WO2025141398A1

WO2025141398A1 - Bacterial biopolymer formulation suitable for the treatment of wastewater with suspended solids, methods of producing and using the same

Info

Publication number: WO2025141398A1
Application number: PCT/IB2024/062827
Authority: WO
Inventors: Ana Paula KUAN YON CHUNG; Paula Maria DE MELIM VASCONCELOS DE VITORINO MORAIS
Original assignee: Solancis Exploradora De Pedreiras SA Soc; Universidade de Coimbra
Current assignee: Solancis Exploradora De Pedreiras SA Soc; Universidade de Coimbra
Priority date: 2023-12-27
Filing date: 2024-12-18
Publication date: 2025-07-03
Anticipated expiration: 2026-06-27

Abstract

The present application relates to a biopolymer produced by a bacterial strain, Duganella sp. NCIMB 44166, which has never been reported as a biopolymer producer before. A bacterial biopolymer formulation comprising said biopolymer is also disclosed herein, which is a formulation suitable for the treatment of wastewater with suspended solids. Herein disclosed is also a method of obtaining the biopolymer and the formulation, and a method of using said formulation in the treatment of wastewater. The formulation disclosed is environmentally friendly and has easy natural degradation. The residues produced by the formulation flocculation enable the use of the resulting sludge as secondary raw materials in the production of new composite materials.

Description

Title of Invention: BACTERIAL BIOPOLYMER FORMULATION SUITABLE FOR THE TREATMENT OF WASTEWATER WITH SUSPENDED SOLIDS, METHODS OF PRODUCING AND USING THE SAME |

Technical Field

[0001] This application relates to a bacterial biopolymer formulation suitable for the treatment of wastewater, particularly wastewater comprising suspended solids resulting from the stone-cutting process. The application also relates to a method of obtaining said biopolymer and formulation and a method of using the same.

Background Art

[0002] Currently, organic synthetic flocculants, such as polyacrylamide, are used for the sedimentation of suspended solids in wastewater from the stone-cutting process, thus producing stone-cutting sludge. However, the environmental incompatibility of these organic flocculants is a matter of great concern, since their degradation produces toxic residues (acrylamide monomers) that can enter the food chain and cause hazardous effects on human health. Thus, the use of synthetic flocculants can hardly be considered an ecologically sustainable approach to water treatment.

[0003] There are several extracellular polymeric substances (EPS) produced by bacterial strains that are potentially applicable to different industrial wastewater treatments (brewery, soy sauce brewing, meat processing, and pulp and paper). For example, EPS synthesized by Bacillus mucilaginosus was reportedly able to remove more than 85% of suspended solids (SS) and 68.5% of dissolved organic carbon (COD) from starch wastewater. The EPS was also used to treat swine wastewater with turbidity and COD removal efficiency of 91% and 42%, respectively. However, reports on biopolymers used to treat wastewater resulting from the stone-cutting process are scanty. Most of the studies about EPS applications in wastewater treatment were carried out only on a laboratory scale.

[0004] The use of bio-based materials, such as bacterial biopolymers, to treat wastewater is a sustainable and environmentally friendly approach that contributes to the well-being and safety of the population. The current wastewater treatment process using polyacrylamide can cause health issues due to the release of acrylamide monomers [1], and the sludge decantation system based on the use of bacterial biopolymers will benefit stone transformation companies from an environmental, energy, economic, and operational point of view. Sludges resulting from the flocculation with a biopolymer do not contain toxic polyacrylamide monomers. This enables the promotion of new synergies and industrial symbioses, promoting sludge as a secondary raw material, and contributing to the transformation of the stone industry into a zero-waste industry.

[0005] Some patent applications already disclose technologies that relate to the production of polymers for use as flocculants. However, none of the following examples discloses all the technical features of the technology disclosed in the present application.

[0006] W08800948 A1 relates to a method for identifying, characterizing, utilizing and modifying a set of genes which interact to produce a specific polymer. The method is demonstrated for the production of an exocellular polysaccharide produced by the bacteria Zoogloea ramigera. The isolated polysaccharide is useful as a viscosity modifier, oil field chemical, drag-reducing agent, dispersant or flocculant. Modification of the isolated genes, for example, by insertion of a regulatable promoter, provides a means for alteration of the enzymes responsible for the synthesis of the polysaccharide and its resulting structure.

