Wastewater treatment

Abstract

The present invention relates to the use of carbon nanodots to treat wastewater. Specifically, the invention relates to the use of carbon nanodots for improving the efficiency of microorganism-mediate

Description

The present invention relates to the use of carbon nanodots to treat wastewater. Specifically, the invention relates to the use of carbon nanodots for improving the efficiency of microorganism-mediated wastewater treatment. Methods for treating wastewater and wastewater treatment solutions containing carbon nanodots perse are also contemplated. BACKGROUND OF INVENTION Wastewater is typically classified depending on its origin as domestic, industrial or agroindustrial. Domestic wastewater is generated from human activity and tends to contain relatively low levels of organic matter and nutrients. Industrial wastewater is usually industry specific, for example, wastewater from breweries tends to contain relatively high concentrations of sugar and alcohol, whereas wastewater generated by mining industries tends to contain relatively high levels of heavy metals. Agro-industrial wastewater is typically produced by animal production (e.g. cattle, sheep, pigs, poultry and fish), and tends to have a relatively high carbon and ammonium content. Conventional chemical treatments for wastewater include chlorination, chloroamination, ozonation and use of UV light. Biological approaches can also be used, which make use of the metabolic activities of microorganisms to decompose and convert pollutants of wastewater into biomass and associated gases (e.g. CO2, CH4, N2 and SO2). Typical biological treatments for agro-industrial wastewater includes anaerobic digestion (AD) and activated sludge treatment. AD is a sequence of processes by which microorganisms break down biodegradable material (typically manure) in the absence of oxygen. The process can be used in domestic or industrial settings to manage waste by providing both volume and mass reduction of the waste, and by reducing its chemical oxygen demand (COD). The outputs of AD, biogas and digestate, can also be of use as fuel or fertilizer, respectively. However, although AD is capable of removing carbon from the wastewater, the microorganisms are not capable of assimilating nitrogen and phosphorus, and the solid sludge that is generated still has to be disposed of in landfill sites. Furthermore, the timing of the process is not compatible with many filtration / wastewater processing plants, as the water has to be sat for many days to go through the process, necessitating large storage areas. Activated sludge treatment is an aerobic process which involves the oxidation of carbonaceous and nitrogenous biological matter, and the removal of nutrients such as nitrogen and phosphorous, by microorganisms. The aerobic microorganisms involved in the treatment process naturally predominate or are activated in the treatment environment. The microorganisms involved are composed of a diverse range of bacteria, protozoa, and sometimes even metazoa. In a typical activated sludge treatment system, raw wastewater first enters an aeration tank where microorganisms digest organic matter in the wastewater and clump together by flocculation, entrapping fine particulate matter as they do so. A liquid results which is relatively free from suspended solids and organic material, and the flocculated particles readily settle out and can be removed from a settling tank. The activated sludge is then sent back to the aeration tank to maintain the necessary microbial concentration, and any excess sludge is removed and disposed of. However, although activated sludge processes are used worldwide for the treatment of domestic wastewater, the processes have a high energy demand due to the need for mechanical aeration, and relatively low carbon capture efficiency. The efficiency can be improved by adding certain mineral-derived materials and silicon to promote the proliferation of certain bacteria such as Bacillus bacteria which secrete proteases, thereby further degrading organic matter to provide lower molecular weight substances which improves treatment time. However, minerals are only sparingly soluble in water and hence insufficient amounts of the minerals are supplied to the bacteria, and increasing amounts of sludge are created, which increases the cost of disposal. Again, due to the length of time required for completion of the process, large system areas or multiple parallel systems are required. It is therefore desirable to improve the efficiency of activated sludge processes, without increasing waste byproducts. An alternative group of photosynthetic microorganisms can be used for wastewater treatment. Photosynthetic microorganisms can broadly be divided into two groups: photosynthetic bacteria (PSB) and microalgae. Photosynthetic bacteria (PSB) include a diverse range of microorganisms capable of using light as an energy source to fix carbon dioxide. This group encompasses various types of bacteria, including but not limited to cyanobacteria, green sulfur bacteria (Chlorobiaceae), purple sulfur bacteria (Chromatiaceae), purple non-sulfur bacteria (Rhodospirillaceae), and green non-sulfur bacteria (Chloroflexaceae). PSB grow phototrophically, meaning they derive energy from sunlight and fix carbon dioxide, though they generally do not produce oxygen, with the exception of cyanobacteria. On the other hand, microalgae are eukaryotic organisms that also perform photosynthesis but differ from cyanobacteria in their cellular structure. Microalgae produce oxygen during photosynthesis and are highly efficient in nutrient removal, making them effective for wastewater