NL2029147B1

NL2029147B1 - A method for preparing a composition comprising extracellular polymeric substances from aerobic granular sludge and a plasticizer

Info

Publication number: NL2029147B1
Application number: NL2029147A
Authority: NL
Inventors: Christiaan Verschoor Daniël; Aad George Binneveld Robbert; Jacobus Van Stralen Nicolaas
Original assignee: Haskoningdhv Nederland Bv
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2023-03-21

Abstract

The present invention relates to a method for preparing a composition comprising EPS and a plasticizer, comprising mixing EPS and a plasticizer wherein, the composition is obtained by adding a plasticizer to the obtained EPS, wherein the plasticizer is chosen from the group consisting of: esters or sugar based polymers. The invention further relates to a composition comprising EPS and a plasticizer, obtained or obtainable by the method of the present invention, as well as a film obtained by drying the composition obtained or obtainable by the method of the present invention. The invention finally relates to the use of the composition according to the present invention, or the film according to the present invention, as a liquid, moisture, vapor and/or gas barrier, coating material for fertilizer granules, film coating of seeds, paper and textile sizing, and flame retardant coating or adhesive.

Description

Title: A method for preparing a composition comprising extracellular polymeric substances from aerobic granular sludge and a plasticizer

Description

The present invention relates to a method for preparing a compasition comprising extracellular polymeric substances from aerobic granular sludge (EPS) and a plasticizer, comprising obtaining and treating EPS and mixing EPS and a plasticizer.

The present invention further relates to a composition comprising EPS and a plasticizer, a film obtained by drying said composition, the use of said composition, or said film as a liquid, moisture, vapor and/or gas barrier, coating material for fertilizer granules, film coating of seeds, paper and textile sizing, and flame retardant coating or adhesive.

Biobased polymeric substances, such as extracellular polymeric substances, can be recovered by extraction from aerobic granular sludge and can be produced in large guantities in reactors designed for this purpose. EPS are substances secreted by microorganisms, e.g. in wastewater, during growth consisting of a mixture of organic substances such as polysaccharides, proteins, nucleic acids and lipids. Examples of such production methods can be found in WO2015/057067 A1, and WO2015/050449

A1. Examples of extraction methods for obtaining biobased polymers can be found in

Dutch Patent application NL2016441 and in WO2015/190927 A1. Specific examples of obtaining these substances, such as aerobic granular sludge and anammox granular sludge, and the processes used for obtaining them are known from Water

Research, 2007, doi : 10.1016/ waters .2007.03.044 (anammox granular sludge) and

Water Science and Technology, 2007, 55(8-9), 75-81 (aerobic granular sludge).

Further, Li et al. in "Characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot plant", Water Research, Elsevier, Amsterdam, NL, Vol. 44, No. 11 {June 1 2010), pp. 3355-3364) recites specific alginates in relatively raw form. Details of the biopolymers can also be found in these documents, as well as in

Dutch Patent applications NL20118098, NL2011542, NL2011852, and NL2012089.

These documents, and there contents, are incorporated by reference in their entirety.

It is appreciated that EPS is a biobased product and is as such non-toxic and biodegradable. EPS can be applied in several ways; for example as a bio-stimulant,

as a binding agent for fertilizer products, and as a slow release coating for fertilizer.

And since it is biodegradable product, pollution is avoided. EPS is seen as a sustainable, green and economically viable alternative to conventional products. Such products often depend on fossil raw materials. EPS, on the other hand, comes from wastewater. The use of EPS in agricultural applications also contributes to an increased crop yield and/or a reduction in fertilizer use.

However, EPS without additives is too brittle, causing pieces of the composition to break off during production. Further, the use of well-known plasticizers such as glycerol and sorbitol results in a water-soluble end product, which makes it unusable for many purposes such as a slow release coating for fertilizer.

The aforementioned drawbacks, among others, are overcome by the invention as defined in the appended claims.

Summary of the invention

The present invention relates to a method for preparing a composition comprising EPS and a plasticizer, comprising mixing EPS and a plasticizer wherein EPS is obtained by a method comprising the steps of: a) providing an aerobic granular sludge comprising solid EPS in an aqueous solution; b) heating of said aerobic granular sludge; c) addition of a base to aerobic sludge, before or after heating of said aerobic granular sludge in step b) to dissolve the solid EPS; d) separating the basic aqueous solution comprising EPS from the remaining solids; e) adding an acid to the basic aqueous solution comprising solubilized EPS to form a hydrogel from said EPS; f) separating the precipitated EPS from the remaining aqueous solution to obtain EPS; and wherein, the composition is obtained by; g) adding a plasticizer to the obtained EPS, wherein the plasticizer is chosen from the group consisting of: esters, acids or sugar based polymers.

