NL2028485B1 - Photogranule - Google Patents

Abstract

The invention provides a method for providing a photogranule (10) for nutrient recovery, wherein: the method comprises subjecting a plurality of micro-organisms (20) in a sequencing batch reactor (110) to one or more cultivation cycles (200) to provide the photogranule (10), wherein the sequencing batch reactor (110) has a reactor height H, the plurality of micro-organisms (20) comprise a filamentous cyanobacterium, a polyphosphate- 10 accumulating organism, and an alga, the one or more cultivation cycles (200) comprise a feeding stage (210), a cultivation stage (220), a settling stage (230), and an effluent removal stage (240), the feeding stage (210) comprises providing a growth medium (30) to the plurality of micro-organisms (20), the cultivation stage (220) comprises providing alternating dark phases (221) and light phases (222), Wherein the plurality of microorganisms (20) are exposed 15 to a first average photosynthetic photon flux density P1 during the dark phases (221) and to a second average photosynthetic photon flux density P2 during the light phases (222), wherein P1/P2 5 0.05, wherein the cultivation stage (220) comprises imposing anaerobic conditions on the growth medium (30) during the dark phases (221), and Wherein the cultivation stage (220) comprises passing a gas (40) through the growth medium (30) during the light phases (222), 20 wherein the gas (40) comprises C02, wherein the settling stage (230) has a settling duration T2 selected from the range of 0.01 — 0.05 min/cm * H, and the effluent removal stage (240) comprises removing a remainder of the growth medium (30) and non-settled micro-organisms. Fig. 1A 25

Description

Photogranule

FIELD OF THE INVENTION The invention relates to a method for providing a photogranule. The method further relates to a photogranule. The invention further relates to a treatment method. The method further relates to a use of the photogranule.

BACKGROUND OF THE INVENTION Photogranules are known in the art. For instance, US2016318782 describes a granular or particulate composition of matter that includes algae and bacteria. The algal-sludge granules are generated by incubating a wastewater system with algae under specific quiescent conditions with illumination. The methods described include ab initio generation of the algal- sludge granules, use of the algal-sludge granules to remediate wastewater, and use of the algal- sludge granules to generate biomass.

SUMMARY OF THE INVENTION The global population may be growing at unprecedented rates and may be expected to reach 11.2 billion people in 2100. This growing population may lead to a larger requirement of various resources, including water and food, a large part of which may need to be recovered from waste(water).

However, waste(water) treatment may generally be an energy intensive process, despite many nutrients not being recovered from the waste(water). In particular, current technologies for removing organic carbon and nutrients from waste(water) may need mixing, such as via aeration, which may imply a high energy consumption. Further, current technologies for removing organic carbon and nutrients from waste(water) may produce a biomass (sludge) that is mostly not recycled. Hence, such processes may result in losses of carbon, macronutrients (N, P, K) and micro elements (Zn, Co, Cu, Fe, Mo, etc.).

For example, for phosphate recovery, a commonly applied technology may be struvite precipitation. Although struvite precipitation may be relatively efficient at P recovery, it may require the addition of Mg as well as a high pH (>9). Further, although struvite precipitation may be suitable for P recovery, it may not or barely recover other valuable nutrients, such as micro elements, which may, for instance, be valuable for soil enrichment.

Further, for prior art methods, harvesting of the microbial biomass, particularly of algal biomass, may be challenging and/or expensive.

Hence, current technologies may be wasteful with regards to energy use, may be inefficient with regards to nutrient recovery, may provide a biomass that is challenging or cumbersome to harvest, and may be limited in the removal of organic carbon.

Hence, it is an aspect of the invention to provide an alternative photogranule, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Hence, in a first aspect, the invention may provide a method for providing a photogranule, especially a photogranule for nutrient recovery, such as for recovery of phosphate, nitrogen and/or other elements. The method may comprise subjecting a plurality of micro-organisms in a sequencing batch reactor to one or more cultivation cycles, especially thereby providing the photogranule. The sequencing batch reactor may have a reactor height H (in cm). In embodiments, the plurality of micro-organisms may comprise a filamentous cyanobacterium, a polyphosphate-accumulating organism, and a (eukaryotic) alga. In embodiments, the one or more cultivation cycles may comprise a feeding stage, a cultivation stage, a settling stage, and/or an effluent removal stage. The feeding stage may comprise providing a (liquid) growth medium to the plurality of micro-organisms, especially to the sequencing batch reactor. The cultivation stage may comprise cultivating the plurality of microorganisms in the growth medium. In embodiments, the cultivation stage may comprise providing alternating dark phases and light phases, especially wherein the plurality of microorganisms is exposed to a first average photosynthetic photon flux density P1 during the dark phases and to a second average photosynthetic photon flux density P2 during the light phases, more especially wherein P1/P2 < 0.05. In further embodiments, the cultivation stage may comprise imposing anaerobic conditions on the growth medium during the dark phases. In further embodiments, the cultivation stage may comprise passing a gas through the growth medium during the light phases, especially wherein the gas comprises CO: and/or O2, such as especially at least CO. The settling stage may have a settling duration T2 selected from the range of 0.01 — 0.05 min/cm * H. The effluent removal stage, especially temporally arranged after the settling stage, may comprise removing a remainder of the growth medium and non- settled micro-organisms.

The method of the invention may provide a photogranule with beneficial properties, especially with regards to the treatment of waste(water). In particular, the photogranule of the invention may comprise a filamentous cyanobacterium, a polyphosphate- accumulating organism, and a (eukaryotic) alga, as well as optionally additional micro- organisms such as a nitrifier and a denitrifier. This photogranule may be suitable for removing organic carbon and removing and recovering various nutrients by incorporating the carbon and/or (other) nutrients into biomass. In particular, the photogranule may be suitable for recovering phosphate. Further, the photogranule may comprise both organisms consuming O2 and providing carbon dioxide, such as the polyphosphate-accumulating organism, as well as organisms consuming carbon dioxide and providing O2, such as the alga. Thereby, the photogranule may be self-sustaining with respect to an oxygen cycle, and may not rely on aeration with externally supplied O2 and/or CO, which may otherwise be relatively energy- intensive. Further, the photogranule may settle relatively quickly, which may facilitate a faster process as well as easy retrieval of the biomass (comprising the nutrients).

Hence, the invention may provide a method for creating/assembling a (micro)algae-bacteria photogranule that can remove pollutants such as organic carbon and nutrients (nitrogen, phosphate) from nutrient-rich waters (polluted waters). Further, the invention may provide a method for maintaining a microalgae-bacteria photogranule for removing and recovering a nutrient (as the N and P will be assimilated into the biomass) without external aeration needed (also see below). This may be achieved by kindling a symbiotic relation between O: producing microorganisms and CO: producing microorganisms using the method of the invention.

In particular, the photogranule of the invention may provide the benefits that (1) no or very little aeration may be needed for waste(water) treatment, (ii) a variety of elements, including C, P, and N, as well as micro-nutrients, may be recovered from the photogranule biomass, (iii) the biomass may be easily harvested due to the formation of a fast-settling granule, (iv) the system may be robust due to the species diversity, and (v) persistent pollutants may be removed due to a relatively large retention time in the photogranule.

In specific embodiments, the invention may provide a method for providing a photogranule for nutrient recovery, wherein: the method comprises subjecting a plurality of micro-organisms in a sequencing batch reactor to one or more cultivation cycles to provide the photogranule, wherein the sequencing batch reactor has a reactor height H; the plurality of micro-organisms comprise a filamentous cyanobacterium, a polyphosphate-accumulating organism, and an alga; the one or more cultivation cycles comprise a feeding stage, a cultivation stage, a settling stage, and an effluent removal stage; the feeding stage comprises providing a growth medium to the plurality of micro-organisms; the cultivation stage comprises providing alternating dark phases and light phases, wherein the plurality of microorganisms are exposed to a first average photosynthetic photon flux density P1 during the dark phases and to a second average photosynthetic photon flux density P2 during the light phases, wherein P1/P2 < 0.05, wherein the cultivation stage comprises imposing anaerobic conditions on the growth medium during the dark phases, and wherein the cultivation stage comprises passing a gas through the growth medium during the light phases, wherein the gas comprises COs; the settling stage has a settling duration T2 selected from the range of 0.01 — 0.05 min/cm * H; and the effluent removal stage comprises removing a remainder of the growth medium and non-settled micro- organisms.

Hence, the invention may provide a method for providing (or “assembling”) a photogranule. The term “photogranule” may herein especially refer to a biological aggregate, especially a granule, comprising a plurality of micro-organisms, especially of which at least one organism performs (oxygenic) photosynthesis. In embodiments, the plurality of micro- organisms may comprise at least a (motile) filamentous cyanobacterium, a polyphosphate- accumulating organism, and an alga. In embodiments, (at least part of) the plurality of micro- organisms may have a syntrophic relationship, especially wherein photosynthetic autotrophic micro-organisms provide Oz for heterotrophic micro-organisms, and wherein the heterotrophic micro-organisms provide CO; for the photosynthetic autotrophic micro-organisms.

In particular, (photo)granules may be discrete well-defined microbial aggregates formed by cell-to-cell attraction with regular dense and strong structure, and excellent settleability.

In embodiments, the photogranule may have an approximately ellipsoidal shape, especially approximately a spherical shape. The properties of a photogranule may depend on the conditions, especially the selective pressures, employed to drive granulation and to select for granule functions. In particular, the method of the invention may provide a photogranule for nutrient recovery, especially (specialized) for phosphate and/or nitrogen recovery, more especially for phosphate recovery. In particular, the photogranule may provide improved N removal and recovery properties compared to aerobic granular sludge, and may provide improved P removal and recovery, similar N recovery, and better N removal compared to prior art microalgae-based wastewater systems.

The method may comprise subjecting a plurality of micro-organisms in a sequencing batch reactor to one or more cultivation cycles, especially thereby providing the photogranule.

In embodiments, the one or more cultivation cycles may comprise at least 2 cultivation cycles, such as at least 4 cultivation cycles, especially at least 6 cultivation cycles. In further embodiments, the one or more cultivation cycles may comprise at most 20 cultivation cycles, especially at most 14 cultivation cycles, such as at most 8 cultivation cycles.

5 The term “sequencing batch reactor” may herein especially refer to a reactor suitable for waste(water) treatment. In particular, the sequencing batch reactor may have a time- based reactor configuration that operates in filling-reaction-settle-drain cycles to exert positive selection pressure towards self-settling biomass. The sequencing batch reactor may especially have a bottom inlet and an outlet. The bottom inlet may be configured to provide a fluid, especially a gas, or especially a liquid, to (a reactor space of) the sequencing batch reactor. The outlet may especially be configured for removing a fluid, such as left-over growth medium, optionally comprising micro-organisms, from (the reactor space of) the sequencing batch reactor. In further embodiments, the sequencing batch reactor may comprise a second inlet for providing a fluid, especially a liquid, such as a growth medium, to the (reactor space of the) sequencing batch reactor. In embodiments, at least part of the sequencing batch reactor may have a conical shape tapering towards the bottom inlet. In further embodiments, the sequencing batch reactor may have a flat bottom.

In embodiments, the sequencing batch reactor may have (a reactor space having) a reactor height H (in cm). In further embodiments, the reactor height H may especially be selected from the range of 0.2 — 8m, such as from the range of 0.4 — 4m, especially from the range of 0.5 — 3m.

In embodiments, the sequencing batch reactor may comprise a mixing element, especially a mechanical mixing element, such as a stirrer, configured to mix a fluid, especially a growth medium, in (the reactor volume of) the sequencing batch reactor.

In further embodiments, the sequencing batch reactor may be configured for mixing a fluid, especially a growth medium, in (the reactor volume of) the sequencing batch reactor via aeration. In particular, the bottom inlet may be configured for mixing the fluid in (the reactor volume of) the sequencing batch reactor via aeration, especially by providing a gas to (the reactor volume of) the sequencing batch reactor.

In embodiments, the plurality of micro-organisms may comprise a filamentous cyanobacterium, a polyphosphate-accumulating organism and a (eukaryotic) alga. As will be understood by the person skilled in the art, the method may not be limited to specific species or even genera of micro-organisms, but may rather relate to specific (ecosystem) functions provided for by (specific members of) the plurality of micro-organisms.

The filamentous cyanobacterium may provide a (filamentous) structure facilitating photogranule formation. In particular, the filamentous cyanobacterium may provide an interwoven (mat-like) structure, which may provide rigidity to the photogranule. Further, the filamentous cyanobacterium may provide (oxygenic) photosynthesis for the photogranule.

Hence, the filamentous cyanobacterium may provide a beneficial function for the syntrophic relation in the photogranule, especially by providing Oz and energy for other micro-organisms in the photogranule. In embodiments, the filamentous cyanobacterium may especially comprise a motile filamentous cyanobacterium. In further embodiments, the filamentous cyanobacterium may be selected from the group comprising the orders Synechococcales, Pseudanabaenales and Oscillatoriales. In further embodiments, the filamentous cyanobacterium may be selected from the group comprising the families Leptolyngbyacaea, Oscillatoriaceae, Phormidiaceae and Pseudanabaenaceae. In further embodiments, the filamentous cyanobacterium may be selected from the group comprising the genera Alkalinema, Cephalothrix, Leptolyngbya, Pseudanabaena, Linmothrix. In specific embodiments, the filamentous cyanobacterium may be selected from the group comprising Alkalinema pantanalense (CENA528), Leptolyngbya boryana (PCC-6306), Cephalothrix komarekiana (SAG 75.79), Pseudanabaena biceps (PCC 7429) and Limnothrix sp..

