, and nitrifying microorganisms and/or Mn-oxidising microorganisms. When adding nitrifying and/or Mn-oxidising microorganisms to the first containers, the nitrifying and/or Mn-oxidising microorganisms may be added together with a filter material.

In the context of the present invention, the term "filling a plurality of containers" should be understood as process of adding a substance and/or an element and/or a liquid and/or others, to the plurality of containers. The containers need not be fully filled after the process.

To facilitate execution of the selection process, the plurality of first containers may be equally filled. This may also be the case for the subsequent filling of the second containers and third containers. The containers may have a volume in the range of 1-5000 litres, such as 5-100 litres. It should however be understood that containers of another size may also be used, as the size as an example may depend on the amount of microbial starter culture to be manufactured. To facilitate manufacturing of the microbial starter culture, the plurality of first containers may be identical, or at least of the same volume. In one embodiment, the plurality of first container may be 4, such as 6, such as 10, such as 12, or even more. It should however be understood that another number of containers may also be used.

The nitrifying microorganisms and/or the Mn-oxidising microorganisms may be extracted from a filter in an apparatus for treating raw water, which apparatus may be in use at a water treatment plant. The nitrifying microorganisms and/or the Mn-oxidising

microorganisms may be extracted together with a part of the filter material. The nitrifying microorganisms and/or the Mn-oxidising microorganisms may be in a biofilm which may be built up on the outer surface of the particles of the filter material, i.e. a biofilm comprising microorganisms which can remove ammonium by nitrification and/or comprising microorganisms which can remove Mn by oxidisation. The biofilm may comprise a layer of iron hydroxides in which the biomass/microorganisms are situated. This layer may also comprise other inorganic constituents, such as Mn, Ca, and Mg. Thus, the nitrifying microorganisms and/or the Mn-oxidising microorganisms may be added to the first containers as a biofilm on the filter material.

The water may be raw water which has to be treated before being used as drinking water to thereby expose the nitrifying microorganisms and/or the Mn-oxidising microorganisms to conditions similar to the condition in a filter in an apparatus for treating raw water.

In the context of the present invention "raw water" should be understood as water to be treated to be used as drinking and/or process water, i.e. water having a sufficiently high quality to be used as drinking water and/or used as process water in processing plants. In the context of the present invention "microbial nitrification" should be understood as processes in which ammonium is oxidised to different oxidation products, such as nitrate. In the context of the present invention "Mn oxidisation" should be understood as processes in which Mn is oxidised to different oxidation products, such as manganese dioxide (Mn0 ₂ ).

A mineral medium is added to the first containers. The mineral medium may be selected so that the growth of the nitrifying microorganisms and/or the Mn-oxidising microorganisms is enhanced.

The method also comprises a step of filling a plurality of second containers with a filter material and water. The plurality of second containers may be filed with a filter material which does not comprise nitrifying microorganisms or does not comprise Mn-oxidising microorganisms. The water may be raw water.

The number of second containers may be identical to the number of first containers. It should however be understood, that the number of second containers may also be lower or higher. To facilitate manufacturing of the microbial starter culture, the plurality of second containers may be identical, or at least of the same volume. It should be understood, that the size and/or volume of the second containers may be different from the first containers, as the first containers may be smaller or larger than the second containers. A mineral medium is added to the second containers. The mineral medium may be selected so that the growth of the nitrifying microorganisms and/or the Mn-oxidising microorganisms is enhanced.

The method also comprises a step of executing a selection process which comprises a step of defining a growth rate for each first container, the growth rate expressing the growth of microorganisms taking place in the first containers by, and a step of defining a first group of microorganisms such that the microorganisms in the first group have a higher growth rate than microorganisms not being in the first group. After defining the first group of

microorganisms, the first group of microorganisms is transferred to the second containers by transferring a part of the first group of microorganisms to each of the second containers. To be able to subsequently compare the second containers, the part transferred to each of the second containers may be of the same size and/or weight.

In one embodiment, the growth rate may be defined as at least one of a nitrification rate and a Mn-oxidisation rate, the nitrification rate expressing the nitrification taking place in the first containers by the nitrifying microorganisms and the Mn-oxidisation rate expressing the Mn- oxidisation taking place in the first containers by the Mn-oxidising microorganisms. Thus, the selection process comprises a step of defining a growth rate expressing the growth of microorganisms taking place in the first containers by. The step of defining the growth rate may comprise a step of determining as at least one of: a reduction in ammonium content in the first containers, a reduction of dissolved oxygen content in the first containers, an increase of nitrate and nitrite content in the first containers, a reduction of Mn content in the first containers, the number of nitrifying and/or Mn oxidising microorganisms in the first containers, genera of the microorganisms in the first containers, and the density of microorganisms in the first containers. In a specific embodiment, the growth rate may be determined as the nitrification rate which may be defined as the reduction of ammonium content in the containers.