[0007] CN114751495 A relates to a composite biological flocculant and a preparation method thereof. The composite biological flocculant is prepared from polyaluminium chloride, CaCh and a biopolymer.

[0008] CN111995026 A discloses a composite biological flocculant and a preparation method thereof. The method comprises adding an acrylamide monomer and an aqueous locust bean gum solution. The composite biological flocculant provided has very high flocculation performance, can reduce the dosage of the flocculant, and is safe and non-toxic to use and free of secondary pollution.

[0009] WO2011073874 A2 discloses a microbial biopolymer comprising fucose in its composition. This biopolymer consists of a polysaccharide comprising fucose, which represents at least 10% of its composition. This fucose-containing polysaccharide also contains non-sugar components, namely, acyl group substituents. The biopolymer is obtained by cultivation of the bacterium Enterobacter A47 (DSM 23139), using glycerol or glycerol-rich mixtures as carbon sources. The fucose-containing biopolymer may be used in several industrial applications (e.g. pharmaceutical, cosmetics and agro-food industries) and the treatment of industrial wastes (e.g. oil and metal recovery).

[0010] CN114773531 A discloses a modified chitosan flocculant as well as a preparation method and application thereof and belongs to the technical field of wastewater treatment. The prepared modified chitosan flocculant is loose, porous and excellent in adsorption performance and has the advantages of being good in water solubility, wide in pH use range, high in charge density and large in molecular weight.

Summary of Invention

[0011] The present invention relates to a bacterial biopolymer formulation comprising:

[0012] - Between 10% and 20% w/v of a biopolymer obtained from a Duganella strain NCIMB 44166;

[0013] - Between 0.5% and 1 % w/v of agarose;

[0014] - Between 0.1% and 0.2% w/v of glycerol.

[0015] The invention also relates to a method of obtaining the bacterial biopolymer, comprising the steps of:

[0016] - Producing inoculum of Duganella strain NCIMB 44166 by batch growth in xanthan medium at a temperature between 25 and 30°C for a time between 3 to 4 days;

[0017] - The growth and production of the bacterial biopolymer is carried out in the same condition as the inoculum;

[0018] - Extracting the bacterial biopolymer by alkaline treatment at a temperature between 75 and 80°C;

[0019] - Precipitation with ethanol in a ratio between 2:1 to 3:1 v/v to obtain the bacterial biopolymer. [0020] In one embodiment, the alkaline treatment is carried out with NaOH between 0.1 and 0.2 N.

[0021] In one embodiment, the bacterial biopolymer obtained is freeze-dried.

[0022] The invention also relates to a method to produce the bacterial biopolymer formulation comprising the following steps:

[0023] - Adding between 10 and 20% w/v of the bacterial biopolymer obtained from a Duganella strain NCIMB 44166 to a gelified solution in a proportion between 1 :10 to 1 :15 w/v, wherein the gelified solution comprises agarose 0.5 to 1% w/v, and glycerol 0.1 to 2% w/v;

[0024] - Allowing the previous mixture to solidify at a temperature between 20^sC and 30^sC to obtain the bacterial biopolymer formulation.

[0025] In one embodiment, the bacterial biopolymer formulation obtained is freeze- dried.

[0026] The invention also relates to a method for the treatment of wastewater with suspended solids comprising the following steps:

[0027] - Mixing wastewater with the bacterial biopolymer formulation disclosed above in a concertation between 0.05 to 0.2 mg/ml;

[0028] - Adding a solution comprising calcium between 1% and 2% w/v to have a final concentration between 5 and 10 mM of calcium and mixing between 200 and 400 rpm for a time between 5 and 10 minutes;

[0029] - Allowing the mixture to rest between 20 to 30 minutes without agitation to allow sedimentation of solid particles.

[0030] In one embodiment, the solution comprising calcium is made of CaCh.

[0031] In one embodiment, the bacterial biopolymer formulation is for use in the treatment of wastewater with suspended solids.

General description

[0032] The present application relates to a formulation comprising a bacterial biopolymer, wherein the formulation efficiently promotes the flocculation and settling of solid particles present in wastewater. The bacterial biopolymer formulation herein disclosed is suitable to be used as a substitute for polyacrylamide for the sedimentation of suspended solids in wastewater.