treatment. PSB are known to be of use in environmental waste control, wastewater treatment, waste and disposal, by consuming nutrients and absorbing carbon dioxide. Heavy metals and organic pollutants can also be absorbed, and the resulting biomass can contain high-value byproducts such as carotenoid, co-enzyme Q10 and proteins (Chen et al., 2020). Processes using PSB are considered to be more efficient that activated sludge treatment, because while activated sludge treatment relies on the carbon cycle (hence limiting its carbon capture efficiency), PSB have a special metabolic mode which in effect breaks the carbon cycle. However, although processes using PSB are generally quicker and more efficient than activated sludge treatments, they are still too slow (average of 1 hour to treat 1 L with 1 kg of PSB) for certain applications e.g. the average wastewater treatment plant processes 450-2500 m3 of wastewater per hour. It is therefore desirable to enhance the metabolic rate of PSB, thereby improving rates of wastewater treatment, and indeed the rate of any processes which employ PSB. Microalgae also grow efficiently in wastewater and have potential as treatment agents. In particular microalgae exhibit great tolerance against toxins in wastewater, as well as removing phosphorous and nitrogen components, and hence have gained significant attention. Another promising approach is the co-culturing of microalgae with other microorganisms to enhance wastewater treatment efficiency (Abdelfattah et al., 2023). In summary, it is desirable to improve the efficiency of all wastewater treatments involving the use of microorganisms. SUMMARY OF THE INVENTION According to a first aspect of the present invention, there is provided a wastewater treatment solution comprising: - wastewater; - microorganisms suitable for wastewater treatment; and - carbon nanodots. According to a further aspect of the invention, there is provided a method for treating wastewater, comprising the following step: preparing a solution comprising: - wastewater: - microorganisms suitable for wastewater treatment; and - carbon nanodots. According to a further aspect of the invention, there is provided the use of carbon nanodots for improving the efficiency of microorganism-mediated wastewater treatment. According to a further aspect of the invention, there is provided the use of carbon nanodots for controlling flocculation and / or sedimentation times in microorganism-mediated wastewater treatment. Embodiments and preferences described hereinbelow with respect to the wastewater treatment solution apply equally to the method and uses of the invention. BRIEF DESCRIPTION OF FIGURES Figure 1A shows a comparison of organic dye treatment with PSB and carbon nanodots (left) versus PSB alone (right) at time zero. Figure 1B shows a comparison of organic dye treatment with PSB and carbon nanodots (left) versus PSB alone (right) at two minutes. Figure 1C shows a comparison of organic dye treatment with PSB and carbon nanodots (left) versus PSB alone (right) at about 5 minutes and about 40 minutes, respectively. Figure 1D shows distinct layers formed after five minutes. Figure 1E shows a mixture of organic dye treatment and carbon nanodots at time zero and after 40 minutes. DETAILED DESCRIPTION The present inventors have discovered that carbon nanodots improve the efficiency of wastewater treatment, where the wastewater treatment involves the use of microorganisms. In particular, carbon nanodots have been found to improve wastewater treatment involving bacteria, such as activated sludge treatment and treatment with photosynthetic bacteria, and also in treatments using microalgae. Carbon nanodots (also known as “carbon quantum dots” and “carbon quantum nanodots” and “C-dots”; and commonly abbreviated to C-dots, CQDs, CNDs and CDs), are carbon nanoparticles which are typically less than 10 nm in diameter and usually have some form of surface passivation. Depending on the nature of any surface passivation, the carbon nanodots may have diameter of greater than 10 nm. Carbon nanodots are particulate and approximately spherical. Suitably, the carbon nanodots have an aspect ratio (longest dimension / shortest dimension) of between about 5 and about 1, preferably between about 3 and about 1, in particular between about 2 and about 1, and most preferably about 1. These aspect ratios distinguish carbon nanodots from carbon nanotubes, carbon flakes, carbon fibres and graphene sheets, all of which have a significantly higher aspect ratio. The structure of the carbon nanodots is predominantly sp3 hybridised carbon atoms, although some degree of sp2 hybridisation may also be present (e.g. less than about 50%). Typically, the carbon nanodots will have a crystalline core of sp2 or sp3 hybridised material with a surface layer that is formed from amorphous carbon and may include embedded aromatic and / or polyaromatic carbon regions. Carbon nanodots are typically chemically stable, biocompatible, have excellent water dispersibility, large area-to-volume ratio and tuneable surface chemistry. Carbon nanodots are photoluminescent under the influence of UV or visible light. This is also a distinguishing feature compared with carbon nanotubes, carbon flakes, carbon fibres and graphene sheets. Although the fundamental mechanisms responsible for the fluorescence capability are not well understood, it is generally accepted that the mechanism involves the core composition, surface states, and their combined effects, conjugation and particle size. It is known that the fluorescence can be tuned by varying the precursor materials used to form the structural core, and also the surface passivation (Kaur