By the selection process, it may be possible to select the one container of the first containers comprising the microorganisms with the highest growth rate; i.e. the first group of microorganisms and thus the microorganisms which may best be capable of reducing the ammonium content and/or the Mn content. Consequently, the microorganisms with the highest growth rate may be transferred to the plurality of second containers.

By selecting from a set of containers the one container comprising the microorganisms with the highest growth rate, it may be possible to select the most fast-growing microorganisms, as transfer of microorganisms to a new container/a new set of containers with new filter material will provide the microorganisms with more space to compete for, which competition may prioritise the fast-growing microorganisms.

It should be understood, that the selection process may result in selection of more than one container, as the first group of microorganisms having a higher growth rate than

microorganisms not being in the first group may be identified in more than one container. It should further be understood, that the transfer of microorganisms may include the transfer of at least a part of the filter material from the relevant container(s).

The growth rate may as an example be determined in one of the following ways:

1. Measurement of the ammonium (NH ₄ ⁺ ) content in the beginning and after a

predetermined time step (t); then determining the difference (i.e. the reduction in

NH ₄ ⁺ content) :

Δ N H ₄ ⁺ = N lVstart - N H ₄ ⁺ _t

The first group may be selected by selecting the container(s) with the highest Δ NH ₄ ⁺ , and a part of this first group will be transferred to each of the second containers.

By assuming that the start concentration N H ₄ ⁺ _S tart is substantially the same in all first containers, only NH ₄ ⁺ _t may be measured. And the container(s) of the plurality of first containers with the lowest NH ₄ ⁺ _t may be selected for transferring a part hereof to each of the second containers. In one embodiment, also the start concentration may be measured.

2. Nitrification is an oxidation reaction which consumes oxygen. Therefore, as a measure for the nitrification rate, the change in dissolved oxygen concentration may be used : Measurement of the dissolved oxygen (0 ₂ ) content in the beginning and after a predetermined time step (t); then determining the difference (i.e. the reduction in 0 ₂ content, as the removal of ammonium consumes oxygen) :

Δ 0 ₂ = 0 ₂ ,start - 0 ₂ ,t

The first group may be selected by selecting the container(s) with the highest Δ 0 ₂ and a part of this first group will be transferred to each of the second containers.

By assuming that the start concentration 0 _2iSt art is substantially the same in all first containers, only 0 _2it may be measured. And the container(s) of the plurality of first containers with the lowest 0 _2it may be selected for transferring a part hereof to each of the second containers. In one embodiment, also the start concentration may be measured .

Nitrification is the oxidation of ammonium to nitrite (N0 ₂ ^~ ) and (then further) to nitrate (N0 ₃ ^~ ) . The removal of ammonium may therefore also be expressed as the formation of N0 ₂ ^" + N0 ₃ ^~ . Nitrite and nitrate (N0 ₂ ^~ + N0 ₃ ^~ ) _s t _art may be measured at the beginning of a step and again (N0 ₂ ^~ + N0 ₃ ^~ ) _t may be measured at a given time (t) within the step. The difference may be calculated :

Δ (N0 ₂ ^" + N0 ₃ ^" ) = (N0 ₂ ^" + N0 ₃ ^" ) _t - (N0 ₂ ^" + N0 ₃ ^" ) _start

The first group may be selected by selecting the container(s) with the highest Δ (N0 ₂ ^~ + N0 ₃ ^" ) and a part of this first group will be transferred to each of the second containers.

By assuming that the start concentration (N0 ₂ ^" + N0 ₃ ^~ ) _start is substantially the same in all first containers, only (N0 ₂ ^" + N0 ₃ ^" ) _t may be measured . And the container(s) of the plurality of first containers with the highest (N0 ₂ ^" + N0 ₃ ^" ) _t may be selected for transferring a part hereof to each of the second containers. In one embodiment, also the start concentration may be measured.

Measurement of the manganese (Mn) content in the beginning and after a

predetermined time step (t) ; then determining the difference (i .e. the reduction in Mn content) :

Δ Mn = Mn _start - Mn _t

The first group may be selected by selecting the container(s) with the highest Δ Mn, and a part of this first group will be transferred to each of the second containers.

