Extracellular polymeric substances as a biobased flame-retarding binding material

Abstract

The present invention is in the field of organic macromolecular compounds, their preparation, and compositions based thereon, in particular biobased extracellular polymeric substances, a method of forming said biobased extracellular polymeric substances, and a composite material comprising said biobased extracellular polymeric substances.

Description

[0001] P100977PC00 EXTRACELLULAR POLYMERIC SUBSTANCES AS A BIOBASED FLAME-RETARDING BINDING MATERIAL

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of organic macromolecular compounds, their preparation, and compositions based thereon, in particular biobased extracellular polymeric substances, a method of forming said biobased extracellular polymeric substances, and a structural material comprising said biobased extracellular polymeric substances.

[0004] RELATED APPLICATIONS

[0005] The present application claims the benefit of priority from Dutch Patent Application NL2039514, filed on December 27, 2024, in the name of Technische Universiteit Delft, The Netherlands.

[0006] The entire contents of the above-referenced applications and of all priority documents referenced in the Application Data Sheet filed herewith are hereby incorporated by reference for all purposes.

[0007] BACKGROUND OF THE INVENTION

[0008] It is known that microorganisms are capable of producing biochemical compounds, such as lactic acid. Thereto typically a microbial culture is used. Therein microbial organisms reproduce in predetermined culture medium under controlled laboratory conditions. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. Remarkably also non-pure cultures are capable of producing chemical substances. Bacteria are capable of producing a wide variety of chemical substances, such as lactic acid, but also methane, and are therefore used in food industry, in waste treatment, and so on.

[0009] With the term “microbial process” here a microbiological conversion is meant.

[0010] Recently it has been found that biobased polymeric substances, such as extracellular polymeric substances (EPS), in particular polysaccharide and proteins comprising materials, obtainable from granular sludge can be produced in large quantities. These substances relate to biobased carboxylic acid-like chemicals, 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 alginate-like EPS in relatively raw form. Though initially the term “alginate-like”, or “alginate-like EPS” (ALE) was used, the biobased polymeric substances are found to be more complex in terms of chemical building blocks present therein. Therefore the term EPS is preferred. Details of the biopolymers can also be found in these documents, as well as in Dutch Patent applications NL2011609, NL2011542, NL2011852, NL2017470, and NL2012089. Also reference can be made to the Nereda® process. These documents, and there contents, such as characteristics (e.g. molecular weights, dynamic viscosity, shear rate, tensile strength, and flexural strength) of the biobased polymers, are incorporated by reference.

[0011] In general it has been found difficult to further process products of biological origin, or make use thereof, such as (these) microbial products, in particular biopolymers such as EPS, either in pure form as obtained from a reactor, or purified or extracted as indicated above.

[0012] The present invention relates to a specific biobased extracellular polymeric substance, in particular a use thereof as a flame retardant, either as such or in a structured material, and further aspects thereof, which overcomes one or more of the above disadvantages, without jeopardizing functionality and advantages.

[0013] SUMMARY OF THE INVENTION

[0014] In a first aspect, the invention relates to a biobased extracellular polymeric substance (EPS) for use as a flame retardant comprising 1-20 wt.% biopolymers, in particular wherein the biopolymers are selected from biopolymers with a relatively small molecular weight, more in particular wherein the biopolymers are selected from the group consisting / comprising of monomers, dimers, and trimers, and mixtures thereof, 0.1-10 wt.% phosphate, in particular 1-9 wt.% phosphate, more in particular 3, 5-8.5 wt.% phosphate, even more in particular 4, 5-7.5 wt.% phosphate, wherein in particular the phosphate comprises condensed phosphate, and 60-98 wt.% water, in particular 80-98 wt.% water, wherein all weight percentages are calculated based on a total weight of the EPS.

[0015] In a second aspect, the invention relates to a method of forming such a biobased extracellular polymeric substance (EPS), by exposing microorganisms to specific conditions. More specifically, the invention relates to a method of forming a biobased extracellular polymeric substance (EPS) according to the invention by exposing microorganisms at a pH of between 5 and 9 and to saline conditions, in particular wherein saline conditions are salt water conditions with 1 -25 g / L of NaCl, in particular 5-20 g / L of NaCl, or synthetic seawater conditions, in particular with 1-40 g / L, preferably 20-38 g / L, of aquatic salt, at a growth temperature of between 10 and 45°C, in particular of between 15 and 40°C, at a sludge retention time of 5-100 days, in a batch reactor, under cyclic operation between anaerobic and aerobic conditions, at a volume exchange ratio between cycles of 40-60%, with feeding of a carbon source, such as polyalcohol or fatty acid.

[0016] In another aspect, the invention relates to a structural material comprising 10-30 wt.% ofthe EPS according to the invention and 70-90 wt.% composite material, wherein all weight percentages are calculated based on a total weight of the structural material. The composite material is preferably selected from the group consisting of paper, cardboard, fibers, cellulose fibers, wood, textiles, and plastics.

[0017] Further, the invention relates to a method of preparing the present structural material. Finally, the invention relates to the use of EPS comprising 1-20 wt.% biopolymers, 0.1 -10 wt.% phosphate, wherein in particular the phosphate comprises condensed phosphate, and 60 - 98 wt.% water, as flame-retardant, wherein all weight percentages are calculated based on total weight of the EPS. It is noted that the 0.1 - 10 wt.% phosphate refers to the total amount of phosphates present in the EPS.

[0018] 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.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates in a first aspect to a specific biobased extracellular polymeric substance (EPS) for use as flame retardant.

[0021] In an exemplary embodiment of the present biobased extracellular polymeric substance, the EPS is obtained from a wastewater treatment plant, in particular from wastewater sludge, more in particular from aerobic granular sludge and / or activated granular sludge.

[0022] Aerobic granular sludge (AGS) is an upcoming technology for wastewater treatment, capable of simultaneously removing organic carbon, nitrogen, and phosphorous typically in a single process unit. The sludge granules consist of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS). Activated granular sludge is another common method for wastewater treatment wherein the activated sludge is composed of microbial groups and organic and inorganic substances. When applying oxygen to the activated sludge the microbial community is activated which promotes the degradation of compounds in water. Besides providing a structural matrix in which cells can grow, EPS may also serve as a protection against adverse conditions in the bulk liquid. EPS are found in all kinds of sludge, and a multitude of properties and compositions have been described with different operating conditions. The EPS for use as a flame retardant according to the present invention is obtained under specific saline conditions.

