WITH SAID PROCESS AND FIBROUS ALGINATE AMIDE

FIELD OF THE INVENTION

The present invention is in the field of extraction of a biopolymer from a granular sludge, a fibrous biopolymer ob- tained by said process, in particular alginate or bacterial alginate, and a use of said biopolymer.

BACKGROUND OF THE INVENTION

In a prior art reactor set up a (non-axenic) bacteria culture may be fed with a suitable carbon sources, in an aque- ous environment. Therein dense aggregates of microorganisms are formed, typically in or embedded in an extracellular ma- trix. Such may relate to granules, to sphere like entities having a higher viscosity than water, globules, a biofilm, etc .

Some years ago the sludge produced from e.g. wastewater treatment processes, including granular sludge, was considered as a waste product, having no further use. On top of that, costs of waste disposal are high.

Recently it has been found that extracellular polymer- ic substances, in particular linear polysaccharides, obtaina- ble from granular sludge can be produced in large quantities.

Granules making up granular sludge are (dense) aggre- gates of microbial cells self-immobilized through extracellu- lar polymeric substances into a spherical form without any in- volvement of carrier material. A characterizing feature of granules of granular sludge is that they do not significantly coagulate during settling (i.e. in a reactor under reduced hy- drodynamic shear) . Extracellular polymeric substances make up a significant proportion of the total mass of the granules.

Extracellular polymeric substances comprise high- molecular weight compounds (typically >5 kDa, see also Li be- low} secreted by microorganisms into their environment. Extra- cellular polymeric substances are mostly composed of polysac- charides and proteins, and may include other macro-molecules such as DNA, lipids and humic substances.

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 sub- stances 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 re- quired for isolation of the extracellular polymeric substanc- es .

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 ex- tracellular polymeric substances are obtained from granular sludge the extracellular polymeric substances are preferably isolated from bacteria (cells) and/or other non-extracellular polymeric substances.

However, for various applications the extracellular polymeric substances, in this document also referred to as

"biopolymers", can not be used directly, e.g. in view of in- sufficient purity, a typical (brownish) coloring of the extra- cellular polymeric substances, etc. In this respect it is not- ed that the present invention is in principle equally suited for biopolymers in general.

With the term "microbial process" here a microbiologi- cal conversion is meant.

Some documents recite isolation of alginate from aero- bic granular sludge.

Li et al. in "Characterization of alginate-like ex- opolysaccharides isolated from aerobic granular sludge in pi- lot plant", Water Research, Elsevier, Amsterdam, NL, Vol. 44, No. 11 (June 1 2010), pp. 3355-3364) recites specific algi- nates in relatively raw form. These alginates are typically not suited for further use as these are at least partially colored. In the process active carbon may be used, but that is mainly for removing impurities from the solution. Also the li- pid content of the alginates is rather low (< 1 wt.%).

In WO2015/190927 Al an extraction method of biopoly- mers is recited for extracting biopolymers from dense aggre- gates formed by microbial organisms. The method specifically relates to extracting an anionic biopolymer. In a step of the method the pH is increased by addition of at least 1-20 % v/v of at least one of CI ₂ , 0C1 ^~ and H ₂ O ₂ . The addition of the CI ₂ , 0C1 ^~ or H ₂ O ₂ is found to cause discoloration of the biopolymer. In terms of efficiency this method is capable of extracting only 20% of the biopolymers being available in the aggregates being available in an aqueous solution. Also the quality of an end-product (biopolymer) is not very good, as sticky and some- times hydrophilic products are formed; as a result thereof a further use, without an extra process step for improvement, is limited .

Incidentally some documents recite alginate suspen- sions and the like.

US 2,902,446 Al recites methods of preparing suspen- sions of (sodium) alginate in concentrated preparations for the production of foams for fire extinguishing, though the document is not directly clear on amounts used.