The polyphosphate-accumulating organism (PAO) may especially accumulate (relatively) large amounts of polyphosphate, which may facilitate recovery of phosphate from waste(water). In particular, the polyphosphate-accumulating organism may have the capability to consume simple carbon compounds without the presence of an external electron acceptor (such as nitrate or oxygen) by generating energy from internally stored polyphosphate and/or glycogen. In particular, under anaerobic conditions, a PAO may hydrolyze internally stored poly-P. The PAO may use energy released upon the hydrolysis to take up organic carbon, such as volatile fatty acids (VFAs), which may be converted to an energy-storage compound, such as a polyhydroxyalkanoate (PHA). Under aerobic conditions, the PAO may generate energy from the energy-storage compound for P uptake, and cellular growth. In particular, for the PAO, the uptake of P during the aerobic phase may be larger than its release during the anaerobic phase, which may result in a net P accumulation. Hence, the PAO may provide a phosphate- accumulating mechanism to the photogranule.

In embodiments, the PAO may be selected from the group comprising the orders Betaproteobacteria, Micrococcales, Rhodocyclales, Oceanospirillales, Corynebacteriales and Obscuribacteriales. In further embodiments, the PAO may be selected from the group comprising the families Rhodocyvclaceae, Intrasporangiaceae, Azonexacecae,

Halomonadaceae, Corvnebacteriaceae and Obscuribacteraceae. In further embodiment, the PAO may be selected from the group comprising Candidatus Accumulibacter, Tetrasphaera sp., Dechloromonas sp., Halomonas sp., Corynebacterium sp., and Candidatus Obscuribacter.

The alga may provide (oxygenic) photosynthesis for the photogranule. Hence, the alga may provide a beneficial function for the syntrophic relation in the photogranule, especially by providing O: and energy for other micro-organisms in the photogranule. Hence, in embodiments, the alga may be configured to perform oxygenic photosynthesis.

In embodiments, the alga may be selected from the group comprising the orders Chlorellales, Chlorococcales, Sphaeropleales. In further embodiments, the alga may be selected from the group comprising the families Chlorellaceae, Chlorococcaceae, Scenedesmaceae. In further embodiments, the alga may be selected from the group comprising the genera Chlorella, Chlorococcum, Desmodesmus and Botrvosphaerella. In further embodiments, the alga may be selected from the group comprising Chlorella sorokiniana, Chlorococcum vacuolatum, Desmodesmus sp., and Botryosphaerella sp. .

The term “filamentous cyanobacterium” may also refer to a plurality of (different) filamentous cyanobacteria, especially to two or more filamentous cyanobacteria belonging to different genera, such as belonging to different families. Similarly, the term “polyphosphate-accumulating organism” (or “alga”) may also refer to a plurality of (different) polyphosphate-accumulating organisms (or algae), especially to two or more polyphosphate- accumulating organisms (or algae) belonging to different genera, such as belonging to different families.

In embodiments, the plurality of micro-organisms may further comprise a nitrifier. The nitrifier may especially be configured to (or “capable of”) convert ammonium into nitrite and/or to convert nitrite into nitrate. In particular, in embodiments, the plurality of micro-organisms may further comprise a nitrifier configured to convert ammonium into nitrate. The term “nitrifier” may also refer to a plurality of (different) nitrifiers.

In further embodiments, the plurality of micro-organisms may further comprise a denitrifier. The denitrifier may especially be configured to (or “capable of’) converting nitrate ammonium into nitrite, and/or to convert nitrite into nitric oxide, and/or to convert nitric oxide into nitrous oxide, and/or to convert nitrous oxide into Na. In particular, in embodiments, the plurality of micro-organisms may further comprise a denitrifier configured to convert nitrate into Na. The term “denitrifier” may also refer to a plurality of (different) denitrifiers.

The presence of the nitrifier and the denitrifier in the photogranule may be particularly advantageous for the treatment of waste(water) with a high N:P ratio, such as for waste(water) with a N:P ratio of at least 10:1.

However, the presence of the nitrifier and the denitrifier in the photogranule may also be disadvantageous as the formation of N2 may result in a loss of nitrogen, i.e., the nitrogen converted to N: may generally not be recovered. Hence, in further embodiments, the plurality of micro-organisms may be devoid of a nitrifier and/or of a denitrifier.

In further embodiments, the nitrifier may be selected from the group comprising the orders Nitrosomonadales, Nitrospirales, Rhizobiales and Burkholderiales. In further embodiments, the nitrifier may be selected from the group comprising the families Nitrosomonadaceae, Nitrospiraceae, Bradyrhizobiaceae and Gallionellaceae. In further embodiments, the nitrifier may be selected from the group comprising the genera Nitrosomonas, Nitrospira, Candidatus Nitrotoga and Nitrobacter. In further embodiments, the nitrifier may be selected from the group comprising Nitrosomonas oligotropha, Nitrospira sp., Nitrobacter sp. and Candidatus Nitrotoga.

In further embodiments, the denitrifier may be selected from the group comprising the orders Rhodocyclales, Corynebacteriales, Rhodobacterales, Burkholderiales and Gammaproteobacteria. In further embodiments, the denitrifier may be selected from the group comprising the families Zoogloeaceae, Corynebacteriaceae, Rhodobacteraceae, Comamonadaceae, and Competibacteraceae. In further embodiments, the denitrifier may be selected from the group comprising the genera Thauera, Zoogloea, Corynebacterium, Paracoccus, Hvdrogenophaga, Candidatus Accumulibacter, Dechloromonas and Candidatus Contendobacter. In further embodiments, the denitrifier may be selected from the group comprising Thauera aminoaromatica, Zoogloea resiniphila, Zoogloea caeni, Zoogloea oleivorans, Corynebacterium marinum, Paracoccus homiensis, Hvdrogenophaga taeniospiralis, Candidatus Accumulibacter sp, Dechloromonas sp. and Candidatus Contendobacter sp. In further embodiments, the plurality of micro-organisms may comprise an extracellular polymeric substances producing organism (or: “EPS-producing organism”). The EPS-producing organism may be configured to produce extracellular polymeric substances (EPS), including extracellular polysaccharides and proteins. The formation/presence of EPS may benefit the formation and/or maintenance of the photogranule, as the EPS may provide functional and structural integrity. In embodiments, the plurality of micro-organisms may comprise a dedicated EPS-producing organism. However, generally, the (other) micro-

organisms may provide sufficient EPS. For instance, the cyanobacterium, the PAO and the alga may (together) produce sufficient EPS for granule assembly and maintenance.

In embodiments, the method may comprise providing (at least part of) the micro- organisms as free (or “non-aggregated”’) cells.

In further embodiments, the method may comprises providing (at least) part of the plurality of micro-organisms as a granule, i.e, (at least) part of the plurality of micro- organisms may be present as a pre-existing granule. The presence of a pre-existing granule may provide a springboard for development of the photogranule, i.e., other members of the plurality of micro-organisms may settle in/on the pre-existing granule, which may provide in a more rapid assembly of the photogranule.

As indicated above, the method may comprise subjecting the one or more micro- organisms to one or more cultivation cycles comprising a feeding stage, a cultivation stage, a settling stage, and/or an effluent removal stage.

In embodiments, the feeding stage may comprise providing a (liquid) growth medium to the plurality of micro-organisms, especially to the (reactor volume of the) sequencing batch reactor.

The growth medium may especially comprise nutrients suitable to support the growth of the plurality of micro-organisms. In particular, the growth medium may at least comprise a carbon source, a nitrogen source, and a phosphor source. It will be clear to the person skilled in the art how to provide a growth medium comprising suitable nutrients for the (selected) plurality of micro-organisms.

In embodiments, the growth medium may especially comprise wastewater or synthetic wastewater, especially wastewater, or especially synthetic wastewater. The term “synthetic wastewater” may refer to a growth medium selected to approximate the nutrient composition of (typical) wastewater, especially of wastewater the photogranule will be used to treat.

In further embodiments, the growth medium provided during the feeding stage may comprise < 50 ppm dissolved oxygen, such as < 10 ppm dissolved oxygen, especially <2 ppm dissolved oxygen. By providing little dissolved oxygen via the growth medium, a (larger) selective pressure may be imposed on (an alga and/or a cyanobacterium in) the photogranule to generate O2, which may thus provide a further selective pressure on establishing syntrophic relationships in the photogranule. In addition, little dissolved oxygen in the influent may also favor anaerobic carbon uptake by PAOs over carbon respiration with Oz. Hence, the limited dissolved oxygen may also increase the selective pressure for (inclusion of) PAOs.

In embodiments, the cultivation stage may comprises cultivating the plurality of microorganisms in the growth medium. Hence, the cultivation stage may comprise exposing the plurality of microorganisms to growth conditions suitable for the growth of the plurality of micro-organisms.

In further embodiments, the cultivation stage may comprise providing alternating dark phases and light phases. Alternating dark and light phases may simulate diurnal rhythms, which may provide a selective pressure on the photogranule to thrive both during day and night conditions. In further embodiments, the plurality of microorganisms may be exposed to a first average photosynthetic photon flux density P1 during the dark phases and to a second average photosynthetic photon flux density P2 during the light phases, especially wherein P1/P2 < 0.05, such as < 0.03, especially < 0.01. Essentially, the first average photosynthetic photon flux density P1 may essentially be insufficient for (substantial) photosynthesis, whereas the second average photosynthetic photon flux density P2 may facilitate photosynthesis. Hence, as (oxygenic) photosynthesis may provide O as a (by-)product, the O2 concentration may (at least partially) also vary as a function of the dark and light phases. In particular, in further embodiments, the cultivation stage may comprise imposing anaerobic conditions on the growth medium during the dark phases, especially by not providing external oxygen, such as by providing a fluid separation to external oxygen. Hence, during the dark phases the plurality of micro-organisms may essentially be exposed to anaerobic conditions, whereas during the light phases the plurality of micro-organisms may essentially be exposed to aerobic conditions. The alternating aerobic and anaerobic conditions may especially provide a selective advantage to the polyphosphate-accumulating organisms (see above), and may thereby promote their presence and growth in the photogranule.

In particular, in embodiments, each cultivation stage may comprise at least one light phase and at least 1 dark phase. In further embodiments, (each of) the cultivation stage(s) may comprise two or more light phases and/or two or more dark phases, wherein the light phases and the dark phases are configured to alternate.

In further embodiments, a dark phase may be temporally arranged (directly) after the feeding stage, and/or a light phase may be temporally arranged (directly) prior to the settling stage, optionally with one or more dark phases and one or more light phases temporally arranged in between. However, in further embodiments, a light phase may be temporally arranged (directly) after the feeding stage, and/or a dark phase may be temporally arranged (directly) prior to the settling stage. In further embodiments, the cultivation stage may comprise an identical number of dark phases and light phases. In yet further embodiments, the cultivation stage may comprise an unequal number of dark phases and light phases.

The term “average photosynthetic photon flux density” may herein refer to the photosynthetic photon flux density averaged over the duration of the respective (dark/light) phase.

In embodiments, the photosynthetic photon flux density may be an incidence area photosynthetic photon flux density. In such embodiments, the photosynthetic photon flux density may refer to the number of photons in pmol having a A in the range of 400 — 700 nm incident on 1 m? per second. The incidence area (in m?) may especially refer to a surface of the sequencing batch reactor.

In further embodiments, the first average photosynthetic photon flux density P1 may be selected from the range of < 100 umol/m?/s, such as < 50 umol/m?/s, especially < 10 umol/m?/s, such as < 1 umol/m?/s.

In further embodiments, the second average photosynthetic photon flux density P2 may be selected from the range of 150 — 1800 umol/m?/s, especially 250 — 1500 umol/m?s, such as 500 - 1000 pmol/m?/s.

In further embodiments, the photosynthetic photon flux density may be a biomass-related photosynthetic photon flux density. In such embodiments, the photosynthetic photon flux density may refer to the number of photons in mol having a A in the range of 400 — 700 nm as a function of grams of biomass per day (mol/gVSS/d). In further embodiments, the first average photosynthetic photon flux density P1 may be selected from the range of <0.1 mol/gVSS/d, such as < 0.05 mol/gVSS/d, especially < 0.02 mol/gVSS/d. In further embodiments, the first average photosynthetic photon flux density Pl may be selected from the range of < 0.01 mol/gVSS/d, such as < 0.005 mol/gVSS/d, especially < 0.002 mol/gVSS/d.

In further embodiments, the second average photosynthetic photon flux density P2 may be selected from the range of 0.02 — 15 mol/gVSS/d, such as 0.03 — 13 mol/gVSS/d, especially 0.05 — 10 mol/gVSS/d. In further embodiments, the second average photosynthetic photon flux density P2 may be selected from the range of 0.07 — 8 mol/gVSS/d, especially from the range of 0.09 - 5.4 mol/gVSS/d.

In embodiments, the cultivation stage may comprise exposing the plurality of micro-organisms to (natural) sunlight during the light phases.

In further embodiments, the cultivation stage may comprise exposing the plurality of micro-organisms to (artificial) light source light during the light phases. For instance, the cultivation stage may comprise providing light source light using a light source, such as a solid state light source, especially a light-emitting-diode.

In further embodiments, the cultivation stage may comprise — during the light phases — supplementing (natural) sunlight with artificial light to provide the second average photosynthetic photon flux density.

In further embodiments, the cultivation stage may comprise mixing the growth medium, especially during the light phases, such as with aeration, or such as with mechanical mixing.