[0023] The biobased EPS for use as a flame retardant according to the invention comprises: (a) 1-20 wt.% biopolymers;

[0024] (b) 0.1-10 wt.% phosphate, preferably 3.5-9 wt.%, more preferably 4-8 wt.%, even more preferably 5-7 wt.% phosphate, in particular comprising condensed phosphate;

[0025] (c) 60-98 wt.% water,

[0026] wherein all weight percentages are calculated based on a total weight of the EPS.Said bacterial biopolymers are in particular biopolymers selected from the group consist-ing / comprising of monomers, dimers, rtimers, and mixtures thereof. Said bacterial biopolymers are in particular biopolymers selected from the group consisting of (poly)saccharides, and / or wherein the EPS fraction comprises molecules selected from monosaccharides, proteins, lipids, nucleic acids, carboxylic acids, and mixtures thereof. The phosphate according to the invention in particular comprises condensed phosphate. The wording “condensed phosphate” in this respect has its conventional meaning. Thus, the wording “condensed phosphates” as used throughout the specification denote multiple orthophosphate molecules combined or “condensed” together. The condensed phosphates may contain salts or metals. Such salts or metals can be any salts or metals as known in the art as conventionally present in such compounds and present in the EPS. Exemplary examples include potassium, sodium, magnesium, and calcium. The condensed phosphate may exist as linear, cyclic or branched structures.

[0027] It is noted that the wording “(poly)saccharides” means “saccharides, disaccharides and polysaccharides”. It is furthermore noted that the biobased EPS of the invention in particular comprises polysaccharides (i.e. saccharides with tri- or more saccharide groups).

[0028] In an embodiment of the invention the biobased EPS fraction comprises molecules selected from aromatic moieties, such as monocyclic molecules and bicyclic molecules, such as indole, in particular wherein the aromatic moiety comprises a heterocycle, wherein the heterocycle comprises at least one of B, N, S, O, and P, and / or wherein the heterocycle comprises a 5 -membered ring or a 6-membered ring such as but not limited to pyrrole, pyrroline, pyrrolidine, oxolane, furan, phospholane, phosphole, thiolane, thiophene, borolane, borole, Pyrazolidine, imidazolidine pyrazole (pyrazoline), imidazole (imidazoline), oxazolidine, isoxazolidine, oxazole, isoxazole, thizolidine, thiazole, oxothiolane, oxathiole, dioxolane, dioxole, dithialane, dithiole, borinine, boratabenzene anion, piperidine, pyridine, pyridinium cation, oxane, pyran, pyrilium cation, phos-phinane, phosphinene, phosphinium cation, thiane, thiopyran, thiopyrilium cation.

[0029] In an embodiment the biobased EPS fraction comprises molecules comprising at least one of a carboxylic acid group, and a hydroxy group. In an embodiment the biobased EPS fraction comprises molecules with an atomic mass of < 10 kDa, in particular < 5 kDa, more in particular < 3 kDa, such as < 1 kDa. Examples of such molecules with an atomic mass of e.g. <10kDa are, but not limited to, indoles .

[0030] In an embodiment the biobased EPS fraction comprises particles with a particle size < 2 pm, in particular a particle size of 0.2-2 pm, such as 0.4-1.6 pm, 0.6-1.2 pm, 0.8-1.0 pm. The biobased EPS is passing over a membrane with such pore size to separate the fraction into a fraction comprising particles with the specified particle sizes. A particle size is therefore defined as a size of a particle, in any direction, allowing the particle to pass over the according membrane with pore sizes equal to said size; e.g. if a membranewith pore sizes equal to 2 pm, particles passing over the membrane have a particle size of <2 pm. Particles can with the use of a series of membranes be separated from one and another based on their respective size without much effort, e.g. by using a series of membranes with pore sizes of e.g. 2 pm, 1.6 pm, 1.2 pm, 1.0 pm, 0.8 pm, 0.6 pm, 0.4 pm, and 0.2 pm.

[0031] In an embodiment the biobased EPS is obtained from a wastewater treatment plant, in particular from a municipal wastewater or sewage treatment plant, more in particular obtained from wastewater sludge, even more in particular from aerobic granular sludge and / or activated sludge, or wherein the biobased EPS is obtained under saline conditions, in particular wherein saline conditions are salt water conditions with 1-25 g / L, in particular 5-20 g / L, of NaCl or synthetic seawater conditions, in particular with 1 - 40 g / L, preferably 20-38 g / L, of aquatic salt. Activated sludge is composed of microbial groups and organic and inorganic substances. When applying oxygen to the activated sludge the microbial community is activated which promotes the degradation of compounds in water. Aerobic granular sludge is a community of microbial organisms that grow in flocs. Flocs is defined as a mass of microorganisms held together by filaments, which helps with aerobic decomposition and trapping of particles.

[0032] The biobased EPS is produced by microorganisms, in particular by polyphosphate accumulating microorganisms, such as Pseudomonadota (previously denoted as Proteobacteria) and Actinomycetota and more particularly by microorganisms selected from the families Rhodocy-clales, Propionibacteriaceae, and / or Micrococcaceae, and most particularly by microorganisms selected from the genera Candidatus Accumulibacter, Tessaracoccus, and / or Micropruina.

[0033] In one embodiment, the biobased EPS is produced by microorganisms including at least the microorganism Candidatus Accumulibacter Phosphatis.

[0034] In an embodiment of the invention, the biobased EPS has a granular form, having a particle size of between 1-200 pm, 20 - 100 pm as measured by SEM, particles size average of largest and smallest cross-section observable. The skilled person will know how to cany out scanning electron microscopy (SEM) for EPS samples. A typical procedure is as follows, a sample is fixed with a calcium carbonate buffer, then it is dehydrated with acetone or ethanol of increasing concentrations, and freeze-dried. Finally, the biobased EPS samples are sputter-coated with a conductive metal such as gold to ensure electrical conductivity.

[0035] The biobased EPS of the invention in particular comprises

[0036] - at least one biopolymer selected from the group consisting of polysaccharides, preferably selected from trioses, tetroses, pentoses, hexoses, heptoses, octoses, and dodecyloses; amino sugars, preferably selected from galactosamine, glucosamine, sialic acid, and N-acetylglucosa-mine; sulfosugars, preferably selected from sulfoquinovose and carrageenan; mannitol; polyu-ronic acids, preferably selected from glucuronic acid, d-Galacturonic acid, and mannuronic acid; (poly)sugar acids, preferably selected from aldonic acid, ulosonic acid, uronic acid, aldaric acid, Glyceric acid (3C), Xylonic acid (5C), Gluconic acid (6C), Ascorbic acid (6C), Neu-raminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), Ketodeoxyoc-tulosonic acid (KDO or 3-deoxy-D-manno-oct-2-ulosonic acid), Glucuronic acid (6C), Galac-turonic acid (6C), Iduronic acid (6C), Tartaric acid (4C), meso-Galactaric acid (Mucic acid) (6C), and D-Glucaric acid (Saccharic acid) (6C); polymers comprising nonulosonic acid, such as sialic acid; alginate; inulin; starch; celluloses; nitropolysaccharides; and guaran;

[0037] - and at least one second biopolymer comprising at least one anionic polymer, wherein said second biopolymer preferably comprises 30-200 % free OH-groups, in particular 50-150%, and / or comprises 5-30% free COOH groups, in particular 10-25%, and / or comprises 1-10% free NH2 groups, in particular 5-8%,

[0038] and / or wherein the second biopolymers have a number average molecular weight of 10-1000 kDa, have a number average molecular weight of 20-800 kDa, or have a number average molecular weight of 100-500 kDa. The number average molecular weight of the biopolymers can be determined using size exclusion chromatography as conventionally used in the art.