The invention further relates to a composition comprising EPS and a plasticizer, obtained or obtainable by the method of the present invention.

The invention furthermore relates to a film obtained by drying the composition obtained or obtainable by the method of the present invention.

The invention finally relates to the use of the composition according to the present invention, or the film according to the present invention, as a liquid, moisture, vapor and/or gas barrier, coating material for fertilizer granules, film coating of seeds, paper and textile sizing, and flame retardant coating or adhesive.

Brief description of drawings

The present invention is described hereinafter with reference to the accompanying drawing in which embodiments of the present invention are shown. Figure 1 depicts a process schedule EPS extraction from sludge, comprising the steps of i) providing an aerobic granular sludge comprising solid extracellular polymeric substances (EPS) in an aqueous solution; ii) heating of and addition of an alkaline to said aerobic granular sludge to dissolve the solid EPS; iii) separating the basic aqueous solution comprising

EPS (centrate) from the remaining solids (sludge cake) iv) addition of acid to the centrate; v) separating the precipitated EPS from the remaining aqueous solution to obtain EPS.

Detailed description of the invention

The EPS obtained from the aerobic granular sludge consists of proteins, polysaccharides and other organic components, including humic acids. In an exemplary composition, the organic matter content of EPS is approximately 70%, between 50 — 70% of this organic matter consists of proteins, and about 25% consists of polysaccharides. The largest part concerns neutral polysaccharides (20%), a smaller part (5%) concerns uronic acid {polysaccharides with a carboxyl group) (Kaumera Nereda Gum; samenvatting NAOP onderzoeken 2013 — 2018 https://edepot.wur.nl/501893). It is however understood that organic matter content and composition may vary depending on the source of the aerobic granular sludge.

For example the organic matter content of EPS may be between 50% and 90% or between 60% and 80%. The organic matter may consist of approximately between 50% to 70% of protein, approximately between 20% and 30% of polysaccharides and approximately between 2% and 10% uronic acid. The average molecular weight of the polymers in EPS is derived from viscosity (as determined using a Ubbelohde type viscometer which uses a capillary based method of measuring viscosity) and is approximately 70 - 100 kDa. The polysaccharides in particular have similarities with alginate, but also with, for example, ‘arabic gum’. The nutrient contents (nitrogen and phosphate) are in the same order of magnitude as for sludge; and may be about 2-3%

P, 6-9% N as a percentage of dry matter in EPS. The dry matter (DM) content of precipitated EPS is approximately 5-12%. The aforementioned EPS characteristics may vary from batch to batch, depending on, for instance, the aerobic granular sludge the EPS is extracted from, that is the type and condition of the starting material, the exact extraction parameters, and how the extracted EPS is handled and stored.

Therefore viscosity, nutrient content and dry matter content of individual samples of

EPS may deviate from the above referenced values.

EPS can be extracted in a similar way to the extraction of alginate from seaweed. EPS may be extracted from the granular sludge by heating the sludge to between 70 - 90 °C, preferably between 75 - 85 °C and raising the pH to pH 9 — 11. EPS dissolves in the water phase and may then be separated from the sludge with a centrifuge. The centrate (with the dissolved EPS) may then be acidified to pH 2 - 4 to allow the EPS to flocculate. The flocculated EPS can be separated using a plate centrifuge. About 30% of the organic matter in the granular sludge can be separated in this way as EPS (Kaumera Nereda Gum; samenvatting NAOP onderzoeken 2013 - 2018 https://edepot.wur.nl/501893).

The inventors have now developed a novel method of preparing a composition comprising EPS and a plasticizer which is flexible and resilient and is more or less water insoluble. The inventors found that in the protonated form of EPS, being an anionic polymer containing, i.e. carboxylic acids, crosslinking takes places to form a hydrogel, and that this hydrogel may be purified and dried. The inventors further found that the use of an ester in combination with said cross-linked hydrogel resulted in a water-insoluble composition. The inventors furthermore found that in the deprotonated form of EPS no cross-linking occurs and esters don’t act as plasticizer, resulting in a water-soluble composition. However, the combination of deprotonated EPS and a sugar based polymer does lead to a water-insoluble composition. It was further found that both the composition comprising protonated EPS, and an ester, as well as the 5 composition comprising deprotonated EPS, and a sugar based polymer, can be dried to obtain a water-insoluble, flexible film, and that both said compositions and said film can be used as a liquid, moisture, vapor and/or gas barrier, as a coating material for fertilizer granules, film coating of seeds, paper and textile sizing, and flame retardant coating or adhesive. For most applications it is important that the EPS is not soluble.

