Process of producing extracellular polymeric substances

Abstract

The invention relates to an economic process of producing extracellular polymeric substances (EPS), said process comprising: • contacting an aqueous stream comprising a carbon source with a biofilm generating system (BGS) under aerobic conditions to develop an EPS-containing biofilm on the BGS, the BGS comprising (i) an inert carrier and (ii) EPS-producing bacteria attached to the surface of the inert carrier; and • subjecting the BGS comprising the EPS-containing biofilm to a harvesting step in which the EPS-containing biofilm is separated from the inert carrier, the separated biofilm having an EPS content of at least 30% by weight of the organic matter that is contained in the separated biofilm; wherein the BGS is provided in the form of discrete pieces comprising an inert carrier body having a diameter in the range of 0.2-30 cm or wherein the BGS is provided in the form of a layer comprising a sheet of inert carrier.

Description

[0001] PROCESS OF PRODUCING EXTRACELLULAR POLYMERIC SUBSTANCES

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention provides an economic process for the production of extracellular polymeric substances (EPS), especially EPS that can suitably be applied, for instance, as a flocculant, as a soil improver (water retention) or in bioremediation (e.g. removal of heavy metals and / or toxic organic chemical removal).

[0004] BACKGROUND OF THE INVENTION

[0005] Flocculation is a process by which colloidal particles come out of suspension to settle in the form of floc or flake. Flocculation is widely employed in the purification of drinking water as well as in sewage treatment, storm-water treatment and treatment of industrial wastewater streams. In order to induce flocculation, flocculants are employed to cause suspended solids to clump together and form flocs. These flocs are then easily removed by e.g. sedimentation and / or filtration.

[0006] There are a number of different flocculants that can be used, including inorganic flocculants, organic flocculants, and natural flocculants. Inorganic flocculants are typically salts of metal ions, such as aluminium sulfate and ferric chloride. Organic flocculants are typically polymers, such as polyelectrolytes or uncharged non-ionic polymers. Natural flocculants are typically biopolymers, such as chitosan, starch, cellulose, tannin and gelatine.

[0007] As the demand for eco-friendly flocculation processes continues to grow, so does the need for natural biodegradable flocculants.

[0008] WO 2019 / 009708 describes a method for production of natural, biodegradable polymeric flocculants from a fluid, such as a waste fluid, the method comprising the steps of:

[0009] providing a reactor comprising:

[0010] o at least one reactor vessel;

[0011] o at least one fluid inlet;

[0012] o at least one fluid outlet;

[0013] o at least one flocculant outlet; and

[0014] o at least one membrane provided in the reactor vessel between the fluid inlet and the fluid outlet;

[0015] inoculation of the reactor;

[0016] providing feed to the at least one fluid inlet;

[0017] forming the flocculants; separating the flocculants using the membrane; and

[0018] providing the flocculants at the flocculant outlet.

[0019] Bahgat et al. (Integrated resource recovery from aerobic granular sludge plants, Water Research, Volume 234, 1 May 2023, 119819) investigated the combined phosphorus, nitrogen, methane, and extracellular polymeric substances (EPS) recovery from aerobic granular sludge (AGS) wastewater treatment plants. About 30% of sludge organics were recovered as EPS and 25-30% as methane by integrating alkaline anaerobic digestion (AD). The authors developed a laboratory protocol mimicking demonstration-scale EPS extraction conditions.

[0020] US 5,595,893 describes a method of producing a fermentation product within a biofilm reactor, comprising the steps of:

[0021] (a) providing a solid support comprised of 50-95 wt-% of a polyolefin synthetic polymer in admixture with about 5-50 wt-% of an organic polymeric plant material, the surface of the support having cells of a film-forming microorganism attached as a film over a substantial proportion of the surface of the support to form a biofilm reactor;

[0022] (b) placing the biofilm reactor in a liquid medium comprising a substance to be fermented by the microorganism; and

[0023] (c) allowing the microorganism to substantially ferment the substance to produce the fermentation product.

[0024] Moga et al. (Polyethylene Based Materials for Biofilm Carriers Used in Wastewater Treatment, I OP Conf. Series: Materials Science and Engineering 374 (2018) 012080 doi: 10.1088 / 1757-899X / 374 / 1 / 01208) propose a novel biofilm carrier to be used in tertiary treatment for tannery and paper-mill wastewaters. The biological treatment is based on fungal activity. The selected fungal strains will be grown on innovative polyethylene carriers containing cellulose. The carrier will be designed to be exploited in a moving bed bioreactor and to favour fungal growth in the presence of competing bacteria.

[0025] Zhe Liu et al (A comparison between exogenous carriers enhanced aerobic granulation under low organic loading in the aspect of sludge characteristics, extracellular polymeric substances and microbial communities, Bioresource Technology 346 (2022) 126567) describe a study in which three exogenous carriers (polymeric ferric sulfate, aluminium sulfate, and diatomite were added to enhance aerobic granulation under low organic loading rate, and their effects of aerobic granule formation, extracellular polymeric substances (EPS) secretion and microbial community were investigated. Zixuan Liang et al (Formation, extracellular polymeric substances, and structural stability of aerobic granules enhanced by granular activated carbon, Environmental Science and Pollution Research (2019) 26:6123-6132) describe a study in which the impact of granular activated carbon (GAC) on the formation of EPS was investigated. Raw GAC was pulverized at high speed and then passed through a screen to yield a refined GAC with a diameter of 0.2 ± 0.025 mm. The refined GAC was added to a reactor while inoculating the reactor with activated sludge.

