NL2017470B1 - Extraction of biopolymers from aerobic granular sludge by denaturation of proteins using urea - Google Patents

Abstract

The present invention is in the field of a process of extraction of biopolymers and proteins from extracellular biopolymeric substances. Recently it has been found that biobased polymeric substances, such as extracellular polymeric sub- stances, in particular linear polysaccharides, obtainable from granular sludge can be produced in large quantities and hence a process of extraction is desired.

Description

Title Extraction of biopolymers from aerobic granular sludge by denaturation of proteins using urea

FIELD OF THE INVENTION

The present invention is in the field of a process of extraction of biopolymers and proteins from extracellular biopolymeric substances.

BACKGROUND OF THE INVENTION

Recently it has been found that biobased polymeric substances, such as extracellular polymeric substances, in particular linear polysaccharides, obtainable from granular sludge can be produced in large quantities. These substances relate to biobased carboxylic acid, which may be present in an ionic form (e.g. cationic or anionic). Examples of such production methods can be found in W02015/057067 Al, and W02015/050449 Al, whereas examples of extraction methods for obtaining said biobased polymers can be found in Dutch Patent application NL2016441 and in WO2015/190927 Al. 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/j.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 NL2011609, NL2011542, NL2011852, and NL2012089. These documents, and there contents, are incorporated by reference.

Advantageously, granules of granular sludge can be readily removed from a reactor by e.g. physical separation, settling, centrifugation, cyclonic separation, decantation, filtration, or sieving to provide extracellular polymeric substances in a small volume. Compared to separating material from a liquid phase of the reactor this means that neither huge volumes of organic nor other solvents (for extraction), nor large amounts of energy (to evaporate the liquid) are required for isolation of the extracellular polymeric substances .

Extracellular polymeric substances obtainable from granular sludge (preferably obtained from granular sludge) do not require further purification or treatment to be used for some applications, hence can be applied directly. When the extracellular polymeric substances are obtained from granular sludge the extracellular polymeric substances are preferably isolated from bacteria (cells) and/or other non-extracellular polymeric substances.

Extraction of biopolymers from aerobic granular sludge is an emerging topic, and so far amongst others extraction with alkaline substances, such as Na2C03, NaOH or NaOCl have been suggested and implemented. The processes provide acceptable results for some further applications, but still produce a mixture of components present and need relatively harsh conditions. Na2C03 is found to provide biopolymers with an average number molecular weight of 50-75 kDa, NaOH at a pH of 11-12 provides biopolymers with an average number molecular weight of 20-30 kDa, and NaOCl provides biopolymers with an average number molecular weight of about 120 kDa(100-150 kDa).

However, for various applications the extracellular polymeric substances, in this document also referred to as "biopolymers" or "biobased carboxylic acids", cannot be used directly, e.g. in view of insufficient purity, a typical (brownish) coloring of the extracellular polymeric substances, etc .

With the term "microbial process" here a microbiological conversion is meant.

Some applications of ionic biopolymeric substances per se or in extracted form have been considered. For instance application of the polymers in paper as a sizing agent, and application on a concrete or metal surface have been found beneficial. Further uses and applications, however, are still limited in extent. From another perspective use and application of biobased substances instead of e.g. chemically based substances is nowadays considered an advantage, especially in view of sustainability. Hence there is a need for further fully biobased applications, methods and products.

The present invention relates to a process for extraction of biopolymers and proteins, which overcomes one or more of the above disadvantages, without jeopardizing functionality and advantages.

SUMMARY OF THE INVENTION

The present invention relates in a first aspect to a process according to claim 1.