etal. (2022)). Methods of synthesising carbon nano-dots are broadly split into two categories : top-down and bottom-up routes. Top-down synthetic routes involve breaking down bulk carbon structures such as graphite, carbon nanotubes and nanodiamonds using techniques such as laser ablation, arc discharge and electrochemical techniques. Bottom-up routes involve synthesising carbon nanodots from carbon-based molecular precursors, through techniques such as ultrasonication, or thermal, combustion and microwave heating. Various bottom-up syntheses are described in Examples 1a-1e. The surface of carbon nanodots are typically functionalised (which can also be described as passivation) in order to modify their properties e.g. to enhance the stability of the carbon nanodot surface; and / or increase their shelf life (by preventing the surface undergoing unwanted reactions); and in some cases to improve fluorescence intensity. The nature of the surface of the carbon nanodots is firstly dictated by the carbon-based precursors used in the initial carbon nanodot formation reaction (i.e. to form a “naked” carbon nanodot). Depending on the precursors used, the naked carbon nanodot may be immediately suitable for purpose, or it may require functionalisation. Such functionalisation tends to be carried out using a polymer or an organic compound, e.g. via amide conjugation of linear diamines (polyethyleneimine or polyethylene glycol (PEG)-type diamines) to surface-bound carboxylic acids in either the carbon nanodot formation step or in a subsequent step (Hill et al. 2016). One or more pre-modification steps may be carried out on the naked carbon nanodot, e.g. as illustrated in Example 2, where a carbon nanodot of formula CND-NH2 was modified to bear carboxyl groups, which can then be further modified e.g. as carried out in Example 4b. Essentially any carbon-containing material can be used to synthesise carbon nanodots, including (but not limited to) natural gas, grass, bell pepper, milk, coffee grinds, beer yeast powder, carbohydrates (including monosaccharides such as glucose, disaccharides such as sucrose, amino sugars (such as glucosamine) and polysaccharides (such as dextran, chitosan and cellulose)), heteroatom-containing polymers (such as polyethylene glycol and polyethyleneimine), heteroatom-containing oligomers (such as 4,7,10-trioxa-1,13-tridecanediamine (TTDDA)), amino acids, beta-amino acids, other small organic molecules (such as N-acetylcysteine, carbon tetrachloride, glycerol, ethylene glycol, ascorbic acid, urea, glutathione, phenylenediamine, ethylenediamine, citric acid and citrate). Typically, carbonbased precursor(s) used to form carbon nanodots have polar, particularly oxygen and nitrogen functionalities (e.g. amine, hydroxyl or carboxyl functionality). Combinations of starting different carbon starting sources may also be used, as well as combinations of a carbon source and non-carbon source material (e.g. ammonia, a silica composite, nitric acid) (Kahn et al., 2021). Organic carbon-rich by-products from wastewater biological treatment, such as sludge, microalgae, photosynthetic bacteria, and organic matter present in the water to be treated, can be repurposed as precursors for the synthesis of carbon nanodots. This innovative approach establishes a closed-loop system by converting waste materials into new, functional additives, reducing reliance on external inputs and promoting a sustainable, circular model for wastewater management. In one embodiment, the carbon nanodots are synthesised from materials selected from the group consisting of monosaccharides, disaccharides, amino sugars, polysaccharides, heteroatom-containing polymers, heteroatom-containing oligomers, amino acids, beta-amino acids, and other small organic molecules (including mixtures thereof). Suitably, the carbon nanodots contain hydrophilic surface groups, such as amine (primary, secondary and / or tertiary), hydroxyl, carboxyl and / or carbonyl groups. In one embodiment, the carbon nanodots are derived from PEG, or contain surface PEG-functionality, or are derived from PEI, or contain surface PEI-functionality. Polyethylene glycol (PEG) is a polymer with a repeating subunit of ethylene glycol (H-[-O-CH2CH2-]n-OH). Suitably, PEG has average molecular weight of between about 100 Da and about 5,000,000 Da, for example between about 100 Da and about 100,000 Da, between about 100 Da and about 10,000 Da, or between about 2,000 Da and about 5,000 Da. Polyethyleneimine (PEI) is a polymer with a repeating subunit of ethyleneimine (-[NH-CH2CH2]n-). Suitably, PEI has average molecular weight between about 100 Da and about 5,000,000 Da, for example between about 100 Da and about 100,000 Da, between about 100 Da and about 10,000 Da, or between about 2,000 Da and about 5,000 Da. Thus, in one embodiment the carbon nanodots contain surface PEG functionality, and / or surface PEI functionality. Both PEG and PEI have been approved by the FDA for various biomedical uses due to their being biocompatible and biodegradable, and hence carbon nanodots derived from / containing PEG and / or PEI are generally benign to the environment. In one embodiment, the carbon nanodots have a diameter of between about 0.2 nm and about 200 nm, such as between about 0.2 nm and about 150 nm, between about 0.2 nm and about 100 nm, between about 0.2 nm and about 50 nm, between about 0.2 nm and about 25 nm, between about 0.2 nm and about 20 nm, between about 0.2 nm and about 15 nm, between about 0.2 nm and about 10 nm, in particular between about 1 nm and about 8 nm. The diameter of the carbon nanodot will depend on the nature of the surface functionality e.g. carbon nanodots with high molecular