Vallee et al. in "Synthesis and rheological properties of hydrogels based on amphophilic alginate-amide deriva- tives.", Carbohydr. Res. 2009 Jan 26 ; 344 ( 2 ) : 223-8 , recite am- phophilic derivatives of sodium alginate were prepared by co- valent attachment of dodecylamine onto the polysaccharide via amide linkages at different substitution ratios, using 2- chloro-l-methylpyridinium iodide (CMPI) as coupling reagent for activation of the carboxylate groups of the alginate. Do- decylamine is considered to have a low reactivity, hence the need for CMPI, as well as poor solubility, hence DMF. Alginate was dissolved in DMF. The method of Vallee is considered com- plex and not environmentally friendly. It is performed under cooling to 0 °C.

Chejara et al. in "Facile synthesis of new sodium al- ginate-anthracene based photosensitizers", in Polymer Bulle- tin, Vol. 72, No. 1, p. 35-48, obtain water-soluble sodium al- ginate (Na-Alg) derivatives employing anthracene moieties (Anth) . Na-Alg amide (Alg-Anth amide) , and Na-alg ester (Alg- Anth ester) derivatives were synthesized using 2-amino anthra- cene and 9-chloromethyl anthracene (9-CMA), respectively;

hence always comprising anthracene. It is not clear of water is separated during said synthesis. The synthesis is carried out at room temperature (-25 °C) .

Sailaja et.al in "Preparation of sodium alginate nano- particles by desolvation technique using iso propyl alcohol as desolvating agent", Asian J. Pharmaceutical and Clinical Re- search, 2012, Vol. 5, No. 2, p. 132-134, amongst others by continuous addition of an otherwise unknown (relative) amount of acetone at a pH of 4, in order to load a medicament, such as Ibuprofen. As mentioned only nanoparticles are formed.

WO 00/29449 A recites Biopolymer salts with low endo- toxin levels, Biopolymer compositions thereof and methods of making the same, having an endotoxin content less than about 100 endotoxin units per gram. Because of their low endotoxin content, the biopolymer salts and biopolymer compositions of this invention may be administered parenterally to a patient. In an example such a precipitation of commercially available biopolymer (alginate) , with high endotoxin levels, with metha- nol is shown, using 2/3 methanol and 1/3 alginate solution, aimed at reducing the endotoxin level. The obtained product was dried and milled. A further example shows a use of acetone instead of methanol, but the example is silent of the charac- teristics of the product formed. It is further not clear what the efficiency of the method is.

The present invention relates to a method of extrac- tion of a biopolymer from a granular sludge, a biopolymer ob- tained by said process, and a use of said biopolymer, 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. There with a simple and ef- fective process is presented for extracting biopolymers from e.g. dense aggregates formed by microbial organisms, and spe- cifically alginates. The process specifically relates to ex- tracting an ionic biopolymer, such as an anionic biopolymer, and specifically mono-valent alginate (alg) or bacterial algi- nate (ALE) from an aqueous solution. In a step an aqueous mono-valent anionic biopolymer such as alginate or bacterial alginate (ALE) solution is provided, wherein the alginate is present in an amount of 0.1-30 wt.%. Thereafter 35-60 vol . % (or vol. %) non-solvent, such as acetone (dimethyl ketone), is added under mixing, such as by stirring or shaking, to the so- lution; steps (ii) and (iv) of claim 1 may be interchanged. The acetone may typically have a water content of 5-25 %v/v. It is preferred to use relatively dry acetone having a water content as low as possible, e.g. 1-5 %v/v water/acetone mix- ture, and likewise for other non-solvents. The mixing is pref- erably a vigorous mixing, e.g. using an impellor at relatively high speeds (600-1000 rpm) . Surprisingly thereafter the bi- opolymer can be extracted from the solution with a relatively high yield. Throughout the description weight percentages are relative to a total mass of the solution, unless indicated otherwise; likewise volume percentages are calculated. The steps may be performed at an elevated temperature (e.g. 310- 375 K) , or at an (close to) environmental temperature (e.g. 280-300 K) . The present process is typically carried out in a reactor .