In the present process a biopolymeric material is provided wherein the biopolymeric substance is present in an amount of 0.1-40 wt.%, preferably 0.2-30 wt.%, more preferably 0.5-10 wt.%, even more preferably 1-8 wt.%, such as 3-5 wt.%; the amount of biopolymeric material is preferably not too high if an aqueous solution is formed in view of viscosity thereof, and is preferably not too low in view of yield; the biopolymeric material may be in the form of an aqueous solution, as a suspension of e.g. granules, as a dry material typically having a water content of 5-20 wt.%, etc. As a biopolymer an acidic or ionic biopolymer may be used, such as an anionic biopolymer, and specifically alginate (alg) or bacterial algi-nate-like exopolysaccharide (ALE) c.q. the acid form thereof, from an aqueous solution. In an example the present biopolymeric substance is of biological origin; it typically comprises further substances, such as proteins and lipids; a lipid content of the present biopolymers may be much higher than those of prior art comparable biopolymers, namely 2-5 wt.%, such as 3-4 wt. %; an analysis of an exemplary biopolymer using a PerkinElmer 983 double beam dispersive IR spectrometer showed approximately 3.2 wt.% peaks that are attributed to lipids; a protein content of the present biopolymers may be 0.1-30 wt.%, typically 0.5-20 wt.%, such as 1-10 wt. %; the present biopolymeric substance may relate to an aerobic granular sludge which are dense and spherical and which may be formed in a wastewater treatment process. The sludge comprises biofilms that are composed of extracellular polymeric substances that are directly linked together and form a three-dimensional network. The present process is specifically suited for said sludge and the like; it is considered that extraction of the polymeric substances takes place by breaking the tightly bonded polymeric network by a process of denaturing the proteins present in the biofilm. The biopolymers and proteins are in such examples present in a weight ratio of 1:1-5:1, such as 2:1-3:3 w/w. It has been found that in order to make the present biopolymeric substance sufficiently soluble, typically in water, a material comprising at least two amine groups needs to be added. Thereof 0.05-70 wt. % is added, preferably 1-60 wt. %, more preferably 2-50 wt. %, such as 5-40 wt. %; the amount may also be expressed in relative terms of maximum solubility at a given temperature; it is preferred to provide the material comprising at least two amine groups in an amount >50% of the maximum solubility, preferably 60-100% of the maximum solubility, such as 75-95%. In an example urea is provided in an amount of about 6-8M, at room temperature, which is 75-100% of the maximum soluble amount. In view of solubility and speed of the reaction an increased temperature is somewhat preferred, e.g. 303-273 K (30-100 °C); the yield of biopolymers and proteins is found not to increase significantly. The weight percentages are relative to a total mass of the final system. The material is preferably a biobased amine comprising material and is added under forming a solution, typically under stirring and/or mixing. The amine comprising material and the biopolymeric material are considered to interact, thereby making the proteins and the biopolymers in the biopolymeric material available for further processing. An advantage of the present process is that reaction conditions are typically close to neutral, e.g. at a pH of 5-9; in a preferred example the pH is from 6-8, such as slightly acidic (pH 6-6.5) to slightly alkaline (pH 7.5).

Then the biopolymer and proteins are extracted from the solution with relative ease, with distinctly identifiable fractions, and with good quality and purity. For instance urea is found to provide biopolymers with an average number molecular weight of above 150 kDa. These biopolymers may find application in fibers, as a thickener, and so on.

Typically in the supernatant the biopolymer is present, whereas the proteins are present in a gel like fraction (on the bottom). Upon heating, e.g. to 313-363 K (40-90 °C), a more or less homogenous mixture is formed. Impurities can be removed from the mixture, such as by filtering, by centrifugation, etc. The temperature is preferably not too high, such as below 70 °C, and preferably below 65 °C. After cooling down the two fractions are obtained again. The supernatant can be separated by e.g. decantation. After separation the protein can be recovered, such as under addition of acetone.

The average number molecular weight is for the present polymers typically determined by using a dilute (< 0.1 mol/1) solution of the extracted biopolymer in an alkaline monovalent solution of pH 9, e.g. with NaOH. Impurities are removed by filtering. Than dynamic light scattering is used to determine de molecular weight distribution. It has been found that the polydispersity of the extracted biopolymers is relatively low, indicating a relatively homogeneous distribution of molecular weights, i.e. molecules of similar weight.