[0026] SUMMARY OF THE INVENTION

[0027] The inventors have designed an economic process for the production of extracellular polymeric substances (EPS). The process employs a biofilm generating system (BGS) comprising (i) an inert carrier and (ii) EPS-producing bacteria attached to the surface of the inert carrier. The BGS is contacted with an aqueous stream comprising a carbon source to develop an EPS-containing biofilm on the inert carrier. Once a substantial biofilm layer has developed, the EPS-containing biofilm can easily be harvested by removing the biofilm from the carrier. The BGS employed in the present process is provided in the form of discrete pieces comprising an inert carrier body or in the form of a layer comprising a sheet of inert carrier.

[0028] Accordingly, a first aspect of the invention relates to a process of producing extracellular polymeric substances (EPS), said process comprising:

[0029] • contacting an aqueous stream comprising a carbon source with a biofilm generating system (BGS) under aerobic conditions to develop an EPS-containing biofilm in the BGS, the BGS comprising (i) an inert carrier and (ii) EPS-producing bacteria attached to the surface of the inert carrier; and

[0030] • subjecting the BGS comprising the EPS-containing biofilm to a harvesting step in which the EPS-containing biofilm is separated from the inert carrier, the separated biofilm having an EPS content of at least 30% by weight of the organic matter that is contained in the separated biofilm;

[0031] wherein the BGS is provided in the form of discrete pieces comprising an inert carrier body having a diameter in the range of 0.2-30 cm or wherein the BGS is provided in the form of a layer comprising a sheet of inert carrier.

[0032] The process of the present invention offers the advantage that it is very robust and efficient. Unlike the process of WO 2019 / 009708, for instance, it does not require continuous concentration of polymers and bacteria, from a dilute aqueous stream, using an ultrafiltration membrane that needs to be cleaned at regular intervals. In the present process, EPS-containing biofilm is obtained in highly concentrated form by simply collecting the biofilm-comprising BGS, and thus separating it from aqueous liquid. Also harvesting of the biofilm from the BGS is easily achieved by removing the biofilm from the inert carrier, yielding a biofilm material that can contain over 60% EPS by weight of the organic matter that is contained in the separated biofilm.

[0033] Another aspect of the invention relates to EPS-containing biofilm obtained by the process of the present invention.

[0034] Yet another aspect of the invention relates to the use of the aforementioned EPS-containing biofilm, or of an EPS extract obtained from said biofilm, as a flocculant, as a soil improver or in bioremediation.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The invention relates to a process of producing extracellular polymeric substances (EPS), said process comprising:

[0037] • contacting an aqueous stream comprising a carbon source with a biofilm generating system (BGS) under aerobic conditions to develop an EPS-containing biofilm on the BGS, the BGS comprising (i) an inert carrier and (ii) EPS-producing bacteria attached to the surface of the inert carrier; and

[0038] • subjecting the BGS comprising the EPS-containing biofilm to a harvesting step in which the EPS-containing biofilm is separated from the inert carrier, the separated biofilm having an EPS content of at least 30% by weight of the organic matter that is contained in the separated biofilm;

[0039] wherein the BGS is provided in the form of discrete pieces comprising an inert carrier body having a diameter in the range of 0.2-30 cm or wherein the BGS is provided in the form of a layer comprising a sheet of inert carrier.

[0040] The term “extracellular polymeric substances” as used herein refers to biopolymers that are secreted by bacteria into their environment, said biopolymers having a molecular weight of at least 10 kDa. Examples of such biopolymers include polysaccharides, proteins and lipopolysaccharides.

[0041] The term “inert carrier” as used herein refers to a carrier that remains essentially unchanged in the present process in that it does not dissolve and does not undergo chemical reactions with components of the aqueous stream. EPS-producing bacteria are not part of the inert carrier.

[0042] The term “biofilm” as used herein refers to a community of EPS-producing bacteria that are embedded in a matrix of EPS, produced by these bacteria, that is stuck to the surface of the inert carrier. The term “carbon source” as used herein refers to carbon-containing substances that can be digested by the EPS-producing bacteria. Examples of carbon sources include sugars, alcohols, glycerol and volatile fatty acids.

[0043] The term “under aerobic conditions” means that sufficient oxygen is provided to allow aerobic and / or micro-aerophilic EPS-producing bacteria to grow.

[0044] The EPS concentration expressed “by weight of the organic matter of the separated biofilm” is determined on the basis of the total amount of dry organic matter that is contained in the separated biofilm. The organic matter content of the separated biofilm can be determined by determining the weight of biofilm sample after drying the separated biofilm for 24 hours at 105°C (dry matter content) minus the weight of the biofilm after another 4 hours incineration at 550°C (ash content).

[0045] The term “or” as used herein should be construed as “and / or”, unless specified otherwise.

[0046] The term “a” or “an” as used herein is defined as “at least one” unless specified otherwise.