weight PEG and / or PEI functionality will typically have larger diameters. A “naked” carbon nanodot will typically have a diameter of between about 0.2 nm and about 10 nm. In one embodiment, the carbon nanodots are of formula (I): CND-[(L)y-X]z wherein, CND is a carbon nanodot having a diameter of between about 0.2 nm and about 10 nm; L is a linker selected from the group consisting of C1-20 alkylene, C2-20 alkenylene and C2-20 alkynylene; any of which optionally includes 1-5 heteroatoms independently selected from -O-, -NR’- and -S-, wherein L is optionally substituted with one or more =0 moieties; wherein R’ is H or C1-6 alkyl; X is selected from the group consisting of -PEG-NR”2, -PEI-NR”2, -OH, -CO2R” and -NR”2; wherein each R” is independently selected from the group consisting of H, Ci-6 alkyl, and a moiety comprising one or more -OH and / or -CO2H groups; y is 0 or 1; wherein when y is 1 each of the CND and X moieties is attached to the L group either directly or via an amide bond; and wherein when y is 0 then each of the X moieties is attached to the L group via an amide bond; z is at least 1. The term "C1-20 alkylene" refers to a straight or branched fully saturated hydrocarbon chain having from 1 to 20 carbon atoms. Suitably alkylene is C1-12 alkylene, C1-10 alkylene, C1-8 alkylene, C1-6 alkylene, C1-5 alkylene, C1-4 alkylene, C1-3 alkylene, or C1-2 alkylene. Examples of alkylene groups include -CH2-, -CH2CH2-, -CH(CH3)-CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH2CH3)- and -CH2CH(CH2CH3)CH2-. The term "C2.2o alkenylene" refers to a straight or branched hydrocarbon chain having from 2 to 20 carbon atoms and containing at least one carbon-carbon double bond. Suitably alkenylene is C2-12 alkenylene, C2-10 alkenylene, C2.3 alkenylene, C2-6 alkenylene, C2-s alkenylene, C2-4 alkenylene, or C2.3 alkenylene. Examples of alkenylene groups include -CH=CH-, -CH=C(CH3)-, -CH2CH=CH-, -CH=CHCH2-, -CH2CH2CH=CH-, -CH2CH=C(CH3)-and -CH2CH=C(CH2CH3)-. The term C2.2o alkynylene refers to a straight or branched hydrocarbon group having from 2 to 20 carbon atoms and containing at least one carbon-carbon triple bond. Suitably alkynylene is C2-12 alkynylene, C2-10 alkynylene, C2.3 alkynylene, C2-6 alkynylene, C2-s alkynylene, C2-4 alkynylene, or C2.3 alkynylene. Examples of alkynylene groups include -C=C-, -CH2C=C-, -C=C-CH2-, -CH2CH2CeC-, -CH2CeCCH2- and -CH2CeC-CH2CH2-. The term C1-6 alkyl refers to a straight or branched fully saturated hydrocarbon group having from 1 to 6 carbon atoms. The term encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl and t-butyl. Other alkyl groups, for example C1-5 alkyl, C1-4 alkyl, Ci-3 alkyl, or Ci-2 alkyl are as defined above but contain different numbers of carbon atoms. “PEG” refers to a polyethylene glycol residue which is bonded to both group -NR”2 and to group L / directly to CND. The PEG may be unbranched or branched. In one embodiment, PEG has average molecular weight of between about 100 Da and about 5,000,000 Da, for example between about 100 Da and about 100,000 Da, between about 100 Da and about 10,000 Da, or between about 2,000 Da and about 5,000 Da. “PEI” refers to a polyethyleneimine residue which is bonded to both group to group L or directly to CND. The terminal end has functionality -NR”2. The PEI may be unbranched or branched, but is preferably branched. In one embodiment, PEI has weight average molecular weight of between about 100 Da and about 5,000,000 Da, for example between about 100 Da and about 100,000 Da, between about 100 Da and about 10,000 Da, or between about 2,000 Da and about 5,000 Da. In one embodiment, R’ is H. In one embodiment, L is a linker selected from C1-20 alkylene, or C1-20 alkenylene groups either of which optionally includes 1-5 heteroatoms selected from -O-, -N(H)-, and -S-, wherein L is optionally substituted with one or more =0 moieties. Preferably, L is -NH-C=O-. In one embodiment, R” is H or C1-6 alkyl. In one embodiment, R” is a moiety comprising one or more -OH and / or -CO2H groups e.g. a sugar or biomolecule. In one embodiment, X is selected from the group consisting of-PEG-NH2, -PEI-NH2, -CO2H and -NH2. There are no particular limitations as to the wastewater to be treated by the present invention. Examples of wastewater include domestic wastewater, industrial wastewater, and agroindustrial wastewater such as, household wastewater, wastewater from meat product production, livestock food production, dairy product production, marine food product production, cereal starch production, sugar production, meat production, livestock farming and brewing processes, and wastewater from organic chemical and inorganic chemical industries. In one embodiment, the wastewater comprises organic matter. In another embodiment, the wastewater contains inorganic matter. In another embodiment, the wastewater contains organic matter and inorganic matter. The wastewater treatment typically involves the use of microorganisms to achieve one or more of the following effects: - the decomposition and / or conversion of pollutants to biomass and associated gases; - the sedimentation of solids waste. There are no particular limitations as to the microorganisms that enable the wastewater treatment, as long as the microorganisms have ability to purify the wastewater of a pollutant included in the wastewater. In one embodiment, the microorganisms comprise bacteria. In one embodiment, the microorganisms comprise sulphur reducing bacteria. In one embodiment, the microorganisms comprise gram positive bacteria. In one embodiment, the microorganisms comprise gram negative bacteria. In one embodiment, the microorganisms comprise anaerobic bacteria. In one