[0033] The bacterial biopolymer formulation described is environmentally friendly, has easy natural degradation, and complies with safety material rules. Residues produced by the bacterial biopolymer formulation flocculation enable the use of the resulting sludge as secondary raw materials in the production of new composite materials, without the need for deposition in landfills, since it does not contain toxic residues as the acrylamide in the formulation.

[0034] The bacterial biopolymer formulation herein described efficiently promotes the flocculation and settling of solid particles. The formulation comprises a biopolymer that is produced by a bacterial strain, Duganella sp. NCIMB 44166, isolated from a mine environment. The biopolymer is a metabolic product of the bacteria grown on sucrose as a carbon source. The bacterial biopolymer formulation is extracted by alkali treatment and ethanol precipitation and is obtained as a powder after freeze-drying.

[0035] The Duganella sp. NCIMB 44166 bacterial strain herein reported has never been reported as a biopolymer producer before and belongs to the private collection of the Bacterial Culture Collection of the University of Coimbra. Moreover, based on the phylogenetic characterization, this strain is a potential new species of the genus Duganella. The functional characteristics of the biopolymer indicate it is different from previously described bacteria.

[0036] This bacterial biopolymer formulation is particularly suitable for use as a flocculant in the sedimentation of suspended solids in wastewater resulting from the stone-cutting process.

[0037] There are several advantages to the bacterial biopolymer formulation disclosed herein:

[0038] 1 - It has high efficiency in the sedimentation of solid particles in suspension compared to polyacrylamide;

[0039] 2- It is non-toxic, enabling the development of an ecologically sustainable strategy for wastewater treatment of stone transformation companies;

[0040] 3- It is biodegradable, innocuous, and free of secondary pollutants; [0041] 4- Enables the use of biopolymer-flocculated sludge in the production of new composite materials, and its recovery in innovative products, instead of its deposition in landfills.

Brief Description of Drawings

[0042] For an easier understanding of this application, figures are attached in the annexes that represents the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

Fig.1

[0043] [Fig.1 ] shows the percentage of flocculation of the biopolymer obtained from Duganella sp. NCIMB 44166, in assays with different concentrations of biopolymer, 0.05 mg/ml (A), 0.1 mg/ml (B) and 0.2 mg/ml (C) in the presence and absence of CaCh (5 mM);

Fig.2

[0044] [Fig.2] shows the flocculation activity of the biopolymer and the formulation at different concentrations (0.05 mg/ml or 0.1 mg/ml) and in the presence of different calcium concentrations (5; 7.5; and 10 mM). (A) 0.05 mg/ml biopolymer; (B) 0.1 mg/ml biopolymer; (C) 0.05 mg/ml formulation;

Fig.3

[0045] [Fig.3] shows the comparison of the flocculation activity of the formulation comprising Duganella sp. NCIMB 44166 biopolymer, with polyacrylamide, and with other bacterial composites at different sedimentation times;

Fig.4

[0046] [Fig.4] shows the comparison of the maximum flocculation efficiency of the biopolymer and formulation of Duganella sp. NCIMB 44166, with other biopolymers, composites, and polyacrylamide, under the flocculation conditions tested;

Fig.5

[0047] [Fig.5] shows the comparison of the flocculation efficiency of different batches of the formulation of Duganella sp. NCIMB 44166, and polyacrylamide over a 20 minute trial period, using 0.5 L of stone-cutting water; [0048] [Fig.6] shows the flocculation efficiency of the formulation in up-scale assays, with 2.5 L of stone cutting water, where the formulation was used without and with prior hydration. In the assays were used 0.05 mg/ml of this biopolymer and two different concentrations of CaCI2 5 and 7.5 mM;

Fig.7

[0049] [Fig.7] shows the flocculation efficiencies obtained for the biopolymer of Duganella sp. NCIMB 44166 in up-scale assays with 2.5 L of stone cutting water, where two different concentrations of biopolymer, 0.05 mg/ml (A), and 0.1 mg/ml (B) were used;

Fig.8

[0050] [Fig.8] shows the flocculation efficiencies obtained for the biopolymer of Duganella sp. NCIMB 44166 (A) and polyacrylamide (B) in up-scale assays (2.5 L of stone cutting water), with a higher concentration of polymer 0.5 mg/ml;