In further embodiments, the cultivation stage may comprise passing a gas through the growth medium during the light phases, especially wherein the gas comprises CO: and/or Oz, especially CO:. In particular, the cultivation stage may comprise providing the gas at a bottom inlet of the sequencing batch reactor, which may provide an upwards air flow in the sequencing batch reactor, which may result in flotation of the plurality of micro-organisms, especially of the photogranule.

In further embodiments, the gas may comprise 0.01 — 20 vol.% CO, such as

0.03 — 5 vol.% COs, especially 0.5-4 vol.% CO: In further embodiments, the gas may comprise at least 0.5 vol.% CO:, such as at least 1 vol.%, especially at least 2 vol.%. In further embodiments, the gas may comprise at most 20 vol.% CO, such as at most 12 vol %, especially at most 5 vol.%.

In further embodiments, the gas may comprise 0 - 35 vol.% Os, such as 0-21 vol .% Oa, especially 1-15 vol.% O2. Especially, the gas may (essentially) be devoid of O2, such as comprise < 0.1 vol% Oa.

As indicated above, the gas may primarily be provided during light phases of the cultivation stage. In particular, in general, (essentially) no gas is provided during the dark (anaerobic) phases of the cultivation stage. However, if there is a dark phase temporally arranged (directly) after a feeding stage, it may be beneficial to mix contents of the reactor, which may be done by providing the gas, which is preferably devoid of oxygen. Hence, in embodiments, the gas may be provided during (part of) the dark phase of the cultivation stage (for mixing), especially during a dark phase temporally arranged directly after a feeding stage, and the gas (provided during the dark phase) may (essentially) be devoid of O2, such as comprise < 0.1 vol% Oa.

In general, because of respiration the photogranules may quickly go anaerobic during a (non-aerated) dark phase. However, the sequencing batch reactor may be sparged with N: to remove residual oxygen.

Hence, in further embodiments, the cultivation stage may comprise imposing the anaerobic conditions by sparging with N». Especially, the cultivation stage may comprise providing N; to the sequencing batch reactor via the bottom inlet.

The light phases may, in embodiments, have independently selected durations from the range of 0.5 — 15h, such as from the range of 1 — 12 h, especially from the range of 2- 6h. In further embodiments, the light phases may have independently selected durations of at most 21h, such as at most 18h, especially at most 15h.

In further embodiments, the dark phases may have independently selected durations from the range of 0.1 — 18 h, especially from the range of 0.2 — 12 h, such as from the range of 0.5 — 6h.

In further embodiments, in a single cultivation cycle of the one or more cultivation cycles, the dark phases may have a combined first duration D1, and the light phases have a combined second duration D2, especially wherein 0.5 <D1/D2 <2.

The cultivation stage may, in embodiments, comprise controlling a temperature inside the sequencing batch reactor. In particular, the cultivation stage may comprise controlling the temperature the plurality of micro-organisms is exposed to. As different micro- organisms may be more tuned to growth in different temperatures, the microbial composition (and the photogranule functions) may be steered by the selection of the temperature. In embodiments, the temperature may especially be selected from the range of 10 — 40 °C, which may be particularly suitable for the growth of the alga.

Further, in embodiments, the cultivation stage may comprise controlling a pH inside the (contents of the) sequencing batch reactor. In particular, the cultivation stage may comprise controlling the pH the plurality of micro-organisms is exposed to. In particular, at a high pH also a high NH: concentrations may be present, which may inhibit the (micro)alga.

However, a low pH may be detrimental to the micro-organisms which may tend to die at pH <

5.5 - 6 (dependent on the species). Hence, in embodiments, the cultivation stage may comprise controlling the pH in the range of 6 — 8.5, especially in the range of 6.5 — 8, such as in the range of 6.8 - 7.5.

In embodiments, the settling stage may comprise allowing the photogranule to settle. In particular, the settling stage may comprise providing non-aerating conditions to the sequencing batch reactor. Especially, the settling stage may comprise fluidically separating a bottom inlet of the sequencing batch reactor from a gas supply.

As the settling stage may be temporally arranged prior to the effluent removal stage (see below), the duration of the settling may be tuned to provide a selective pressure on well-settling granules. In particular, well-settling granules may be dense granules. If the duration of the settling stage is too short, then (relatively) large amounts of biomass may be lost, reducing the efficiency of providing the photogranule. If, however, the duration of the settling stage is too long, insufficient selective pressure may be applied for well-settling granules. The suitable duration may, however, further depend on a reactor height H of the sequencing batch reactor.

Hence, in embodiments, the settling stage may have a settling duration T2 selected from the range of 0.01 — 0.05 min/cm * H, wherein H is the reactor height in cm. In further embodiments, T2 may be at least 0.005 min/cm * H, such as at least 0.01 min/cm *H.

In further embodiments, T2 may be at least 0.015 min/cm * H, especially at least 0.02 min/cm *H, such as at least 0.03 min/cm *H. In further embodiments, T2 may be at most 0.1 min/cm *H, such as at most 0.05 min/cm *H, especially at most 0.04 min/cm *H.

In further embodiments, the settling duration T2 may be selected from the range of 2 — 10 minutes, such as from the range of 3-7 minutes, especially from the range of 4-6 minutes.

In embodiments, the effluent removal stage may comprise removing a remainder of the growth medium and non-settled micro-organisms. In particular, the sequencing batch reactor may have a reactor volume Vg, and the effluent removal stage may comprise removing a volume (of remainder of the growth medium and non-settled micro-organisms) of at least

0.4*Vg from the sequencing batch reactor, especially at least 0.5% Vg, such as at least 0.6* Vr. In further embodiments, the effluent removal stage may comprise removing a volume (of remainder of the growth medium and non-settled micro-organisms) of 0.3*Vg — 0.75* Vr from the sequencing batch reactor, such as 0.4*Vr — 0.7% Vg, especially 0.5 — 0.67% Vr.

In embodiments, the method may further comprise harvesting, such as recovering (or “obtaining” }, the photogranule from the sequencing batch reactor. In particular, the method may comprise harvesting the photogranule after the one or more cultivation cycles.

In embodiments, the method may comprise subjecting the plurality of micro- organisms to feast-famine conditions. By imposing feast/famine conditions with regards to a specific nutrient, a selective pressure may be applied on the photogranule to include a micro- organism that stores that nutrient. For instance, feast/famine conditions may be applied with respect to N, P, or chemical oxygen demand (COD), which may promote the presence of micro- organisms that produce storage compounds such as EPS, lipids, polyhydroxyalkanoate, glycogen and polyphosphate.

Hence, in embodiments, the feeding stage may comprise providing the growth medium comprising a primary nutrient at a first nutrient concentration C1, and wherein the cultivation stage continues at least until the primary nutrient in the growth medium is reduced to a predetermined second nutrient concentration C2, wherein C2/C1 < 0.05, such as < 0.02, especially < 0.01. In particular, the cultivation stage may have a famine duration during which C2/C1 £0.05, such as < 0.02, especially < 0.01. In embodiments, the famine duration may be at least 0.25h, such as at least 0.5h, especially at least 1h.

In further embodiments, the famine duration may be at most 10h, such as at most Sh, especially at most 2h.

In further embodiments, the primary nutrient may comprise one or more carbon sources.

In further embodiments, the primary nutrient may comprise one or more nitrogen sources.

In further embodiments, the primary nutrient may comprise one or more phosphor sources.

In embodiments, the method may comprise operating the sequencing batch reactor during the one or more cultivation cycles at a solids retention time (SRT). In particular, a too low SRT may result in biomass washout, small photogranules and low biomass concentrations, whereas a too high SRT may result in nutrient limitation, large (and inefficient) photogranules and light limitation.

Hence, in embodiments, the SRT may be selected from the range of 3 — 21d, such as 4 — 18d, especially 7 — 14d.

In further embodiments, the SRT may be at least 7d, such as at least 10d, especially at least 13d.

In further embodiments, the SRT may be at most 21d, such as at most 18d, especially at most 16d.

The term “solids retention time” may indicate the average time a solid, such as a microbial cell, especially such as a photogranule, may spend in the sequencing batch reactor.

In particular, the SRT may correspond to the quantity of solids maintained in the sequencing batch reactor divided by the quantity of solids coming out of the sequencing batch reactor in a given timeframe: SRT = Vr*Cd/Qout*Cout.

Where Vr is the reactor volume; Cd is the solids concentration in the reactor space; Qout is the volume of the effluent, and Cout is the solids concentration of the effluent.

Hence, the SRT may relate to (or partially dictate) the settling duration and the volume removed during the effluent removal stage.

In further embodiments, the method may comprise operating the sequencing batch reactor during the one or more cultivation cycles at a hydraulic retention time (HRT). In particular, if HRT is too low, the micro-organisms may lack nutrients for growth.

However, if the HRT is too high, the formation of photogranules may be hampered.

Hence, in embodiments, the HRT may be selected from the range of 0.1 — 6d, such as from the range of 0.15 — 4d, especially from the range of 0.2 - 3d.

The term “hydraulics retention time” may refer to the average time a soluble compound may spend in the sequencing batch reactor. In particular, the HRT in a given timeframe may correspond to Vr/Vg, wherein Vg is the volume of added growth medium (in that timeframe).

In general, in embodiments, the method may comprise subjecting the plurality of micro-organisms to a plurality of cultivation cycles. In particular, the plurality of micro- organisms may be provided as free (“non-aggregated”) micro-organisms or as a pre-existing granule (see above), and may form the photogranule over a plurality of cultivation cycles. The plurality of cultivation cycles may, however, differ with regards to operational conditions. In particular, conditions during initial cultivation cycles may be selected to stimulate the formation of a photogranule, whereas conditions during subsequent (or “terminal”) cultivation cycles may be selected to provide a desired selective pressure on the photogranule(s). In particular, if the conditions of the initial cultivation cycles are maintained, the photogranule may not have all desired features, whereas if the conditions of terminal cultivation cycles are imposed directly the selective pressure may be too high, resulting in large loss of biomass or, potentially, no (photo)granule formation in the first place.

Hence, in embodiments, the plurality of cultivation cycles may comprise an initial cultivation cycle and a terminal cultivation cycle temporally arranged after the initial cultivation cycle.

In further embodiments, the initial cultivation cycle may have an initial settling duration Ti, and the terminal cultivation cycle may have a terminal settling duration Tt, especially wherein T; > 2*T,. In particular, the terminal settling duration T: may be T2, whereas Ti may be > 2*T2. Hence, a selective pressure on well-settling (dense) granules may be increased over time.

In further embodiments, the initial cultivation cycle may have an initial average second photosynthetic photon flux density P2; during the (respective) light phases, and the terminal cultivation cycle may have a terminal average second photosynthetic photon flux density P2; during the (respective) light phases, especially wherein P2 > 2*P2;. In particular, too much light during the initial cultivation cycle may lead to photoinhibition of the algae in suspension during assembly from single cells in suspension to granules. Hence, a selective pressure on photosynthetic activity may beneficially be increased over time.

In further embodiments, the initial cultivation cycle may have an initial ratio R; of total duration of dark phases to total duration of light phases, and the terminal cultivation cycle has a terminal ratio R+ of total duration of dark phases to light phases, especially wherein

R¢ > 2* R;. Hence, a selective pressure on survival during anaerobic/dark conditions may be increased over time.

Similarly, in further embodiments, the initial cultivation cycle may have an initial longest dark phase duration di, and the terminal cultivation cycle has a terminal longest dark phase duration d, especially wherein dt > 2*d..

Applying a selective pressure on increased (relative) duration of dark phases may facilitate applications of the photogranule without artificial/supplemental lighting. In particular, applications relying on natural sunlight may have variable light and dark phases, and may, for instance, have dark phases lasting more than 12 hours. Hence, selection for photogranules that can withstand such long dark/anaerobic phases may be beneficial in view of such applications.

Hence, the method may comprise adjusting an operational parameter over successive cultivation cycles. In particular, in embodiments, the method may comprise decreasing a settling duration over successive cultivation cycles. In further embodiments, the method may comprise increasing an average second photosynthetic photon flux density over successive cultivation cycles. In further embodiments, the method may comprise increasing a ratio R of total duration of dark phases to total duration of light phases over successive cultivation cycles. In further embodiments, the method may comprise increasing a longest dark phase duration over successive cultivation cycles.

The term “stage” and similar terms used herein may refer to a (time) period (also “phase”) of a method and/or an operational mode. The different stages may (partially) overlap (in time). For example, the effluent removal stage may, in general, be initiated prior to the feeding stage, but may partially (such as fully) overlap in time therewith, such as during plug- flow feeding (see below). However, for example, the settling stage may typically be completed prior to the effluent removal stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time. For example, the effluent removal stage may occur simultaneously with the feeding stage such that plug-flow conditions are provided, which may facilitate providing anaerobic conditions during feeding.

Hence, in embodiments, for at least part of the plurality of cultivation cycles may apply that the effluent removal stage temporally overlaps with the feeding stage of a subsequent cultivation cycle, especially wherein the effluent removal stage and the feeding stage overlap for at least 50% of their total duration.

In a further aspect, the invention may provide a photogranule. Especially, a photogranule obtainable using the method of the invention.

The photogranule may comprise a plurality of micro-organisms, especially wherein the plurality of micro-organisms comprises (at least) a filamentous cyanobacterium, a polyphosphate-accumulating organism, and a (eukaryotic) alga.

It will be clear to the person skilled in the art, that the microbial composition of the photogranule may be indicative, especially causative, of the performance features of the photogranule.