embodiment, the microorganisms comprise aerobic bacteria. In one embodiment, the microorganisms comprise Bacillus bacteria. In one embodiment, the microorganisms comprise cyanobacteria. In each case the bacteria may be mutated or not mutated. For activated sludge treatment, the treatment can be enabled by microorganisms, in particular bacteria, that are naturally present in the treatment environment or have been made to predominate. There are typically five major groups of microorganisms found in the aeration basin of the activated sludge process: 1. bacteria-aerobic bacteria (which remove organic nutrients); 2. protozoa (which remove and digest dispersed bacteria and suspended particles); 3. metazoa (which dominate longer age systems including lagoons); 4. filamentous bacteria (which bulk the sludge); and 5. algae and fungi. Thus, in one embodiment the treatment solution comprises activated sludge. Put another way, the microorganisms in the treatment solution enable activated sludge treatment. As described herein “to predominate” means that the microorganisms are present in predominating amounts in the biological flora, living with the treatment region (e.g. tank). Whether or not microorganisms predominate can be ascertained by techniques that are well known in the art, such as by DNA sequencing. Alternatively or additionally, microorganisms can be supplied from outside of the treatment system by seeding e.g. by addition to the wastewater before it enters the treatment (aeration) tank, or after the wastewater has been added to the treatment tank, or by addition to introduced seed sludge. Seeding can help to ensure that optimal microorganisms predominate during treatment. Suitable microorganisms that can be added include Bacillus bacteria, such as Bacillus methylotrophicus CBMB205T (Ell 194897), Bacillus subtilis subsp. subtilis DSM 10T (AJ 276351), Bacillus subtilis subsp. subtilis NBRC 3009, Bacillus subtilis subsp. subtilis ATCC 6051, or combinations thereof. In one embodiment, the microorganisms comprise photosynthetic bacteria. The photosynthetic bacteria can be gram positive or gram negative, and can be anaerobic or aerobic. Types of photosynthetic bacteria include purple sulfur bacteria (Chromatiaceae, purple non-sulfur bacteria (Rhodospirillaceae), green sulfur bacteria (Chlorobiaceae), and green non-sulfur bacteria (Chloroflexacea). In one embodiment, the microorganisms comprise microalgae. Suitably, the microalgae comprises chlorophyta (green algae) and / or cyanobacteria (blue green algae). Specific types of microalgae include Anabaena sphaerica, Botryococcus sp., Chlamydomonas Mexicana, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Chlorella variabilis, Chlorella sp., Chlorella sorokiniana, Chlorella vulgaris, Coelastrella sp., Cosmarium sp., Desmodesmus sp., Desmodesmus subspicatus, Galdieria sulphuraria, Haematococcus sp., Halochlorella rubescens, Maugeotia genuflexa, Nannochloris sp., Nostoc sp., Oscillatoria sp., Pseudochlorococcum typicum, Scenedesmus abundans, Scenedesmus bijuga, Scenedesmus sp., Scenedesmus spp. Scenedesmus obliquus, Spirogyra sp. Spirulina platensis, Spirulina sp., Vaucheria sp. and combinations thereof. Co-cultures of microorganisms are also of use, for example co-cultures of bacteria and microalgae e.g. a co-culture of activated sludge and microalgae. The wastewater treatment can be monitored by observing the water quality during the treatment process, such as by monitoring the dissolved oxygen content, oxidation-reduction potential, hydrogen ion concentration and sludge concentration, examples of which are described in Evaluation Methods below. The activity of microorganisms can also be monitored by measuring the activity of enzymes derived from the microorganisms. Furthermore, the wastewater treatment can be monitored by using a dye as a visual measure of pollutant content, for example as described in Example 5. Without wishing to be bound by theory, the present inventors believe that the addition of carbon nanodots to wastewater improves efficiency of treatment using microorganisms by acting as metabolic supplements. It is believed that the carbon nanodots act as supporting carriers with large area-to-volume ratios thereby providing additional surface area and nutrients that support the metabolic processes. In the case of photosynthetic microorganisms, it is thought that the carbon nanodots also work as light-capture enhancers. By capturing and transferring light energy more effectively, the carbon nanodots boost the photosynthetic activity of photosynthetic bacteria, leading to faster and more efficient breakdown of pollutants, as shown in Example 5. The carbon nanodots are capable of flocculating and then being sedimented along with the biomass produced by the microorganisms. Indeed, the present inventors have observed that carbon nanodots speed up the rate of microorganism growth (which results in more ingestion of matter) and the formation of e.g. biofilms, which results in faster flocculation. If required, the carbon nanodots can be removed from flocculated material by centrifugation, and reintroduced into the wastewater purification system. In this sense, the carbon nanodots function as catalysts to speed up purification processes whilst not undergoing any permanent chemical change. Thus, in one aspect of the invention, there is provided the use of carbon nanodots for improving the efficiency of microorganism-mediated wastewater treatment. In another aspect of the invention, there is provided the use of carbon nanodots for controlling flocculation and / or sedimentation times in microorganism-mediated wastewater treatment. “Controlling” typically involves enhancing