The finding that a combination of EPS extraction method and choice of plasticizer may result in a more or less insoluble EPS product prompted the inventors to investigate further. It was found that good results (i.e. an insoluble EPS) may be obtained by choosing an ester or sugar based polymer as plasticizer, as demonstrated by the examples below.

A first aspect of the present invention provides herewith a method for preparing a composition comprising EPS and a plasticizer, comprising mixing EPS and a plasticizer wherein EPS is obtained by a method comprising the steps of: a) providing an aerobic granular sludge comprising solid extracellular polymeric substances (EPS) in an aqueous solution; b) heating of said aerobic granular sludge; c) addition of a base to the heated aerobic sludge to dissolve the solid EPS; d) separating the basic aqueous solution comprising EPS from the remaining solids; e) adding an acid to the basic aqueous solution comprising solubilized EPS to form a hydrogel from said EPS; f) separating the precipitated EPS from the remaining aqueous solution to obtain EPS; and wherein, the composition is obtained by; g) adding a plasticizer to the obtained EPS, wherein the plasticizer is chosen from the group consisting of: esters, acids or sugar based polymers.

Sludge is understood to be a semi-solid slurry that can be produced from a range of industrial processes, from water treatment, wastewater treatment or on-site sanitation systems. Aerobic granular sludge is understood to mean a type of sludge that can self- immobilize flocs and microorganisms into spherical and strong compact structures.

The advantages of aerobic granular sludge are settleability, high biomass retention, simultaneous nutrient removal and tolerance to toxicity. Aerobic granular sludge is applied as a wastewater treatment.

Solid extracellular polymeric substances (EPS) is understood to mean substances secreted by microorganisms during growth consisting of various organic substances such as polysaccharides, proteins, nucleic acids and lipids.

EPS is understood to mean a mixture of organic polymers that can be released from aerobic granular sludge by extraction at a temperature of between 70 - 90 °C, preferably between 75 and 85 °C, and a pH in the range of 9 — 11. The average molecular weight (EPS contains a mixture of different length polymers) of the extracted biopolymers is determined on the basis of the intrinsic viscosity of EPS at different dilutions and the so-called Mark Houwink equation, an empirical formula with which the average molecular weight of organic polymer mixtures can be calculated. With extraction time of 0.5 — 6 hours and a temperature of 80 °C, the molecular weight of the extracted EPS is approximately 70-100 kDa, with an extraction time of 24 hours or a 25% higher lye dosage the molecular weight is approximately 20-40 kDa. It is therefore clear to the skilled person that the average molecular weight of EPS is dependent on the type of sludge and the extraction conditions. Therefore the average molecular weight can be varied depending on the intended application. In general, a higher molecular weight results in a better coating of materials. EPS with a lower molecular weight, and lower viscosity, is more soluble.

It is appreciated that because EPS may be extracted at a high temperature between 70-90 °C, preferably between 75 — 85 °C, the structure of the sludge changes and disintegrates and therefore has a low viscosity.

Another typical feature of EPS is its ability to bond with calcium and other polyvalent cations. Alginate from seaweed is known to bind very strongly to calcium. Compared to seaweed alginate, EPS has less affinity with calcium but more with magnesium. The binding with multivalent cations by EPS is useful in several applications, for example in pelleting calcium/magnesium fertilizers, and in building materials based on calcium lime hemp blocks.

It is further appreciated that EPS can retain approximately 15 times its own weight in dry matter in water, which is a useful property for agricultural and horticultural applications, food production, gardening, horticulture, forestry, recreational areas such as play grounds, and golf-courses, for example to improve water conservation in soils, to improve soil drainage, as moisture absorber in soil, to maks the soil surrounding seeds moist. Use of EFS to soil can result in earlier seed germination and/or blooming, decreased irrigation requirements, increased propagation, increased crop growth, increased crop production, a reduction in fertilizer and pesticide use and decreased soil crusting. EPS can further be used as a bio-stimulant, a binding agent for fertilizer products, and as a slow release coating for fertilizer.

It is also appreciated that adding a coating of EPS to paper and textile makes it hydrophobic. After coating, the paper or textile absorbs less water compared to uncoated paper. The reduction achieved with a EPS coating is close to that of normally applied sizing agents.