[0047] Numerical ranges expressed in the format “from x to y” are understood to include x and y.

[0048] Whenever components A and B are said to be present in a weight ratio of x:y, what is meant is that the concentration of component A in wt.% divided by the concentration of component B in wt.% equals x:y.

[0049] Ratios mentioned herein are based on weight / weight, unless indicated otherwise. Similarly, all percentages are percentages by weight (w / w) unless otherwise indicated.

[0050] When multiple preferred ranges are described in the format “from x to y” for a specific feature, it should be understood that all ranges combining the different endpoints are also contemplated.

[0051] If, for a particular component, a range of 0% to y% or less than y% is recited, said ingredient may be absent.

[0052] In the present process, the BGS comprising an inert carrier and EPS-producing bacteria attached to the surface of the inert carrier may be produced by contacting the inert carrier with aqueous liquid containing the EPS-producing bacteria under conditions stimulating the bacteria to attach themselves to the surface of the inert carrier, e.g. by forming a biofilm onto the inert carrier. In an embodiment of the present process this may be achieved by contacting an aqueous stream comprising EPS-producing bacteria and a carbon source with the inert carrier under aerobic conditions.

[0053] In a preferred embodiment, prior to the harvesting step, the process comprises the step of collecting BGS comprising the EPS-containing biofilm. By collecting the BGS comprising the EPS-containing biofilm, the BGS is separated from aqueous liquid, allowing the biofilm to be harvested very efficiently. The collecting of the BGS comprising the EPS-containing biofilm may be achieved by separating the BGS comprising the EPS-containing biofilm from the aqueous stream. This may be achieved, for instance, by removing BGS comprising the EPS-containing biofilm together with some aqueous stream liquid and by subsequently separating the BGS comprising EPS-containing biofilm from the aqueous stream liquid. The separated aqueous stream liquid is then preferably returned to the aqueous stream. The collecting of the BGS comprising the EPS-containing biofilm may also be achieved by removing BGS comprising the EPS-containing biofilm from the aqueous stream, e.g. by lifting or rotating it out of the aqueous stream.

[0054] In yet another preferred embodiment, the present process is carried out using two or more reactors in a parallel arrangement, each reactor comprising the biofilm generating system. This arrangement offers the advantage that the aqueous stream can be diverted from one reactor to another reactor, thus allowing easy harvesting of the EPS-containing biofilm in the reactor from which the aqueous stream has been diverted. The process may suitably be carried out using multiple reactors in a parallel arrangement by feeding the aqueous stream to all but one of the reactors and by simultaneously carrying out the harvesting step in the remaining reactor. This arrangement offers the advantage that the aqueous stream does not need to be interrupted for harvesting.

[0055] The inert carrier that is employed in accordance with the present invention preferably is a synthetic carrier.

[0056] In accordance with one advantageous embodiment, the BGS employed is provided in the form of discrete pieces. Preferably, these BGS pieces comprise an inert carrier body.

[0057] The inert carrier body preferably has a diameter in the range of 0.2-30 cm, more preferably the range of 0.5-20 cm and most preferably the range of 1-10 cm.

[0058] The inert carrier body preferably has a high surface to volume ratio, e.g. a surface to volume ratio of at least 25 nr1, more preferably of at least 100 nr1, still more preferably of at least 200 nr1and most preferably of 300-3000 nr1. The surface to volume ratio of the carrier body is determined e.g. by completely filling a cylindrical vessel with a diameter of approximately 1 m and a volume of 1 m3with the carrier bodies. The surface to volume ratio (<t>) of the carrier body is calculated on the basis of the following equation:

[0059] = (n xA) / V

[0060] wherein:

[0061] n = the total number of carrier bodies in the vessel

[0062] A = the surface area of a single carrier body (in m2)

[0063] V = the internal volume of the vessel (in m3).

[0064] In the embodiment of the present process wherein BGS pieces are employed, the aqueous stream comprising a carbon source preferably is contacted with a bed of the BGS pieces. The aqueous stream may be contacted with the BGS pieces by passing it through a bed of the BGS pieces. The bed of BGS pieces can be a moving bed. In the present process, a moving bed of BGS pieces may suitably be realised by passing the aqueous stream upwards through a bed of BGS pieces that is moving downwards under the influence of gravity. In an alternative arrangement, the bed of BGS pieces is rotated through the aqueous stream, e.g. by employing rotating chambers filled with BGS pieces, which chambers comprise multiple openings through which the aqueous stream can pass.

[0065] According to a particularly preferred embodiment of the present process, BGS pieces comprising EPS-containing biofilm are removed from the bed of BGS pieces and inert carrier bodies are added to the bed to replenish the inert carrier bodies that were removed as part of the BGS pieces. This embodiment enables continuous or semi-continuous operation of the present process. The BGS pieces comprising the EPS-containing biofilm that are removed from the bed can be subjected to the harvesting step in which the biofilm is removed from the inert carrier bodies while the flow of the aqueous stream through the bed of BGS pieces continues. Furthermore, the inert carrier bodies obtained after the harvesting step can be returned to the bed of BGS pieces to replenish the BGS pieces that were removed. Preferably, the BGS pieces comprising the EPS-containing biofilm are removed from the bottom of the bed of BGS pieces and the inert carrier bodies obtained after the harvesting step are returned to the top of the bed.