It has been found that the extracted polymeric substances behave as hydrogels; a hydrogel is considered to relate to a network of polymer chains that are hydrophilic in which water (hence hydro) is a dispersion medium. Hydrogels may contain large amounts of water, e.g. 10-95%. By nature such gels are very soft.

The present gel may find application in absorbents, adhesives, breast implants, for cell culture, for contact lenses, in a dressing, e.g. for healing a skin or of a wound, in (medical) electrodes, in granules, e.g. for improving soil moisture, a microenvironment for biological cells, in scaffolds such as in tissue engineering, in sensors, such as for pH, temperature, in biosensors, such as for metabolite or antigen concentration, glucose detection, and in a sustained-release drug delivery system.

The present method may be further optimized by removing part of the water being present, thereby increasing an amount of biopolymeric substances. In an example, after providing the sludge, water is removed to a 1-40% w/v content of the wet sludge, more preferably 2-38 % w/v, even more preferably 3-35 % w/v, even more preferably 5-30 % w/v, such as 10-20 % w/v. For better understanding also a solid contents fraction may be used. In the latter case solid contents are typically in the range of 0.1-30 % w/w, preferably 1-10 % w/w, most preferably 4-10 % w/w, such as 5-8 % w/w. The present method is found to be more effective if part of the water is removed at an initial stage.

For the present process the temperature need not be increased and can be maintained at 10-40 °C, such as at 15-30 °C; the present process is typically performed during a period of 10-60 min, such as 20-30 min, preferably under stirring. In certain cases however elevated temperatures such as 40-90 °C are beneficial for ease of processing, such as for separation of fraction and removal of impurities; it is preferred to use a temperature in the range of 45-70 °C, such as 50-65 °C, e.g. in view of reaction time.

It is noted that some of the steps may be performed in a different sequence, and/or at a later or earlier stage.

Thereby the present invention provides a solution to one or more of the above mentioned problems.

Advantages of the present invention are detailed throughout the description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in a first aspect to a process according to claim 1.

It has been found that a compound comprising at least two amine groups causes the tightly bonded biopoly-meric networks contained within granular sludge to break by denaturing the proteins present therein. This results in solubilized aerobic granules that can be further processed downstream.

In an exemplary embodiment of the invention the biopolymeric substance used in the process comprises extracellular polymeric substances, consisting of proteins, polysaccharides and other macromolecules, such as humic acids, DNA and lipids. These polymeric substances are considered to function as an adhesive between different microorganisms and their environment and therefore provide integrity of a biofilm formed by these microorganisms.

Preferably the biopolymeric substance is alginate or bacterial alginate-like exopolysaccharide (ALE). Other biopolymeric substances could be provided as well, such as acetan, cellulose, chitosan, curdlan, cyclosophorans, dex-tran, emulsan, galactoglucopolysaccharides, galacotsamino-galactan, gellan, glucuronan, N-acetylglucosamine, N-acetyl-heparosan, hyaluronic acid, indican, kefiran, lenti-nan, levan, pullulan, scleroglucan, schizophyllan, stewar-tan, succinoglycan, xanthan, welan or any combination thereof .

In an exemplary embodiment of the invention the biopolymeric substance is granular sludge being one or more of aerobic granular sludge and anammox granular sludge. Aerobic granular sludge contains a relatively large amount of proteins as compared to algae. A large amount of proteins results in a potentially large amount of extractable biopolymeric substance after the compound comprising at least two amine groups has been added to the sludge.

Preferably the extracellular polymeric substance is dissolved in an aqueous solution at a concentration in the range of 0.1-30 % w/w, preferably 0.2-10 % w/w, more preferably 0.3-5 % w/w, even more preferably 0.5-3 % w/w, such as 1-2 % w/w. Such a concentration is high enough to enable efficient downstream processing.

Preferably the extracellular polymeric substance comprises a first weight percentage consisting of exopolysaccharides, and a second weight percentage consisting of lipids and other components being more hydrophobic than the exopolysaccharides, wherein the first weight percentage is larger than the second weight percentage, preferably 10 wt. % or more larger. As a lower weight percentage of hydro-phobic components as compared to the exopolysaccharides allows for a dissolved product to be captured in subsequent downstream processes.