[0039] It is noted that with the wording “20-300% free OH-groups” is meant that the biopolymer on average comprises 0.3 - 2 OH groups per molecule. The amount of free OH-groups can be determined via conventionally used extraction method, typically performed at high pH (9-11) and typically adding 1.2- 1.8 mol OH / gVS. (VS being volatile solids, i.e. the biomass fraction).

[0040] Said EPS typically has an average particle size of 1-100 pm as measured by SEM (particles size average of largest and smallest cross-section observable), more in particular 5-50 pm An Fourier-transform infrared spectroscopy (FTIR) spectrum, in particular measured using a PerkinElmer Spectrum 100 spectrometer, of the biobased EPS of the invention has specific peaks in the 1090 - 1200 cm'1region of the spectrum. More particularly, in the FTIR spectrum peaks are observed at 1735, 1650, 1535, 1230, 1150, 975 and 900 cm'1. It is noted that a peak at 900 cm'1is absent in case EPS is grown without the saline conditions.

[0041] Preferably, the biobased EPS of the invention comprises 0.2-7 wt.% condensed phosphate, in particular 0.5-5 wt.% condensed phosphate, more in particular 1-4 wt.% condensed phosphate, such as 1.2-2.5 wt.% condensed phosphate, with the weight percentages being calculated based on the total weight of the biobased EPS. The biobased EPS may in particular comprise 150-200 mg glucose eq. / g VS-EPS and / or 350-420 mg BSA eq. / g VS-EPS.

[0042] The biobased EPS in particular comprises a polysaccharide to protein ratio of 0.35-0.6, such as 0.38-0.50.

[0043] In a further aspect, the invention relates to a method of forming the specific biobased extracellular polymeric substance (EPS) of the invention. Said method comprises exposing microorganisms at a pH of between 5 and 9 to saline conditions with a combined salt concentration of 0.1-1 mol / 1, more in particular a saline environment comprising a sodium chloride concentration of 1-25 g / L, in particular 2-23 g / L, more in particular 5-20 g / L, at a growth temperature of between 10 and 45°C, in particular of between 15 and 40°C, at a sludge retention time of 5-100 days, typically 5 days - 12 weeks, in a batch reactor, under cyclic operation between anaerobic and aerobic conditions, at a volume exchange ratio between cycles of 40-60%, underfeeding of a carbon source such as polyalcohols or fatty acids, for example glycerol, acetate, and proprionate.

[0044] In an embodiment, the anaerobic conditions are controlled by maintaining a dissolved oxygen concentration between 0-5% saturation, in particular between 0-1% saturation, such as between 1 ppm-0.1% saturation, and / or the aerobic conditions are controlled by maintaining a dissolved oxygen concentration between 40-6% saturation, in particular between 45-55% saturation, such as between 48-52% saturation.

[0045] Typically, each operational cycle comprises of 3-7 min of nitrogen sparging before feeding, 3-7 min of feeding, 3-7 min of settling, 3-7 min of effluent withdrawal, 50-70 min of nitrogen sparging (anaerobic phase), and 100-140 min of aeration.

[0046] In an exemplary embodiment of the invention, said method further comprises

[0047] - adding a carbon comprising medium, wherein the carbon comprising medium comprises a carbon source, said carbon comprising medium in particular comprises 20 - 50 mM glycerol, 2- 8 mM MgSO4.7H2O, and 3- 9 mM KC1,

[0048] and / or

[0049] adding a nutrient medium comprising a carbon source, said nutrient medium in particular comprising 20-60 mM NH4C1, 0.5 - 10 mM K2HPO4, 0.5 - 10 mM KH2PO4, 0.1 - 2 mM Allythiourea (ATU) and 2-25 mL / L of trace element solution comprising 3 - 7 g / L FeSO4.7H2O, 1 - 5 g / L Zn.SO4.7H2O, 4 - 10 g / L CaCl2.2H2O, 2 - 7 g / L MnSO4.H2O, 1 - 5 g / L Na2MoO4.2H2O, 0.5 - 4g / L CuSO4.5H2O, 0.5 - 4 g / L COC12.6H2O and g / L EDTA, and / or

[0050] wherein a final influent concentration has a Chemical Oxygen Demand (COD) of between 200 and 1000 mg O2 / L, 30-100 mg / L of NH4+-N, and 3 - 25 mg / L of PO43-P.

[0051] The biobased EPS can be extracted from the aerobic granular sludge via any conventionally used method known to the person skilled in the art.

[0052] In a further aspect, the invention relates to a structural material comprising

[0053] 1-5 wt.% of the biobased EPS according to the invention, and

[0054] 95-99 wt.% composite material,

[0055] wherein all weight percentages are calculated based on a total weight of the composite. The composite material is preferably selected from the group consisting of paper, cardboard, fibers, cellulose fibers, wood, textiles, and plastics.

[0056] The biobased EPS is preferably provided as a coating, in particular as a coating with a thickness of 1-200 pm, such as 10-100 pm.

[0057] The composite material of the invention is preferably formed by a mold.

[0058] The inventors have surprisingly found that the composite of the invention typically exhibits very good flame retardancy, which is clear from visual inspection but can also be determined using an ASTM flame application testing method, viz. ASTM D3801 -20a. It is noted that the flame retardancy is significantly better than in case of EPS without the required amount of phosphate according to the invention.In yet another aspect, the invention relates to a method of preparing the structural material of the invention. More particularly, the invention relates to a method of preparing the structural material comprising

[0059] applying an aqueous solution, emulsion, or suspension of the biobased EPS of the invention onto the surface of a composite material, and

[0060] allowing the material to dry.

[0061] Typically, for this method an 1 - 10% w / w EPS solution, emulsion, or suspension is applied onto the surface of the composite material. This step can be repeated multiple times to reach the required thickness of the biobased EPS on the composite material. Typically, application is performed in 2-10 consecutive times, preferably 3-5 times. It is not needed to let the material dry in-between the application steps. Preferably, an aqueous solution of the biobased EPS according to the invention is used. Application thereof onto the composite material can take place in any conventional manner known to the skilled person, such as by spraying, soaking, brushing, etc.