or speeding up the rate of the particular process. In one embodiment, carbon nanodots are present in the wastewater treatment solution (or added to the wastewater treatment solution) at a concentration of between about 0.01 mg / mL and about 10 mg / mL. In one embodiment, in the wastewater treatment solution the ratio of carbon nanodots to microorganisms is about 1 mg per 104'8 CFU. ADVANTAGES The use of carbon nanodots in wastewater treatments, in at least in some embodiments, is expected to have one or more of the following merits or advantages: • enhance the efficacy of microorganisms that facilitate wastewater treatment; • enhance the metabolic activity of microorganisms that facilitate wastewater treatment; • enhance the rate of flocculation and / or sedimentation during wastewater treatment; • enhance the light-capture of photosynthetic microorganisms that facilitate wastewater treatment. It should be noted that in the context of the present application, when referring to a range of between about “AA” and about “BB”, the point values of AA and BB are intended to be included as possible values in the range. The invention is further illustrated by means of the following non-limiting examples. ABBREVIATIONS AD anaerobic digestion CFU colony forming units COD chemical oxygen demand DLS dynamic light scattering PEG polyethylene glycol PEI polyethyleneimine PSB photosynthetic bacteria rpm TTDDA revolutions per minute 4,7,10-trioxa-1,13-tridecanediamine EXAMPLES Chemicals Chemicals were purchased and used without further purification. Glucosamine hydrochloride, beta-alanine, glutamine, N-acetylcysteine, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide poly(ethyleneglycol) diamine, polyethyleneimine and acid violet 17 were purchased from Merck. Evaluation methods Laser diffraction The diameter of carbon nanodots can be measured using a laser diffraction method. Dynamic light scattering (DLS) is well-established technique for measuring the size and size distribution of molecules and particles based on the Brownian motion of dispersed particles. DLS typically measures the size of particles in the submicron region, and with the latest technology, lower than 1nm. Measuring dye distribution An industrial dye can be used to model a pollutant in wastewater. The dye provides a visual indication for assessing the extent of flocculation and sedimentation, which is indicative of microorganism activity. Suitable dyes include Acid Violet 17, direct red, blue 92, methylene blue and Rhodamine b. The dye concentrations are evaluated using solution Uv:Vis methodology as each dye absorbs at a specific wavelength in the region and at a specific intensity related to the dye concentration. Measuring dissolved oxygen content The dissolved oxygen content of wastewater is an indication for assessing whether the required oxygen content is excessive or insufficient for optimum microorganism activity. Dissolved oxygen content can be measured using a commercially available dissolved oxygen meter (preferably using the membrane potential method using diffusion of oxygen against the membrane of the meter). Measuring hydrogen ion content The hydrogen ion content of wastewater can be used to assess whether the nitrogen in the wastewater is being removed, because when nitrogen content in wastewater is oxidised from ammonia ions to nitrate and nitrite ions, the pH of the wastewater drops. The pH of the wastewater can be monitored using a pH electrode meter. Example 1a - Carbon nanodot formation from glucosamine hydrochloride and betaalanine (CND-CO2H) Glucosamine hydrochloride (1.20 g, 5.58 mmol) was mixed with 20 mL of deionized water. Beta-alanine (0.62 g, 6.97 mmol) was then added to the solution and stirred at 300 rpm for 30 min to ensure homogeneity. The mixture was heated under microwave irradiation (5 min (split into 1 min intervals), 500 Watts) until the solution changed to a brown-yellow viscous solution, indicating the formation of carbon nanodots bearing terminal carboxylic groups (CND-CO2H). Example 1b - Carbon nanodot formation from glucosamine hydrochloride and glutamine (CND-CO2H) Glucosamine hydrochloride (1.20 g, 5.58 mmol) was mixed with 20 mL of deionized water. Glutamine (1.02 g, 6.97 mmol) was then added to the solution and stirred at 300 rpm for 30 min to ensure homogeneity. The mixture was heated under microwave irradiation (5 min (split into 1 min intervals), 500 Watts) until the solution changed to a brown-yellow viscous solution, indicating the formation of carbon nanodots bearing terminal carboxylic groups (CND-CO2H). Example 1c - Carbon nanodot formation from glucosamine hydrochloride and N-acetylcysteine (CND-CO2H) Glucosamine hydrochloride (1.20 g, 5.58 mmol) was mixed with 20 mL of deionized water. N-acetylcysteine (1.14 g, 6.97 mmol) was then added to the solution and stirred at 300 rpm for 30 min to ensure homogeneity. The mixture was heated under microwave irradiation (5 min (split into 1 min intervals), 500 Watts) until the solution changed to a brown-yellow viscous solution, indicating the formation of carbon nanodots bearing terminal carboxylic groups (CND-CO2H). Example 1d - Carbon nanodot formation from glucosamine hydrochloride and poly(ethylene glycol) diamine (CND-NH2) Glucosamine hydrochloride (1.20 g, 5.58 mmol) was mixed with 20 mL of deionized water. A poly(ethylene glycol) diamine (6.97 mmol) selected from a molecular weight between 400 to 20,000 Da (e.g., 2.78 g of poly(ethylene glycol) diamine molecular weight of 400 Da) was then added to the solution and stirred at 300 rpm for 30 min to ensure homogeneity. The mixture was heated under microwave irradiation (5 min (split into 1 min intervals), 