[0066] In the present process the bed of BGS pieces may be completely submerged in the aqueous stream comprising a carbon source. In a more preferred embodiment, the aqueous stream percolates through the bed of BGS pieces. During percolation only a fraction of the volume between the BGS pieces in the bed of BGS pieces is filled with liquid from the aqueous stream. Percolation offers the advantage that the BGS pieces can be removed from the aqueous stream without also removing a substantial amount of aqueous liquid. According to a particularly preferred embodiment, the aqueous stream is passed through the bed of the BGS pieces at a flow rate of at least 2 m / hr, more preferably at least 3 m / hr and most preferably of 5 to 30 m / hr.

[0067] In accordance with another advantageous embodiment, the BGS is provided in the form of a layer comprising a sheet of inert carrier and wherein the aqueous stream is passed through and / or along the BGS layer. More preferably, the aqueous stream is passed along the BGS layer.

[0068] In the embodiment in which the aqueous stream is passed along the BGS layer, it can be advantageous to employ a sheet of inert carrier with an irregular surface so as to maximise the surface area.

[0069] The layer comprising a sheet of inert carrier may suitably be provided in the form of plates or rotatable discs. The sheet of inert carrier may be provided as a coating on the surface of the layer. Alternatively, the layer may consist of a sheet of inert carrier.

[0070] In the embodiment in which the BGS comprises one or more sheets of inert carrier, the step of collecting the BGS comprising the EPS-containing biofilm may be carried out by removing the sheets carrying EPS-containing biofilm from the aqueous stream before subjecting these one or more biofilm-carrying sheets to the harvesting step. The collecting of the BGS comprising the EPS-containing biofilm may be achieved by moving the layer comprising the sheet of inert carrier out of the aqueous stream. This way of collecting the BGS comprising the EPS-containing biofilm may suitably be carried out using a rotating biological contactor (RBC). Once the BGS comprising the EPS-containing biofilm is removed from the aqueous stream, the EPS-containing biofilm may be separated from the one or more sheets of inert carrier, e.g. by scraping. After removal of the EPS-containing biofilm, the sheets of inert carrier material can be moved back into the aqueous stream.

[0071] The aforementioned layer of inert carrier preferably has a thickness in the range of 0.1-5,000 microns, more preferably in the range of 50-5,000 microns and most preferably in the range of 100-3,000 microns.

[0072] According to a particularly preferred embodiment, the aqueous stream is passed through and / or along the BGS layer at a flow rate of at least 1 m / hr, more preferably at a flow rate of 2-50 m / hr and most preferably at a flow rate of 5-30 m / hr.

[0073] Examples of inert carriers that can be suitably used in the present process are synthetic polymer, ceramic, minerals, metal and combinations thereof. Preferably, the inert carrier is a synthetic polymer. Examples of suitable synthetic polymers include polyester, polypropylene, polytetrafluoroethylene, polyvinyl chloride and polydimethylsiloxane.

[0074] The inert carrier may suitably be made from a (slowly) biodegradable material, e.g. biodegradable synthetic or biologically produced polymer. Examples of biodegradable polymers include polyesters selected from polycaprolactone, polylactic acid polybutylene succinate, polyhydroxy alkanoates, poly(butylene adipate-co terephthalate), polypropylene carbonate, poly(lactic-co-glycolic acid), poly(butylene succinate-co-adipate), poly(ethylene adipate), poly(salicylic glycolide) and poly(salicylic methyl glycolide) and combinations thereof.

[0075] Various carbon sources can be used to produce EPS. Preferably the carbon source is an organic compound that contains 1 to 18 C atoms. Examples of suitable organic compounds include glycerol, ethanol, fatty acids, sugars, lactic acid and combinations thereof. Wastewater with biodegradable and preferably soluble organic compounds can be used as a feedstock.

[0076] The chemical oxygen demand (COD) is a measure of the amount of carbon source that is present in the aqueous stream. COD is expressed in mass of oxygen consumed per volume of aqueous stream (mg / L). The aqueous stream employed in the present process preferably has a COD of 500 to 75,000 mg / L, more preferably of 1,000 to 30,000 mg / L and most preferably of 2,000 to 10,000 mg / L.

[0077] The inventors have observed that production of EPS is stimulated if the aqueous stream is nutrient (e.g. nitrogen, phosphor and / or sulphur) and / or oxygen limited.

[0078] In accordance with one preferred embodiment, the aqueous stream is nitrogen limited. Preferably, the COD / N (wt / wt) ratio is at least 50:1, more preferably at least 70:1 and most preferably at least 100:1

[0079] In accordance with another preferred embodiment, the aqueous stream is phosphorous limited. Preferably, the COD / P (wt / wt) ratio is at least 100:1, more preferably at least 200:1 and most preferably at least 400:1

[0080] In accordance with yet another preferred embodiment, the aqueous stream is sulphur limited. Preferably, the COD / S ratio is at least 2000:1, more preferably at least 4000:1 and most preferably at least 6000:1 The present process is preferably carried out under conditions that allow aerobic and micro-aerophilic EPS-producing bacteria to grow. Preferably, the aqueous stream has a dissolved oxygen content of at least 0.01 mg / L, more preferably of 0.1-10 mg / L and most preferably of 0.5-4 mg / L.