[0062] In yet another aspect, the invention relates to the use of a biobased extracellular polymeric substance (EPS) as flame-retardant, said EPS comprising

[0063] 1-20 wt.% biopolymers, in particular comprising (poly)saccharides, proteins, nucleic acids, carboxylic acids, and mixtures thereof,

[0064] 0.1-10 wt.% phosphate, wherein in particular the phosphate comprises condensed phosphate,

[0065] 60-98 wt.% water,

[0066] wherein all weight percentages are calculated based on a total weight of the biobased EPS.

[0067] As described above, said EPS is obtained from a wastewater treatment plant, in particular from waste-water sludge, more in particular from aerobic granular sludge, and has been obtained under saline conditions as specified above. All other characteristics as mentioned above for the biobased EPS apply in this respect as well.

[0068] The invention is further detailed by the accompanying 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.

[0069] EXAMPLES / EXPERIMENTS

[0070] The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying examples.

[0071] Description of the figures:

[0072] Figure 1 : In this figure, a Stereozoom picture of the biomass obtained from each reactor condition at the end of each study period (A-E) as described in detail in the examples isdepicted. The scale bars are equal to 1 mm in A, D, E, and 3 mm in B and C. Phase contrast images of the abundant taxon representing the respective granules. The scale bars in phase contrast images are equal to 10 pm.

[0073] Figure 2: In this figure, normalized FTIR spectra of the biobased EPS at different salinities, are shown.

[0074] Figure 3: Polysaccharide (PS) to protein (PN) ratio of the samples are depicted in Figure 3. The PS and PN content are expressed as glucose and BSA equivalents. Error bars indicate standard deviation.

[0075] Figure 4: Pictures of the biobased EPS-coated cellulose (tissue) fibres before (A) and after flame application (B) are shown in Figure 4. Picture C shows the flame application setting.

[0076] Figure 5: In this figure, a sketch of the lab-scale ceramic microfiltration is shown. Figure 6: In this figure, the heat release rate of the control cotton (average) and the biobased EPS cotton is depicted.

[0077] Figure 7: In this figure, FTIR spectra of the biobased EPS permeate composition is shown.

[0078] Material and methods

[0079] Reactor operation

[0080] A batch reactor was operated as described in detail in:

[0081] Elahinik, A., de Clercq, F., Pabst, M., Xevgenos, D., van Loosdrecht, M. C. M., & Pronk, M. (2024). Effects of Salinity on Glycerol Conversion and Biological Phosphorus Removal by Aerobic Granular Sludge. Water Research, 121737. https: / / doi.org / 10.1016ZJ.WATRES.2024.121737 (which is hereby incorporated by reference).

[0082] In summary, the biobased EPS granules were cultivated in a bubble column reactor operating in sequencing batch mode. The reactor had a working volume of 3.1 L. After effluent withdrawal at the end of each cycle, a volumetric exchange ratio of about 50% was maintained. The pH was automatically controlled at 7.1 ± 0.1 by dosing 0.5 M NaOH or HC1. The dissolved oxygen (DO) concentration was controlled at 0% and 50% saturation (~ 0 - 4.5 mg / L) during the anaerobic and aerobic phases by a mixture of nitrogen gas and air. The off-gas was recirculated with a flow of 5 L / min to maintain the DO concentration. The DO probes were calibrated for each salt concentration. The room temperature where the reactor was located was controlled at ~ 20 °C. The reactor was inoculated with about 600 mL of a mixture of AGS from a municipal WWTP located in Hamaschpolder, the Netherlands and a glycerol-adapted sludge performing phosphate removal from the lab. Each operational cycle consisted of 5 min of nitrogen sparging before feeding, 5 min of feeding, 5 min of settling, 5 min of effluent withdrawal, 60 min of nitrogen sparging (anaerobic phase), and 120 min of aeration.

[0083] The synthetic influent fed at the beginning of each cycle with a total volume of 1,500 mL consisted of 1,200 mL of (salt) water, 150 mL of nutrient medium and 150 mL of carbonmedium. The synthetic seawater with a final concentration of 35 g / L was made with Instant Ocean® sea salt crystals. 35 g / L sea salt was used to account for the mass of other elements in the mixture and to achieve a 30 g / L NaCl equivalent. Carbon medium contained 35.7 niM glycerol, 3.6 mM MgSO4.7H2O, and 4.7 mM KC1. No extra MgSO4.7H2O and KC1 were added when sea salt crystals were used. The nutrient medium contained 41.1 mM NH4CI, 1.95 mM K2HPO4, 1.98 mM KH2PO4, 0.6 mM Allythiourea (ATU; CAS 109-57-09) to inhibit nitrification and 10 mL / L of trace element solution. The trace element solution contained 4.99 g / L FeSO4.7H2O, 2.2 g / L ZnSO4.7H2O, 7.33 g / L CaCl2.2H2O, 4.32 g / L MnSO4.H2O, 2.18 g / L Na2MoO4.2H2O, 1.57 g / L CuSO4.5H2O, 1.61 g / L COC12.6H2O and 50 g / L EDTA. These feed streams combined resulted in a final influent concentration of 400 mg / L COD, 58 mg / L NH4+-N, and 12 mg / L of PO43"-P. The reactor conditions were distinguished based on their salinity and are referred to as R0 (0 g / L NaCl), R10 (10 g / L NaCl), R20 (20 g / L NaCl), R30 (30 g / L NaCl), and R35x (35 g / L sea salt).

[0084] To monitor the reactor performance, samples from the supernatant were frequently analysed. The samples were filtered through 0.45 pm PVDF filters and the concentrations of PO43" -P and NH4+-N were measured using a Gallery Discrete Analyser (DA) (ThermoFisher Scientific, USA). Volatile fatty acids and sugars were measured using an HPLC (Vanquish, ThermoFisher Scientific, USA) equipped with an RI and UV detector, Aminex HPX-87H column (BioRad, USA) using 0.0015M phosphoric acid as eluent. The visualization of the morphology of the granules was done with a stereo zoom microscope (M205 FA, Leica Microsystems, Germany) equipped with Qwin image analysis software (V3.5.1, Leica Microsystems, Germany).

[0085] Extracellular polymeric substances extraction

[0086] The biobased EPS extraction was performed according to the method described by Chen, L. M., Keisham, S., Tateno, EL, van Ede, J., Pronk, M., van Loosdrecht, M. C. M., & Lin, Y. (2024). Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions. ACS ES and T Water, 4(1), 279-286. https: / / doi.org / 10.1021 / ACSESTWATER.3C00625 / ASSET / IM-AGES / LARGE / EW3C00625 0005.JPEG (which is hereby incorporated by reference).

[0087] In short, freeze-dried granules were placed in 0.1 M NaOH for 30 min at 80 °C while stirring at 400 rpm (1% volatile solids w / v). The solution was centrifuged at 4000 x g for 20 min and cooled down at 4 °C. The collected supernatant was dialysed against demi-water overnight in dialysis bags with a molecular weight cut-off of 3.5 kDa MWCO (Snakeskin™, ThermoFisher Scientific, Landsmeer). The dialysed EPS solution was then freeze-dried for further analysis.