By assuming that the start concentration Mn _start is substantially the same in all first containers, only Mn _t may be measured . And the container(s) of the plurality of first containers with the lowest Mn _t may be selected for transferring a part hereof to each of the second containers. In one embodiment, also the start concentration may be measured .

In the above examples of determining NH ₄ ⁺ , 0 ₂ , N0 ₂ ^" , N0 ₃ ^" , and Mn, the concentration of the mentioned substances may be measured in the water phase of the containers. The determination of growth rate; e.g. the reduction of the content of ammonium/the nitrification rate and or the reduction of Mn is not limited to the above measurements and calculations. Each calculation and/or measured parameter may as an example be normalized by time and/or for example filter material volume and/or the number of microorganisms in the container. The microorganisms may contain microorganisms being attached to the filter material and/or microorganisms in the water phase. Thus, the determination of the growth rate may be based on measurements/assessments carried out/performed in the water phase and/or based on measurements/assessments carried out/performed at the filter material.

Normalizing by time would give a more specific removal rate, normalizing by the filter material volume would give a volumetric removal expression, and normalizing by the number of microorganisms within a certain group would give a cell-specific removal expression. It should further be understood, that the growth rate may additionally or alternatively be determined by other measures, such as real-time polymerase chain reaction (Real-time PCR), also known as quantitative polymerase chan reaction (qPCR) by which the number of nitrifying microorganisms and/or Mn-oxidising microorganisms may be determined.

Furthermore, the selection process of defining a first group of microorganisms and transferring said first group of microorganisms may be based on certain genera of the microorganisms and/or the density of microorganisms with the genera in question, such as genera Nitrosomonos and/or Nitrotoga which may be identified by DNA sequencing. Methods used for DNA sequencing may be one of the following, but not limited to the following methods; i.e. high-throughput sequencing such as 454 pyrosequencing, sequencing by synthesis (e.g. illumina), Nanopore sequencing, etc.

The manufactured microbial starter culture may be used during start-up of a water treatment filter. By use of a microbial starter culture manufactured according to the above described method, it may be possible to reduce commissioning time for a new granular filter in a water treatment plant down to e.g. two to four weeks.

Thus, water loss and consumption of chemicals and energy may be reduced considerably.

The microbial starter culture may constitute below 10 percent of the total granular filter material, such as below 6 percent, such as below 3 percent.

The start-up time may by the use of the microbial starter culture be reduced from about 2-3 months to below 1 month. The step of adding nitrifying microorganisms and/or Mn-oxidising microorganisms to the first containers may comprise a step of extracting the nitrifying microorganisms and/or Mn- oxidising microorganisms from a water treatment filter. This may ensure that microorganisms capable of nitrifying raw water and/or oxidising Mn are added. Furthermore, the

microorganisms may be added together with a filter material suitable for water treatment.

The method may further comprise a step of filling a plurality of third containers with a filter material and water. The plurality of third containers may be filed with a filter material which does not comprise nitrifying microorganisms or does not comprises Mn-oxidising

microorganisms. The water may be raw water. The number of third containers may be identical to the number of first and/or second containers. It should however be understood, that the number of third containers may also be lower or higher.

To facilitate manufacturing of the microbial starter culture, the plurality of third containers may be identical to each other, or at least of the same volume. It should be understood, that the size and/or volume of the third containers may be different from the first and/or second containers.

A mineral medium is added to the third containers. The mineral medium may be selected so that the growth of the nitrifying microorganisms and/or Mn-oxidising microorganisms is enhanced. The method may comprise a step of executing a selection process which comprises a step of defining a growth rate for each second container, the growth rate expressing the growth of microorganisms taking place in the second containers, and a step of defining a second group of microorganisms such that the microorganisms in the second group have a higher growth rate than microorganisms not being in the second group, and transferring the

microorganisms from the second group to the third containers by transferring a part of the second group of microorganisms to each of the third containers.

Thereby it may be possible to select the one container of the second containers comprising the group of microorganisms with the highest growth rate; i.e. the second group of microorganisms, and thus the microorganisms which may best be capable of reducing the ammonium content and/or the Mn content. Consequently, the microorganisms with the highest growth rate may be transferred to the plurality of third containers. To be able to subsequently compare the content of the third containers, the part of the second group of microorganisms transferred to each of the third containers may be of the same size and/or weight.