[0081] The aqueous stream used in the present process may suitably comprise wastewater. The aqueous stream may consist of a stream of waste water or it may be a combination of two or more streams, including a wastewater stream.

[0082] The aqueous liquid of the aqueous stream may also be prepared by combining one or more carbon source containing materials, e.g. liquid organic waste materials, with water.

[0083] In a preferred embodiment of the present process, the aqueous stream is composed of a feed stream comprising a carbon source, a recirculated stream and optionally a dilution stream. The recirculation stream is produced by recirculating aqueous liquid that becomes available after the aqueous stream has been contacted with the BGS. In this embodiment, the feed stream is used to provide fresh carbon source.

[0084] According to a particularly preferred embodiment, the aqueous stream of the present process is prepared by combining recirculated aqueous stream and feed stream in a weight ratio of at least 1 :2, preferably in a weight ratio of 1 : 1 to 20: 1. The use of a dilution stream can be advantageous in case the feed stream is highly concentrated (e.g. COD of more than 75,000 mg / L).

[0085] Nutrients and / or caustic may suitably be introduced in the aqueous stream. In case the aqueous stream is composed of two or more combined streams, nutrients and / or caustic may be introduced into the aqueous stream via one or more of the donating streams.

[0086] According to a preferred embodiment, the retention time of the aqueous stream in the present process is 1-12 hours, more preferably 1-6 hours. The retention time of the aqueous stream can be calculated by dividing the total internal water volume of the system in which the present process is carried out by the total flow of the incoming stream(s).

[0087] The retention time of the BGS in the present process preferably is in the range of 0.5 to 5 days, more preferably of 1 to 3 days. The retention time of the BGS refers to the average residence time of the BGS in the system in which the present process is carried out.

[0088] In a particularly preferred embodiment of the invention, the retention time of the aqueous stream is at least 2 times shorter than the retention time of the BGS, more preferably 3 to 120

[0089] times shorter than the retention time of the BGS. The aqueous stream preferably has a pH in the range of 6 to 9. More preferably, the aqueous stream has a pH in the range of 6 to 8.

[0090] The aqueous stream in the reactor preferably has a temperature in the range of 15 to 60 °C, more preferably in the range of 18 to 35 °C and most preferably of 20 to 25 °C when it is contacted with the BGS.

[0091] The EPS-producing bacteria employed in the present process preferably are aerobic or micro-aerophilic EPS-producing bacteria.

[0092] The EPS content of the separated biofilm, calculated as a ratio by weight of the organic matter that is contained in the separated biofilm, preferably is 20-98% by weight of organic matter, more preferably 25-95% by weight of organic matter and most preferably 30-90% by weight of organic matter.

[0093] In the present process, the step of separating the EPS-containing biofilm from the inert carrier may be carried out batchwise or continuously. Preferably, the harvesting step is carried out continuously.

[0094] The step of separating the EPS-containing biofilm from the inert carrier preferably comprises mechanical separation and / or chemical separation.

[0095] Mechanical separation of the EPS-containing biofilm from the inert carrier may suitably be achieved by exposing the BGS comprising the EPS-containing biofilm to a stream of liquid (e.g. water) or gas (e.g. air, nitrogen, carbon dioxide). Alternatively, the biofilm may be separated mechanically e.g. by exposing the EPS-containing biofilm to vibrations.

[0096] Chemical separation of the EPS-containing biofilm from the inert carrier may suitably be achieved by contacting the BGS comprising the EPS-containing biofilm with a caustic aqueous solution.

[0097] In case the BGS employed in the present process is a layer comprising a layer of inert carrier, the EPS-containing biofilm is preferably separated from the inert carrier by mechanical action, e.g. by vibrations or scraping.

[0098] According to a particularly preferred embodiment, after separation of the EPS-containing biofilm from the inert carrier, the inert carrier is re-used in the present process. In the present process the EPS with a molecular weight of at least 20 kDa preferably represents at least 10 wt.%, more preferably 30-95 wt.% and most preferably 40-90 wt.% of the organic matter that is contained in the separated biofilm.

[0099] The EPS in the separated biofilm preferably have an average molecular weight of at least 50 kDa, more preferably of 500-30,000 kDa and most preferably of 1,000-10,000 kDa.

[0100] Polysaccharides preferably represent 10-95 wt.%, more preferably 25-80 wt.% and most preferably 30-60 wt.% of the organic matter that is contained in the separated biofilm.

[0101] Proteins preferably represent 10-95 wt.%, more preferably 25-80 wt.% and most preferably 30-60 wt.% of the organic matter that is contained in the separated biofilm.

[0102] The separated biofilm preferably has a water content of 80-99.5 wt.%, more preferably of 85-98 wt.% and most preferably of 90-97 wt.%.

[0103] Preferably, the EPS in the separated biofilm have an absolute charge density of 0.2 to 7 meq / g at pH 7, more preferably of 0.5 to 5 meq / g at pH 7, and most preferably of 1 to 4 meq / g at pH 7.