[0088] Polysaccharides, protein, and phosphate measurements

[0089] The phenol sulfuric acid method was used to measure total polysaccharides. Phenol 5% (w / v) and concentrated sulfuric acid (95%) were added to lyophilised EPS dissolved in waterand incubated for 20 min at room temperature. The samples were then measured with a spectrophotometer at 490 nm wavelength. The measurements were done in duplicate.

[0090] To measure and quantify the monosaccharides, lyophilised EPS was hydrolysed in 1 M HC1 at 105 °C overnight. The hydrolysed sample was centrifuged and the supernatant was neutralised with 1 M NaOH. The neutralised sample was filtered through a 0.45 pm PVDF filter and analysed by high-performance anion-exchange chromatography with pulsed amperometry detection (HPAEC-PAD) on a Dionex CarboPac PA20 column (Thermo Fisher, USA) using NaOH and NaHAc as the eluent.

[0091] To quantify total proteins, BCA analysis was performed according to the manufacturer’s protocol with the commercially available kit (Pierce BCA protein assay Kit, Thermo Scientific). The absorbance was measured at 562 nm wavelength with a 96-well plate (TECAN Infinite M200 PRO, Mannedorf, Switzerland). The measurements were done in duplicate.

[0092] Lyophilised EPS was dissolved in ultrapure water (~1 mg / mL) filtered through 0.22 pm filters, and the total phosphate content of the solution was measured using Hach Lange cuvette tests following the instructions of the kit (LCK350, Hach, USA). The orthophosphate concentrations were measured using the DA (ThermoFisher Scientific, USA). Hydrolysable phosphate was measured after mild sulfuric acid hydrolysis by skipping the persulfate oxidation step using the LCK350 kit. The measurements were performed in triplicates. The organic phosphate was calculated by subtracting acid hydrolysable phosphate from total phosphate and the condensed phosphate was calculated by subtracting orthophosphate from acid hydrolysable phosphate.

[0093] Fourier- transform infrared and 2-D correlation spectroscopy

[0094] FTIR spectroscopy was performed on a Spectrum 100 spectrometer (PerkinElmer, Shelton, CT). The spectra of the lyophilised samples (extracted EPS and granules) were recorded at room temperature over the range of 600 - 4000 cm1wavenumber with 8 accumulations and 2 cm1resolution. Normalisation was done to correct baseline shifts and intensity variations. After baseline correction of the IR spectra, 2D-COS was performed using an in-house MATLAB script provided in the supplementary material. In short, a series of spectra were collected and laid out in a matrix format where each row represented a spectrum and each column represented a point in the spectrum. Correlation analysis was then performed by comparing intensities at each point in the spectrum change as the conditions change. Finally, a 2D map was created where both axes represented points in the spectra. Each point on the map shows how strongly the corresponding points in the original spectra are correlated.

[0095] Burning test

[0096] To qualitatively assess the impact of EPS as a biobased flame-retardant coating, EPS-coated cellulose fibers were prepared as follows Tissue papers (purchased from a local supermarket) were soaked in water, blended to disintegrate the fibers and make a pulp solution, and dried at 105 °C. The dried tissue pulp (~ 200 mg) was then mixed with an EPS solution of 3%w / w, pressed into a flat plastic container, and let air dry at room temperature for 72 hrs. The biobased EPS solution was made by dissolving ~45 mg of EPS from each sample in 1.5 mL demineralised water. In terms of mass distribution, EPS accounted for approximately 20% of the sample weight. A blow torch was placed 10 cm from the samples and a blue flame was applied for 12 s. A cellulose fibre sample with no added EPS was used as a control. The flame extinguishing and afterglow time, dripping, and burning of the samples were monitored. The residual mass of each sample was measured in triplicates. The results are depicted in Figures 6a-c.

[0097] Samples collected

[0098] The flocculent sludge was collected from municipal wastewater treatment plants using the activated sludge process in Delft, the Netherlands. The biobased EPS was extracted from the flocculent sludge according to the previously reported extraction protocol (Li et al., 2021, Water. Res., 205 (2021), Article 117706, 10.1016 / j.watres.2021.117706), i.e. 3.0 g of dried sludge was resuspended in 100 mL demi-water with adding 0.5 g of sodium carbonate. The mixtures were then heated at 80 °C for 30 min.

[0099] Membrane set-up

[0100] (X-AI2O3 ceramic membranes, manufactured by atech innovations GmbH (Germany), were used in this study. These membranes are available with various pore sizes: 0.2 pm, 0.1 pm , 0.05 pm, 100 kDa, 25 kDa, and 5 kDa.

[0101] Two types of membranes were used in the experiments. Both types featured a tubular configuration. The first type was a single-channel membrane with an outer diameter (OD) of 10 mm and an inner diameter (ID) of 6 mm, providing a filtrate surface area of 0.00019 m2 / cm and an effective length of 28 cm.

[0102] The second type was a 19-channel membrane with an OD of 25.4 mm and individual channel diameters of 3.3 mm. This configuration provided a total surface area of 0.002 m2 / cm and the same effective length of 28 cm.

[0103] Filtration experiments

[0104] Figure 5 shows the experimental apparatus constructed for the filtration trials. The system was designed to operate with up to four membranes in series and could accommodate both single-channel and multi-channel membrane configurations. It allowed for a range of flow patterns and pressure conditions.

[0105] 2 gear pumps operating in parallel circulated the feed emulsion from a glass feed reserv ir through the vertically mounted membrane units. The retentate was either returned to the feed reservoir or partially bypassed to the pump inlet. A diaphragm valve was used to regulate system pressure.

[0106] The permeate was collected on a balance, and its weight was monitored to determine themembrane flux. Additionally, a backflush line was integrated into the system to facilitate membrane cleaning and to support future studies on backflushing and back-pulsing techniques.

[0107] After each run, the apparatus containing the ceramic membranes was rinsed with demineralized water (DW). The ceramic membrane was then restored using a cleaning-in-place (CIP) procedure consisting of the following steps: alkaline cleaning with a 20 g / L NaOH solution at 40 °C for 50 minutes, acid cleaning with a 10 g / L HNO3 solution at 40 °C for 30 minutes, and a final rinse with DW at 20 °C for 20 minutes. The alkaline step targeted organic and some inorganic colloidal foulants, while the acid step dissolved inorganic deposits such as salts.

[0108] To verify membrane cleanliness and integrity, permeability was measured using DW at 20 °C and 1 bar before every experiment. The result was compared to both the manufacturer's reference value (within a 5% margin) and the value recorded prior to the microfiltration runs, ensuring that no unexpected variation occurred beyond typical experimental fluctuations.

[0109] The filtration was ran in the mode of retentate recycling and permeate was removed. Tangential velocity was 3.34 m / s at the pressure of 0.4 MPa.