A step in the extraction process is treatment of the alginate solution to be able to extract the polymer as a solid material. It is found that of a range of possible extraction solvents acetone (dimethylketone) is by far the best in terms of subsequent sample removal via filtering and solvent evapo- ration to obtain a monovalent (e.g. sodium) salt of the ex- tracted polymer and is very effective/efficient; other suita- ble non-solvents are mono- or dialkyl ketones, such as methyl ethyl ketone, and alkoxy alkanols, such as iso propoxy etha- nol, giving similar results as acetone. In an example the ad- dition of about 50% acetone to a 3% sodium alginate solution in water yields about 80% extraction efficiency via filtration of the formed fibrous mass of precipitated Na-alg/Na-ALE . The precipitate is not sticky/gel like contrary to the more com- monly used precipitation with ethanol (known from current al- ginate extraction processes applied to seaweed harvesting and by analogy is intended to be used for Nereda ^® sludge) . No doubt other (non) solvents could in principle be used, but surpris- ingly the results thereof were found to be relatively poor: ethanol forms a sticky precipitate, which is difficult to work up; ether, such as diethyl ether, does not form a precipitate, and it does not mix with water; butanol forms a two phase sys- tem, with a gel like consistency; DMSO (dimethyl sulfoxide) lets alginate precipitates, but it forms a sticky gel, and DMSO is not volatile hence can not be separated easily from the alginate; ethylene glycol shows no precipitation and forms a homogenous gel; and methanol shows a similar behaviour to ethanol, and is in fact even more sticky. The acetone extrac- tion also works extremely well for other monovalent anionic biopolymers such as ammonium alginate solutions and for drying of gel-like acid ALE. Acetone is considered to be a rather op- timal treatment method as it is easy to work-up again via dis- tillation/membrane separation and analogous methods; also it is a naturally occurring compound so it does not provide any major issue if trace amounts remain in the product. Part of the acetone recovery can be combined with the drying of the biopolymer precipitate, which may involve heat such as with distillation. Other processes are available to get rid of the remaining acetone/water supernatant.

The obtained biopolymers resemble those of the prior art, but are different in certain aspects, such as character- ized in the claims; i.e. compared to e.g. algae alginate chem- ical and physical characteristics are found to be different, such as a lipid content is much higher {2-5 wt.%) and the pol- ymers have a fibrous structure comparable to cotton wool. Typ- ical fibres obtained are a few (1-2) mm to a few (1-5} cm long having a thickness of 2-250 μm (such as 10-100 μπι) and fibres may have a yellowish appearance. The different characteristics result in different applications of the present biopolymers now being possible, or likewise being impossible, compared to those of the prior art.

It is noted that numbering of the various steps are given for a better understanding of an optional sequence of the steps. 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 through- out the description.

DETAILED DESCRIPTION OE THE INVENTION

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

The present process may be further optimized by in an initial stage removing larger particles, such as by siev- ing. Preferably particles with a diameter larger than 500μπ\ are removed, more preferably particles larger than 300μm, such as larger than 200μm. Therewith it has been found that the present process is more effective, less energy consuming, and an even higher yield of biopolymer is obtained.

The present process may be further optimized by remov- ing part of the water being present, thereby increasing an amount of aggregates. In an example, after providing the sludge, water is removed to a 1-40% w/v content of the wet sludge, more preferably 5-38 % w/v, even more preferably 10-35 % w/v, even more preferably 20-32 % w/v, such as 25-30 % w/v. For better understanding also a solid contents fraction may be used. The present process is found to be more effective if part of the water is removed in an initial stage.

In an example the present process comprises the step of (i) extracting the mono-valent biopolymer such as alginate or bacterial alginate (ALE) from an aqueous solu- tion with e.g. extracellular polymeric substances obtainable from granular sludge. In the latter case the alginate is ob- tained from granular sludge in a mono-valent form. Suitable cations are K ⁺ , Na ⁺ , and NH ₄ ⁺ , preferably NH ₄ ⁺ . One might consider K ⁺ and NH ₄ ⁺ to be of a similar physical and chemical nature, e.g. in terms of radius, valence, solubility of salts containing said cation, etc. It is therefore unex- pected that NH ₄ ⁺ performs much better in terms of extraction efficiency of the biopolymer from the aqueous solution, i.e. about 20% for K ⁺ versus up to 40 % for NH ₄ ⁺ , i.e. about twice as much.