The DM content of precipitated EPS is 5-12% DM. The extraction conditions and the settings of the centrifuges will determine the DM content together.

Approximately 50%-70% of the organic matter in EPS consists of proteins. About 25% of the organic matter in EPS consists of polysaccharides. The main part concerns neutral polysaccharides (20%), a smaller part (5%) concerns uronic acids (polysaccharides with a carboxyl group).

It is appreciated that because EPS is a complex mixture of organic substances, it is difficult to determine exactly what organic compounds it is made up of. A rough estimate of dry matter content of various organic substances can however be made.

See example 1.

A plasticizer is understood to mean a substance that is added to a compound to make it softer and more flexible and to increase its plasticity. In the EPS compositions according to the invention, the plasticizer is selected from an ester, acid, or sugar based polymer. A sugar based polymer is understood to mean a polysaccharide, where the sugar is the bulk polymer composition, or derivatives thereof. The use of an ester or an acid in combination with protonated EPS and the use of an sugar based polymer in combination with deprotonated EPS results in a flexible and resilient and water-insoluble composition. It is understood that an ester is formed from an acid wherein at least one -OH group is replaced with an -O-alkyl group, for example through substitution reaction with of a carboxylic acid and an alcohol. In a preferred embodiment, the ester is an alkyl, di-alkyl or tri-alkyl ester formed from an acid, wherein the alkyl is each individually selected from methyl, ethyl, propyl, butyl or isobutyl, and wherein the acid is selected from a linear or branched, preferably linear,

C4 — C10 mono-, di- or tri-carboxylic acid, or combinations thereof. For example, the carboxylic acid may by a C4, C5, C6, C7, C8, C9 or C10 carboxylic acid (carboxylate).

Preferably the alkyl is saturated. Preferably the carboxylic acid is saturated. In a further preferred embodiment, the ester is selected from the group consisting of dibutyl sebacate, dimethyl adipate, dimethyl pimelate, ethyl heptanoate, ethyl hexanoate, methyl butyrate, triethyl citrate, diethyl phthalate, ethyl benzoate, or a combination thereof. In a preferred embodiment the acid is 2-ethylbutyric acid.

It is appreciated that the concentration of EPS can be increased, to at least 25% by evaporating water, and that a plasticizer may be added to the concentrated form of

EPS.

In a preferred embodiment, the sugar based polymer is selected from Chitosan, hydroxyethyl cellulose, starch, or a combination thereof.

The method of extraction of EPS is based on alginate extraction from seaweed and is extensively researched (Extraction of structural extracellular polymeric substances from aerobic granular sludge. Felz et al. 2018). The extraction may involve heating the sludge, adding a base, extraction for a predesignated time period, separation between sludge and centrate with dissolved EPS, acidification, and separation of the precipitated EPS.

It is appreciated that both gravitationally thickened sludge and mechanically thickened sludge can be extracted. The heating of the sludge can be done in different ways, for instance, heating with hot water via a heat exchanger or injection of steam into the thickened sludge. It will be clear for the skilled person which method is the most appropriate under given circumstances and given the end result to be achieved. It is further appreciated that the sludge can be separated from the centrate with a centrifuge and that the centrate is collected in a tank to which an acid (such as hydrochloric acid) is added.

Itis further appreciated that after a basic extraction, the dissolved biopolymers can also be separated using a so-called non-solvent, such as acetone. Due to the non- solvent, the solubility of the EPS decreases, which makes settling or centrifuging easier. Using a non-solvent such as acetone also makes it easier to separate the EPS from humic acids, which are largely soluble in the non-solvent.

It is further appreciated that adding ammonium carbonate to the sludge and increasing the temperature is also a possible extraction technique. Due to the high pH, ammonium can be evaporated in the form of ammonia and optionally recovered via an acid washing step. The precipitated EPS has with this method a higher organic matter content compared to the alkaline extraction with caustic soda.

It is appreciated that the skilled person may select an appropriate base used to separate the basic aqueous solution comprising EPS from the remaining solids. A base may be any type of base suitable for performing the method of the present invention, such as for example sodium hydroxide (lye) (NaOH), potassium hydroxide (KOH), ammonium hydroxide (NHsOH) and ammonium carbonate ((NH4)2CO3). Instead of a base, or in combination with a base, a bleaching agent may be used, such as NaOCl,

KOCI, H2O2.