[0110] Sample preparation for the flame retardant tests.

[0111] Around 25 g of cotton fibers were filled in a plastic mold with the size of 100 mm* 100 mm*25mm and pressed to fit for the mold. For the control sample, demi water was added into the cotton. For the sample with the tested fraction of the biobased EPS, around 2.2 g of the permeate (organic fraction of 1 g) was added into the cotton.

[0112] Cone calorimeter test

[0113] The fire reaction characteristics of the samples under heat radiation were investigated using a cone calorimeter (FTT Ltd., East Grinstead, UK), according to ASTM El 354 / ISO 5660-1:2002. A sample was exposed to 50 kW / m2 heat flux to measure various properties including time to ignition (TTI), heat release rate (HRR), total smoke release (TSR) and CO yields. A gas analyser (Servomex 1440) using a paramagnetic and infrared systems in the cone calorimeter detected oxygen, CO and CO2. The sample’s exposure area to a conical heater and thickness were 88.4 cm2 and 3 mm, respectively. Moreover, all samples were preconditioned at 23 °C and relative humidity of 50%.

[0114] EPS fraction composition measurement

[0115] FT-IR spectroscopy studies

[0116] The FT-IR spectra of the extracted melanin- like material, the commercial synthetic melanin and humic acid was recorded on a FT-IR spectrometer at room temperature, with the wavenumber range from 650 cm-1 to 4000 cm-1. Resolution of 1 cm-1 and accumulation of 8 scans were applied to each sample.

[0117] Monosaccharide composition was analysed following acid hydrolysis process and quan-tified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The detailed information on analytical column and standards referred to the previous study (Li et al., 2025).

[0118] Results

[0119] Reactor performance

[0120] Aerobic sludge granules were cultivated under different salinities using glycerol as the sole carbon source at an average sludge retention time (SRT) of 12 ± 4 days. The salinities were obtained by varying the NaCl concentration or using sea salt for medium preparation. Reactor conditions with 0, 10, 20 g / L NaCl and 35 g / L sea salt (RO, RIO, R20, and R35x), showed successful granulation, EBPR performance, and complete anaerobic glycerol removal. In the reactor operated with 30 g / L NaCl (R30), glycerol was only partially converted anaerobically and EBPR performance gradually decreased until completely lost. The incomplete anaerobic substrate uptake led to COD overflow to the aerobic phase and the growth of filamentous biomass. Glycerol uptake and phosphate release and uptake during a representative cycle under pseudo-steady-state conditions at each salinities are shown in the supplementary materials. The microscopic and stereoscopic picture of the biomass obtained from each reactor condition at the end of each study period is shown in Figure 1.

[0121] Metaproteomic analysis in combination with microscopic observations of the microbial community revealed the dominant presence of Ca. Accumulibacter Phosphatis, a well-known polyphosphate accumulating organism (PAO), under stable reactor conditions performing EBPR (R0, R10, R20, R35x). Additionally, the genera Tessaracoccus and Micropruina, two actinobacteria classified as fermentative glycogen accumulating organisms (fGAO) responsible for glycerol conversion, were detected in the biomass. In R30, characterised by slightly worse EBPR performance and granulation, the genus Zoogloea was the predominant taxon. Overall, the microbial community composition was similar across all reactor conditions except in R30.

[0122] FTIR analysis of the biobased EPS

[0123] FTIR spectroscopy was utilised to provide quick and qualitative information about the functional groups and chemical bonds present in the samples (Figure 2). The assignment of infrared spectra was based on existing literature The manual analysis of the spectra revealed that the samples contain functional groups likely corresponding to lipids (-2930-2965 cm ' polysaccharides and phosphates (-1000 - 1200 cm-1), proteins (1535 and 1650 cm-1), nucleic acids (975 cm-1), and carboxylic acids (-1735 cm-1) based on the vibration of the bonds.

[0124] The normalised IR spectra were analysed for correlations using 2D-COS, a well-known technique used to unveil hidden relationships among IR spectra. 2D-COS revealed that the main differences were in the 860 - 950 cm'1and 1090 - 1200 cm'1regions based on a change in spectral intensity as a function of external perturbation which was salinity.. It's important to note that 860 - 950 cm'1falls within the fingerprint region of the infrared spectrum, which isknown for its complex and often overlapping absorption bands.

[0125] Total (poly)saccharide, protein, and phosphate

[0126] Lyophilised aerobic sludge granules underwent digestion according to a thermal-alkaline protocol, enabling the extraction of alkaline soluble polymers from the samples. The granules extracted EPS samples were then analysed for total protein, (poly)saccharide, and phosphate content. Notably, sample R0 exhibited a significantly lower phosphate content compared to the other samples. The biobased EPS yield of sample R30 was found to be almost half compared to the rest.

[0127] Polysaccharides (PS) and proteins (PN), which are among the main components of EPS, were quantified and their respective ratios are displayed in Figure 3. The PS / PN ratio of the samples showed a slight increase in the samples from saline conditions, compared to the freshwater sample.

[0128] The total, hydrolysable, and ortho-phosphate contents in the biobased EPS were determined as indicated above and the results are given in Table 1. The hydrolysable fraction accounted for the majority of the total phosphate content while orthophosphate was only a minor fraction. The condensed and the organic phosphates were calculated and presented in 1. Overall, the total phosphate concentration of the samples from saline conditions was significantly higher than the sample from freshwater conditions.

[0129] Table 1. Total, hydrolysable, and orthophosphate of the samples. Organic and condensed phosphates are calculated as described in the material and methods. The values are given in mg / gVS-EPS with their standard deviations.

[0130] Samples Total-P Organic-P Hydrolysable-P Ortho-P Condensed-P RO 34 ± 0.0 7 ± 0.6 27 ± 0.6 23 ± 3.8 4 ± 3.9 RIO 91 ± 0.7 43 ± 0.9 47 ± 0.6 12 ± 2.3 36 ± 2.3 R20 101 ± 0.6 39 ± 0.7 62 ± 0.3 22 ± 2.7 40 ± 2.7 R30 45 ± 0.1 12 ± 0.1 33 ± 0.1 8 ± 0.4 25 ± 0.4 R35x 64 ± 1.2 31 ± 1.3 33 ± 0.3 10 ± 0.1 23 ± 0.3

[0131] Flame retardancy of the biobased EPS

[0132] To understand the impact of biobased EPS origin on its flame retardant properties, a burning test was performed with biobased EPS-coated cellulose fibers. The flame extinguishing and afterglow time and burning of the samples were assessed. The control sample (cellulose fibers with no EPS coating) was completely burned to ash after exposure to fire with >99% mass loss, as expected. The biobased EPS-coated samples had a higher residual mass than their uncoated counterpart. The samples with higher residual mass (R10, R20, and R35x) had a significantlyshorter after-glow and extinguishing time (Table 2) with visible char formation (Figure 4) compared to R0 and R30. The cellulose fibers coated with R10, R20, and R35x, maintained better structural integrity due to char formation compared to R0 and R30, where more ash formation was observed.