Fig.9

[0051] [Fig.9] shows the flocculation efficiency of biopolymer (A) and formulation (B) of Duganella sp. NCIMB 44166 in up-scale assays with 0.05 mg/ml of biopolymer and in the presence of different calcium concentrations. (5; 7.5; and 10 mM of CaCI₂);

Fig.lOA

[0052] [Fig.l OA] shows the comparison of the flocculation efficiency of biopolymer and formulation of Duganella sp. NCIMB 44166 with polyacrylamide in trials with 0.02 L (A) of stone-cutting water;

Fig.lOB

[0053] [Fig.l OB] shows the comparison of the flocculation efficiency of biopolymer and formulation of Duganella sp. NCIMB 44166 with polyacrylamide in trials with 0.5 L (B) of stone-cutting water;

Fig.lOC

[0054] [Fig.l OC] shows the comparison of the flocculation efficiency of biopolymer and formulation of Duganella sp. NCIMB 44166 with polyacrylamide in trials with 2.5 L (C) of stone-cutting water; Fig.lOD

[0055] [Fig.l OD] shows the comparison of the flocculation efficiency of biopolymer and formulation of Duganella sp. NCIMB 44166 with polyacrylamide in trials with 1000 L (D) of stone-cutting water.

Description of Embodiments

[0056] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0057] The present application relates to a bacterial biopolymer formulation comprising a biopolymer obtained from a strain of Duganella. The bacterial biopolymer formulation is suitable for use in the treatment of wastewater comprising suspended solids.

[0058] The strain was deposited on 20/06/2023 according to the Budapest Treaty on the National Collection of Industrial, Food and Marine Bacteria (NCIMB) for the Purposes of Patent Procedure in the Culture Collection, Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland, with the accession number NCIMB 44166.

[0059] In a preferred embodiment, the bacterial biopolymer formulation is suitable for use in the treatment of wastewater resulting from the stone-cutting process.

[0060] The bacterial biopolymer formulation disclosed herein efficiently promotes the flocculation and settling of solid particles present in wastewater.

[0061] The bacterial biopolymer formulation comprises:

[0062] Between 10% and 20% w/v of a biopolymer obtained from a Duganella sp. NCIMB 44166;

[0063] Between 0.5% and 1% w/v of agarose;

[0064] Between 0.1% and 0.2% w/v of glycerol.

[0065] The present application also discloses a method to obtain a bacterial biopolymer from a Duganella sp. NCIMB 44166 comprising the following steps:

[0066] - Producing inoculum of Duganella sp. NCIMB 44166 by batch growth in xanthan medium at a temperature between 25 and 30°C for a time between 3 to 4 days; [0067] - The growth and production of the bacterial biopolymer is carried out in the same condition as the inoculum;

[0068] - Extracting the bacterial biopolymer by alkaline treatment with at a temperature between 75 and 80°C;

[0069] - Precipitation with ethanol in a ratio between 2:1 to 3:1 v/v to obtain the bacterial biopolymer.

[0070] In one embodiment, the alkaline treatment is carried out with NaOH between 0.1 and 0.2 N.

[0071] In one embodiment, the bacterial biopolymer obtained is freeze-dried.

[0072] In one embodiment, the xanthan medium is prepared according to reference [2]-

[0073] The present application also discloses a method to produce a bacterial biopolymer formulation comprising the following steps:

[0074] - Adding between 10 and 20% w/v of the bacterial biopolymer obtained from a Duganella sp. NCIMB 44166 to a gelified solution in a proportion between 1 :10 to 1 :15 w/v, wherein the gelified solution comprises agarose 0.5 to 1% w/v, and glycerol 0.1 to 2% w/v;

[0075] - Allowing the previous mixture to solidify at a temperature between 20^sC and 30^sC to obtain the bacterial biopolymer formulation.

[0076] In one embodiment, the bacterial biopolymer formulation obtained is freeze- dried.

[0077] The present application also discloses a method for the treatment of wastewater with suspended solids comprising the following steps:

[0078] - Mixing wastewater with the bacterial biopolymer formulation disclosed above in a concertation between 0.05 to 0.2 mg/ml;

[0079] - Adding a solution comprising calcium between 1% and 2% w/v to have a final concentration between 5 and 10 mM of calcium and mixing between 200 and 400 rpm for a time between 5 and 10 minutes;

[0080] - Allowing the mixture to rest between 20 to 30 minutes without agitation to allow sedimentation of solid particles. [0081] In one embodiment, the solution comprising calcium is made of CaCI2.