It should be understood, that the first group of microorganisms transferred to the plurality of second groups of containers and/or the second group of microorganisms transferred to the plurality of third group of containers may be transferred together with an amount of filter material. In one embodiment, the first and/or second group of microorganisms may be in a biofilm which it built up on the outer surface of the particles of the filter material.

By each time executing a selection process comprising a step of defining a growth rate, it may be possible to each time select the microorganisms with the highest growth rate and transfer these to the plurality of next set of containers; i.e. to select the container with the microorganisms with the highest growth rate and transfer these to the plurality of next set of containers. In one embodiment, the growth rate may be in the form of a nitrification rate.

In one embodiment the selection process may additionally comprise a step of determining the nitrite content in the containers.

The section process may be executed a plurality of times, and each time the microorganisms with the highest growth rate, such as nitrification rate may be selected for further cultivation; i.e. for further growth and subsequent selection. Thus, the steps of filling a plurality of containers, adding a mineral medium, and executing a selection process may be repeated a plurality of times, such as repeated at least three times, such as four times, such as five times.

The method may further comprise a step of adjusting the temperature in at least one of the containers to be in the range of 5 degrees to 15 degrees, such as in the range of 7 degrees to 12 degrees. This temperature may be selected to adjust the conditions of the at least one container to the conditions in a water treatment to thereby select the nitrifying

microorganisms and/or Mn-oxidising microorganisms based on conditions to which they will be exposed to in a treatment plant.

To be able to compare the growth rate in one container with the growth rate in another container of the first containers and subsequently be able to compare the growth rate in one container with the growth rate in another container of the second containers it may be an advantage if the temperature in each of the first containers is adjusted to the same temperature, and an advantage if the temperature in each of the second containers is adjusted to the same temperature, or at least that the temperature in some of the first and/or second containers are adjusted to the same value. It should be understood, that the temperature in the first containers may be different from the temperature in the second containers.

The method may further comprise a step of adjusting the pH value in at least one of the containers to be in the range of 5.5-9.5, such as in the range of 6.5-8.5. This pH value may be selected to adjust the conditions of the at least one container to the conditions in a water treatment to thereby select the nitrifying microorganisms and/or Mn-oxidising

microorganisms based on conditions to which they will be exposed to in a treatment plant.

To be able to compare the nitrification rate in one container with the growth rate in another container of the first containers and subsequently be able to compare the growth rate in one container with the growth rate in another container of the second containers it may be an advantage if the pH value in each of the first containers is adjusted to the same pH value, and an advantage if the pH value in each the second containers is adjusted to the same pH value, or at least that the pH value in some of the first and/or second containers are adjusted to the same value. It should however be understood, that the pH value in the first containers may be different from the pH value in the second containers.

As the nitrifying microorganisms may subsequently be used to remove ammonium in a water treatment plant, the mineral medium added to the first and/or second and/or third containers may comprise ammonia and nitrite. The mineral medium may comprise nitrite to avoid a Nitrite peak in the containers, as test has revealed that a Nitrite peak may occur in the containers if Nitrite is not added.

The mineral medium may further comprise manganese(II). This may be an advantage as supply of manganese may facilitate removal of manganese from the raw water.

As the Mn-oxidising microorganisms may subsequently be used to remove Mn in a water treatment plant, the mineral medium added to the first and/or second and/or third containers may comprise manganese.

The step of adding a filter material may comprise a step of selecting a filter material comprising particles having an effective size in the range of 0.4-6 millimetres. The particles may be quartz sand, anthracite coal, limestone, expanded clay, activated carbon, activated carbon, calcium carbonate, or other similar particles of natural or human made origin.

To avoid clogging of a filter in a water treatment plant e.g. due to biofilm growth and accumulation of different impurities, and thus avoid decreased efficiency, such a filter may comprise a backwash structure for reversing the flow direction of the filter thereby enabling that water is forced through the filter in a direction opposite to the flow direction from the inlet to the outlet.

To select nitrifying microorganisms and/or Mn-oxidising microorganisms with the ability of being kept at the filter material, the method may further comprise a step of moving the filter material in at least one of the containers after adding the nitrifying microorganisms and/or Mn-oxidising microorganisms, and wherein the selection process is further based on the ability of keeping the microorganisms at the filter material during the movement.

In the context of the present invention, the term "moving the filter material" should be understood as covering both a step of vibrating the at least one container to thereby move the filter material. Moving of the filter material may alternatively or additionally be achieved by stirring in the at least one container, or by tilting the at least one container back and forth. Other ways to achieve moving the filter material may also be applied.