In an example of the present process the biopolymer is bacterial aerobic granular sludge or anammox granular sludge, and is selected form exopolysaccharide, preferably comprising mannuronic acid and guluronic acid residues, block-copolymers comprising uronic acid residues, alginate, lipids, and combi- nations thereof, or wherein the biopolymer is an algae biopol- ymer. In an example, the exopolysaccharides are block- copolymers comprising uronic acid (e.g. mannuronic acid and guluronic acid) residues. Especially bacterial aerobic granu- lar sludge or anammox granular sludge has been found to pro- duce high amounts of biopolymers, in good quality. By nature the biopolymers produced as such vary in their characteris- tics, e.g. composition, molecular weight, etc.

Aerobic granular sludge and anammox granular sludge, and the processes used for obtaining them are known to a person skilled in the art. For the uninitiated, reference is made to Water Research, 2007, doi:10.1016/j.watres.2007.03.044 {anam- mox granular sludge) and Water Science and. Technology, 2007, 55(8-9), 75-81 (aerobic granular sludge).

In an example of the present process 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 Planctomycetales (anammox granular sludge) , such as Brocadia anammoxidans, Kuenenia Stuttgartiensis or Brocadia fulgida; or, combinations thereof.

In an example of the present process the extracellular polymeric substances are in aqueous solution at a concentra- tion 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.

In an example of the present process the extracellular polymeric substances comprise a major portion consisting ex- opolysaccharides, and a minor portion, such as less than 30 % w/w, typically less than 10 %w/w, consisting of lipids and/or other components more hydrophobic than the exopolysaccharides. Extracellular polymeric substances obtained from granular sludge having a major portion of exopolysaccharides and a mi- nor portion of lipids have been found to provide very effec- tive water resistance.

In an example of the present process the extracellular polymeric substances comprise at least 50 % w/w exopolysaccha- rides, preferably at least 60 % w/w exopolysaccharides, most preferably at least 75 % w/w exopolysaccharides, such as at least 90 % w/w exopolysaccharides, more preferably wherein an exopolysaccharide content is less than 100 % and the isolated extracellular polymeric substances further may comprise 0.1-10 w/w% lipids, such as 0.2-5 w/w%. The exopolysaccharide content is preferably not 100 %, as a remainder has been found to con- tribute to the present advantageous effects.

In an example the present process comprises the step of, directly before adding a non-solvent as acetone, (iii) dis- solving alginate under addition of 1-5 wt . % of a mono-valent salt of at least one of carbonate, hydroxide, hypochlorite, peroxide, such as hydrogen peroxide, such as Na ⁺ , K ⁺ , and NH ₄ ⁺ - salts, e.g. and ammonia, preferably ammonia.

In an example of the present process the amount of non-solvent, such as acetone, is 40-55 vol.%. The amount of acetone is determined taking in mind any water typically being present in the acetone. It has been found that too much ace- tone does not improve the process, and too low amounts of ace- tone do not yield biopolymer. Optimal results have been found in the ranges given.

In an example of the present process extraction is performed by filtering.

In an example the present process comprises (vi) a work up step for retrieving acetone, such as by distillation, de- cantation, membrane separation, or evaporation.

In an example the present process comprises (vii) a step of redispersing biopolymer, such as alginate, by wetting with 0.1-10 vol.% acetone. Unexpectedly the present biopolymer such as alginate can be wetted very well and thereafter redispersed or redissolved. In the prior art redispering or redissolving alginates is found to be cumbersome.

In an example the present process comprises (viii) a step of sedimentation. Therewith polymer obtained by the present process can be removed easily.

In an example the present process comprises a step of (ix) heating the alginate to a temperature above 340 K, pref- erably above 350 K, such as above 360 K, during a period suf- ficient to irreversibly transfer the alginate into alginate- amide under separation of water (condensation) , wherein the alginate is ammonium-alginate . Surprisingly a fully biobased thermoset resin can be formed as a result. It has been found that a period of 1-10 minutes is typically sufficient, at a temperature of 365 K.

In an example of the present process comprises the step of removing the sludge by one or more of physical separation, settling, centrifugation, cyclonic separation, decantation, filtration, sieving, and flotation, under suitable conditions. Especially good results have been obtained by centrifuging the sludge .