[0082] All flocculation assays for proof of concept were performed in duplicate, and in the presence of a control, composed only by the wastewater. The flocculation activity (FA) was expressed as a percentage (%), using the following equation:

[0083]

[0084] Where A, is the absorbance value at 550 nm of the control and B, is the absorbance value at 550 nm of the samples.

[0085] The flocculation assays were performed with different volumes of wastewater: 20, 500 and 2500 ml, different concentration of biopolymer (0.05; 0.1 and 0.2 mg/ml), and different concentrations of CaCI2 (5; 7.5 and 10 mM) at a temperature between 20 and 30^sC.

Examples

[0086] Several bacterial biopolymer formulations were prepared from the biopolymer obtained from Duganella sp. NCIMB 44166. The conditions for production and extraction were established, and laboratory scale flocculation of solid particles from wastewater (0.02, 0.5, and 2.5 L) was tested.

[0087] In addition, a comparison of the efficiency of the biopolymer, formulation comprising the biopolymer, and polyacrylamide was also carried out.

[0088] 1 . Study of the treatment, on a laboratory scale, of wastewater from the stonecutting process by bioflocculation

[0089] A bacterial strain Duganella sp. NCIMB 44166 was identified as having a high capacity to produce a biopolymer. Based on this biopolymer, a sample of the formulation was prepared by adding this biopolymer to a mixture of agarose 0.5% (v/v) and glycerol 0.1% (v/v), in the proportion of 1 :10 (w/v) of biopolymer and mixture. This sample formulation comprising the biopolymer showed a high flocculation capacity and potential for use in the treatment of stone-cutting wastewater. Flocculation conditions for the biopolymer produced by Duganella sp. NCIMB 44166, as well as, the formulation comprising it, were determined in small-scale (0.02 L) laboratory assays.

[0090] Flocculation conditions were determined by varying the following parameters: io [0091] a) Stirring conditions;

[0092] b) Concentration of biopolymer/formulation (0.05; 0.1 ; 0.2 mg/ml);

[0093] c) Calcium concentration (5; 7.5; 10 mM of CaCI2);

[0094] d) Different sedimentation times (5 to 30 minutes).

[0095] a) Stirring conditions

[0096] A constant stirring of 300 rpm, in a magnetic stirrer for 5 minutes showed good results in the small-scale flocculation trials for both biopolymer and formulation.

[0097] b) Biopolymer/formulation concentration (0,05; 0,1 ; 0,2 mg/ml)

[0098] Lower concentrations of biopolymer/formulation (0.05 or 0.1 mg/ml) promoted a higher percentage of flocculation. The biopolymer of Duganella sp. NCIMB 44166 and the formulation comprising it showed a higher flocculation capacity in the presence of calcium (5 mM CaCI2) when compared with its performance in the absence of calcium. In the concentration of 0.2 mg/ml, the biopolymer did not show flocculation activity in the absence of calcium (Fig 1 C).

[0099] c) Calcium concentration for flocculation (5; 7.5; 10 mM Ca)

[0100] Assays with 0.05 mg/ml of biopolymer showed that the presence of 10 mM Ca promotes higher flocculation efficiency (Fig 2 A). In the presence of higher concentrations of biopolymer 0.1 mg/ml and with 0.05 mg/ml of the formulation, the flocculation efficiency is similar in the presence of the three calcium concentrations tested (Fig 2 B and 2C).

[0101 ] d) Different settling times (5 to 30 minutes)

[0102] The flocculation efficiency of the biopolymer/formulation was evaluated for a period of 30 minutes. The flocculation efficiency was determined at 5-minute intervals until the end of the trial. For the biopolymer and under the conditions tested, the maximum flocculation efficiency was seen in the first 15 minutes (85- 90%). After, this time the flocculation efficiency was maintained or slightly decreased. The maximum efficiency of the formulation comprising the biopolymer of Duganella sp. NCIMB 44166 (97% flocculation) was obtained at 10 min, comparable to that of polyacrylamide, a flocculant currently used in stone processing companies (Figure 3).

[0103] The polyacrylamide in the tested conditions presented its maximum efficiency at 5 min (93.2%) (Figure 3). The formulation comprises the biopolymer of Duganella sp. NCIMB 44166 also presented higher flocculation efficiency than the other two tested formulations comprising biopolymers obtained from other bacteria strains (composite 1 and 2), which were produced with biopolymers of other bacterial strains.