To be able to compare the growth rate in one container with the growth rate in another container of the first containers and subsequently be able to compare the growth rate in one container with the growth rate in another container of the second containers it may be an advantage if the filter material in each of the first and/or second and/third containers is moved, or at least that the filter material in some of the first and/or second containers is moved. In order to assure that the nitrifying microorganisms and/or Mn-oxidising microorganisms are only exposed to typical water treatment plant conditions, the method may further comprise a step of sterilising the filter material before filling at least one of the second, third or subsequent containers with the filter material.

According to a second aspect, the invention provides a microbial starter culture for a water treatment filter, the starter culture being manufactured by a method according to the first aspect of the invention, and comprising a filter material with particles having an effective size in the range of 0.4-6 millimetres and a group of nitrifying microorganisms comprising at least 5 percent Betaproteobacteria, such as Nitrosomonas and Candidatus Nitrotoga.

As, the microbial starter culture is manufactured by the method according to the first aspect of the invention, it should be understood, that a skilled person would readily recognise that any feature described in combination with the first aspect of the invention could also be combined with the second aspect of the invention, and vice versa. The method according to the first aspect of the invention is very suitable for providing a microbial starter culture according to the second aspect of the invention. The remarks set forth above in relation to the method are therefore equally applicable in relation to the microbial starter culture.

According to a third aspect, the invention provides a method of microbial inoculating an apparatus for treating raw water by microbial nitrification and/or Mn oxidisation; the method comprising the steps of;

- providing a filter in a fluid flow path from an inlet to an outlet, the filter comprising a porous filter material and biomass;

- providing a fluid flow of raw water in the flow path through the filter;

- manufacturing a microbial starter culture by the method of the first aspect of the invention; and

- adding the microbial starter culture to the filter.

It should be understood, that a skilled person would readily recognise that any feature described in combination with the first and second aspects of the invention could also be combined with the third aspect of the invention, and vice versa.

The method according to the first aspect of the invention is very suitable for providing a microbial starter culture according to the second aspect of the invention. And the microbial starter culture according to the second aspect of the invention is very suitable for the method according to the third aspect of the invention. The remarks set forth above in relation to the method and the microbial starter culture are therefore equally applicable in relation to the method of microbial inoculating.

The microbial starter culture may constitute below 10 percent of the total granular filter material, such as below 6 percent, such as below 3 percent.

The start-up time may by the use of the microbial starter culture be reduced from about 2-3 months to below 1 month. Brief description of the drawings

Embodiments of the invention will now be further described with reference to the drawings, in which:

Fig. 1 illustrates an embodiment of a method of manufacturing a microbial starter culture, Fig. 2 illustrates steps of the manufacturing method, Fig. 3 illustrates laboratory-scale tests, Fig. 4 schematically illustrates pilot tests, Figs. 5 and 6A-6C illustrate test results,

Fig. 7 illustrates the content of an embodiment of a mineral medium, Fig. 8A illustrates ammonium oxidation during manufacturing, Fig 8B illustrates nitrite oxidation during manufacturing,

Fig.9 illustrates a comparison between the composition of a filter of a water treatment plant with the composition of a manufactured microbial starter culture, and

Fig. 10 illustrates the development of bacterial cultures in containers from pilot tests. Detailed description of the drawings

It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Fig. 1 illustrates an embodiment of a method of manufacturing a microbial starter culture. A first group of microorganisms from an existing water treatment plant (left container) that has a broad diversity (several colours) as a start is transferred to a new filter material and new mineral media several times, resulting in a reduction of diversity (fewer colours), but increased part of the wanted/selected microorganisms. The more transfers the more selected Figs. 5 and 6A-6C illustrate test results.

Fig. 5 illustrates the results from the pilot sand filter test illustrated in Fig. 4 and described above. Two columns were inoculated with 3% of the "biosand" and two columns were left un- inoculated. Ammonium, nitrite and nitrate were measured in the outlet of the columns. Figs. 6A-6C illustrates three nitrogen species were measured in different depths of the columns showing a successful colonization of the "biosand" throughout the filter columns at days 17, 28 and 56.

Fig. 7 illustrates the content of an embodiment of a mineral medium; i.e. an ammonium- containing mineral media used for enrichment of nitrifying microorganisms and the production of "biosand". Optimized ammonium (1 mM) mineral media were supplied with different initial concentrations of nitrite (0, 0.3, 0.6, 1.0 mM).