[0133] Table 2. Samples mass residue with standard deviation, afterglow time, and extinguish time.

[0134] SampleMass residue [%] Afterglow time [s] Extinguish time [s]

[0135] RO 4.0 ± 0.4 40.0 8.5

[0136] RIO 15 ± 0.5 24.0 4.5

[0137] R20 14 ± 1.0 29.5 4.5

[0138] R30 5.0 ± 0.6 43.5 5.5

[0139] R35x 11 ± 1.3 40.0 6.5

[0140] EPS as a biobased flame retardant

[0141] The impact of biobased EPS of the invention as a biobased flame retardant was evaluated by coating cellulose fibers with the biobased EPS and subjecting them to controlled burning tests. Cellulose was chosen as a earner for conducting burning tests. Cellulose can also be recovered from tissue papers collected in WWTPs in large quantities. It is widely used in the construction industry as an adhesive or building material. The biobased EPS-coated cellulose fibers can therefore be used as biobased flame retardant fillers in the construction industry. After flame application, the cellulose fibers coated with EPS (R10, R20, and R35x,) retained their structural integrity, exhibited char formation, and experienced less mass loss compared to the other samples.

[0142] The yield of the biobased EPS fraction in the permeate is 5% of the organic fraction of the total sludge.

[0143] The results of the cone calorimeter test are listed in Table 2. It was noticed that the MARHE (maximum average rate of heat emission) of the cotton with the biobased EPS addition is much lower than the one without addition of the biobased EPS. The time for the effective heat of combustion was delayed from 115 seconds to 590 seconds. The peak CO2 and CO yield was tremendously reduced. As shown in Figure X, the total heat release rate was reduced as well.

[0144] Table 3. Cone calorimeter test

[0145] Without the addition of EPS fraction With the addition of the biobased EPS fractionTest results (between 4 and 605 s)

[0146] Total heat release 37.2 MJ / m2

[0147] Total oxygen consumed 25.1 g

[0148] Mass lost 2736,2 g / m2

[0149] Av. specific MLR (mA.io-90) 5.69 g / (s*m2)

[0150] Total smoke release 25.8 m2 / m2

[0151] Total smoke production 0.2 m2

[0152] MARHE 129.0 kW / m2

[0153] Test results (between 4 and 625 s)

[0154] Total heat release 37.2 MW / m2

[0155] Total oxygen consumed 25.1 g

[0156] Total oxygen consumed 24.5 g

[0157] Mass lost 2817.7 g / m2

[0158] Total oxygen consumed 25.1 g

[0159] Av. specific MLR (mA.io-9o) 5.24 g / (s*m2

[0160] Total oxygen consumed 25.1 g

[0161] Total smoke release 25.6 m2 / m2

[0162] Total smoke production 0.2 m2

[0163] MARHE 74.6 kW / m2

[0164] MLR- mass lost rate, MARHE- maximum average rate of heat emission

[0165] Mean Peak at time (s) Heat release rate (kW / m2) 61.25 153.04 10 Effective heat of comb. (MW / kg) 13.38 62.16 115 Class loss rate (g / (s* m2)) 4.51 33.80 10 Specific extinction area (m2 / kg) 7.65 372.09 500 Carbon monoxide yield (kg / kg) 0.0168 126.3837 575 Carbon dioxide yield (kg / kg) 1.23 1333.16 575

[0166] Mean Peak at time (s) Heat release rate (kW / m2) 59.74 92.64 10 Effective heat of uoii>b. (MJ / kg) 13.41 61.96 590 Mass loss rate (g / ( s* m2)) 4.45 11.31 15 Specific extinction area (m2 / kg) 9.24 86.30 580 Carbon monoxide yield (kg / kg) 0.03190.1906 590 Carbon dioxide yield (kg / kg) 1.06 3.13 590

Claims

CLAIMS1. A biobased extracellular polymeric substance (EPS) for use as a flame retardant, said EPS comprising1-20 wt.% molecules selected from bacterial biopolymers, in particular wherein the biopolymers are selected from the group consisting / comprising of monomers, dimers, and trimers, and mixtures thereof;0.1-10 wt.% phosphate, wherein in particular the phosphate comprises condensed phosphate;60-98 wt.% water, in particular 80-98 wt.% water;wherein all weight percentages are calculated based on a total weight of the biobased EPS.

2. The biobased extracellular polymeric substance (EPS) according to claim 1, comprising 1-20 wt.% biopolymers selected from the group consisting of (poly)saccharides, and / or wherein the biobased EPS fraction comprises molecules selected from monosaccharides, proteins, lipids, nucleic acids, (poly)carboxylic acids, and mixtures thereof.

3. The biobased EPS according to any of claim 1-2, wherein the fraction comprises molecules selected from aromatic moieties, such as monocyclic molecules and bicyclic molecules, in particular wherein the aromatic moiety comprises a heterocycle, wherein the heterocycle comprises at least one of B, N, S, O, and P, and / orwherein the heterocycle comprises a 5-membered ring or a 6-membered ring.

4. The biobased EPS according to any of claim 1-3, wherein the fraction comprises molecules comprising at least one of a carboxylic acid group, and a hydroxy group.

5. The biobased EPS according to any of claim 1-4, wherein the fraction comprises molecules with an atomic mass of < 10 kDa, in particular < 5 kDa, more in particular < 3 kDa, such as < 1 kDa (considering indole per se 117 gr / mol).

6. The biobased EPS according to any of claim 1-5, wherein the fraction comprises particles with a particle size < 2 pm, in particular a particle size of 0.2-2 pm. (passing over a membrane with such pore size).

7. The biobased EPS according to any of claim 1-6, wherein the biobased EPS is obtained from a wastewater treatment plant, in particular from wastewater sludge, more in particular from aerobic granular sludge and / or activated sludge, orwherein the biobased EPS is obtained under saline conditions, in particular wherein saline conditions are salt water conditions withl-25 g / L, in particular 5-20 g / L, of NaClorsynthetic seawater conditions, in particular with 1 - 40 g / L, preferably 20-38 g / L, of aquatic salt.

8. The biobased EPS according to any of claim 1-7, wherein the biobased EPS is produced by microbes, in particular by polyphosphate accumulating organisms, such as Pseudo-monadota and Actinomycetota, more particularly by microorganisms selected from the families Rhodocyclales, Propionibacteriaceae, and / or Micrococcaceae, and most particularly by microorganisms selected from the genera Candidatus Accumulibacter, Tessaracoccus, and / or Mi-cropruina.

9. The biobased EPS according to any of claim 1-8 wherein the biobased EPS is produced by microorganisms including at least the microorganism Candidatus Accumulibacter Phospha-tis.