[0104] The small-scale flocculation tests (0.02 L) showed that the conditions for flocculation of suspended solid particles from cutting wastewater using the biopolymer and formulation of Duganella sp. NCIMB 44166 can be:

[0105] - Constant stirring (300 rpm) of the samples with the biopolymers and formulations for a period of at least 5 minutes;

[0106] - Lower concentrations of biopolymer and formulation tested (0.05 mg/ml or 0.1 mg/ml) promoted higher flocculation efficiency;

[0107] - An efficient flocculation of water suspended particles (similar to that obtained with polyacrylamide) was obtained with biopolymer and the formulation of Duganella sp. NCIMB 44166 in the presence of calcium;

[0108] - The maximum flocculation efficiency occurred in the first 10-15 minutes for Duganella sp. NCIMB 44166 biopolymer and formulation (94-95%) (Figure 4).

[0109] This biopolymer and formulation have a high potential to be used as a bioflocculant, showing flocculation efficiency similar to that of polyacrylamide, a flocculant currently used in the industry (Figure 4).

[01 10] 2. Study of large-scale (0.5 L and 2.5 L) laboratory-scale treatment of wastewater from stone-cutting process by bioflocculation

[01 11 ] Once the flocculation parameters for the Duganella sp. NCIMB 44166 biopolymer and formulation were optimised in small-scale trials (0.02 L), it was intended to up-scale the flocculation process in larger volumes. The volumes of stone-cutting water in the flocculation assays were increased to 0.5 and 2.5 L.

[01 12] A) Flocculation efficiency of the different batches of biopolymer produced [0113] Several batches of biopolymer and formulation were produced in order to have sufficient amounts of biopolymer for the upscale trials with larger volumes of stone-cutting water. Flocculation trials were conducted with the different batches produced in 0.5 L volumes of stone-cutting water to determine if the different polymer batches showed similar flocculation efficiency.

[0114] The tests with the different batches of formulation of Duganella sp. NCIMB 44166 produced were performed with the lowest concentration of biopolymer (0.05 mg/ml) and in the presence of the lowest concentration of CaCk (5 mM) tested. These assays showed that the flocculation efficiency is similar for the different batches, including the flocculation efficiency of the mixture, which is nothing more than a mixture, 20 to 30 mg of each of the different batches of the produced formulation of Duganella sp. NCIMB 44166 (batch 3, 4 and 6) used in the concentration of 0.05 mg/ml in the assay (Figure 5).

[0115] Table 1 shows the maximum flocculation values obtained for the different batches of biopolymer formulations and for polyacrylamide. The maximum flocculation efficiency of the different batches of biopolymer formulation and its mixture, ranged between 91 .5% and 97.9%, comparable to those of polyacrylamide, which ranged between 97.1% and 97.5%. However, the maximum efficiency of the formulation of Duganella sp. NCIMB 44166 was obtained later between 10-15 minutes of testing, while polyacrylamide shows its maximum flocculation at 5-10 minutes.

[0116] Table 1 . Maximum flocculation values

[0117]

[0118] B) Hydration of biopolymer/formulation in upscale flocculation tests [0119] In the upscale flocculation assays with volumes of 2.5 L of stone-cutting water, the methodology used was the one optimized in the small-scale trials (0.02 L). However, it was found that the introduction of the dry biopolymer formulation, without prior hydration, in the upscale assays resulted in a lower flocculation efficiency. The maximum flocculation percentage of biopolymer formulation of Duganella sp. NCIMB 44166 (obtained in assays with 0.05 mg/ml and two different CaCI2 concentrations (5 and 7.5 mM) was only 63.5-63.9 % (Figure 6), much lower than the values presented in the small-scale assays (0.02 L).

[0120] Hydration of biopolymer formulation of Duganella sp. NCIMB 44166 for 15 minutes with 5 ml of the CaCh solution to be used in the assay, increased the maximum flocculation efficiency values of this biopolymer to values of 81 .5- 92.5 % (Figure 6), similar to those obtained in the small-scale assays. Thus, in all upscale trials, the polymer was hydrated with the CaCh solution for 15 minutes before being added to the assay.