500 Watts) until the solution changed to a brown-yellow viscous solution, indicating the formation of carbon nanodots bearing primary amine groups (CND-NH2). Example 1e - Carbon nanodot formation from glucosamine hydrochloride and TTDDA The synthesis of carbon nanodots from glucosamine hydrochloride and 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) is described in Hill et al. (2016). Example 2 - Carbon nanodot functionalisation to form CND-L-CO2H Carbon nanodots obtained according to Example 1d (based on glucosamine hydrochloride and poly(ethylene glycol) diamine) were mixed with ionized water (20 mL), succinic anhydride (0.87 g, 8.71 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.91 g; 10 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was then concentrated under vacuum and purified by dialysis in distilled H2O for 24 hours employing 500 Da dialysis membranes to yield intermediate carbon nanodots bearing carboxylic acid groups (CND-NH-C(O)CH2CH2CO2H). Example 3a - Carbon nanodot surface functionalisation to form CND-PEGIOOO-NH2 Carbon nanodots obtained according to Example 1a (CND-CO2H, based on glucosamine hydrochloride and beta-alanine) were mixed with ionized water (20 mL), and polyethylene glycol having a weight average molecular weight of 1000 Da, bearing terminal amine groups (PEGIOOO-diamine; 8.71 g, 8.71 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.91 g; 10 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was then concentrated under vacuum and purified by dialysis in distilled H2O for 24 hours employing 500 Da dialysis membranes to yield carbon nanodots bearing PEG moieties terminating in primary amine groups (CND-CGNH-PEG1000-NH2). Example 3b - Carbon nanodot surface functionalisation to form CND-L-PEG1000-NH2 Carbon nanodots obtained according to Example 2 (CND-NH-C(O)CH2CH2CO2H) were mixed with ionized water (20 mL), and polyethylene glycol having a weight average molecular weight of 1000 Da, bearing terminal amine groups (PEG1000-diamine; 8.71 g, 8.71 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (1.91 g; 10 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was then concentrated under vacuum and purified by dialysis in distilled H2O for 24 hours employing 500 Da dialysis membranes to yield carbon nanodots bearing PEG moieties terminating in primary amine groups (CND-NH-C(0)CH2CH2CONH-PEG1000-NH2). Example 4a - Carbon nanodot surface functionalisation to form CND-PEI1000-NH2 Carbon nanodots obtained according to Example 1a (CND-CO2H, based on glucosamine hydrochloride and beta-alanine) were mixed with ionized water (20 mL), and polyethyleneimine having a weight average molecular weight of 1000 Da (PEI1000; 8.71 g, 8.71 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.91 g; 10 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was then concentrated under vacuum and purified by dialysis in distilled H2O for 24 hours employing 500 Da dialysis membranes to yield carbon nanodots bearing PEI moieties terminating in primary amine groups (CND-C(O)NH-PEHOOO-NH2). Example 4b - Carbon nanodot surface functionalisation to form CND-L-PEI1000-NH2 Carbon nanodots obtained according to Example 2 (CND-NH-C(O)CH2CH2CO2H) were mixed with ionized water (20 mL), and polyethyleneimine having a weight average molecular weight of 1000 Da (PE11000; 8.71 g, 8.71 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.91 g; 10 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was then concentrated under vacuum and purified by dialysis in distilled H2O for 24 hours employing 500 Da dialysis membranesto yield carbon nanodots bearing PEI moieties terminating in primary amine groups (CND-NH-C(0)CH2CH2CONH-PEI1000-NH2). Example 5 - Use of carbon nanodots and photosynthetic bacteria to purify wastewater solution The effect of adding carbon nanodots to a wastewater solution treatment using photosynthetic bacteria was assessed using an industrial dye, as described in Evaluation Methods above. 0.1 mL of Acid Violet 17 was dissolved in deionized to water to create a 40 mL solution. 1 g of commercially available photosynthetic bacteria was added, and the resulting solution thoroughly mixed by shaking. Control solutions containing Acid Violet 17 and photosynthetic bacteria only, and Acid Violet 17 and the carbon nanodots only, were also prepared in an analogous manner. All three solutions were left under natural light for two hours. The results are shown in Figures 1A-1D: Figure 1A shows a comparison of Condition 1: organic dye treatment with PSB and carbon nanodots (left) vs. Condition 2: PSB alone (right) at time zero; Figure 1B comparison of Condition 1: organic dye treatment with PSB and carbon nanodots (left) vs. Condition 2: PSB alone (right) at 2 minutes; Figure 1C shows comparison of Condition 1: organic dye treatment with PSB and carbon nanodots (left) at 5 minutes 42 seconds vs. Condition 2: PSB alone (right) at 40 minutes 33 seconds. Figure 1D shows a zoomed-in view of Condition 1 of Figure 1C showing organic dye recovery after 5 minutes. The dashed lines highlight the distinct layers forming within the solution, suggesting enhanced sedimentation or aggregation of the microorganisms facilitated by the carbon nanodots. Figure 1E shows a mixture of organic dye and carbon nanodots mixture from time zero to 40 minutes, showing no direct interaction in the absence of microorganisms. REFERENCES Abdelfattah et al. Environmental Science and Ecotechnology 2023, 13, 100205 Chen etal. Environmental International 2020, 137, 105417 Hill etal. Nanoscale 2016, 8, 18630 Kahn et al. International Journal of Molecular Sciences 2021,22, 6786 Kaur etal. Materials Today Sustainability 2022, 18, 100137