In an example of the present process a suspension of the sludge is centrifuged, such as at 2000-6000 rpm, during 10-45 minutes, and a supernatant is collected for further pro- cessing ,

In an example of the present process after step an acidic gel is centrifuged, such as at 2000-6000 rpm, during 10-45 minutes, and a supernatant is collected for further pro- cessing.

In an example of the present process the extracted bi- opolymer is further treated, such as by precipitation, such as by addition of an alcohol, by desalination, by osmosis, by re- verse osmosis, by salt-formation, such as Na-salt, by neutral- ising, by adding a base, by drying, by storing, and by freez- ing. Therewith a product is obtained that can be used in a further application, that can be sold, and that can be stored.

In a second aspect the present invention relates to fi- brous polymer, such as alginate or ALE, obtainable by the pre- sent process. It has been found that the alginate has differ- ent characteristics from prior art alginates, such as being fibrous, as is further detailed throughout the description. It is noted that the present alginate typically comprises trace amounts of non-solvent such as 0.0001-0.1 wt . % acetone.

In an example of the present fibres as monovalent cation

NH ₄ ⁺ is present.

The present biopolymers may be characterized by various (further) parameters. They may be different in various aspects from e.g. known comparable chemically or otherwise obtainable polymers, such is in viscosity behaviour, molecular weight, hydrophobicity, lipid content, microstructure {as can be ob- served under an electron microscope) , etc. For instance, the lipid content of the present biopolymers is much higher than those of prior art comparable biopolymers, namely 2-5 wt.%, such as 3-4 wt. %. Analysis of an exemplary biopolymer using a PerkinElmer 983 double beam dispersive IR spectrometer shows approximately 3.2 wt.% peaks that are attributed to lipids. Typically the present biopolymers are also less pure, i.e. a mixture of polymers is obtained. The present biopolymer may relate to an alginate, such as ALE. This is different from the alginates e.g. obtainable by the above pilot plant alginates in various aspects. For in- stance it may have a decreasing dynamic viscosity with in- creasing shear rate (@ 25 °C) , wherein a relative decrease is from 5-50% reduction in dynamic viscosity per 10-fold increase in shear rate. It may have a dynamic viscosity of > 0.2-1 Pa*s (@ 25 °C, @ shear rate of l/sec) . It may have a number aver- aged weight of > 10,000 Dalton, preferably > 50,000 Da, such as > 100,000 Da. It may have a hydrophilic part and hydropho- bic part. It may have a tensile strength (according to ISO 37; DIN 53504) of 1-150 MPa. It may have a flexural strength (ac- cording to ISO 178) of 5-250 MPa. And it may relate to combi- nations of the above.

In an example of the present biopolymer it may have > 30% with a molecular weight of > 300,000 Da, > 10% with a molecu- lar weight of > 100,000 Da, > 15% with a molecular weight of > 5,000 Da, and < 10% with a molecular weight of < 5,000 Da.

In a third aspect the present invention relates to a use of 0.1-10 wt . % biopolymer, such as alginate or ALE, in a solu- tion, preferably the present alginate or ALE, preferably in an aqueous solution, for fire extinguishing, optionally compris- ing 0.1-10 wt . % clay mineral. It has been found that fires are extinguished more efficiently, using less water, and the algi- nate and optional clay are considered to form a coating, which coating does not set fire. It is preferred to use 0.2-5 wt.% alginate, such as 1-2.5 wt.%. It is preferred to use 0.2-5 wt.% clay mineral, such as 1-2.5 wt.%. In an example the pre- sent clay minerals are one or more of a natural or artificial clay, the clay preferably being a monovalent cation clay. The clay preferably has a cationic exchange capacity of 2-200 meq/100 grams clay at a pH of 7, more preferably 5-150 meq/100 grams, even more preferably 10-120 meq/100 grams. It has been found that clays having a relatively higher CEC perform better in terms of relevant characteristics for the present inven- tion. The clay may comprise one or more of H+, Na+, K+, Li+. The clay may be a tetrahedral-octahedral-tetrahedral (TOT)- clay (or 2:1 clay), such as a kaolin clay, such as kaolinite, dickite, halloysite and nacrite, a smectite clay, such as ben- tonite, montmorillonite, nontronite and saponite, an illite clay, a chlorite clay. Also a silicate mineral, such as mica, such as biotite, lepidolite, muscovite, phlogopite, zinnwal- dite, clintonite, and allophane, are applicable as well as platelet like particles.