[0104] According to a particularly preferred embodiment, the charge density of the EPS in the separated biofilm is in the range of -0.2 to -7 meq / g at pH 7, more preferably of -0.5 to -5 meq / g at pH 7, and most preferably of -1 to -4 meq / g at pH 7. The charge density of the EPS can be determined by colloid titration using a Mutek Particle Charge Detector (PCD03) described by Ajao (Flocculants from wastewater, PhD thesis, Wageningen University (2020)). The charge density is determined at a sample solution pH of 7.0 and calculated from the titrant (polydiallyldimethylammonium chloride) consumption according to the equation:

[0105] c ■ V

[0106] q = — m

[0107] where q is the specific charge quantity (eq / g), c is the titrant concentration (eq / L), V is the consumed titrant volume and m is the mass of the sample (g).

[0108] The separated biofilm preferably has a flocculation performance of at least 0.06 mg biofilm / g clay, more preferably of 0.1-10 mg biofilm / g clay and most preferably of 0.2-1 mg biofilm / g clay as determined by the method described by Ajao (Flocculants from wastewater, PhD thesis, Wageningen University (2020)). In a preferred embodiment of the present process, EPS is extracted from the EPS-containing biofilm during or after separation of the biofilm from the inert carrier.

[0109] In a particularly advantageous embodiment of the present process, the separation of the biofilm and extraction of EPS from the biofilm are carried out simultaneously. This may be achieved, for instance, by contacting the BGS comprising the EPS-containing biofilm with an extraction liquid under conditions that detach the biofilm from the inert carrier into the extraction liquid and that allow the EPS to be extracted from the biofilm into the extraction liquid. Accordingly in a preferred embodiment, the separation of the biofilm and extraction of EPS from the biofilm are carried out simultaneously by contacting the BGS comprising the EPS-containing biofilm with an extraction liquid to detach the biofilm from the inter carrier into the extraction liquid and to extract the EPS from the biofilm into the extraction liquid. The extraction liquid containing extracted EPS is preferably subjected to a solidliquid separation step to remove non-dissolved solids. Examples of solid-liquid separation techniques that may be used include centrifugation, decanting, filtration and separation in hydrocyclones.

[0110] Simultaneous harvesting and EPS-extraction may suitably be achieved by using two or more reactors in a parallel arrangement as described above. Whilst in one reactor the aqueous stream is contacted with the BGS to develop an EPS-containing biofilm, in another reactor BCG comprising the EPS-containing biofilm is contacted with extraction liquid to simultaneously detach the biofilm from the inert carrier and extract EPS from the biofilm. Simultaneous harvesting and EPS-extraction may also be achieved using a rotating biological contactor (RBC). Once the BGS comprising the EPS-containing biofilm is removed from the aqueous stream, it may be contacted with an extraction liquid to detach the biofilm from the inter carrier and to extract EPS from the biofilm into the extraction liquid.

[0111] In embodiments of the present process in which harvesting and extraction are carried out simultaneously, the EPS content of the separated biofilm equals the EPS-content of the EPS-containing biofilm immediately before harvesting and extraction.

[0112] In another embodiment, the present process comprises an additional step of extracting EPS from the separated EPS-containing biofilm to produce an EPS extract. In this embodiment, first the BGS comprising the EPS-containing biofilm is separated from the inert carrier and next the separated EPS-containing biofilm is extracted. In this embodiment, the separation of the EPS-containing biofilm from the inert carries is preferably achieved by mechanical action. Extraction of EPS from the separated biofilm is preferably achieved by contacting the separated biofilm with an extraction liquid. The extraction liquid employed in the present process preferably is an aqueous liquid, more preferably an aqueous liquid containing at least 50 wt.% water, more preferably at least 75 wt.% water and most preferably at least 90 wt.% water.

[0113] The EPS are preferably extracted from the biofilm by a method comprising heating of the biofilm to a temperature of at least 60°C for at least 30 minutes, more preferably to a temperature of at least 80°C for at least 60 minutes.

[0114] The extraction of the EPS from the biofilm preferably comprises contacting with a caustic substance, e.g. in the form of concentrated aqueous caustic solution. Examples of caustic substances that may be used include sodium hydroxide, potassium hydroxide, sodium carbonate, calcium hydroxide and combinations thereof.

[0115] EPS with a molecular weight of at least 10 kDa preferably represents at least 80-100 wt.%, more preferably 90-100 wt.% and most preferably 95-99.5 wt.% of the organic matter that is contained in the EPS extract.

[0116] The EPS in the EPS extract preferably have an average molecular weight of at least 50 kDa, more preferably of 500-30,000 kDa and most preferably of 1,000-10,000 kDa.

[0117] In one embodiment of the invention the EPS extract is enriched in extracellular polysaccharides. In accordance with this embodiment, polysaccharides preferably represent 10-100 wt.%, more preferably 40-100 wt.% and most preferably 50-98 wt.% of the organic matter that is contained in the EPS extract.

[0118] In another embodiment of the invention the EPS extract is enriched in extracellular proteins. Proteins preferably represent 10-100 wt.%, more preferably 35-100 wt.% and most preferably 40-95 wt.% of the organic matter that is contained in the EPS extract.