In an exemplary embodiment of the invention, the extracellular polymeric substance comprises at least 50 % w/w exopolysaccharides, and 1-25 % w/w proteins, such as 2-15 % w/w proteins, and the isolated extracellular polymeric substance further may comprise 0-15 % w/w lipids.

More preferably, the granular sludge has been substantially produced by bacteria belonging to the order Pseudomonadaceae, such as pseudomonas and/or Acetobacter bacteria (aerobic granular sludge); or, by bacteria belonging to the order Plancto-mycetales (anairanox granular sludge), such as Brocadia anammoxidans, Kuenenia stuttgartiensis or Brocadia fulgida; or, combinations thereof. Bacterial granular sludge is advantageous over algae sludge or plant exopolymeric substance, as bacteria in general grow faster and require less sophisticated and costly growth conditions. Exopolysaccharides produced by bacteria are almost identical to the gums that are currently used, and thus provide a suitable alternative.

In an exemplary embodiment of the invention the extracellular polymeric substance has been produced by algae, such as brown algae. Besides bacteria, algae are also known to synthesize a wide array of extracellular polymeric substances .

In an exemplary embodiment of the invention the material comprising at least two amine groups is selected from one or more of primary and secondary diamines, such as alkyl diamine (R(NHR')2), alkanol diamine(ROH(NHR')2) , aldehyde diamine (R=0(NHR')2) r imine diamine (R=N (NHR') 2) , aromatic diamine, such as phenylenediamine, and phenol-diamine, urea (C=0 (NH2) 2) , N, N' -di a Iky lure a ( (NRH) C=0 (NR' H) ) , aldehyde N,N'-diaIkylurea ((NRH)R=0(NR'H)), N-monoalkylurea-( (NHR' ) C=0 (NH2) ) , aldehyde N-monoalkylurea ( (NHR' ) R=0 (NH2) ) wherein each R and R' is independently selected from C1-C12 alkyls, and R' is also selected from H, preferably C1-C6 alkyls, such as methyl, ethyl, propyl, iso-propyl, butyl, pentyl, and hexyl, such as N-methylurea, N-ethylurea, N,N'-dimethylurea, N,N'-diethylurea, N,N'-methylethylurea, and ( (H2N)C-C=0(NH2) ) , with the proviso that the at least two amine groups are preferably not at a same carbon of the alkyl. A variety of molecules containing at least two amine groups can be used to denature proteins within the extracellular polymeric substance. The resulting soluble extracellular polymeric substance can thereafter be further processed as desired. Preferably at least one of the amine groups is attached to the same carbon as the aldehyde or imine, preferably two or more amine groups are attached to the same carbon as the aldehyde or imine. Such a configuration of the amine groups is preferred, as this seems to be the most effective configuration for the denaturation of proteins from the extracellular polymeric substance.

In an exemplary embodiment of the invention the amount of the material comprising at least two amine groups is 0.5-70 wt. %, preferably 1-60 wt.%, more preferably 2-50 wt.%, even more preferably 5-50 wt.%, such as 10-45 wt.% or 20-40 wt.%, depending on the molecular weight of the material. Typical an amount of 0.1-12 mole/1 of diamine material is used, preferably 0.5-10 mole/1, more preferably 1-8 mole/1, such as 2-5 mole/1. At elevated temperatures an amount in the higher range may be used. Also at higher amounts of biopolymeric substance higher amounts of amine comprising material may be used. In an example urea is used in a range of 2-45 wt.%, such as 3-42 wt.%, e.g. 5-32 wt.%. A relatively large amount of the material is required to efficiently denature proteins. Around 30-50 wt. % is found to be the most advantageous mass %.