Fig. 8A illustrates ammonium oxidation during manufacturing. The graphs illustrate the effect of producing a microbial starter culture in optimized ammonium (1 mM) mineral media with an initial supply on nitrite (0, 0.3, 0.6, 1.0 mM) aimed at reducing the nitrite peak during production.

Fig 8B illustrates nitrite oxidation during manufacturing. The graphs illustrate nitrite oxidation during manufacturing of a microbial starter culture.

Fig.9 illustrates a comparison between the composition of a filter of a water treatment plant with the composition of a manufactured microbial starter culture. Analysis of the bacterial composition of the lab (A) and pilot (B) columns by DNA amplicon sequencing. The composition of the filter of a water treatment plant ("Org") shows a clear dominance of Nitrospira-type bacteria, known as Comammox-bacteria (capable of complete nitrification, slow growing, high cell yield). Columns with the two enrichment cultures developed from this source (Cl_Top (B2B-31), C4_Top (B2B-33)) had a clear different composition than the starting material, and were dominated by Betaproteobacteria in various degrees in the top, middle and bottom of the inoculated lab columns.

Fig. 10 illustrates the development of bacterial cultures in containers from pilot tests and illustrates dominance of Betaproteobacteria in the beginning of the sand filter operation - and then a gradually shift towards Alphaproteobacteria and Nitrospira-type bacteria. Embodiments

The invention may e.g. be covered by the following embodiments:

Embodiment 1. A method of manufacturing a microbial starter culture for a water treatment filter, the method comprising the step of: - filling a plurality of first containers with water and a filter material comprising nitrifying microorganisms;

- adding a mineral medium to the first containers;

- filling a plurality of second containers with a filter material and water;

- adding a mineral medium to the second containers; and - executing a selection process which defines a first group of microorganisms in the first containers, and transferring the first group of microorganisms to the second containers by transferring a part of the first group of microorganisms to each of the second containers; wherein the selection process comprises a step of defining a nitrification rate expressing the nitrification taking place in the first containers by the microorganisms. Embodiment 2. A method according to embodiment 1, wherein the step of adding nitrifying microorganisms to the first containers comprises a step of extracting the nitrifying microorganisms from a water treatment filter.

Embodiment 3. A method according to embodiment 1 or 2, further comprising the steps of:

- filling a plurality of third containers with a filter material and water; - adding a mineral medium to the third containers; and

- executing a selection process which defines a second group of microorganisms in the second containers, and transferring the second group to the third containers by transferring a part of the second group to each of the third containers. Embodiment 4. A method according to any of the preceding embodiments, wherein the steps of filling a plurality of containers, adding a mineral medium, and executing a selection process are repeated at least three times.

Embodiment 5. A method according to any of the preceding embodiments, further comprising a step of adjusting the temperature in at least one of the containers to be in the range of 5 degrees to 15 degrees.

Embodiment 6. A method according to any of the preceding embodiments, further comprising a step of adjusting the pH value in at least one of the containers to be in the range of 5.5- 9.5.

Embodiment 7. A method according to any of the preceding embodiments, wherein the mineral medium comprises ammonia and nitrite.

Embodiment 8. A method according to any of the preceding embodiments, wherein the mineral medium comprises manganese(II).

Embodiment 9. A method according to any of the preceding embodiments, wherein the step of adding a filter material comprises a step of selecting a filter material comprising particles having an effective size in the range of 0.4-6 millimetres.

Embodiment 10. A method according to any of the preceding embodiments, further comprising a step of moving the filter material in at least one of the containers after adding the nitrifying microorganisms, and wherein the selection process is further based on the ability of keeping the microorganisms at the filter material during the movement.

Embodiment 11. A method according to any of the preceding embodiments, further comprising a step of sterilising the filter material before filling at least one of the second, third or subsequent containers.

Embodiment 12. A microbial starter culture for a water treatment filter, the starter culture comprising a filter material with particles having an effective size in the range of 0.4-6 millimetres and a group of nitrifying microorganisms comprising at least 5 percent

Betaproteobactera, such as Nitrosomonas and Candidatus Nitrotoga.

Embodiment 13. A starter culture according to embodiment 12, wherein the starter culture is manufactured by a method according to any of embodiments 1-11. Embodiment 14. A method of microbial inoculating an apparatus for treating raw water by microbial nitrification; the method comprising the steps of;

- providing a filter in a fluid flow path from an inlet to an outlet, the filter comprising a porous filter material and biomass; - providing a fluid flow of raw water in the flow path through the filter; and

- adding a microbial starter culture to the filter, wherein the microbial starter culture is manufactured by a method according to any of embodiments 1-11.