[0119] The EPS extract is preferably dried to produce a free-flowing powder. Preferably, the EPS extract has a water content of 0-15 wt.%, more preferably of 0-12 wt.% and most preferably of 0-10 wt.%.

[0120] The EPS in the EPS extract preferably has an absolute charge density of 0.2 to 7 meq / g at pH 7, more preferably of 0.5 to 5 meq / g at pH 7, and most preferably of 1 to 4 meq / g at pH 7.

[0121] According to a particularly preferred embodiment, the a charge density of the EPS in the EPS extract is in the range of -0.2 to -7 meq / g at pH 7, preferably of -0.5 to -5 meq / g at pH 7, and most preferably of -1 to -4 meq / g at pH 7. The EPS extract preferably has a flocculation performance of at least 0.03 mg extract / g clay, more preferably of 0.06-5 mg extract / g clay and most preferably of 0.1 -0.5 mg extract / g clay.

[0122] According to a particularly preferred embodiment, the present process is carried out in wastewater treatment facility and the EPS-containing biofilm or the EPS extract obtained by the process is used in the same wastewater treatment facility, preferably as a flocculant.

[0123] Another aspect of the present invention relates to an EPS-containing biofilm obtained by a process as described above.

[0124] According to a particularly preferred embodiment, the EPS-containing biofilm is used as a flocculant, a soil improver or in bioremediation.

[0125] The EPS-containing biofilm may advantageously be used as a flocculant, e.g. in the treatment of wastewater, dredging water or rinsing water. The EPS-containing biofilm may also suitably be used, e.g. as a replacement of polyacrylamide, in the preparation of drinking water or in the treatment of sludge derived from the preparation of drinking water.

[0126] The EPS-containing biofilm may also suitably be used as a soil improver as it has a positive effect on water and multivalent cations I nutrient retention and binds loose soil particles into stable aggregates.

[0127] Bioremediation is another purpose for which the EPS-containing biofilm may be used. Examples of such bioremediation include removal of heavy metals or other cations such as ammonia, removal of polycyclic aromatic hydrocarbons (PAHs) and clean-up of oil spills.

[0128] Yet another aspect of the invention relates to an EPS extract obtained by a process as described above.

[0129] According to a particularly preferred embodiment, the EPS extract is used as a flocculant, a soil improver or in bioremediation.

[0130] The EPS extract may advantageously be used as a flocculant, e.g. in the treatment of wastewater.

[0131] The EPS extract may also suitably be used as a soil improver as it has a positive effect on water and multivalent cations / nutrient retention and binds loose soil particles into stable aggregates. Bioremediation is another purpose for which the EPS extract may be used. Examples of such bioremediation include removal of heavy metals, removal of polycyclic aromatic hydrocarbons (PAHs) and clean-up of oil spills.

[0132] BRIEF DESCRIPTION OF THE DRAWING

[0133] Figure 1 schematically depicts an installation for the production and extraction of EPS in accordance with the present invention.

[0134] DESCRIPTION OF EMBODIMENTS

[0135] Figure 1 schematically depicts an installation for use in the process according to the invention.

[0136] The aerobic reactor unit (1) comprises a bed (2) composed of pieces of biofilm carrier (3). Aqueous liquid (4) comprising a carbon source (e.g. glycerol, glucose, volatile fatty acids, ethanol etc.) is supplied via aqueous stream line (5) to the top of bed (2). The reactor unit (1) is aerated or supplied with oxygen containing gas by means of an aeration device (6). The air or oxygen containing gas flow is introduced below the bottom of the bed (2) and moves upward through the bed (2) before exiting the aerobic reactor (1) near or at the top via outlet (7).

[0137] During start-up of the process, the aqueous liquid (4) is inoculated with EPS-producing bacteria. These EPS-producing bacteria attach themselves to the pieces of biofilm carrier (3) while the aqueous liquid (4) percolates through the bed (2). Once attached to the pieces of biofilm carrier (3), the EPS-producing bacteria start producing an EPS-containing biofilm (8) that sticks to the surface of the pieces of biofilm carrier (3).

[0138] At the bottom of the bed (2) aqueous effluent (9) is continuously withdrawn and transferred to effluent tank (10).

[0139] At the bottom of the bed (2), once sufficient biofilm (8) has developed on the surface of the pieces of biofilm carrier (3), a stream comprising pieces (3) carrying biofilm (8) as well as some aqueous liquid is continuously or discontinuously withdrawn and transferred to separator (11). In separator (11), aqueous liquid is separated from the pieces (3) carrying biofilm (8). The aqueous liquid separated in separator (11) is transferred via effluent line (12) to effluent tank (10).

[0140] From the effluent tank (10), aqueous liquid is continuously removed via effluent outlet (13) and recirculation water is continuously pumped via recirculation line (14) into aqueous stream line (5) where it is combined with the feed stream (15) from feed tank (16) before entering the top of the reactor unit. If required, nutrients (not shown in figure) and / or dilution water are pumped into the aqueous stream line (5). A dilution stream (not shown) may be employed in case the feed stream (15) is very concentrated.