In an exemplary embodiment of the invention the biopolymer is present in an amount of 1-30 % w/v, preferably 2-20 % w/v, more preferably 3-10 % w/v, such as 5-8 % w/v. A high weight per volume percentage of extracellular biopolymeric substance provides an efficient denaturation process and a high output of soluble extracellular biopolymeric substance.

In an exemplary embodiment of the invention the process comprises the step of (iv) separating proteins and biopolymers. Depending on the desired purity of the extracellular biopolymers and proteins, respectively, a separation step can be included in the process to separate proteins from biopolymers and enhance the purity.

In an exemplary embodiment of the invention the process comprises the step of (v) recovering the biobased amine comprising material. As a relatively large amount of amine comprising material, such as urea, is required, this represents significant costs in terms of materials and waste removal. To alleviate this problem, the biobased amine comprising material can be recovered and reused.

In an exemplary embodiment of the process the bi-opolymeric material is provided on a substrate, such as on a filter; the present process may be used to clean such substrates; typically the substrate needs to be removed from an installation or the like for cleaning, or harsh chemicals need to be used, typically involving a (scheduled) down time of an installation and being labor intensive, which is unwanted. By using the present relatively mild chemicals cleaning can be performed in shorter times and without a need to remove parts.

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 the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

EXAMPLES/EXPERIMENTS

The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying examples and figures.

Details of the present methods for obtaining and characterizing e.g. ALE and alginate can be found in the documents cited in the introductory part, which documents are also in this respect incorporated by reference.

FIGURES

Figures 1 and 2 represent alginate extraction processes .

DETAILED DESCRIPTION OF THE FIGURES

Fig. 1 represents a prior art extraction process of alginate, and specifically of ALE. Two routes are shown, a first wherein alginate is extracted (step (1)) from a sludge using Na2C0s, and a second wherein NaOH is used. Alginate may be obtained in medium density (M, 50-75 kDa) or low density (L, 20-30 kDa) form (step (2)). Thereafter, in step (3), ALE may be obtained in acid form (H-), in monovalent form (e.g.

Na+) , or in divalent form (e.g. Ca2+) .

Fig. 2 represents a recent extraction processes of alginate, and specifically of ALE. Three routes are shown, a first wherein alginate is extracted (step (1)) from a sludge using NH3/ (NH4)2C03, a second wherein NaOCl is used, and a third (present invention) wherein urea is used. Alginate may be obtained in M, L, high density (H, about 120 kDa) or mixed M/H form (step (2)). Thereafter, in step (3), ALE may be obtained e.g. by precipitation in acid form (H-), in monovalent form (e.g. NH4+ and Na+) such as by evaporation, in divalent form (e.g. Ca2+) such as in a gel, or in the present invention in urea-form (U-).

RESULTS OF EXPERIMENTS

In a flask of 300 ml 6 gr of the granular sludge was added. To the granular sludge suspension 200 ml of 8M urea was added. After the extraction at 60 °C for 1 hour the sample was centrifuged and we observed a supernatant comprising predominantly ALE, and a gel-like fraction at the bottom of the flask comprising predominantly proteins. After removal of the supernatant the ALE-fraction was dried. The dried fraction comprises mainly ALE (about 30 wt. % of the added ALE and urea) . The gel fraction comprises proteins (about 20 wt. % of the added ALE), which may be considered as an extra fraction compared to prior art methods. In comparison, when using e.g. Na2C03 about 20 wt. % of the initial ALE in the form of polysaccharides was obtained.