However, as will also be clear to the person skilled in the art, it may be challenging to determine an exact community composition in terms of, e.g., wt.% or sheer numbers as the plurality of different micro-organisms have formed a single photogranule.

An approach to estimate a community composition may be 16S and/or 18S ribosomal RNA sequencing, which may be common amplicon sequencing techniques used to taxonomically classify the members of a community.

In particular, 16S rRNA sequencing may be used to identify/classify bacterial species, whereas 18S rRNA sequencing may be used to identify/classify eukaryotic species.

In particular, the photogranule, especially the microbial composition, may comprise bacterial members.

In embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 5% of the polyphosphate-accumulating organism, especially at least 10%, such as at least 15%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 20% of the polyphosphate-accumulating organism, especially at least 25%, such as at least 30%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 60% of the polyphosphate- accumulating organism, especially at most 55%, such as at most 50%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 45% of the polyphosphate-accumulating organism, especially at most 40%, such as at most 35%. The phrase “based on 16S rRNA sequencing, the bacterial members may comprise at least X% of organism Y” and similar phrases may herein refer to at least X% of a total number of reads in a (16S/18S) dataset corresponding to an amplicon sequencing variant (ASV) corresponding to organism Y, which may serve as a proxy for the relative prevalence of organism Y in the bacterial members.

In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 5% of the filamentous cyanobacterium, especially at least 10%, such as at least 20%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 30% of the filamentous cyanobacterium, especially at least 35%, such as at least 40%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 65% of the filamentous cyanobacterium, such as at most 62%, especially at most 60%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 55% of the filamentous cyanobacterium, such as at most 50%, especially at most 45%. As indicated above, in embodiments, the plurality of micro-organisms may further comprise a nitrifier and/or a denitrifier. Hence, in further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 5% of the denitrifier, especially at least 10%, such as at least 15%. Hence, in further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 20% of the denitrifier, especially at least 25%. In particular, a (relatively large) proportion of PAOs may also perform denitrification. Hence, in further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 65% of the denitrifier, such as at most 60%, especially at most 50%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 45% of the denitrifier, such as at most 40%, especially at most 35%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 25% of the denitrifier, such as at most 23%, especially at most 20%.

In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at least 0.5% of the nitrifier, such as at least 1%, especially at least 1.5%. In further embodiments, based on 16S rRNA sequencing, the bacterial members may comprise at most 5% of the nitrifier, such as at most 4%, especially at most 3%.

In particular, the photogranule, especially the microbial composition, may comprise eukaryote members. In embodiments, based on 18S rRNA sequencing, the eukaryote members may comprise at least 50% of the alga, such as at least 55%, especially at least 60%. In further embodiments, based on 18S rRNA sequencing, the eukaryote members may comprise at least 65% of the alga, such as at least 70%. In embodiments, the alga may be the only eukaryote present, i.e, the eukaryote members may comprise at most 100% of the alga. In further embodiments, the eukaryote members may comprise at most 99% of the alga, such as at most 98%, especially at most 95%. In further embodiments, the eukaryote members may comprise at most 90% of the alga, especially at most 85%, such as at most 80%.

Hence, the photogranule, especially the eukaryote members, may further comprise one or more other (eukaryotic) species, such as fungi, and such as predatory organism, including Rotifer, Ciliates, and Amoebae.

In embodiments, based on 18S rRNA sequencing, the eukaryote members may comprise at least 5% of fungi, such as at least 7%, especially at least 10%. In further embodiments, based on 18S rRNA sequencing, the eukaryote members may comprise at most 20% of the fungi, especially at most 18%, such as at most 15%.

In further embodiments, based on 18S rRNA sequencing, the eukaryote members may comprise at least 5% of predatory organisms, such as at least 7%, especially at least 10%. In further embodiments, based on 18S rRNA sequencing, the eukaryote members may comprise at most 20% of the predatory organisms, especially at most 18%, such as at most 15%.

In embodiments, the photogranule may approximate an ellipsoidal shape, especially a spherical shape. The term “approximate” and its conjugations herein, such as in “to approximate a shape”, refers to being nearly identical to, especially identical to, the following term, for example nearly identical to an ellipsoidal shape or a spherical shape. For example, a photogranule may define an ellipsoidal shape but for some irregularities. In particular, an object approximating a first shape may herein refer to: a first shape realization encompassing the object, wherein the first shape realization is defined as the smallest encompassing shape of the (3D) object wherein the first shape realization has the shape of the first shape, wherein a ratio of the volume of the first shape realization to the volume of the object is < 1.2, especially < 1.1, such as <1.05, especially <1.02. For instance, a photogranule may approximate an ellipsoidal shape, wherein the first shape realization may be defined as the smallest encompassing ellipsoidal shape of the photogranule, wherein a ratio of the volume of the first shape realization to the volume of the photogranule is < 1.2, especially, especially <

1.1, such as <1.05, especially <1.02, including 1.

Different micro-organisms may thrive at different locations of the photogranule, especially with regards to a distance to a surface of the photogranule. For instance, the surface of the photogranule may receive the most light, and may directly be exposed to the growth medium, or, in a treatment application, to waste(water). If, for example, the alga is arranged on the surface of the photogranule, and thus produces O; at the surface of the photogranule, then there may be an oxygen gradient from the surface to a core of the photogranule.

Hence, as different organisms may thrive at different locations of the photogranule, the prevalence of these different locations may beneficially be balanced in a desired range, which may depend on the photogranule size. Hence, in embodiments, the photogranule may have a (smallest) circularly equivalent diameter of a most 6 mm, such as at most 5 mm, especially at most 3 mm. In further embodiments, the photogranule may have a (smallest) circularly equivalent diameter of a most 2.5 mm, such as at most 2.2 mm, especially at most 2 mm. In further embodiments, the photogranule may have a (smallest) circularly equivalent diameter of at least 0.1 mm, such as at least 0.15 mm, especially at least 0.2 mm. In further embodiments, the photogranule may have a (smallest) circularly equivalent diameter selected from the range of 0.08 — 2.2 mm, especially from the range of 0.1 — 2 mm, such as of

0.15-1.7 mm. In particular, the photogranule may pass through a sieve having circularly equivalent pore sizes of 6 mm, especially of 5 mm, such as of 2 mm, and the photogranule may be retained by a sieve having circularly equivalent pore sizes of 0.08 mm, such as of 0.1 mm, especially of 0.15 mm.

The photogranule may be well-settling. The settleability of the photogranule may be determined using the sludge volume index (SVI). The SVI may be defined as the total volume of one gram of settled sludge after a set amount of time. A lower SVI may indicate a better settleability of the biomass as the photogranule occupies less volume per gram compared to a higher SVI. Specifically, in embodiments, the photogranule may have a sludge volume index after five minutes of settling SVIs < 100 mL/g, such as <85 mL/g, especially <70 mL/g.

In particular, the SVI may be determined by placing IL of a sample ,e.g. reactor content, of which the biomass concentration is known (TSS/VSS) into a graded IL measurement cylinder and measuring sludge height after a predetermined amount of time, as described in Chapter 6 of Loosdrecht et al, “Experimental Methods in Wastewater Treatment, IWA publishing, 2016”, which is hereby herein incorporated by reference. Hence, for determining the SVL the sludge height may be measured after 5 min.

In embodiments, the photogranule may have an elemental composition. The elemental composition may comprise at least 35 wt.% carbon (relative to total weight of the photogranule), such as at least 40 wt.%, especially at least 42 wt.%.

In further embodiments, the elemental composition may comprise at least 7 wt.% nitrogen, such as at least 8 wt.% nitrogen, especially at least 8.5 wt.% nitrogen.

In further embodiments, the elemental composition may comprise at least 2.7 wt.% phosphorus, such as at least 3 wt.%, especially at least 3.3 wt.%.

In further embodiments, the photogranule may comprises at least 0.01 wt.% of chlorophyll (relative to total weight of the photogranule), especially at least 0.05 wt.%, such as at least 0.1 wt.%.

In a further aspect, the invention may provide a treatment method for treating water and/or waste using the photogranule of the invention, especially for treating water, such as wastewater, or such as nutrient-rich water. The treatment method may have similarities to the method for providing the photogranule. In particular, the method for providing the photogranule may be tuned in view of conditions that the photogranule will be exposed to during the treatment method. Hence, in particular, the terminal cultivation cycles of the method for providing the photogranule may approximate cultivation cycles of the treatment method. Specifically, the treatment method may comprises subjecting a photogranule in a sequencing batch reactor to one or more cultivation cycles to treat the waste(water). The sequencing batch reactor may have a reactor volume having a reactor height H (in cm). In embodiments, (each of) the one or more cultivation cycles may comprise a feeding stage, a cultivation stage, a settling stage, and an effluent removal stage. The feeding stage may comprise providing the waste(water) to the photogranule. The cultivation stage may comprise exposing the waste(water) to the photogranule to provide a treated product, such as treated water, especially wherein the cultivation stage comprises providing alternating dark phases and light phases. In embodiments, the photogranule may be exposed to a first average photosynthetic photon flux density P1 during the dark phases and to a second average photosynthetic photon flux density P2 during the light phases, especially wherein P1/P2 < 0.05. The settling stage may, in embodiments, have a settling duration T2 selected from the range of

0.001 — 0.05 min/em * H, especially from the range of 0.01 — 0.05. The effluent removal stage may comprise removing the treated product, especially the treated water, from the sequencing batch reactor. The photogranule of the invention may provide the advantage that limited or no oxygen needs to be externally supplied for the treatment of waste(water). Hence, in embodiments, an amount of externally supplied oxygen to the sequencing batch reactor — especially excluding DO in the waste(water) — may be < 5 mmol/L/d, such as <3 mmol/L/d, especially <2 mmol/L/d, i.e., less than 5 mmol of oxygen may be provided to the sequencing batch reactor per L of a reactor volume Vr of the sequencing batch reactor per day. The oxygen produced by photosynthetic micro-organisms, such as the alga, in the photogranule may (at least partially) be consumed by heterotrophic micro-organisms, such as the PAO, in the photogranule. However, part of the oxygen may also leave the photogranule and rise to a headspace of the sequencing batch reactor. In embodiments, the cultivation stage may comprise recirculating gas from a headspace of the sequencing batch reactor to a bottom inlet of the sequencing batch reactor. During the treatment method, the photogranule may not only clean the waste(water) to provide a treated product, such as treated water, but the photogranule may also accumulate nutrients, which may be valuable for further processes. In embodiments, the treatment method may comprise operating the sequencing batch reactor during the one or more cultivation cycles at a solids retention time (SRT). Hence,

in embodiments, the SRT may be selected from the range of 3 — 21d, such as 4 — 18d, especially 7 — 14d. In further embodiments, the SRT may be selected from the range of 2 — 10d, such as 3 — 7d. In further embodiments, the SRT may be at least 2d, such as at least 2.5d, especially at least 3d. In further embodiments, the SRT may be at least 7d, such as at least 10d, especially at least 13d. In further embodiments, the SRT may be at most 21d, such as at most 18d, especially at most 16d. In further embodiments, the SRT may be at most 10d, such as at most 7d, especially at most 6d. In particular, in embodiments, the treatment method may have a lower SRT than the method for providing the photogranule. Hence, in embodiments, the treatment method may comprise harvesting the (enriched) photogranule after the one or more cultivation cycles.

In particular, the (elemental composition of the) harvested photogranule may comprise at least 2.7 wt.% phosphorus, especially at least 4 wt.% phosphorus (relative to total weight of the harvested photogranule), such as at least 5 wt.% phosphorus, especially at least 6 wt. % phosphorus. In further embodiments, the harvested photogranule may comprise at most wt.% phosphorus, such as at most 15 wt.% phosphorus.

Hence, in a further aspect, the invention may provide a harvested (enriched) photogranule obtainable by the treatment method.

In a further aspect, the invention may provide a use of the photogranule according to the invention or obtainable with the method according to the invention for 20 treatment of waste or water, especially for water treatment, such as for wastewater treatment, or especially for waste treatment. Hence, in embodiments, the water treatment may comprise wastewater treatment. In further embodiments, the water treatment may comprise the treatment of nutrient-rich (surface) water, such as of water from a eutrophic lake.

The invention may herein primarily be described in the context of wastewater treatment. However, it will be clear to the person skilled in the art that the invention is not limited to such application and may further apply for treatment of other (aqueous) sources, such as for nutrient-rich water.

The embodiments described herein are not limited to a single aspect of the invention. For example, an embodiment describing the method may, for example, further relate to the treatment method, especially to a stage of the treatment method. Similarly, an embodiment of the treatment method describing a stage of the treatment method may further relate to the method. In particular, an embodiment of the photogranule describing a feature (of the photogranule) may indicate that the method may, in embodiments, be configured for and/or be suitable for providing a photogranule having such feature.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1A-B schematically depict embodiments of the method of the invention. Fig. 2 schematically depicts embodiments of cultivation cycles. Fig. 3A-J schematically depict the formation of a photogranule from the plurality of micro-organisms using the method of the invention. The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS Fig. 1A schematically depicts a method for providing a photogranule 10 for nutrient recovery, especially for phosphate and/or nitrogen recovery. The method comprises subjecting a plurality of micro-organisms 20 in a sequencing batch reactor 110 to one or more cultivation cycles 200 to provide the photogranule 10. Specifically, Fig. 1A depicts a single cultivation cycle 200, wherein, for visualizational purposes, at the start (at a feeding stage 210) a plurality of micro-organisms 20 is indicated, at least part of which in the depicted embodiment are provided as a pre-existing granule 25, whereas at the end (at an effluent removal stage 240) a photogranule 10 is indicated. In further embodiments, the plurality of micro-organisms 20 may be provided as free (non-aggregated) micro-organisms, and the plurality of micro- organisms 20 may be subjected to a plurality of cultivation cycles, such as at least 5, especially at least 10, in order to provide the photogranule. As indicated by the arrow from the effluent removal stage 240 to the feeding stage 210, a new cultivation cycle 200 may be initiated by starting a feeding stage 210 during or after the effluent removal stage 240 of the preceding cultivation cycle 200 In the depicted embodiment, the sequencing batch reactor 110, especially a reactor space 115 of the sequencing batch reactor, has a reactor height H. The sequencing batch reactor 110, especially the reactor space 115, may further have a reactor volume Vr. In the depicted embodiment, at least part of the sequencing batch reactor 110 has a conical shape tapering towards a bottom inlet 111. In the depicted embodiment, the plurality of micro-organisms 20 comprise a filamentous cyanobacterium, a polyphosphate-accumulating organism, and a eukaryotic alga. In further embodiments, the plurality of micro-organisms 20 may further comprise a nitrifier and/or a denitrifier.