Itis appreciated that the skilled person may select an appropriate acid used to separate the precipitated EPS from the remaining aqueous solution. An acid may be any type of acid suitable for performing the method of the present invention, such as for example hydrochloric acid (HCL aq), sulphuric acid (H.SQ4), and nitric acid (HNO3).

Instead of an acid, or in combination with an acid, a bleaching agent may be used,

such as NaOCl, KOCI, H2Oz. Further, instead of an acid, divalent cations as crosslinker may be used, such as calcium salts, including CaCl:, CaCOs, Ca(OH); and magnesium salts, including MgCl, MgCO:3, and Mg(OH).. Furthermore, instead of an acid, an antisolvent may be used, such as methanol, ethanol, and aceton.

In a preferred embodiment after step f) of the method of the present invention, as an additional step ff, a base is added to obtain deprotonated EPS. Deprotonation of EPS allows for the use of sugar based polymers, such as chitosan, to be used as a plasticizer, resulting in a flexible, resilient, water-insoluble composition.

It is appreciated that the skilled person may select an appropriate base to deprotonate the EPS. A base may be any type of base suitable for performing the method of the present invention, such as for example sodium hydroxide (lye) (NaOH), potassium hydroxide (KOH), ammonium hydroxide (NH:OH) and ammonium carbonate ((NH4)2CO:s) (the carbonate evaporates to carbon dioxide (COs) in the process).

In a preferred embodiment, in step b) of the method of the present invention, the aerobic granular sludge is heated up to between 70 — 90 °C, preferably between 75 — 85 °C, in order to kill off most (pathogenic) bacteria, viruses and spores, but to prevent heat denaturation and aggregation of proteins.

It is appreciated that the heating of the sludge can be done in different ways, for instance, heating with hot water via a heat exchanger or injection of steam into the thickened sludge. It will be clear for the skilled person which method is the most appropriate under given circumstances and given the end result to be achieved.

In a preferred embodiment, in step c) of the method of the present invention, after the addition of the base the solution comprising the dissolved EPS has a pH of 9 — 12.

Within this pH range and at a high temperature, hydrogen bonds are broken and esters are hydrolyzed sufficiently effective.

It is appreciated that the skilled person may select an appropriate base to in order for the solution to reach a pH of 9 - 12. A base may be any type of base suitable for performing the method of the present invention, such as for example sodium hydroxide

(lye) (NaOH), potassium hydroxide (KOH), ammonium hydroxide {NH3OH) and ammonium carbonate ((NH4)2CO3). Instead of a base, or in combination with a base, a bleaching agent may be used, such as NaOCl, KOCI, H20:2.

In a preferred embodiment, in step d) of the method of the present invention, the aqueous solution comprising EPS is maintained for at least 2 hours. Maintaining EPS for 2 hours or longer will ensure that most pathogenic bacteria, viruses and spores are killed and yield is maximized.

Itis appreciated that the skilled person may determine the appropriate time for maintaining the EPS, as long as it is maintained at least 2 hours.

In a preferred embodiment, in step e) of the method of the present invention, after the addition of the acid the hydrogel has a pH of 1.5 — 5. Addition of the acid in order to achieve a hydrogel with a pH of 1.5 — 5 results in the protonation of carbon groups, forming a conjugate acid, which strongly hydrogen bonds to water, resulting in a crosslinked hydrophilic polymer that does not dissolve in water, is highly absorbent yet maintains its well defined structures.

Itis appreciated that the skilled person may select an appropriate acid to add in order for the hydrogel to reach a pH of 1.5 - 5. An acid may be any type of acid suitable for performing the method of the present invention, such as for example hydrochloric acid (HCl(aq)), sulphuric acid (H2S0s4), and nitric acid (HNO:3). Instead of an acid, or in combination with an acid, a bleaching agent may be used, such as NaOCl, KOCI, H20:2.

Further, instead of an acid, divalent cations as crosslinker may be used, such as calcium salts, including CaCl:, CaCO3, Ca(OH): and magnesium salts, including

MgCl:, MgCOs, and Mg(OH).. Furthermore, instead of an acid, an antisolvent may be used, such as methanol, ethanol, and aceton.

In a preferred embodiment, the ratio of EPS and sugar based polymer is between 1:0.25 and 1:4 more preferably, 1:0.5 and 1:2, most preferably about 1:1, or the ratio of EPS and ester is 1:0.1 and 1:1 more preferably between 1:0.2 and 1:0.5, most preferably about 1:0.25. In case too much plasticizer is used, the composition might become water-soluble. If not enough plasticizer is used, the composition might be too brittle. It has been found that maintaining the aforementioned ratios result in an optimally flexible and resilient and water-insoluble composition.