[0121] C) Concentration of biopolymer/formulation in upscale flocculation tests

[0122] Two different concentrations of biopolymer of Duganella sp. NCIMB 44166, 0.05 mg/ml and 0.1 mg/ml were tested in the upscale flocculation assays. The lowest concentration of biopolymer (0.05 mg/ml) tested promoted higher flocculation efficiency (95.6-96.4%) (Fig 7 A) when compared to assays where the double of concentration of biopolymer (0.1 mg/ml) was used (89.2-92.3%) (Fig 7 B).

[0123] In the industry, the polyacrylamide concentration used to flocculate the solid particles from the stone-cutting water is higher (between 0.5 and 1 mg/ml) than the concentration of biopolymer of Duganella sp. NCIMB 44166 used in these upscale tests. Thus, flocculation tests were performed with the biopolymer of Duganella sp. NCIMB 44166 and polyacrylamide at a concentration of 0.5 mg/ml. The maximum flocculation values obtained for both biopolymer of Duganella sp. NCIMB 44166 (Fig 8 A) and polyacrylamide (Fig 8 B) were similar, between 70.8% and 77.2%, but lower than those obtained in the assays using lower polymer concentrations (see Figure 5 and Figure 7).

[0124] D) Calcium concentration in upscale flocculation assays [0125] The small-scale flocculation assays had already shown that the biopolymer/formulation of Duganella sp. NCIMB 44166 needed the presence of calcium (CaCI2) to perform more efficiently in flocculating solid particles from stone-cutting water.

[0126] The flocculation tests with biopolymer of Duganella sp. NCIMB 44166 (Fig 9 A) at the concentration of 0.05 mg/ml and in presence of three different calcium concentrations (5; 7.5; and 10 mM of CaCh) showed that the flocculation efficiency is similar in the three calcium concentrations tested, ranging between the values of 82% to 96%. For the formulation of this biopolymer, lower calcium concentrations 5 and 7.5 mM promoted higher flocculation efficiency, between 74% and 93% (Fig 9 B).

[0127] The conditions for flocculation of suspended solid particles from stone-cutting wastewater in large-scale laboratory assays (2.5 L) using biopolymer/formulations were:

[0128] - Constant stirring (300 rpm) to homogenize the stone-cutting water with biopolymer/formulation of Duganella sp. NCIMB 44166 for a period of at least 15 minutes;

[0129] - Solubilisation of biopolymer/formulation of Duganella sp. NCIMB 44166 in 5 ml of CaCh solution for 15 minutes before being used in the tests;

[0130] - Biopolymer/formulation of Duganella sp. NCIMB 44166 required calcium to promote efficient flocculation (5 mM CaCh);

[0131] - A concentration of 0.05 mg/ml biopolymer/formulation of Duganella sp. NCIMB 44166 in the stone-cutting water assays promoted greater flocculation efficiency.

[0132] 3. Comparative study of the flocculation efficiency of polyacrylamide/biopolymer formulation of Duganella sp. NCIMB N^Q 44166 to treat wastewater from stone-cutting process

[0133] In all flocculation tests in the laboratory and also in the field, regardless of the volume of stone-cutting water used, the results showed that the biopolymer/formulation had a flocculation efficiency very similar to polyacrylamide (Figure 10A, 10B, 10C, 10D), the synthetic flocculant currently used in the industry.

[0134] The maximum flocculation efficiency for biopolymer /formulation of Duganella sp. NCIMB 44166 in the "upscale" assays in the laboratory with 2.5 L of stonecutting water occurred at 15-20 minutes. Polyacrylamide showed maximum flocculation at 5 minutes (92.5%), but its maximum efficiency was lower than that of the biopolymer of Duganella sp. NCIMB 44166 (96.4-95.5 %) (Figure 10C). In the pilot field assay with 1000 L of stone-cutting water the biopolymer of Duganella sp. NCIMB 44166, also showed a higher efficiency than the polyacrylamide with a maximum efficiency of flocculation of 99.4-99.8 % that occurred at 30-45 minutes (Figure 10D). The polyacrylamide polymer showed a maximum efficiency of 98.9 % at 30 minutes (Figure 10D).