In embodiments, (each of) the one or more cultivation cycles 200 comprises a feeding stage 210, a cultivation stage 220, a settling stage 230, and an effluent removal stage

240.

The feeding stage 210 may comprise providing a (liquid) growth medium 30 to the plurality of micro-organisms 20. In particular, the feeding stage 210 may comprise providing the growth medium 30 to the sequencing batch reactor 110, especially to the reactor space 115. In embodiments, the feeding stage 210 may comprise filling at least 8% of a reactor volume Vr with growth medium 30, such as at least 15%, especially at least 20%, such as at least 30%, especially at least 40%, such as at least 50%. In further embodiments, the feeding stage 210 may comprise filling at least 50% of a reactor volume Vr with growth medium 30, such as at least 60%, especially at least 70%, such as at least 80%. In further embodiments, the feeding stage 210 may comprise filling at most 95% of a reactor volume Vr with growth medium 30, such as at most 90%, especially at most 80%, such as at least 70%. In particular, in embodiments, the feeding stage 210 may comprise adding a volume of growth medium 30 to the sequencing batch reactor 110 corresponding to at least 8% of a reactor volume Vr with growth medium 30, such as at least 15%, especially at least 20%, such as at least 30%, especially at least 40%, such as at least 50%. In further embodiments, the feeding stage 210 may comprise adding a volume of growth medium 30 to the sequencing batch reactor 110 corresponding to at least 50% of a reactor volume Vr with growth medium 30, such as at least 60%, especially at least 70%, such as at least 80%. In further embodiments, the feeding stage 210 may comprise adding a volume of growth medium 30 to the sequencing batch reactor 110 corresponding to at most 95% of a reactor volume Vr with growth medium 30, such as at most 90%, especially at most 80%, such as at least 70%. In embodiments, the method, especially the cultivation stage, may comprise operating the sequencing batch reactor with 2.5-20% of the reactor space Vr as headspace, especially 3-15% of Vg, such as 5-10% of Vr.

In embodiments, the feeding stage 210 may comprise providing the growth medium 30 via a bottom inlet 111 of the sequencing batch reactor 110. In further embodiments, the feeding stage 210 may comprise providing the growth medium 30 via a second inlet of the sequencing batch reactor 110, which may be arranged spatially removed from the bottom inlet.

The cultivation stage 220 may comprises cultivating the plurality of microorganisms 20 in the growth medium 30. In particular, the cultivation stage 220 may comprise providing alternating dark phases 221 and light phases 222. In the depicted embodiment, for visualizational purposes, the cultivation stage 220 is depicted to start with a dark phase 221 and subsequently have a light phase 222, and the arrow indicates that after the light phase 222 another dark phase 221 may be started. However, in further embodiments, the cultivation stage may start with a light phase 222. In yet further embodiments, the cultivation stage 220 may have an unequal number of light phases 222 and dark phases 221, for instance, the cultivation stage 220 may start and end with dark phases 221.

In embodiments, the plurality of microorganisms 20 may be exposed to a first average photosynthetic photon flux density P1 during the dark phases 221 and to a second average photosynthetic photon flux density P2 during the light phases 222, especially wherein P1/P2 < 0.05. Specifically, the cultivation stage 220 depicted in Fig. 1A may comprise providing light source light 61 with a light source 60 during the light phases 222. In further embodiments, such as depicted in Fig. 1B, the cultivation stage 220 may comprise exposing the plurality of micro-organisms 20 to natural light 65 during the light phases 222. In yet further embodiments, the cultivation stage 220 may comprise exposing the plurality of micro- organisms 20 to light source light 61 to supplement natural light 65 during the light phases 222.

The cultivation stage 220 may further comprise imposing anaerobic conditions on the growth medium 30 during the dark phases 221. In particular, the cultivation stage may comprise (essentially) fluidically separating the reactor space 115 of the sequencing batch reactor 110 from oxygen external to the sequencing batch reactor 110. In further embodiments, the cultivation stage 220 may comprise imposing the anaerobic conditions by sparging with Na.

The cultivation stage 220 may further comprise passing a gas 40 through the growth medium 30 during the light phases 222, especially wherein the gas 40 comprises CO: and/or Og, especially at least CO:. In embodiments, the cultivation stage 220 may comprise providing the gas 40 via the bottom inlet 111, which may result in aeration and mixing of the contents of the reactor space 115. In further embodiments, as depicted in Fig. 1B, the gas 40 may especially be an external gas 41, i.e, a gas provided from a gas supply 120 arranged external to the sequencing batch reactor. In further embodiments, the gas 40 may especially be a headspace gas 42 provided from a headspace 112 of the sequencing batch reactor.

The settling stage 230 may comprise allowing the photogranule to settle for a settling duration T2. In particular, the settling stage 230 may comprise stopping (or “blocking™) a supply of the gas 40 via the bottom inlet 111, thereby stopping aeration. In particular, during the settling stage, (essentially) no gas may be provided via the bottom inlet. Further, during the settling stage, contents of the reactor space 115 may (essentially) not be mechanically mixed.

The settling duration T2 may especially be selected from the range of 0.01 —

0.05, such as from the range of 0.015 — 0.05 min/cm * H, wherein H is the reactor height in cm.

The settling stage 230 may be (directly) followed by the effluent removal stage

240.

The effluent removal stage 240 may comprise removing a remainder of the growth medium 30 and non-settled micro-organisms. In particular, the effluent removal stage 240 may comprise removing an effluent 35 comprising a remainder of the growth medium 30 and non-settled micro-organisms.

In embodiments, the method may comprise harvesting, such as recovering (or: “obtaining”), the photogranule 10 from the sequencing batch reactor 110, especially after the one or more cultivation cycles 200.

In embodiments, the feeding stage 210 may comprise providing the growth medium 30 comprising a primary nutrient at a first nutrient concentration Cl, especially wherein the primary nutrient comprises one or more carbon sources, and wherein the cultivation stage 220 continues at least until the primary nutrient in the growth medium is reduced to a predetermined second nutrient concentration C2, wherein C2/C1 < 0.05. Thereby, feast/famine conditions may be imposed, which may place a selective pressure on the photogranule(s) to produce storage compounds. Similarly, in further embodiments, the primary nutrient may comprise one or more nitrogen sources or one or more phosphor sources, especially the one or more nitrogen sources, or especially the one or more phosphor sources.

In embodiments, the method may comprise operating the sequencing batch reactor 210 during the one or more cultivation cycles 200 at a solids retention time selected from the range of 3 — 21d, such as from the range of 4 - 14d. The solids retention time may, for instance, be tuned by selecting the settling duration T2, the amount of effluent removed during the effluent removal stage, and the total duration of (each of) the cultivation cycles.

In further embodiments, the method may comprise operating the sequencing batch reactor 110 during the one or more cultivation cycles 200 at a hydraulic retention time selected from the range of 0.2 - 3d. The hydraulics retention time may, for instance, be tuned by selecting the amount of growth medium provided in the feeding stage, the amount of effluent removed during the effluent removal stage, and the total duration of (each of) the cultivation cycles.

In further embodiments, the effluent removal stage 240 may comprise removing a volume of effluent 35, especially of remainder of the growth medium 30 and non-settled micro-organisms, of at least 0.08*Vr, especially at least 0.2* Vg, such as at least 0.4*VR, especially at least 0.6% Vg, from the sequencing batch reactor 110.

Fig. 1B schematically depicts an embodiment of the method wherein for at least part of the plurality of cultivation cycles 200 applies that the effluent removal stage 240 temporally overlaps with the feeding stage 210 of a subsequent cultivation cycle 200, especially wherein the effluent removal stage 240 and the feeding stage overlap 210 for at least 50% of the total duration of the two stages. In particular, as feed is provided from the bottom the PAOs in the photogranules may immediately assimilate the carbon and store PHA, as in the settled photogranular biomass the conditions may be almost immediately (essentially) anaerobic. This plug-flow principle may be described in WO2004024638, which is hereby incorporated by reference. Granules that are not suitable for the process may be pushed out with the liquid (effluent). In addition, the microbes may experience the concentrated influent. When filling is done after draining the influent may be mixed with any remaining liquid in the reactor and may thus be diluted. Hence, simultaneous effluent removal and feeding may provide additional control on the exact cultivation conditions the plurality of micro-organisms are exposed to, and may contribute to imposing beneficial selective pressures.

Fig. 1A-B further schematically depict an embodiment of the treatment method of the invention.

Specifically the treatment method may be for treating wastewater 50 using the photogranule 10 of the invention, especially the photogranule obtainable with the method of the invention.

The treatment method may comprise subjecting the photogranule 10 in a sequencing batch reactor 110 to one or more cultivation cycles 200 to treat the wastewater 50, wherein: the sequencing batch reactor 110 has a reactor height H; wherein (each of) the one or more cultivation cycles 200 comprises a feeding stage 210, a cultivation stage 220, a settling stage 230, and an effluent removal stage 240; the feeding stage 210 comprises providing the wastewater to the photogranule 10; the cultivation stage 220 comprises exposing the wastewater 50 to the photogranule 10 to provide treated water 55, wherein the cultivation stage 220 comprises providing alternating dark phases 221 and light phases 222, wherein the photogranule 10 is exposed to a first average photosynthetic photon flux density P1 during the dark phases 221 and to a second average photosynthetic photon flux density P2 during the light phases 222, wherein P1/P2 < 0.05; the settling stage 230 has a settling duration T2 selected from the range of 0.001 — 0.05 min/cm * H; and the treated water removal stage 240 comprises removing the treated water 55 from the sequencing batch reactor 110.

In embodiments, an amount of externally supplied oxygen to the sequencing batch reactor 110 may be < 3 mmol/L/d. In particular, the treatment method may comprise

(essentially) not providing an external gas 41 with oxygen to the sequencing batch reactor 110. In further embodiments, the treatment method may comprise recirculating an headspace gas 42 from a headspace 112 of the sequencing batch reactor 110 into the reactor space 115 of the sequencing batch reactor 110, especially via a bottom inlet 111.

In further embodiments, the method may further comprise harvesting the (enriched) photogranule 10 after the one or more cultivation cycles 200 of the treatment method. The (enriched) photogranule may, after the treatment method, be enriched in nutrients, especially at least in N, or especially at least in P. The (enriched) photogranule may, for instance, be used as (nutrient-rich) fertilizer.

Fig. 1A-B further schematically depict a use of the photogranule for waste(water) treatment.

Fig. 2 schematically depicts embodiments of one or more cultivation cycles 200, especially of three sets of a plurality of successively arranged cultivation cycles 200. In the depicted embodiments, for visualizational purposes, the settling stage 230, the effluent removal stage 240 and the feeding stage 210 are depicted collectively, and the cultivation stage 220 is separated into the dark phases 221 and the light phases 222.

Each of the depicted embodiments in Fig. 2 may correspond (approximately) to 24 hours. Hence, each embodiment may indicate a temporal distribution of six cultivation cycles 200 spread out over 24 hours.

Hence, in the depicted cultivation cycles, the light phases 222 may have independently selected durations from the range of 1 — 12 h, especially from the range of 1 — 4 h, and the dark phases 221 may have independently selected durations from the range of 0.1 — 12 h. In embodiments with plug-flow feeding, a temporal arrangement of the stages of the cultivation cycles 200 may differ and, for instance, a cultivation cycles may comprise (partially) temporally overlapping feeding stage 210, effluent removal stage 240, and dark phase 221 of the cultivation stage 220, followed by the light phase 222 of the cultivation stage 220, followed by the settling phase 230. Specifically, Fig. 2 may schematically depict embodiments of initial cultivation cycles 200a and of terminal cultivation cycles 200b. In particular, the method may comprise subjecting the plurality of micro-organisms 20 to a plurality of cultivation cycles 200, wherein the plurality of cultivation cycles 200 comprise an initial cultivation cycle 200a and a terminal cultivation cycle 200b temporally arranged after the initial cultivation cycle 220a, especially wherein the initial cultivation cycle 200a has an initial settling duration Ti, and wherein the terminal cultivation cycle 220b has a terminal settling duration Ti, wherein T; > 2*T;. Hence, as depicted in Fig. 2, the settling duration T2 may be smaller for the terminal cultivation cycles 200b, resulting in a shorter combined duration of the settling stage 230, the effluent removal stage 240, and the feeding stage.