In a fourth aspect the present invention relates to a use of 0.1-10 wt.% alginate amide in an adhesive, in a thermo- plastic, as an additive in concrete, or in a coating.

The invention is further detailed by the accompany- ing figures and examples, which are exemplary and explanato- ry of nature and are not limiting the scope of the inven- tion. 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 pre- sent claims.

FIGURES

Fig. 1 is a photo of fibrous alginate obtained by the present process.

Figs. 2a-d show SEM-photos of the present alginate. Fig. 3 shows results of variation in amount of ace- tone .

Fig 4. Photo of macroscopic samples from left to right: acetone, methanol.

Fig 5. SEM image of fibrillar Na-Alginate precipi- tated with acetone.

Fig 6. SEM image of Na-Alginate precipitated with methanol. No clear fibrillar structure is observed, rather a dense film, (this is also visible from figure 4).

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 shows a cotton-ball like example of the present fibrous alginate after freeze drying. The length is a few centimeters. First (i) a 3% Na-alginate solution in H20 was prepared, (ii) the solution was left to homogenise and some occasional stirring was applied, (iii) thereafter about 50% acetone to the alginate/H ₂ 0 solution was added, decant on top and shaken vigorously, (iv) then the superna- tant was poured off and replace with 100% acetone. This was repeated several times. A fibrillar mass of Na-alginate in acetone with a low water content was obtained. Then (v) the moistened fibrillar mass was placed in a freezer and then the solvent was evaporated off in vacuum; thereby a freeze dried Na-alginate fibrillar mass was obtained. The freeze drying procedure is found to prevent the sample from col- lapsing so the morphology can be investigated more easily. Overall sample size is about 4 cm long, 1 cm diameter.

Figure 2a-d show SEM photos of the present Na- alginate. Typical fibres obtained are a few (1-2) mm to a few (1-5) cm long having a thickness of 2-250 μηη. Small fragments were imaged using a SEM (see scale bars for magnification and SEM settings) . Various techniques were used, such as sputtered with Au to prevent charging, and also a backscatter image.

EXAMPLES/EXPERIMENTS

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

Inventors found that solutions of NH ₄ -alginate precipitate quantitatively and very efficiently by adding in an example about 50 vol . % acetone. Other (non) solvents work reasonably well, such as ethanol, but acetone has a more dramatic effect. This indicates that a commercial grade ALE alginate can be made from granular sludge via dissolution with a base (e.g. NaOH, NH ₄ OH, KOH etc.) yielding an ammonium ALE or equivalent. This can be easily cleaned from residues via straightforward filtration & sedimentation (especially ammonium is considered optimal) . Adding about 50% acetone under stirring is found to cause a fluffy precipitate of ammonium-alg to form, which can be pressed and dried on filter paper, and left to rest for further desiccation. An alternative procedure may be consid- ered using a shower head, capillary nozzle, spinneret etc. to disperse the alginate solution into acetone as the coagulation bath. By this process it is very easy and cost effective to precipitate and dry large volumes of alginate, at least on a lab scale. Other precipitation inducing non-solvents than ace- tone no doubt exist, also mixing ratios no doubt can be opti- mized.

An exemplary procedure is to take 5 kg granular sludge, add water and technical grade NaOH or ammonia to a total vol- ume of 15 1, dissolve the sodium or ammonium ALE and precipi- tate/filter out unwanted residue, the filtrate (supernatant) can be precipitated by adding about the same volume of ace- tone. Press, dry, and powder yields about 1 kg of dry ALE pow- der .

Detailed example

The extraction of NH ₄ -alginate with acetone and ethanol was performed over the period Q4 of 2015. Stock solution con- tained 0.11081 gr alginate/3.219 gr solution or 0.0344 gr/ml. Inventors precipitated 10 ml of such solutions with about the same amount of ethanol and acetone, the amount of alginate in the system is 10 x 0.0344 = 0.344 gram. Acetone extraction yielded 0.22560 grams or 66 %, and ethanol extraction 0.06346 gram or 6 % which is considered to be a dramatic difference.