10. The biobased EPS according to any of claim 1-9, whereinthe biobased EPS has an average particle size of 1-200 pm,and / or wherein the biobased EPS comprises- at least one biopolymer selected from the group consisting of polysaccharides, preferably selected from trioses, tetroses, pentoses, hexoses, heptoses, octoses, and dodecyloses; amino sugars, preferably selected from galactosamine, glucosamine, sialic acid, and N-acetylglucosa-mine; sulfosugars, preferably selected from sulfoquinovose and carrageenan; mannitol; polyu-ronic acids, preferably selected from glucuronic acid, d-Galacturonic acid, and mannuronic acid; (poly)sugar acids, preferably selected from aldonic acid, ulosonic ac-id, uronic acid, al-daric acid, Glyceric acid (3C), Xylonic acid (5C), Gluconic acid (6C), Ascorbic acid (6C), Neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), Ketodeoxyoc-tulosonic acid (KDO or 3-deoxy-D-manno-oct-2-ulosonic acid), Glucuronic acid (6C), Galac-turonic acid (6C), Iduronic acid (6C), Tartaric acid (4C), meso-Galactaric acid (Mucic acid) (6C), and D-Glucaric acid (Saccharic acid) (6C); polymers comprising nonulosonic acid, such as sialic acid; alginate; inulin; starch; celluloses; nitropolysaccharides; and guaran;.- and at least one second biopolymer comprising at least one anionic polymer, wherein said second biopolymer preferably comprises 30-200 % free OH-groups, in particular 50-150%, and / or comprises 5-30% free COOH groups, in particular 10-25%, and / or comprises 1-10% free NH2 groups, in particular 5-8%,and / or wherein the second biopolymers have a number average molecular weight of 10-1000 kDa, have a number average molecular weight of 20-800 kDa, or have a number aver-age molecular weight of 100-500 kDa.

11. The biobased EPS according to any of claim 1-10, wherein an FTIR spectrum of the biobased EPS includes vibrations in the 1090 - 1200 cm-1 region, and further in particular including FTIR peaks at 1735, 1650, 1535, 1230, 1150, 975, and 900 cm-1.

12. The biobased EPS according to any of claim 1-11, wherein the biobased EPS comprises 0.2-7 wt.% condensed phosphate, in particular 0.5-5 wt.% condensed phosphate, more in particular 1-4 wt.% condensed phosphate, such as 1.2-2.5 wt.% condensed phosphate, and / or wherein the biobased EPS in particular comprises 150-200 mg glucose eq. / g VS-EPS, and / or 350-420 mg BSA eq. / g VS-EPS,and / orwherein the biobased EPS in particular comprises a polysaccharide to protein ratio of 0.35-0.6, such as 0.38-0.50.

13. A method of forming a biobased extracellular polymeric substance (EPS) according to any of claim 1-12, by exposing microorganisms at a pH of between 5 and 9 to saline conditions, in particular wherein saline conditions are salt water conditions with 1 - 25 g / L, in particular 5-20 g / L, of NaCl or are synthetic seawater conditions, in particular with 1-40 g / L, preferably 20-38 g / L, of aquatic salt, at a growth temperature of between 10 and 45°C, in particular of between 15 and 40°C, at a sludge retention time in particular of 5-100 days, in a batch reactor, under cyclic operation between anaerobic and aerobic conditions, at a volume exchange ratio between cycles of 40-60%, under feeding of a carbon source such as polyalcohols or fatty acids.

14. A method of forming a biobased extracellular polymeric substance (EPS) according to any of claim 1-12 by exposing microorganisms15. The method according to claim 13 or 14, wherein anaerobic conditions are controlled by maintaining a dissolved oxygen concentration between 0-5% saturation, in particular between 0-1% saturation, such as between 1 ppm-0.1% saturation,and / orwherein aerobic conditions are controlled by maintaining a dissolved oxygen concentration between 40-60% saturation, in particular between 45-55% saturation, such as between 48-52% saturation.

16. The method according to any of claim 13-15, wherein each operational cycle comprises of 3-7 min of nitrogen sparging 3-7 min of feeding of the carbohydrate source, 3-7 minof settling, 3-7 min of effluent withdrawal, 50-70 min of nitrogen sparging (anaerobic phase), and 100-140 min of aeration.

17. The method according to any of claim 13-16, comprising- adding a carbon comprising medium, wherein the carbon comprising medium comprises a carbon source, said carbon comprising medium in particular comprises 20 - 50 mM glycerol, 2- 8 mM MgSO4.7H2O, and 3- 9 mM KC1,and / oradding a nutrient medium comprising a carbon source, said nutrient medium in particular comprising 20-60 mM NH4C1, 0.5 - 10 mM K2HPO4, 0.5 - 10 mM KH2PO4, 0.1 - 2 mM Allythiourea (ATU) and 2-25 mL / L of trace element solution comprising 3 - 7 g / L FeSO4.7H2O, 1 - 5 g / L Zn.SO4.7H2O, 4 - 10 g / L CaC12.2H2O, 2 - 7 g / L MnSO4.H2O, 1 - 5 g / L Na2MoO4.2H2O, 0.5 - 4g / L CuSO4.5H2O, 0.5 - 4 g / L CoC12.6H2O and g / L EDTA, and / orwherein a final influent concentration has a Chemical Oxygen Demand (COD) of between 200 and 1000 mg 02 / L, 30-100 mg / L of NH4+-N, and 3 - 25 mg / L of PO43— P.

18. A structural material comprising10-30 wt.% of the biobased EPS according to any of claim 1-12,70-90 wt.% composite material,wherein all weight percentages are calculated based on a total weight of the structural mate-rial.

19. The structural material according to claim 18, wherein the composite material is selected from the group consisting of paper, cardboard, fibers, cellulose fibers, wood, textiles, and plastics.

20. The structural material according to any of claim 18-19, wherein the material is formed by a mold.

21. The structural material according to any of claim 18-20, wherein the biobased EPS is pro-vided as a coating, in particular as a coating with a thickness of 1-200 pm, such as 10-100 pm.

22. Method of preparing the structural material of any of claim 18-21 comprising - applying an aqueous solution, emulsion, or a suspension of the biobased EPS according to any of claims 1-14, in particular a 1 - 10% w / w EPS solution, emulsion, or suspension in water, onto the surface of a composite material, and- allowing the material to dry.

23. Use of a biobased extracellular polymeric substance (EPS) as flame-retardant, said EPS comprising1-20 wt.% biopolymers, in particular biopolymers selected from the group consisting of (poly)saccharides, proteins, lipids, nucleic acids carboxylic acids, and mixtures thereof;0.1-10 wt.% phosphate, wherein in particular the phosphate comprises condensed phosphate;60-98 wt.% water;wherein all weight percentages are calculated based on a total weight of the biobased EPS.

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