Similarly, the bottom set of terminal cultivation cycles 220b may schematically depict an embodiment wherein the initial cultivation cycle 200a has an initial ratio R; of total duration of dark phases 221 to total duration of light phases 222b, and the terminal cultivation cycle 200b has a terminal ratio Rt of total duration of dark phases 221 to light phases 222, wherein R; > 2* RL.

In further embodiments, the initial cultivation cycle 200a may have an initial average photosynthetic photon flux density P2; during the (respective) light phases 2224, and the terminal cultivation cycle 200b may have a terminal average photosynthetic photon flux density P2; during the (respective) light phases 222b, wherein P2; > 2*P2;.

Experiments Experiments have been performed to provides photogranules using the method of the invention starting from a plurality of micro-organisms obtained from pre-existing granules. Unless indicated otherwise, the experimental procedures described herein were carried out using the materials and methods described in the following section.

Materials and Methods Plurality of micro-organisms — phosphate-accumulating organisms — For the phosphate-accumulating organisms, pre-existing aerobic granules were grown in saline conditions. The pre-existing aerobic granules correspond to the aerobic granules described in de Graaff et al., “Biological phosphorus removal in seawater-adapted aerobic granular sludge”. Water Research, 2020, which is hereby herein incorporated by reference. The aerobic granules were — where indicated — homogenized prior to inoculation of the system using a Miccra D9 Homogeniser (Miccra GmbH, Heitersheim, Germany).

Plurality of micro-organisms — cyanobacterium and alga — pre-existing second granules comprising the cyanobacterium and the alga adapted to both high (152 mgN L-1 day- 1) and low (23 mgN L-1 day-1) N loading were used. The pre-existing second granules correspond to the photogranules described in Trebuch et al., “Impact of hydraulic retention time on community assembly and function of photogranules for wastewater treatment”, Water Research, 2020, which is hereby herein incorporated by reference.

Growth medium — start-up — A synthetic growth medium was used to simulate wastewater. The synthetic growth medium comprised sodium acetate trihydrate as chemical oxygen demand (COD), KH:PO: and K;HPO: as phosphor sources, NH4Cl as nitrogen source, as well as Mg, Ca, K, EDTA, Fe, B, Mn, Zn, Cu, Mo, and Co.

The concentrations of major constituents such as COD, P, and N were changed over the course of the different experimental phases (Table 1): ep fs Jz 0 Jo Jw Sequencing batch reactor — three sequencing batch reactors, especially glass bubble column reactors (BCRs), with a conical bottom were used to cultivate the photogranules.

The BCRs had a diameter of 10 cm and a total height of 28 cm, with a working volume of 1.6 L being maintained throughout the experimental phases.

The top of each reactor was sealed using a polymethyl methacrylate cap with a total of seven ports for probes or tubes.

Synthetic wastewater was pumped into the reactors via a short glass pipe using a Masterflex L/S peristaltic pump with a Masterflex L/S cartridge pumphead and Masterflex L/S tubing, size (Cole Parmer, Vernon Hills, US). The synthetic wastewater was made by mixing two concentrated stock solutions with tap water drawn from a 100 L tank.

The two concentrated stock solutions were drawn from 1 L glass Schott bottles (DWK Life Sciences GmbH, Mainz, 15 Germany) and added to the tap water using two Minipulse 3 peristaltic pumps (Gilson). The effluent was removed via a longer glass pipe using a single Masterflex L/S peristaltic pump with a Mastertlex L/S Easy Load II or High-Performance pump head (Cole Parmer, Vernon Hills, US) per reactor.

The pump heads were loaded with Masterflex L/S tubing size 25. The amount of effluent removed after each cycle was determined by the height of the glass effluent pipe in the reactor.

A 60 mL syringe (VWR) was attached to the effluent extraction line using a three-way connector to sample the reactor contents.

Separate overflow tanks were connected to ports on the side of each reactor to retain the reactor content in case of overfilling.

A glass heat exchanger, connected to an external water bath, was used to control the temperature within each reactor at 20 + 10C.

The pH in each reactor was controlled at 7.25 + 0.1 using a pH probe (VWR). The pH controllers added 1M NaOH or IM HCL to the reactors to maintain a steady pH value.

Compressed air for mixing or supplying O2 was pumped in from the bottom of each reactor and diffused using a stirring magnet.

The airflow was controlled with a SLA5800 Series mass flow controller (Brooks Instrument LLC, Hatfield, US) and could be adjusted manually with a dedicated rotameter per reactor (Cole Parmer, Vernon Hills, US) to a flowrate of 0.5 L min’. Using a pneumatic switch (Metal Works Pneumatics, Ede, The Netherlands), the incoming gas could be altered between compressed air (OFF position) and N gas (ON position).

The concentration of dissolved oxygen (DO) within the reactors was measured continuously using two pHenomenal OX4100H sensors and one pHenomenal MU6100H DO sensor (VWR). Two of the reactors, PG1 and PG2 (see below), were illuminated on three sides by LED panels (16x Avago ASMT-MY22-NMP00 4000K). The light intensity was adjusted using a custom- made Circle of Light-12 controller (Wageningen University, Wageningen, the Netherlands). Electronic EMT757 timers (EverFlourish Smart Technology Corp., Ltd., Yinzhou, China) were used to operate the reactors.

Inoculation — Two reactors were inoculated with a plurality of micro-organisms comprising a filamentous cyanobacterium, a phosphate-accumulating organism, and an alga. A third reactor was inoculated with pre-existing aerobic granules comprising the phosphate- accumulating organism. The first reactor received homogenized second granules and intact aerobic granules and was labelled ‘PGI’. The second reactor received intact second and aerobic granules and was labelled ‘PG2’. The last reactor was only used to cultivate aerobic granules and was labelled ‘AGSI’. Specifically, On October 26, 2020, reactor AGS1 was inoculated with 100 mL of homogenized aerobic granular sludge (AGS) to reach a starting concentration of 1.38 g volatile suspended solids (VSS) L. The settling time was extended from 5 to 20 minutes on October 30, 2020, to avoid washout of floccular biomass. On November 2, 2020, PG2 was inoculated with second granules to reach a starting concentration of 0.32 g VSS LL. Finally, on November 17, 2020, the reactor contents of AGS1 were split and divided over AGS1 and PGI. During the start-up phase, the SRT of reactors was not controlled. The HRT of each reactor was 0.33 days and the volume exchange ratio was 50%. The light intensity was set to an average (during the light phases) of 325 umol m= st. Reactor operation - All three reactors were operated as sequencing batch reactors and had six cycles of anaerobic influent feeding and reaction, aerobic reaction, settling, and effluent removal per day. In general, each cycle started after the draining of the previous cycle was finished. The reactor contents from the previous cultivation cycle were sparged with Na gas and O; was removed. After 5 minutes of sparging with Nz, the influent (or “growth medium’) was fed into the reactors for another 5 minutes. When the feeding stage was finished, a dark phase was imposed, i.e, the systems were allowed to react under anaerobic conditions with the LED lights turned off. At the end of the dark phase, the LED lights turned back on and the pneumatic switch was turned off to aerate the reactors with compressed air. At the end of the aerobic phase, the LED lights and the mass flow controller were turned off. The biomass was allowed to settle for 5 minutes after cutting off the aeration. Finally, the reactor contents were drained for 3 minutes until the height of the effluent pipe and a new cultivation cycle was initiated.

Dark phase extension - On January 18, 2021, the anaerobic phase of one cycle was extended from 60 to 180 minutes. Subsequent extensions to 350, 530 and 690 minutes were done in intervals of two days until January 25, 2021.

Sampling and kinetics - Samples were taken at the end of each anaerobic phase and from the reactor effluent to study the removal of nutrients per cycle. In addition, several kinetics studies were performed by taking samples every 5 or 10 minutes to gain insight into the hourly removal rates. The maximum uptake and release rates were calculated via linear regression of the kinetics graphs.

Chemical oxygen demand - To observe the removal of acetate during each cycle, COD was measured with Hach Lange COD test-kits (Hach, Loveland, US) in the range of 5 — 60 mg Oz LL. For each measurement, 2 mL reactor content was filtered through a 0.2 pm polyethersulfone (PES) filter (VWR) and pipetted into an LCK 1414 cuvette. The absorbance of the cuvettes was measured using a DR 3900 spectrophotometer (Hach, Loveland, US).

Acetate — HPLC - In addition to using the COD kits, the removal of acetate by the plurality of micro-organisms was analyzed using high-performance liquid chromatography (HPLC). Per time point, 1 mL of filtered reactor content (0.2 um PES) was pipetted into a 2 mL glass HPLC vial (Agilent Technologies, Santa Clara, US) and subjected to HPLC. A calibration curve of 10, 5, 2.5, 1, 0.5, 0.2 and 0.1 mM acetate was made from a 99% acetic acid solution and Milli-Q water. The column was a Hi-Plex H anion exchange column, with a particle size of 8 um, a length of 300 mm and an inner diameter of 7.7 mm (Agilent Technologies, Santa Clara, US). The column temperature was 50 °C, the eluent was 100% 0.01 M Sulfuric acid and the flow was 0.6 mL min-1. A UV detector was used to detect the acetate and was set to 210 nm.

Nitrogen and phosphor measurements - To analyze the operation of the different granules in terms of wastewater treatment, the concentrations of different nutrients was measured with a Skalar SAN" autoanalyzer (Skalar Analytical B.V. Breda, The Netherlands). The concentration of ammonium-nitrogen (NH:*-N) was measured in the ranges 5 — 500 ug L° Land 0.03 —3 mg L*, nitrite-nitrogen (NO>-N) and nitrate-nitrogen (NO+-N) were measured in the range of 1 — 100 ug L°! and 0.03 — 3 mg L“t, and phosphate-phosphorus (PO4-P) was measured in the range 0.05 — 5 mg L'!. Samples taken from the reactors were filtered (0.2 um PES) and diluted appropriately to a total volume of 10 mL according to their expected maximal concentration of 18 mg L™! for NH:*-N, NOz-N or NOs™-N, and 70 mg L*! for PO4-P.

Total inorganic nitrogen (TIN) was calculated as the sum of NHs’-N, NOz°-N and NO;'-N.

Phosphor measurements - To analyze the activity of the PAOs and the P removal for the different granules, the concentration of PO:-P was measured using the PhosVer 3 ascorbic acid method in the range 0.025 - 1.5 mgP L°! (Hach, Loveland, US). Per time point, a sample of filtered reactor content (0.2 um PES) was diluted with demi water to a total volume of 10 mL in a 15 mL tube (VWR or Greiner). The absorbance of the samples was measured at 880 nm using 10 mL demi water as a blank.

Total and volatile suspended solids - To observe the biomass concentration of each reactor and the ash content of the biomass, the total suspended solids (TSS), ash content and volatile suspended solids (VSS) in the mixed liquor were measured.

Per time point, approximately 10-15 mL of mixed liquor was filtered using 47 mm glass fiber filters (Whatman International Ltd., Maidstone, UK; pre-washed, combusted at 550 °C, and weighed). To analyze the biomass concentration in the effluent, two times 50 mL of effluent was filtered on 25 mm glass fiber filters (Whatman International Ltd., Maidstone, UK; pre-washed, combusted at 550 °C, and weighed). The added volumes of sample per time point were noted to determine the original concentrations.

The filters with the collected biomass were subsequently dried at 105 °C overnight.

After weighing the filters, they were combusted at 550 °C for at least two hours and weighed again to measure the ash content of the samples.

The TSS was determined as the weight after drying at 105°C, The Ash was determined as the weight after combustion at 550°C.

The VSS was determined as TSS — Ash.

The Ash content of biomass was determined as Ash/TSS*100%. Chlorophyll - To analyze the integration of algae into the granular biomass, chlorophyll was extracted from mixed liquor samples in PGI and PG2 according to the following procedure: 1. Take 1ml of culture and put it in a 15ml plastic vial; 2. Centrifuge at 4400rpm during 8 min at 4°C; 3. Add Sml of methanol 100% to the pellet; 4. Put 5 min in ultrasound bath to disregard the pellet with methanol; 5. Incubate the cell suspension at 60°C during 50min; 6. Incubate the cell suspension at 0°C during 15 min; 7. Centrifuge the suspension at 4400rpm during 8 min; 8. Add more methanol if the pellet is not white after centrifugation and repeat the extraction; 9. Measure chlorophyll and carotenoid content in a spectrophotometer at 470nm, 652nm and 665nm in a quartz cuvette (blank done with methanol). Specifically, the absorbance at 652 nm and 665 nm was measured to quantify chlorophyll a and b.