The following section is added to support the search to the prior art and it reflects the translation of the claims into English. 1. Process for extraction of biopolymers and proteins comprising the steps of (i) providing a biopolymeric material comprising biopolymers and proteins, and a material comprising at least two amine groups, preferably a biobased amine comprising material, (ii) forming an aqueous solution comprising the biopolymeric material and the amine comprising material, wherein the biopolymeric material is present in an amount of 0.1-20 wt.%, and the amine comprising material is present in an amount of 0.05-70 wt. %, and (iii) extracting the biopolymer and proteins from the solution, wherein weight percentages are relative to a total mass of the aqueous solution. 2. Process according to claim 1, wherein the biopolymeric material comprises extracellular polymeric substances. 3. Process according to claim 1 or 2, wherein the biopolymeric material is alginate or bacterial alginate-like exopolysaccharide (ALE) . 4. Process according to claim 3, wherein the biopolymeric material is granular sludge being one or more of aerobic granular sludge and anammox granular sludge. 5. Process according to one or more of claims 2-4, wherein the extracellular polymeric substances are in aqueous solution at a concentration in the range of 0.1-30 % w/w. 6. Process according to one or more of claims 2-5, wherein the extracellular polymeric substances comprise a first weight percentage consisting of exopolysaccharides, and a second weight percentage consisting of lipids and other components being more hydrophobic than the exopolysaccharides, wherein the first weight percentage is larger than the second weight percentage, preferably 10 wt.% or higher . 7. Process according to one or more of claims 2-6, wherein the extracellular polymeric substances comprise at least 50 % w/w exopolysaccharides, and 1-25 % w/w proteins, and the isolated extracellular polymeric substances further may comprise 0-15 % w/w lipids. 8. Process according to one or more of claims 4-7, wherein the granular sludge has been substantially produced by bacteria belonging to the order Pseudomonadaceae, such as pseudomonas and/or Acetobacter bacteria (aerobic granular sludge); or, by bacteria belonging to the order Plancto-mycetales (anammox granular sludge), such as Brocadia anammoxidans, Kuenenia stuttgartiensis or Brocadia ful-gida; or, combinations thereof. 9. Process according to one or more of claims 1-7, wherein the biopolymeric material have been produced by algae, such as brown algae. 10. Process according to one or more of the preceding claims, wherein the material comprising at least two amine groups is selected from one or more of primary and secondary diamines, such as alkyl diamine (R (NHR')2), alkanol diamine (ROH(NHR')2) , aldehyde diamine (R=0(NHR')2) , imine diamine (R=N(NHR')2) , aromatic diamine, such as phenylenedia-mine, and phenol-diamine, urea (C=0 (NH2) 2) , N,N'-dialkylurea((NRH)C=0(NR'H)), aldehyde N,N'-dialkylurea- ((NRH)R=0(NR'H)), N-monoalkylurea-((NHR')C=0(NH2) ) , aldehyde N-monoalkylurea((NHR')R=0(NH2) ) wherein each R and R' is independently selected from C1-C12 alkyls, and R' is also selected from H, preferably C1-C6 alkyls, such as methyl, ethyl, propyl, iso-propyl, butyl, pentyl, and hexyl, such as N-methylurea, N-ethylurea, N,N'-dimethylurea, N,N'-diethylurea, N,N'-methylethylurea, and ((H2N)C-C=0(NH2)), with the proviso that the at least two amine groups are preferably not at a same carbon of the alkyl. 11. Process according to claim 10, wherein at least one of the amine groups is attached to the same carbon as the aldehyde or imine, preferably two or more amine groups are attached to the same carbon as the aldehyde or imine. 12. Process according to one or more of the preceding claims, wherein the amount of the material comprising at least two amine groups is 1-60 wt.%, preferably 2-50 wt.%, more preferably 5-50 wt.%, even more preferably 10-45 wt.%, such as 20-40 wt.%. 13. Process according to one or more of the preceding claims, wherein the biopolymer is present in an amount in the aqueous solution of 0.2-30 wt.%, preferably 0.5-10 wt.%, more preferably 1-8 wt.%, such as 3-5 wt.%. 14. Process according to one or more of the preceding claims, comprising the step of (iv) separating proteins and biopolymers. 15. Process according to one or more of the preceding claims, comprising the step of (v) recovering the biobased amine comprising material. 16. Process according to any of the preceding claims, wherein the biopolymeric material is provided on a substrate, such as on a filter.