[0135] The different flocculation assays of solid particles from stone-cutting wastewater, carried out in laboratory, both on a small and large scale showed that the biopolymer and formulation comprising the biopolymer of Duganella sp. NCIMB 44166 have great potential to be used as bioflocculants, as an alternative to polyacrylamide, for the production of usable sludge. These biopolymers showed a high flocculation efficiency (about 95 %) and similar to that presented by polyacrylamide (92.5 %). The pilot field assay using 1000 L of stone-cutting water also corroborated this assumption. The biopolymer of Duganella sp. NCIMB 44166 is highly efficient in removing the solid particles of the stonecutting water (about 99.4 to 99.8 %). In large-scale laboratory tests, these biopolymers showed that the minimum concentration required to promote efficient flocculation was 0.05 mg/ml. They required the presence of calcium in the assays (5 mM of CaCh) to have an efficient flocculation and reach maximum values of flocculation between 15 and 20 minutes of the assay. In the pilot field assay the parameters used to promote an efficient flocculation were the ones determined in the large-scale laboratory tests, with higher times of contact between biopolymer and solid particles of the stone-cutting to reach maximum values of flocculation, 30 to 45 minutes.

[0136] Bibliography [0137] [1] Xiong, B., Loss, R.D., Shields, D. et al. Polyacrylamide degradation and its implications in environmental systems, npj Clean Water 1 , 17 (2018): https://doi.Org/10.1038/S41545-018-0016-8.

[0138] [2] Carignatto C.R., Oliveira K.S., de Lima V.M., de Oliva Neto P. New Culture Medium to Xanthan Production by Xanthomonas campestris pv. campestris. Indian J Microbiol. 51 , 283-288 (2011 ) doi: 10.1007/s12088-011 -0171 -9.

[0139] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

Reference to Deposited Biological Material

[0140] The strain was deposited according to the Budapest Treaty on the National Collection of Industrial, Food and Marine Bacteria (NCIMB) for the Purposes of Patent Procedure in the Culture Collection, Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland, with the accession number NCIMB 44166.

Claims

[Claim 1] A bacterial biopolymer formulation comprising:

- Between 10% and 20% w/v of a biopolymer obtained from a Duganella sp. NCIMB 44166;

- Between 0.5% and 1 % w/v of agarose;

- Between 0.1 % and 0.2% w/v of glycerol.

[Claim 2] Method of obtaining the bacterial biopolymer described in claim 1 , comprising the steps of:

- Producing inoculum of Duganella sp. NCIMB 44166 by batch growth in xanthan medium at a temperature between 25 and 30°C for a time between 3 to 4 days;

- The growth and production of the bacterial biopolymer is carried out in the same condition as the inoculum;

- Extracting the bacterial biopolymer by alkaline treatment at a temperature between 75 and 80°C;

- Precipitation with ethanol in a ratio between 2:1 to 3:1 v/v to obtain the bacterial biopolymer.

[Claim 3] Method according to the previous claim, wherein the alkaline treatment is carried out with NaOH between 0.1 and 0.2 N.

[Claim 4] Method according to any of the claims 2 to 3, wherein the bacterial biopolymer obtained is freeze-dried.

Method to produce the bacterial biopolymer formulation described in claim 1 , comprising the following steps:

- Adding between 10 and 20% w/v of the bacterial biopolymer obtained from a Duganella sp. NCIMB 44166 to a gelified solution in a proportion between 1 :10 to 1 :15 w/v, wherein the gelified solution comprises agarose 0.

5 to 1% w/v, and glycerol 0.1 to 2% w/v;

- Allowing the previous mixture to solidify at a temperature between 20^sC and 30^sC to obtain the bacterial biopolymer formulation. [Claim 5] Method according to the previous claim, wherein the bacterial biopolymer formulation obtained is freeze-dried.

[Claim 6] Method for the treatment of wastewater with suspended solids comprising the following steps:

- Mixing wastewater with the bacterial biopolymer formulation disclosed above in a concertation between 0.05 to 0.2 mg/ml;

- Adding a solution comprising calcium between 1% and 2% w/v to have a final concentration between 5 and 10 mM of calcium and mixing between 200 and 400 rpm for a time between 5 and 10 minutes;

- Allowing the mixture to rest between 20 to 30 minutes without agitation to allow sedimentation of solid particles.

[Claim 7] Method according to the previous claims, wherein the solution comprising calcium is made of CaCh.

[Claim 8] Bacterial biopolymer formulation as described in claim 1 for use in the treatment of wastewater with suspended solids, i