The concentration of chlorophyll a was estimated according to 16.72% Ages

- 9.16*Ass2, and the concentration of chlorophyll b was estimated according to 34.09% Ags: -

15.28% Ages. Elemental composition - To study the elemental composition of the biomass granules, the concentrations of C, N and P in the biomass were analyzed on a weekly basis. Per time point, 50 mL of mixed liquor was transferred into a 50 mL tube with a conical bottom and centrifuged in a SL-16 centrifuge at 5000 RPM for 15 minutes (Thermo Fisher Scientific, Waltham, US). The supernatant was discarded, and the tube was stored at -20 °C until the pellet was completely frozen. Next, the pellet was dried in an Alpha 2-4 LD freeze dryer at -80 °C for 3 days (Salm en Kipp, Breukelen, The Netherlands). The dried biomass was transferred to a 2 mL Eppendorf tube and bead beaten in a TissueLyser II (Qiagen, Hilden, Germany) to homogenize the biomass. To analyze C and N, a sample of approximately 2 - 3 mg TSS was transferred into a tin cup and analyzed by a Flash 2000 organic elemental analyzer (Interscience BV, Breda, The Netherlands). The concentration of P in the biomass was analyzed by combusting approximately 2 mg of dried biomass for 30 minutes at 550 °C in Pyrex glass tubes. Next, the combusted biomass was digested with 10 mL of persulfate (2.5%) for 30 minutes at 121 °C. Finally, the concentration of phosphate in the digested solution was measured using a Skalar SAN" autoanalyser (Skalar Analytical B.V, Breda, The Netherlands). Sludge volume index - To observe the settleability of the granules, the sludge volume index (SVI) of the biomass was analyzed. The SVI may be defined as the total volume of one gram of settled sludge after a set amount of time. A lower SVI indicates a better settleability of the biomass as the granules occupy less volume per gram compared to a higher SVI. Specifically, approximately 100 mL of mixed liquor was transferred to a 100 mL measuring cylinder and left to settle for five minutes, after which the sludge volume was recorded. The SVIS (SVI after 5 minutes) was calculated using the settled volume of the sludge, the volume of the sample, and the concentration of biomass. 16S/18S sequencing - DNA extraction from each time point and reactor was conducted in triplicate. Specifically, 2 mL of harvested photogranular biomass was centrifuged at 5500 rpm for 10min at 4°C and the supernatant discarded. The cell pellets were immediately stored at -80 °C until further processing. DNA was extracted using the DNeasy PowerSoil Pro Isolation Kit (Qiagen GmbH, Hilden, Germany). The quantity and quality of DNA were spectrophotometrically determined with a NanoDrop (ThermoFisher Scientific, USA). The 16S rRNA gene V3/V4 variable region was amplified using primer pair 341F and 805R, as described in Herlemann et al., “Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea”, The ISME Journal, 2011, which is hereby herein incorporated by reference. The 18S rRNA gene V4 variable region was amplified using the primer pair 616*f and 1132r, as described in Hugerth et al, “Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia”, PLoS ONE, 2014, which is hereby herein incorporated by reference. Both sets of primers were modified to add Illumina adapter overhang nucleotide sequences to the gene-specific sequences. Sequencing was performed using an [llumina MiSeq system (Illumina MiSeq, USA) with 300-bp reads (v3 chemistry). The raw reads were processed in the following way. After adapter trimming using crfadapt version

1.18, as described in M. Martin, "Cutadapt removes adapter sequences from high-throughput sequencing reads", EMBnet journal, 2011, which is hereby herein incorporated by reference. The R package DADA2 was used to quality filter/trim, merge paired end reads, generate amplicon sequencing variants (ASVs) and do taxonomic alignment of the sequences to the SILVA database (release 138) (hitps://www.arb-silva de), as described in Callahan et al, “dada2: high-resolution sample inference from illumina amplicon data”, Nature Methods, 2020, which is hereby herein incorporated by reference. The 16S and 18S data set was normalized using the cumulative sums scaling (CSS) function of the R package metagenomSeq version 1.24.1, as described in Paulson et al., “differential abundance analysis for microbial marker-gene surveys”, Nature Methods, 2013, which 1s hereby herein incorporated by reference . The analysis of the microbiome data was performed with the R-package p/v/oseg (version 1.26.1), as described in McMurdie and Holmes, “phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data”, PLoS ONE, 2013, which is hereby herein incorporated by reference.

Results During cultivation, the biomass concentration increased steadily for all three reactors. Specifically, the biomass in PGI and PG2 accumulated more rapidly than the aerobic granules in AGS1. Over a period of 53 days, the biomass concentration in PG] increased by

4.1 g volatile suspended solid (gVSS) L"! while the concentration in PG2 increased by 2.9 gVSS Lt. AGS! accumulated 1.7 gVSS L* during the co-cultivation phase. The biomass concentration in PG1 and PG2 did not seem to stabilize at the end of the co-cultivation phase, indicating that the operational settings did not realize a steady-state system in both reactors. The biomass concentration in AGSI did appear to stabilize and an average biomass concentration of 2.3 = 0.1 gVSS L*! was measured from day 49 to day 53. After the SRT control was started on day 8, the average SRT was 13.9 + 1.5 days, 13.1 + 1.7 days and 10.5 + 2.8 days for AGS1, PGI and PG2.

No decrease in biomass was observed during the first week of operation for all three reactors. Subsequently, The settling time of PG1 was gradually decreased from 20 to 5 minutes over the course of a week. As both AGS1 and PG2 only contained intact granular biomass, the settling time for both reactors was immediately decreased from 20 to 5 minutes.

From the kinetics studies (see above), the specific anaerobic P release rates and aerobic P reuptake rates were calculated. The anaerobic release rates were observed to increase while the aerobic release rates decrease for all reactors at the end of the co-cultivation phase. The results from the P measurements indicate that the PAOs maintained their function in PG1 and PG2 after merging the two granule types: AGs1 | 586 | 848 | 403 | 284 | Pez | 199 | 229 [| 146 | 132 | During the first two weeks of the co-cultivation, near complete ammonium removal (>99%) per cycle was observed in PG1 and PG2 while AGS1 removed approximately 33%. On day 15, the loading of N was increased from 45 to 54 mgN L™! day’! and the removal efficiencies in PGI and PG2 remained the same while the efficiency of AGS1 decreased. In AGS 1, low nitrite and nitrate accumulation at the end of the aerobic phase was observed in the range of 0.003 — 0.5 mg N "1, while negligible concentrations of nitrite were found in PG1 and PG2.

Weekly biomass samples were analyzed for their C, N, and P content to observe the effect of the co-cultivation on the elemental composition. As a reference point, also the elemental composition of the second granules were determined. After the first week of cultivation, the fraction of C in the biomass remained stable for all three reactors.

Hence, the photogranules of the invention have both higher nitrogen and phosphor content than the native second granules, and have a higher nitrogen content than the native aerobic granules.

In particular, in embodiments, the photogranule 10 may have an elemental composition comprising at least 40 wt.% carbon, at least 8 wt.% nitrogen, and at least 3 wt.% phosphorus (relative to total weight of the photogranule 10). The SVI5 of the biomass in the reactors was analyzed to gain insight into the settling properties of the granules. In the final week of the cultivation phase, the average SVI; values for each reactor were observed to be: AGS | 759%47 | Pea | 0 78772 Hence, in embodiments, the photogranule 10 may have a sludge volume index after five minutes of settling SVIs < 85 mL/g, such as < 80 mL/g, especially < 70 mL/g, such as < 60 mL/g.

Fig. 3A-J are microscope images taken from reactor samples on a weekly basis to evaluate the formation/integration of the photogranules 10. At the start of the method, PG1 and PG2 were easily distinguishable from each other due to the homogenized and intact second granules (Fig. 3A, Fig. 3G). Towards the end of the co-cultivation phase, the reactor contents of PGI from PG2 were nearly identical, indicating successful integration in both reactors.

Specifically, Fig. 3A depicts the reactor contents of PGI upon merging of homogenized second granules and intact aerobic granules. Fig. 3B depicts aerobic granules with phototrophic biomass from PGI. Fig. 3C depicts an aerobic granule with phototrophs growing in crevices from PGI. Fig. 3D depicts a photogranule in PGI at the end of the cultivation. Fig. 3E depicts aerobic granules with phototrophic biomass from PG2. Fig. 3F depicts a second granule with bacterial outgrowths in PGI. Fig. 3G depicts the reactor contents of PG2 upon merging of intact second and aerobic granules. Fig. 3H depicts a second granule in PG2. Fig. 3I depicts an overgrown aerobic granule in PG2. Fig. 3J depicts a photogranule in PG2 at the end of the co- cultivation phase. The scalebar indicates 1 mm; Fig. 3A and Fig. 3G are not to scale.

Hence, Fig. 3D and 3J schematically depict embodiments of a photogranule 10 obtainable using the method according to the invention. The photogranule 10 may comprise a plurality of micro-organisms 20, wherein the plurality of micro-organisms comprise a filamentous cyanobacterium, a polyphosphate-accumulating organism, and an alga.

As described above, the aerobic granules, the second granule, and the photogranules of the invention were subjected to 16S and 18S amplicon sequencing.

Specifically, during the method of the invention, the photogranules were subjected to the 16S and 18S amplicon sequencing at 6 different timepoints.

Table 2 indicates the microbial community composition of the second granule (PG) and photogranule PG1 based on 16S amplicon sequencing: Roo Ea | Gow Toy [wo [oo [ors [ov [5 [oor [00 Allorhizobium-Neorhizobium- 2% rove OO me OL ie

EL Ee EE [7 [15 [6 Cota Comiter ||| [we [35 Cen Dees || ae OE ee Jw | ron || oma [mm Gomer |E ae poss EE aen | [| ae [| | [| Wei [ee | ene ||| [we [me (we wen | aw | awe OE | | | Teams |e ae OE LLL ae EEE

Rew pe] ows Tow Jw [ow [us [ov [5 [oor [70 Sn | se

EE NO OL ae |E eee OE ws | | Sobor mele | va Oe Table 3 indicates the microbial community composition of the photogranule PG2 and the aerobic granule (AGS) based on 16S amplicon sequencing: Row pe] PS Gow [ow [ow [or [or [us [oor [ow [a0 [05 en NES | eee] Allorhizobium-Neorhizobium- essen | mer OEE ke |E OE ma eee | Co Comptes | || [EE | msg | | [EL | Dee [EEE

KETEN NO me OE Foon WE EE oma Gomer [EEE [EE fe LL

KT in| | |

Rew pe [as] Gown Tow [ow [ur [1 [55 [a7 Jo [wo [00 Tana [we [ve [||] whee me eT [19 19 Te Sw | ee [Jw [me [we lm wen mo ar rons | we || endo (Fm ir || iw mmm Sa sma OE

EEC amen OL an Ws mm |e Cc I Te we | | m Hence, in embodiments, bacterial members of the (microbial composition of the) photogranule 10 may, based on 16S rRNA sequencing, comprise at least 20% of the polyphosphate-accumulating organism, especially at least 25%, such as at least 30%.

Table 4 indicates the microbial community composition of the second granule (PG) and photogranule PGI based on 18S amplicon sequencing: Rew qq] Gown [ow [ow [ws [ors Jor [os Jr [00 Can | || | | |

Rew [fr TT Gown ow [oo [wv [ors [or [us Jr [00

EC NO eae || ww Tamia | nie ea OO me | mepa OL owe | Table 5 indicates the microbial community composition of the photogranule PG2 and the aerobic granule (AGS) based on 18S amplicon sequencing: Row el [ [ [ [aos] Ges Tow [oo [ors [ws [ou [ver [ou [te [en

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EEE Gm Oee Ec ec cic Gen || mw Toone ||| [efor | Pangea |E || Tew [sw [mv nm oane || || | | [wm In the depicted embodiments, the photogranule 10 may approximate an ellipsoidal shape, such as a spherical shape. Further, the photogranule 10 may have a (smallest) circularly equivalent diameter selected from the range of 0.1-2.5 mm.

The term “plurality” refers to two or more. Furthermore, the terms “a plurality of” and “a number of” may be used interchangeably.

The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms “about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” also includes embodiments wherein the term “comprises” means “consists of”.

The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of" but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species”.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims (18)