Claims (16)

A method for extracting biopolymers and proteins comprising the steps of (i) providing a biopolymer material comprising biopolymers and proteins, and a material comprising at least two amine groups, preferably a biological amine comprising material, (ii) forming an aqueous solution comprising the biopolymer material and the amine-containing material, wherein the biopolymer material is present in an amount of 0.1-20% by weight and the amine-containing material is in an amount of 0.05-70% by weight. %, and (iii) extracting the biopolymer and proteins from the solution, weight percentages based on a total mass of the aqueous solution. The method of claim 1, wherein the biopolymer material comprises extracellular polymeric substances. The method of claim 1 or 2, wherein the biopolymer material is alginate or bacterial alginate-like ex ex-polysaccharide (ALE). The method of claim 3, wherein the biopolymer material is granular sludge from one or more of aerobic granular sludge and anammox granular sludge. The method of any one of claims 2 to 4, wherein the extracellular polymeric substances in aqueous solution are in a concentration in the range of 0.1-30% w / w. A method according to any one of claims 2-5, wherein the extracellular polymeric substances comprise a first weight percentage consisting of exopolysaccharides and a second weight percentage consisting of lipids and other components that are more hydrophobic than the exopolysaccharides, the first weight percentage being higher than the second weight percentage, preferably 10% by weight or higher. The method of any one of claims 2-6, wherein the extracellular polymeric substances comprise at least 50% w / w exopolysaccharides, and 1-25% w / w protein, and the isolated extracellular polymeric substances further 0- 15% w / w lipids. A method according to any one of claims 4-7, wherein the granular sludge is mainly produced by bacteria belonging to the order of Pseudomonadaceae, such as pseudomonas and / or Acetobacter bacteria (aerobic granular sludge); or, by bacteria belonging to the order Plancto-mycetales (anammox granular sludge), such as Brocadia anammoxidans, Kuenenia stuttgartiensis or Brocadia fulgida; or combinations thereof. The method of any one of claims 1-7, wherein the biopolymer material is produced by algae, such as brown algae. The method of any one of the preceding claims, wherein the material comprising at least two amino groups is selected from one or more primary and secondary diamines, such as alkyl diamine (R (NHR ') 2), alkanol diamine (ROH (NHR') 2), aldehyde diamine (R = 0 (NHR ') 2), imine diamine (R = N (NHR') 2), aromatic diamine such as phenylenediamine, and phenol diamine, urea (C = 0 (NH 2) 2), N, N 'dialkylurea ((NRH) C = O (NR'H)), aldehyde N, N'-dialkylurea ((NRH) R = 0 (NR'H)), N-monoalkyl urea ((NHR') C = 0 ( NH 2)), aldehyde-N monoalkyl urea ((NHR ') R = O (NH 2)), wherein each R and R' are independently selected from C 1 -C 12 alkyls and R 'is also selected from H, preferably C 1 -C 6 alkyls such as methyl, ethyl, propyl, iso-propyl, butyl, pentyl, and hexyl such as N-methyl urea, N-ethyl urea, N, N'-dimethyl urea, N, N'-diethyl urea, N, N'-methyl ethyl urea, and ((H 2 N) CC = O (NH 2)), provided that the at least two amine groups are preferably not on the same carbon atom of the alkyl. The method of claim 10, wherein at least one of the amine groups is attached to the same carbon atom as the aldehyde or imine, preferably two or more amine groups are attached to the same carbon atom as the aldehyde or imine. 12. A method according to any one of the preceding claims, wherein the amount of the material comprising at least two amine groups is 1-60% by weight, preferably 2-50% by weight, more preferably 5-50% by weight, most preferably 10 -45% by weight, such as 20-40% by weight. A method according to any one of the preceding claims, wherein the biopolymer is present in an amount in the aqueous solution of 0.2-30% by weight, preferably 0.5-10% by weight, more preferably 1-8% by weight. %, such as 3-5% by weight. A method according to any one or more of the preceding claims, comprising the step of (iv) separating proteins and biopolymers. A method according to any one of the preceding claims, comprising the step of (v) recovering the biological amine-containing material. A method according to any one of the preceding claims, wherein in the biopolymer material is provided on a substrate, such as on a filter.