Acetone extraction examples

In the experiment below a solution comprising 3.4447 wt.% Na-alg was formed. The amount of acetone seems to require a critical amount, about 20/80 acetone/water ratio or higher is needed to form a precipitate. The precipitate becomes easy to remove above a 40/60 acetone/water ratio. The following table gives some idea of the tested mixing ratios and extracted per- centages:

Table 1: results of alginate extraction using acetone In this experiment a minimum amount of acetone was found to be about 30/70 acetone/water ratio. At higher percentages of acetone the yield increases. In addition above a ratio of 50/50 acetone/water the improvement in yield dropped in rela- tive terms, and above 60/40 acetone/water not much improvement was obtained. At a ratio of 75/25 all (see 107% above) of the alginate was precipitated; the 7% extra indicates an uncer- tainty in the experimental results, so possibly some more al- ginate was present in the initial solution, some impurities in the final product were present, etc.

Fig. 3 represent results of the above experiments. From left to right (25/75), which is the same as the next (5/15), then (50/50) which is the same as the next (10/10), then the (13.75/6.25), and finally the (75/25) which is the same as (15/5) above. In summary at low relative amounts of acetone no fibers are formed, though possibly nano-particles may be pre- sent. At above about 60% acetone no significant improvement of efficiency was found.

Dispersing obtained alginate

An additional advantage of the present alginate involves acetone being suitability as a dispersant. The dissolution of e.g. prior art Na-alginate in water starting from the powder is found inconvenient due to the tendency for the powder to stick together (there are gel like 'lumps' in the sauce). This can be very effectively eliminated by first moisturizing the Na-alginate powder with acetone. This Na-alg/acetone damp pow- der can be freely dispersed in water by manual stirring. Only after some time does the Na-alginate powder start to swell and dissolve while avoiding the formation of any 'lumps' in the sauce .

Below exemplary embodiments of a process of extraction of a specific biopolymer (microbial alginate; ALE) is given. Note that various steps are optional.

Extraction of ALE from granular sludge

1) Sieve the granular sludge to collect granules diame- ter more than 200μm, then wash with tap water.

2) Remove the excess water using tissue paper placed under the sieve.

3) Before starting the extraction, take ± 1 gram of sludge for dry weight determination. Measure the empty cup, the filled cup and put in the oven (105 °C) to dry. Weigh again if dried.

4) Prepare granules suspension in tap water with a Total Solids TSS content of about 5%. This corresponds with 50-55 gram wet weight sludge in a total volume of 100 mL.

5) The ALE was extracted by addition of ammonium hydrox- ide with final pH at 10.5 for 2 days at 20 °C without any agi- tation .

6) The supernatant was separated by decantation and 30-70 v/v% acetone was added to the solution.

7) The ammonium ALE is precipitated out of the solution as a fibrous mass when the acetone is added.

8) Following the precipitation of the ammonium ALE fur- ther separation can be accomplished by centrifugation, flota- tion or any similar separation methods.

9) The ammonium ALE can be stored at 4 °c or frozen.

ALE molecular weight analysis

Size exclusion chromatography was performed with a Su- perdex 75 10/300 GL column (AKTA Purifier System, GE

Healthcare) . Elution was carried out at room temperature using Phosphate Buffer Saline (PBS) containing 10 mM

with a pH of 7.4, and further having 2.7 mM KC1 and 137 mM NaCl, at a constant 0.4 mL/min flow rate. The detection was monitored by following the absorbance of the eluted molecules at a wavelength of 210 nm.

The Superdex 75 10/300 GL column is capable of sepa- rating molecules of 1,000 to 70,000 Daltons (Da). Measurement of the elution volume of dextran standards (i.e. 1000 Da, 5000 Da, 12000 Da, 25000 Da and 50000 Da) led to the calibration equation:

Log (MW) = 6.212 - 0.1861 Ve;

Wherein MW: Molecular Weight of the molecule in Dalton (Da) , and Ve: elution volume in mL (assayed at the top of the peak) . Chromatogram profiles were recorded with UNICORN 5.1 software (GE Healthcare) . Peak retention times and peak areas were di- rectly calculated and delivered by the program.