ConclusiesConclusions 1. Een werkwijze voor het verschaffen van een fotogranule (10) voor terugwinning van nutriënten, waarbij: - de werkwijze het in een sequencing batchreactor (110) aan één of meer kweekcycli (200) onderwerpen van een veelvoud van micro-organismen (20) om de fotogranule (10) te verschaffen omvat, waarbij de sequencing batchreactor (110) een reactorhoogte H heeft; - het veelvoud van micro-organismen (20) een filamenteuze cyanobacterie, een polyfosfaat-accumulerend organisme en een alg omvat; - de één of meer kweekcycli (200) een voedingsstadium (210), een kweekstadium (220), een bezinkingsstadium (230) en een effluentverwijderingsstadium (240) omvatten; - het voedingsstadium (210) het aan het veelvoud van micro-organismen (20) verschaffen van een groeimedium (30) omvat; - het kweekstadium (220) het voorzien in afwisselende donkere fasen (221) en lichte fasen (222) omvat, waarbij het veelvoud van micro-organismen (20) tijdens de donkere fasen (221) aan een eerste gemiddelde fotosynthetische fotonenfluxdichtheid Pl en tijdens de lichte fasen (222) aan een tweede gemiddelde fotosynthetische fotonenfluxdichtheid P2 blootgesteld wordt, waarbij P1/P2 < 0,05, en waarbij het kweekstadium (220) het tijdens de donkere fasen (221) opleggen van anaërobe omstandigheden aan het groeimedium (30) omvat, en waarbij het kweekstadium (220) het tijdens de lichte fasen (222) leiden van een gas (40) door het groeimedium (30) omvat, waarbij het gas (40) CO: omvat; - waarbij het bezinkingsstadium (230) een bezinkingsduur T2 gekozen uit het bereik van 0,01 — 0,05 min/cm * H heeft; en - het effluentverwijderingsstadium (240) het verwijderen van een restant van het groeimedium (30) en niet-bezonken micro-organismen omvat.A method for providing a photogranule (10) for nutrient recovery, wherein: - the method comprises subjecting a plurality of microorganisms (20) to one or more culture cycles (200) in a sequencing batch reactor (110) to provide the photogranule (10), wherein the sequencing batch reactor (110) has a reactor height H; - the plurality of microorganisms (20) comprises a filamentous cyanobacterium, a polyphosphate accumulating organism and an alga; - the one or more culture cycles (200) comprise a feeding stage (210), a culture stage (220), a settling stage (230) and an effluent removal stage (240); - the nutrient stage (210) comprises providing a growth medium (30) to the plurality of microorganisms (20); - the culture stage (220) includes providing alternating dark phases (221) and light phases (222), wherein the plurality of microorganisms (20) are at a first average photosynthetic photon flux density Pl during the dark phases (221) and during the light phases (222) is exposed to a second average photosynthetic photon flux density P2, where P1/P2 < 0.05, and where the culture stage (220) includes imposing anaerobic conditions on the growth medium (30) during the dark phases (221) and wherein the growth stage (220) comprises passing a gas (40) through the growth medium (30) during the light phases (222), the gas (40) comprising CO:; - wherein the settling stage (230) has a settling time T2 selected from the range of 0.01 - 0.05 min/cm * H; and - the effluent removal stage (240) comprises removing a remainder of the growth medium (30) and unsettled microorganisms. 2. De werkwijze volgens één van de voorgaande conclusies, waarbij het voedingsstadium (210) het verschaffen van het groeimedium (30) omvat dat een primaire voedingsstof met een eerste voedingsstofconcentratie Cl omvat, waarbij de primaire voedingsstof één of meer koolstofbronnen omvat, en waarbij het kweekstadium (220) tenminste voortduurt totdat de primaire voedingsstof in het groeimedium is verminderd tot een vooraf bepaalde tweede nutriëntconcentratie C2, waarbij C2/C1 < 0,05.The method of any one of the preceding claims, wherein the nutrient stage (210) comprises providing the growth medium (30) comprising a primary nutrient having a first nutrient concentration Cl, the primary nutrient comprising one or more carbon sources, and wherein the culture stage (220) continues at least until the primary nutrient in the growth medium has been reduced to a predetermined second nutrient concentration C2, where C2/C1 < 0.05. 3. De werkwijze volgens één van de voorgaande conclusies, waarbij de lichte fasen (222) onafhankelijk gekozen duren in het bereik van 1 — 18 uur hebben, en waarbij de donkere fasen (221) onafhankelijk gekozen duren in het bereik van 0,1 — 12 u hebben.The method of any one of the preceding claims, wherein the light phases (222) have independently selected durations in the range of 1-18 hours, and wherein the dark phases (221) are independently selected in durations in the range of 0.1-18 hours. 12 you have. 4. De werkwijze volgens één van de voorgaande conclusies, waarbij: - de filamenteuze cyanobacterie gekozen is uit de groep omvattende Alkalinema pantanalense, Leptolyngbya boryana, Cephalothrix komarekiana, Pseudanahaena biceps en Limnothrix sp.The method according to any one of the preceding claims, wherein: - the filamentous cyanobacterium is selected from the group comprising Alkalinema pantanalense, Leptolyngbya boryana, Cephalothrix komarekiana, Pseudanahaena biceps and Limnothrix sp. - het polyfosfaat-accumulerende organisme gekozen is uit de groep omvattende Candidatus Accumulibacter, Tetrasphaera sp, Dechloromonas sp. Halomonas sp., Corynebacterium sp. en Candidatus Obscuribacter, - en de alg gekozen is uit de groep omvattende Chlorella sorokiniana, Chlorococcum vacuolatum, Desmodesmus sp. en Botryosphaerella sp..- the polyphosphate accumulating organism is selected from the group comprising Candidatus Accumulibacter, Tetrasphaera sp, Dechloromonas sp. Halomonas sp., Corynebacterium sp. and Candidatus Obscuribacter, - and the alga is selected from the group comprising Chlorella sorokiniana, Chlorococcum vacuolatum, Desmodesmus sp. and Botryosphaerella sp.. 5. De werkwijze volgens één van de voorgaande conclusies, omvattende: het tijdens de één of meer kweekcycli (200) laten werken van de sequencing batchreactor (210) met een vastestofretentietijd gekozen uit het bereik van 4 - 14d.The method of any preceding claim, comprising: operating the sequencing batch reactor (210) during the one or more culturing cycles (200) with a solids retention time selected from the range of 4-14d. 6. De werkwijze volgens één van de voorgaande conclusies, omvattende: het tijdens de één of meer kweekcycli (200) laten werken van de sequencing batchreactor (110) met een hydraulischeretentietijd gekozen uit het bereik van 0,2 -3d.The method of any one of the preceding claims, comprising: operating the sequencing batch reactor (110) during the one or more breeding cycles (200) with a hydraulic retention time selected from the range of 0.2-3d. 7. De werkwijze volgens één van de voorgaande conclusies, waarbij de sequencing batchreactor (110) een reactorvolume Vr heeft, en waarbij het effluentverwijderingsstadium (240) het verwijderen van een volume van ten minste 0,6*VR uit de sequencing batchreactor (110) omvat.The method of any preceding claim, wherein the sequencing batch reactor (110) has a reactor volume Vr, and wherein the effluent removal stage (240) includes removing a volume of at least 0.6*VR from the sequencing batch reactor (110) includes. 8. De werkwijze volgens één van de voorgaande conclusies, waarbij de werkwijze het als korrel (25) verschaffen van een deel van het veelvoud van micro- organismen (20) omvat.The method of any preceding claim, wherein the method comprises providing a portion of the plurality of microorganisms (20) as a bead (25). 9. De werkwijze volgens één van de voorgaande conclusies, waarbij de werkwijze het aan meerdere kweekcycli (200) onderwerpen van het veelvoud van micro-organismen (20) omvat, waarbij het veelvoud van kweekcycli (200) een initiéle kweekcyclus (2004) en een temporeel na de initiële kweekcyclus (2002) gerangschikte terminale kweekcyclus (200b) omvat, waarbij: - de initiële kweekcyclus (200a) een initiële bezinkingsduur Ti heeft, en waarbij de terminale kweekcyclus (220b) een terminale bezinkingsduur Tt heeft, waarbij Ti > 2*T;, en/of - de initiële kweekcyclus (200a) tijdens de lichtfasen (222a) een initiële gemiddelde tweede fotosynthetische fotonenfluxdichtheid P2; heeft, en waarbij de terminale kweekcyclus (200b) tijdens de lichtfasen (222b) een terminale gemiddelde tweede fotosynthetische fotonenfluxdichtheid P2; heeft, waarbij P2; > 2%P2;; - de initiële kweekcyclus (200a) een initiële verhouding Ri van totale duur van donkere fasen (221) tot totale duur van lichte fasen (222b) heeft, en de terminale kweekcyclus (200b) een terminale verhouding R+ van totale duur van donker fasen (221) tot lichtfasen (222) heeft, waarbij Rt > 2*RL.The method of any preceding claim, wherein the method comprises subjecting the plurality of microorganisms (20) to multiple culture cycles (200), the plurality of culture cycles (200) comprising an initial culture cycle (2004) and a comprises a terminal culture cycle (200b) temporally arranged after the initial breeding cycle (2002), wherein: - the initial breeding cycle (200a) has an initial sedimentation time Ti, and wherein the terminal culture cycle (220b) has a terminal sedimentation time Tt, where Ti > 2* T 1 , and/or - the initial breeding cycle (200a) during the light phases (222a) an initial average second photosynthetic photon flux density P 2 ; and wherein the terminal breeding cycle (200b) during the light phases (222b) has a terminal average second photosynthetic photon flux density P2; has, where P2; > 2%P2;; - the initial breeding cycle (200a) has an initial ratio Ri of total duration of dark phases (221) to total duration of light phases (222b), and the terminal breeding cycle (200b) has a terminal ratio R+ of total duration of dark phases (221 ) to light phases (222), where Rt > 2*RL. 10. Een fotogranule (10) verkrijgbaar met de werkwijze volgens één van de voorgaande conclusies, waarbij de fotogranule (10) een veelvoud van micro- organismen (20) omvat, waarbij het veelvoud van micro-organismen een filamenteuze cyanobacterie, een polyfosfaat-accumulerend organisme, en een alg omvat, waarbij op basis van 16S rRNA-sequencing bacteriële leden van de fotogranule (10) ten minste voor 25% het polyfosfaat-accumulerende organisme omvatten.A photogranule (10) obtainable by the method of any preceding claim, wherein the photogranule (10) comprises a plurality of microorganisms (20), the plurality of microorganisms being a filamentous cyanobacteria, a polyphosphate-accumulating organism, and an alga, wherein, based on 16S rRNA sequencing, bacterial members of the photogranule (10) comprise at least 25% of the polyphosphate-accumulating organism. 11. De fotogranule (10) volgens conclusie 10, waarbij de fotogranule (10) een cirkelvormige equivalente diameter gekozen uit het bereik van 0,1 - 2 mm heeft.The photogranule (10) according to claim 10, wherein the photogranule (10) has a circular equivalent diameter selected from the range of 0.1 - 2 mm. 12. De fotogranule (10) volgens één van de voorgaande conclusies 10-11, waarbij de fotogranule (10) een slibvolume-index na vijf minuten bezinken SVIS < 85 ml/g heeft.The photogranule (10) according to any of the preceding claims 10-11, wherein the photogranule (10) has a sludge volume index after five minutes of settling SVIS < 85 ml/g. 13. De fotogranule (10) volgens één van de voorgaande conclusies 10-12, waarbij de fotogranule (10) een elementaire samenstelling heeft die ten minste 40 gew.% koolstof, ten minste 8 gew.% stikstof en ten minste 3 gew.% fosfor omvat.The photogranule (10) according to any one of claims 10 to 12, wherein the photogranule (10) has an elemental composition comprising at least 40 wt% carbon, at least 8 wt% nitrogen and at least 3 wt% includes phosphorus. 14. Een behandelingswerkwijze voor het behandelen van afvalwater (50) met behulp van de fotogranule (10) volgens één van de voorgaande conclusies 10-13, waarbij: - de behandelingswerkwijze het in een sequencing batch reactor (110) aan één of meer kweekcycli (200) onderwerpen van de fotogranule (10) omvat om het afvalwater (50) te behandelen, waarbij de sequencing batch reactor (110) een reactorhoogte H heeft; - de één of meer kweekcycli (200) een voedingsstadium (210), een kweekstadium (220), een bezinkingsstadium (230) en een effluentverwijderingsstadium (240) omvatten; - het voedingsstadium (210) het verschaffen van het afvalwater aan de fotogranule (10) omvat; - het kweekstadium (220) het aan de fotogranule (10) onderwerpen van het afvalwater (50) omvat om behandeld water (55) te verschaffen, waarbij het kweekstadium (220) het afwisselen van donkere fasen (221) en lichte fasen (222) omvat, waarbij de fotogranule (10) tijdens de donkere fasen (221) aan een eerste gemiddelde fotosynthetische fotonenfluxdichtheid P1 en tijdens de lichte fasen (222) aan een tweede gemiddelde fotosynthetische fotonenfluxdichtheid P2 blootgesteld wordt, waarbij P1/P2 < 0,05;A treatment method for treating waste water (50) using the photogranule (10) according to any one of the preceding claims 10-13, wherein: - the treatment method involves in a sequencing batch reactor (110) undergoing one or more culture cycles ( 200) subjecting the photogranule (10) to treat the wastewater (50), the sequencing batch reactor (110) having a reactor height H; - the one or more culture cycles (200) comprise a feeding stage (210), a culture stage (220), a settling stage (230) and an effluent removal stage (240); - the feeding stage (210) includes providing the waste water to the photogranule (10); - the culture stage (220) comprises subjecting the waste water (50) to the photogranule (10) to provide treated water (55), the culture stage (220) comprising alternating dark phases (221) and light phases (222) wherein the photogranule (10) is exposed to a first average photosynthetic photon flux density P1 during the dark phases (221) and to a second average photosynthetic photon flux density P2 during the light phases (222), where P1/P2 < 0.05; - het bezinkingsstadium (230) een bezinkingsduur T2 gekozen uit het bereik van 0,001 — 0,05 min/cm * H heeft; en - het effluentverwijderingsstadium (240) het verwijderen van het behandelde water (55) uit de sequencing batchreactor (110) omvat.- the settling stage (230) has a settling time T2 selected from the range of 0.001 - 0.05 min/cm * H; and - the effluent removal stage (240) includes removing the treated water (55) from the sequencing batch reactor (110). 15. De behandelingswerkwijze volgens conclusie 14, waarbij een hoeveelheid van extern toegevoerde zuurstof aan de sequencing batchreactor (110) < 3 mmol/L /d 1s.The treatment method of claim 14, wherein an amount of externally supplied oxygen to the sequencing batch reactor (110) is < 3 mmol/L/d 1s. 16. De behandelingswerkwijze volgens één van de voorgaande conclusies 14- 15, waarbij het kweekstadium (220) het recirculeren van gas (40) uit een kopruimte (112) van de sequencing-batchreactor (110) naar een bodeminlaat (111) van de sequencing batchreactor (110) omvat.The treatment method according to any one of claims 14 to 15, wherein the breeding stage (220) comprises recycling gas (40) from a headspace (112) of the sequencing batch reactor (110) to a bottom inlet (111) of the sequencing batch reactor (110). 17. De behandelwerkwijze volgens één van de voorgaande conclusies 14-16, waarbij de behandelingswerkwijze verder het na de één of meer kweekcycli (200) oogsten van de fotogranule (10) omvat.The treatment method according to any of the preceding claims 14-16, wherein the treatment method further comprises harvesting the photogranule (10) after the one or more cultivation cycles (200). 18. Gebruik van de fotogranule (10) volgens één van de voorgaande conclusies 10-13 voor waterbehandeling.Use of the photogranule (10) according to any one of the preceding claims 10-13 for water treatment.