In a preferred embodiment, as an additional step h) of the method of the present invention, the composition comprising EPS and a plasticizer is dried to obtain a film.

Methods used to obtain such a film may be drum (or pan) coating or fluidized bed spray coating. In drum coating the product to be coated is coated with a continuous thin layer. The advantage of this process is the comparatively short processing time, resulting in a higher processing efficiency. Drum coating is particularly suited for encasing large, non-fluidizable particles. Fluidized bed spray coating produces an optimal surface coating through even application of the composition, even if the particles to be coated have different shapes and sizes. A film thus obtained is useful as a liquid, moisture, vapor and/or gas barrier, coating material for fertilizer granules, film coating of seeds, paper and textile sizing and flame retardant coating or adhesive.

It can further increase shelf life and/or storage stability.

A second aspect of the present invention provides herewith a composition comprising

EPS and a plasticizer, obtained or obtainable by the method of the present invention.

The water-insoluble composition does not or at the most to a small extent penetrate into a surface to which it is applied. The coating formed can't be washed away by water, such as by rain. Surprisingly, the present composition may be applied directly to a surface and provides advantageous effects, such as, as a liquid, moisture, vapor and/or gas barrier, as well as coating material for fertilizer granules, film coating of seeds, paper and textile sizing, flame retardant coating or adhesive. It is an important characteristic of the present composition that once applied onto a surface, the coating is formed immediately, i.e. within a short time frame. As such properties of a surface are not changed significantly, e.g. by penetration of the composition or components thereof into the surface. Once applied to a surface the present composition provides beneficial effects thereto, without deterioration of the surface or optional underlying material.

A third aspect of the present invention provides herewith a film obtained by drying the composition according to the present invention. The film formed can’t be washed away by water. The film is fully integrated with an underlying surface, adheres thereto, and may be considered as a layer having suitable characteristics. The composition may be applied two or several times, e.g. if a thicker film is required, if characteristics of the subsequent coating may or should vary, etc.

It is appreciated that a film can be obtained by drying the composition at 50 — 100 °C for a period of 1 minute — 8 hours, depending on the drying method chosen. In general, temperature and duration within these ranges provide the best characteristics.

A fourth aspect of the present invention provides herewith the use of the composition or the film according to the present invention as a liquid, moisture, vapor and/or gas barrier. The characteristics of the composition and the film are such that they are very well suited to act as a liquid, moisture, vapor and/or gas barrier. The crosslinked hydrophilic polymer doesn’t dissolve in water, is highly absorbent yet maintains its well defined structures. The composition or film is fully integrated with the underlying surface, that is, adheres thereto. The properties of the surface are not changed significantly, e.g. by penetration of the composition or film into the surface. The composition or film doesn’t deteriorate the surface of the underlying material.

A fifth aspect of the present invention provides herewith the use of the composition or the film according to the present invention as coating material for fertilizer granules, film coating of seeds, paper and textile sizing, and the composition as flame retardant coating or adhesive. The characteristics of the composition and the film are such that they can be used as coating material for fertilizer granules, film coating of seeds, paper and textile sizing, flame retardant coating or adhesive. Since the composition or film is biodegradable and has controlled release properties, it is particular well suited to be used as coating for fertilizer granules. Controlling the rate of nutrient release has environmental, economic and yield benefits. The biodegradability of the composition or film is also beneficial when used as seed coating. In addition coating with the present composition or film makes seeds shape, size, weight and seed surface more uniform, which makes it easier to plant them and/or to apply active compounds that improve seed quality. Further, the composition or film, due to its characteristics, protects the seeds from biotic and abiotic stress. Due to its biodegradability and water- insolubility the composition is further ideally suited as paper and textile sizing to act as a protective filler or glaze and to adjust absorption and wear characteristics.

Further, the composition of the present invention may be used as a flame retardant coating or adhesive, due to its high water content, its large heat capacity and enthalpy of vaporization. When exposed to fire, a large amount of energy is absorbed as water heats up and evaporates. The temperature of the composition only exceeds 100 °C until it is fully dehydrated.

The invention is further detailed by the accompanying figures and examples, which are exemplary and explanatory of nature and are not limiting the scope of protection given to the invention. To the person skilled in the art it may be clear that many variants may be conceivable falling within the scope of protection, defined by the present claims.

