FIELD OF THE INVENTION

The present invention is in the field of a process of ex ^¬ traction of biopolymers and proteins from extracellular biopolymeric substances.

BACKGROUND OF THE INVENTION

Recently it has been found that biobased polymeric sub- stances, 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 pro- duction methods can be found in WO2015/057067 Al, and

WO2015/050449 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 exam ^¬ ples 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, Amster ^¬ dam, 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.

WO 2015/076699 Al recites extraction of biopolymers under influence of salt containing water. In a later stage an alka- line re-solubilizing is applied.

US 2006/241287 Al recites methods for using ionic liquids to extract and separate a biopolymer from a biomass containing the polymer.

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 re- quire 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 NaaCC , 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. a2C03 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", can not be used di- rectly, 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 appli ^¬ cation 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 pre- sent 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 bio- films 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 pro- teins 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 3-10 , such as 6-8 , at room temperature, _. which is 75-100% of the maximum soluble amount. In view of solubility and speed of the reaction an increased tem- perature 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 bi- obased 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 frac- tions, 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 centrifuga- tion, 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 sub- stances 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, ad- hesives, 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 pe- riod 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.

Before further processing of the obtained biopolymer and/or proteins the material comprising at least two amine groups, such as urea, may be partly or fully removed, such as by filtering of the biopolymer.

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 biopolymeric networks contained within granular sludge to break by denaturing the proteins present therein. This results in solubil- ized 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 bio- film 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 ac- etan, cellulose, chitosan, curdlan, cyclosophorans, dextran, emulsan, galactoglucopolysaccharides , galacotsaminogalactan, gellan, glucuronan, N-acetylglucosamine , N-acetyl-heparosan, hyaluronic acid, indican, kefiran, lentinan, levan, pullu- lan, scleroglucan, schizophyllan, stewartan, succinoglycan , xanthan, welan or any combination thereof.

In an exemplary embodiment of the invention the biopoly- meric 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 biopoly- meric 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 prefer- ably 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 exopoly-sac- charides, 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 hydrophobic 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 extra- cellular polymeric substance comprises at least 50 % w/w ex- opolysaccharides , 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 Pseudo- monadaceae, 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. 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. Exopolysaccha- rides 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 ) , 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=0 (NR' H) ) , aldehyde N,N'-dial- kylurea ( (NRH) R=0(NR'H) ) , N-monoalkylurea ( (NHR' ) C=0 (NH ₂ ) } , aldehyde N-monoalkylurea ( (NHR' ) R=0 (N¾) ) wherein each R and R' is independently selected from C1-C12 alkyls, and R' is also selected from H, preferably Ci-Ce alkyls, such as methyl, ethyl, propyl, iso-propyl, butyl, pentyl, and hexyl, such as N- methylurea, N-ethylurea, N, N' -dimethylurea, N, N ' -diethylurea, Ν,Ν' -methylethylurea, and ( (H ₂ N) C-C=0 (N¾) } , 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 ma- terial 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 biopoly- mer 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 biopoly- mers. 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 com- prising 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 ma- terial can be recovered and reused.

In an exemplary embodiment of the process the biopoly- meric 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 installa- tion 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. Experiments on industrial filters have resulted in surprisingly good results in terms of cleaning efficiency and use of chemicals .

In an exemplary embodiment the process comprises additional steps of providing an ionic biopolymer aqueous solu- tion, wherein the biopolymer is present in an amount of 0.1-40 wt.%, adding, to the solution, 35-60 vol.% non-solvent, such as acetone (dimethyl ketone) , under mixing, and extracting the biopolymer from the solution, wherein weight c.q. volume percentages are relative to a total mass c.q. volume of the final solution.

In an exemplary embodiment the process comprises extracting a mono-valent alginate or bacterial alginate (ALE) solution from extracellular polymeric substances obtainable from granular sludge.

In an exemplary embodiment of the process the extracellular polymeric substances are in aqueous solution at a concentration in the range of 0.1-30 % w/w.

In an exemplary embodiment the process comprises the step of, directly before adding non-solvent, dissolving al- ginate under addition of 1-5 wt.% of a mono-valent salt of at least one of carbonate, hydroxide, hypochlorite, peroxide, such as hydrogenperoxide, and ammonia, preferably ammonia . In an exemplary embodiment of the process the amount of non-solvent, such as acetone, is 40-55 vol.%.

In an exemplary embodiment of the process extraction is performed by filtering.

In an exemplary embodiment the process comprises a step of redispersing biopolymer by wetting with 0.1-10 wt.% acetone .

In an exemplary embodiment of the process the alginate is ammonium-alginate , and/or wherein the non-solvent is se- lected from mono- or dialkyl ketones, such as methyl ethyl ketone, and alkoxy alkanols, such as iso propoxy ethanol, and further comprises a step of heating the alginate to a temperature above 340 K, during a period sufficient to irreversibly transfer the alginate into alginate-amide under separation of water.

Details and advantages of some of the above steps c.q. measures can be found in PCT application NL2017 /050164 , which application and its contents are incorporated by reference .

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 accom- panying 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 a2C03, 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. Ca ²⁺ ) .

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 N¾/ (NH<3 ) ₂ C03 , 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. NH ₄ ⁺ and Na ⁺ ) such as by evaporation, in divalent form (e.g. Ca ²⁺ ) 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 cen- trifuged 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 super- natant 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.

Na ₂ C0 ₃ about 20 wt . % of the initial ALE in the form of polysaccharides was obtained.