Results

Table 2 : Molecular weight of different fractions in alginate- like exopolysaccharid.es and their percentage.

Rheology experiments

Viscosity is considered to be an important parameter for biopolymers, such as alginate. Rheology studies the phe- nomena that appear during deformation and flow of fluids, sol- ids and of solid systems under the influence of external forc- es. Newton's law is considered to apply for fluids such as to ideal elastic and viscous materials.

Rheological experiments are performed to determine the viscosity versus the shear rate, the critical overlap concen- tration, the thermal stability and the salinity stability. The viscosity is measured as a function of shear rate using an AR- G2 Rheometer.

Materials and method

The rheology experiments are performed in an AR-G2 Rheometer using Couette Geometry. The Rheometer is filled with 20 ml samples of the polymer solution Na-ALE in the desired concentrations and salinity's. The alginic acid is converted to the desired polymer solution (sodium alginate (Na-ALE) ) by adding NaOH and deionized water.

To prepare the polymer solution samples for the rheol- ogy experiments a stock solution of the highest concentration is prepared first. Thereafter the highest concentration stock solution is diluted to the desired (lower) concentrations. The stock solution is prepared as follows:

The amount of alginic acid required is weighted with a mass balance.

Subsequently NaOH (0.1 N) is added gently to the solu- tion to avoid particle agglomeration.

The solution is stirred and the pH is measured contin- uously .

NaOH (0.1 N) is added up to a final pH of approximate- ly 8.3 and the solution is supplemented to the required volume with deionized water.

The beaker is stirred for 30 minutes at high speed and covered with aluminum foil to prevent contact with air.

- Subsequently, the stirrer is reduced to medium speed and the solution is stirred and degassed for at least one day to create a homogeneous polymer solution in equilibrium and to guarantee hydration.

Finally the stock solution is diluted with deionized water to 20 ml of polymer solution to the desired concentra- tions . Results

The viscosity of ALE and alginate solutions at various shear rates is shown in Figure (la-c). The viscosity (vertical axis, Pa*s) of ALE decreases as the shear rate increased (hor- izontal axis, 1/s) . This is shown for four different solu- tions, from top to bottom, having 5%, 3%, 2%, and 1% alginate, respectively. Apparently the present solutions, comprising the present ALE, show non-Newtonian behavior in this respect.

In comparison, in figure lb the viscosity of algae al- ginate is affected less by changing shear rate. This is shown for five different solutions, from top to bottom, having 10%, 5%, 3%, 2%, and 1% algae alginate, respectively.

Such is considered an indication that the solution of ALE is more pseudoplastic than that of comparable algae alginates. This property may provide advantages in processing, such as pumping and filling.

Comparative examples

In order to clarify differences between the biopolymer obtained with the present method and those obtained with prior art methods comparative experiments have been performed. Three samples were prepared using 3 wt.% initial Na-Alginate solu- tion. Following that a 33:66 ratio of Na-Alg solution : solvent (methanol, acetone, and ethanol, respectively) was made. Then the solvent was mixed with the Na-Alginate solution, which was followed by vigorous shaking for 20 seconds. The ethanol and acetone samples were effectively separated, while the methanol system was more slurry (fig 4) .

All of the samples were freezed at -80 °C, followed by freeze drying to completely remove water and solvent. Analysis of the morphology was performed using a scanning electron mi- croscope, where a very fine fibrillar structure for Na- Alginate precipitated by acetone was observed, more coarse structures for the with ethanol precipitated sample, and no fibres for the methanol sample (fig 5-6) .

In conclusion a precipitation with methanol does not result in fibrous alginate; precipitation with ethanol shows some coarse structures, whereas the best results in this re- spect are obtained with acetone providing fluffy structures. It is noted that both the methanol and ethanol precipitation give much lower yields and in addition the products obtained have further disadvantages in comparison with the acetone product, such as being sticky.