Examples

Example 1

TABLE 1. Average composition of EPS from communal sludge from Epe,

Vroomshoop and Dinxperlo (nutrients and metals)

Remarks: Because a significant part of the phosphate and heavy metals in the sludge cake and the acid centrate, the concentrations of most of these components are lower than in secondary sludge. For EPS from industrial sludge, the concentrations of heavy metals can be much lower than for EPS from communal sludge.

The nitrogen and phosphate in EPS are probably mostly bound to the proteins in EPS.

Example 2

Fig. 1 Process schedule EPS extraction from sludge, comprising the steps of i) providing an aerobic granular sludge comprising solid extracellular polymeric substances (EPS) in an aqueous solution; ii} heating of and addition of an alkaline to said aerobic granular sludge to dissolve the solid EPS; iii) separating the basic aqueous solution comprising EPS (centrate) from the remaining solids (sludge cake) iv) addition of acid to the centrate; v) separating the precipitated EPS from the remaining aqueous solution to obtain EPS.

Example 3

Methods and materials 1. Crude EPS was obtained from a commercial production plant utilizing sludge obtained from the treatment of wastewater from a dairy processing plant. The EPS had a concentration between 6.4% and 6.9% 2. To obtain NH4-EPS (non-purified EPS which is dialysed (titrated) with NH4OH but not washed or titrated with HCI), crude EPS was titrated with 2M NH4OH to pH 9.3. 3. Plasticizer was added to either crude or NH4-EPS in a mass ratio of 20%, 25%, 50% and/or 100%, mass ratio defined as: g plasticizer/ g dry EPS. 4. An EPS/plasticizer film was obtained by film casting at 60°C for zê6h. 5. The EPS/plasticizer film was evaluated tactile and visually to identify the additives that work as plasticizer. Evaluation was based on the ability of the film to bend without breaking or deforming. 6. A selection of additives was further evaluated for the EPS/plasticizer film solubility.

For this the EPS was purified with the exception of glycerol and chitosan for which H-

EPS and NH4-EPS respectively were used.

7. One purification method was applied, referred to as 'washing': An equal volume of demi-water was added to the EPS and mixed until a homogenous suspension is obtained. This is centrifuged to obtain a hydrogel pellet. These steps were repeated four times. 8. Another purification method was applied, referred to as 'dialysis': EPS was titrated to a pH 9.3 with 2M NH4OH to obtain 'NH4-EPS'. A dialysis tube (cut-off: <10kDa) was filled with NH4-EPS and placed in a beaker with demi water. The beaker was gently stirred and the demi water was regularly replaced. After 24h the dialyzed NH4-EPS was collected and titrated with 2M HCI to obtain acid EPS again. The excess HCI was removed by dialysis in a similar way NH4-EPS was dialyzed. 9. EPS/plasticizer films were made as described in 3-4 with the only adaptation that the purified EPS from step 8 was used. 10. EPS/plasticizer films were placed in an infuser which was placed in a beaker with demi water. The beaker was gently stirred and the films were incubated overnight.

Films were dried again at 60°C for =6h. Dry film mass was measured before and after incubation to determine the amount of soluble mass in the film, denoted as mass loss. 11. Soluble film mass (mass loss) was measured 1-5 fold. A 'poor' film solubility refers to <10% mass loss, a 'good' film solubility refers to >30% mass loss.

Example 4

Methods and materials 1. Crude EPS was obtained from a commercial production plant utilizing sludge obtained from the treatment of wastewater from a dairy processing plant. The EPS had a concentration between 6.4% and 6.9% 2. The following purification method was applied, referred to as 'washing': An equal volume of demi-water was added to the EPS and mixed until a homogenous suspension is obtained. This is centrifuged to obtain a hydrogel pellet. These steps were repeated four times. 3. Another purification method was applied, referred to as 'dialysis': EPS was titrated to a pH 9.3 with 2M NH4OH to obtain 'NH4-EPS'. A dialysis tube (cut-off: <10kDa) was filled with NH4-EPS and placed in a beaker with demi water. The beaker was gently stirred and the demi water was regularly replaced. After 24h the dialyzed NH4-EPS was collected and titrated with 2M HCI to obtain acid EPS again. The excess HCI is removed by dialysis in a similar way NH4-EPS was dialyzed. 4. Plasticizer was added to either crude or purified EPS (H-EPS) in a mass ratio of 20%, 25%, 50% and/or 100%, mass ratio defined as: g plasticizer/ g dry EPS. 5. An EPS/plasticizer film was obtained by film casting at 80°C for 26h. 6. EPS/plasticizer films were placed in an infuser which was placed in a beaker with demi water. The beaker was gently stirred and the films were incubated overnight.