Claims

1. A wastewater treatment solution comprising:- wastewater;- microorganisms suitable for wastewater treatment; and- carbon nanodots.

2. A method for treating wastewater, comprising the following step: preparing a solution comprising:- wastewater:- microorganisms suitable for wastewater treatment; and- carbon nanodots.

3. Use of carbon nanodots for improving the efficiency of microorganism-mediated wastewater treatment.

4. Use of carbon nanodots for controlling flocculation and / or sedimentation times in microorganism-mediated wastewater treatment.

5. The wastewater treatment solution, method or use according to any one of claims 1 to4, wherein the microorganisms comprise bacteria.

6. The wastewater treatment solution, method or use according to claim 5, wherein the microorganisms comprise photosynthetic bacteria.

7. The wastewater treatment solution, method or use according to claim 5 or claim 6, wherein the microorganisms enable activated sludge treatment.

8. The wastewater treatment solution, method or use according to any one of claims 1 to 7, wherein the microorganisms comprise microalgae.

9. The wastewater solution, method or use according to any one of claims 1 to 8, wherein the wastewater comprises organic matter.

10. The wastewater solution, method or use according to any one of claims 1 to 9, wherein the wastewater comprises inorganic matter.

11. The wastewater solution, method or use according to any one of claims 1 to 10, wherein the carbon nanodots have a diameter of between about 0.2 nm and about 200 nm, such as between about 0.2 nm and about 150 nm, between about 0.2 nm and about 100 nm, between about 0.2 nm and about 50 nm, between about 0.2 nm and about 25 nm, between about 0.2 nm and about 20 nm, between about 0.2 nm and about 15 nm, between about 0.2 nm and about 10 nm, in particular between about 1 nm and about 8 nm.

12. The wastewater solution, method or use according to any one of claims 1 to 11, wherein the carbon nanodots contain surface PEG functionality, and / or surface PEI functionality.

13. The wastewater solution, method or use according to claim 12, wherein the PEG and / or PEI has average molecular weight of between about 100 Da and about 5,000,000 Da, for example between about 100 Da and about 100,000 Da, between about 100 Da and about 10,000 Da, or between about 2,000 Da and about 5,000 Da14. The wastewater solution, method or use according to any one of claims 1 to 11, wherein the carbon nanodots are of formula (I):CND-[(L)y-X]zwherein,CND is a carbon nanodot having a diameter of between about 0.2 nm and about 10 nm;L is a linker selected from the group consisting of C1-20 alkylene, C2-20 alkenylene and C2-20 alkynylene; any of which optionally includes 1-5 heteroatoms independently selected from -O-, -NR’- and -S-, wherein L is optionally substituted with one or more =0 moieties;wherein R’ is H or C1-6 alkyl;X is selected from the group consisting of-PEG-NR”2, -PEI-NR”2, -OH, -CO2R” and -NR”2;wherein each R” is independently selected from the group consisting of H, C1-6 alkyl, and a moiety comprising one or more -OH and / or -CO2H groups;y is 0 or 1; wherein when y is 1 each of the CND and X moieties is attached to the L group either directly or via an amide bond; and wherein when y is 0 then each of the X moieties is attached to the L group via an amide bond;z is at least 1.

15. The wastewater solution, method or use according to claim 14, wherein L is a linker selected from C1-20 alkylene, or C1-20 alkenylene groups either of which optionally including 1-5 heteroatoms selected from -O-, -N(H)-, and -S-, wherein L is optionally substituted with one or more =0 moieties.

516. The wastewater solution, method or use according to claim 14 or claim 15, wherein R’ is H.

17. The wastewater solution, method or use according to any one of claims 14 to 16, 10 wherein X is selected from the group consisting of -PEG-NH2, -PEI-NH2, -CO2H and -NH2.

18. The wastewater solution, method or use according to any one of claims 14 to 17, wherein the carbon nanodots are present in the wastewater treatment solution (or added to the wastewater treatment solution) at a concentration of between about 0.01 mg / mL and about 15 10 mg / mL.

19. The wastewater solution, method or use according to any one of claims 14 to 18, wherein the ratio of carbon nanodots to microorganisms is about 1 mg per 104'8 CFU.s

Images