Films were dried again at 60°C for 26h. Dry film mass was measured before and after incubation to determine the amount of soluble mass in the film, denoted as mass loss. 7. Soluble film mass (mass loss) was measured 1-5 fold. The table shows average mass losses with standard deviations

TABLE 2. Results plasticizer film solubilities - Plasticizer concentration

Plasticizer 0% 17% 20% 33% 50% none 3.3%£2.0% | | ee] glycerol TT 20% + 0.3% 35% + 0.2% 51% + 1.7%

PEG400 562%

Legend: - Percentages plasticizer: concentration of plasticizer added 0%, 17%, 20%, 33%, 50% - Percentages results: percentage of film solubility and standard deviation

Table 3. Results degree of plastication

Plasticizer concentration

Plasticizer 0% 17% 20% 33% 50% none brittle gycerol | | flexible flexible flexible

PEG400 [| flexible flexible

Legend: - Percentages plasticizer: concentration of plasticizer added 0%, 17%, 20%, 33%, 50% - brittle or flexible: can film be bent without breaking it (flexible) or not (brittle)

Claims

CONCLUSIONS

A method of preparing a composition comprising extracellular polymeric substances (EPS) and a plasticizer comprising obtaining and treating EPS and mixing EPS with a plasticizer wherein EPS is obtained by a method comprising the steps of: a ) providing an aerobic granular sludge comprising solid EPS in an aqueous solution; b) heating said aerobic granular sludge; c) adding a base to the aerobic sludge, before or after heating said aerobic granular sludge in step b) to dissolve the solid EPS; d) separating the basic aqueous solution comprising EPS from the remaining solids; e) adding an acid to the basic aqueous solution comprising dissolved EPS to form a hydrogel from the EPS; f) separating the precipitated EPS from the remaining aqueous solution to obtain EPS; and wherein the composition is obtained by; g) adding a plasticizer to the EPS obtained, the plasticizer being selected from the group consisting of: an ester, an acid or a polymer or sugar base.

The method of claim 1 wherein the ester is an alkyl, di-alkyl or tri-alkyl ester formed from an acid, wherein the alkyl is each individually selected from methyl, ethyl, propyl, butyl or isobutyl, and wherein the acid is selected from a linear or branched, preferably linear, C4-C10 mono-, di- or tricarboxylic acid, or combinations thereof, preferably wherein the ester is selected from the group consisting of dibutyl sebacate, dimethyl adipate, dimethyl pimelate, ethyl heptanoate, ethyl hexanoate, methyl butyrate, triethyl citrate, diethyl phthalate, ethyl benzoate or a combination thereof.

3. Process according to claim 1, characterized in that the acid is 2-ethylbutyric acid.

The method of claim 1, wherein the sugar based polymer is selected from chitosan, hydroxyethyl cellulose, starch or a combination thereof.

A method according to any one of claims 1 to 4, wherein after step f) a base is added as an additional step ff to obtain deprotonated EPS.

A method according to any one of claims 1 to 5, wherein in step b) the aerobic granular sludge is heated to between 70-90°C, preferably between 75 and 85°C.

A method according to any one of claims 1 to 8, wherein in step ¢) after addition of the base, the solution containing the dissolved EPS has a pH of 9 - 12.

A method according to any one of claims 1 to 7, wherein in step d} the aqueous solution comprising EPS is maintained for at least 2 hours.

A method according to any one of claims 1 to 8, wherein in step e) the hydrogel has a pH of 1.5 - 5 after addition of the acid.

A method according to any one of claims 1 to 9, wherein the ratio of EPS and sugar-based polymer is between 1:0.25 and 1:4, more preferably 1:0.5 and 1:2, with most preferably about 1:1, or the ratio of EPS and the ratio of EPS and ester is 1:0.1 and 1:1, more preferably between 1:0.2 and 1:0.5, most preferably about 1:0.25.

A method according to any one of claims 1 to 10, wherein in additional step h) the composition comprising EPS and a plasticizer is dried to obtain a film.

A composition comprising EPS and a plasticizer obtained or obtainable by the method according to any one of claims 1 to 11.

A film obtained by drying the composition according to claim 12.

Use of the composition according to claim 12, or the film according to claim 13, as a liquid, moisture, vapor and/or gas barrier, flame retardant coating or adhesive.

Use of the composition according to claim 12, or the film according to claim 13, as coating material for fertilizer granules, film coating of seeds, paper and textiles.