[0141] Pieces (3) carrying biofilm (8) are removed from the separator (11) and transferred to harvesting unit (17). In harvesting unit (17) the pieces (3) carrying biofilm (8) are exposed to a high velocity stream of gas (e.g. air, nitrogen, carbon dioxide) to remove biofilm (8) from the pieces (3). The high velocity gas stream is generated by blower (18) and leaves the harvesting unit (17) via outlet (19). Instead of a high velocity gas stream a high velocity liquid stream of liquid, e.g. water, may be used to remove the biofilm. Removed biofilm is collected in the biofilm collection unit (20) and can optionally be transported via outlet (21) to a downstream processing unit (not shown) in which the biofilm is treated to extract EPS.

[0142] The cleaned pieces of biofilm carrier are returned to the top of the bed (2) via carrier transport line (22).

[0143] The level in the effluent tank is maintained at a constant level by controlling the effluent output (13).

[0144] The pH of the process is controlled by adding caustic. The pH can be measured in the recirculation line or in another part of the installation. Caustic can be dosed into the recirculation stream or elsewhere in the installation to maintain pH at a predetermined level.

[0145] The invention is further illustrated by the following non-limiting examples.

[0146] EXAMPLES

[0147] Example 1

[0148] A laboratory scale experiment was performed to demonstrate the development of an EPS-containing biofilm layer on inert carrier bodies and the flocculating capacity of the formed EPS.

[0149] A lab reactor vessel filled with spherical carrier bodies (polypropylene, diameter appr. 3 cm, a surface to volume ratio of 600 nr1) was used for testing. Water was recirculated over the vessel (230 L / d). At the same time, aqueous solution comprising a carbon source (glycerol and ethanol -1:1 COD-based) and nutrients were continuously pumped into the reactor with a flow of 18.4 L / d. The pH was measured in the recirculation flow and controlled at a pH of 7-7.5 by means of caustic dosing. Air was introduced in the bottom part of the reactor. The composition of the carbon source containing stream is shown in Table 1. Table 1

[0150]

[0151] * Micronutrient composition as described by Ajao (Flocculants from wastewater, PhD Thesis, Wageningen University (2020)).

[0152] A part of the carrier bodies was taken out manually once daily, except during weekends. A portion of the removed carrier bodies was mechanically shaken with water to produce a biofilm solution for analysis. All removed carrier bodies were returned to the reactor after the biofilm had been removed.

[0153] Total COD of the influent and the effluent, the filtered (0.45 pm) COD of the effluent and the COD of the biofilm were measured daily during a period of 15 days. The averaged results are shown in Tables 2 and 3.

[0154] Table 2

[0155]

[0156] Table 3

[0157]

[0158] The bacteria in the biofilm were estimated from the decrease in nitrogen over the system and taking the bacterial COD:N ratio of fresh biomass into account. These results show that about 26 wt.% of influent COD was converted into EPS.

[0159] Example 2

[0160] EPS was extracted from the biofilm solution of Example 1 using the method described by Bou-Sarkis et al. (Bou-Sarkis et al., Effects of alkaline solvents and heating temperatures on the solubilization and degradation of gel-forming Extracellular Polymeric Substances (EPS) extracted from aerobic granular sludge. Biochemical Engineering Journal, 185, 108500).

[0161] The molecular weight of the samples was analyzed using liquid chromatography-organic carbon detection (LC-OCD) following the method described by Ajao (Flocculants from wastewater, PhD thesis, Wageningen University (2020)).

[0162] Charge density measurements were conducted through colloid titration utilizing the Mutek Particle Charge Detector (PCD03, Germany).

[0163] Flocculation performance was tested as described by Ajao (Flocculants from wastewater, PhD Thesis, Wageningen University (2020)) except that Kaolin & Bentonite clay (3 g / L) was used to model wastewater particles.

[0164] The results of these analyses are summarised in Tables 4 and 5.

[0165] Table 4

[0166]

[0167]

[0168] Example 3

[0169] Inert carrier bodies carrying an EPS-containing biofilm were produced as described in Example 1. An extraction vessel with an internal volume of 4 litre was completely filled with the carried bodies carrying the biofilm. Hot water or hot caustic solution (2.2 litre) was introduced into the vessel and circulated across the bed of carrier bodies to simultaneously detach the EPS-containing biofilm from the carrier bodies and extract EPS from the biofilm.

[0170] After 2 hours of circulation, the extraction liquid was collected and centrifuged. The carrier bodies no longer contained biofilm and could be re-used in the production of EPS-containing biofilm.

[0171] The extraction liquids obtained after extraction and the two supernatants obtained after centrifugation of these extraction liquids were analysed for COD. The extraction conditions and the COD results are shown in Table 5.

[0172] Table 5

[0173]

[0174] Temperature decreased during the test

Claims

CLAIMS1. A process of producing extracellular polymeric substances (EPS), said process comprising:• contacting an aqueous stream comprising a carbon source with a biofilm generating system (BGS) under aerobic conditions to develop an EPS-containing biofilm on the BGS, the BGS comprising (i) an inert carrier and (ii) EPS-producing bacteria attached to the surface of the inert carrier; and• subjecting the BGS comprising the EPS-containing biofilm to a harvesting step in which the EPS-containing biofilm is separated from the inert carrier, the separated biofilm having an EPS content of at least 30% by weight of the organic matter that is contained in the separated biofilm;wherein the BGS is provided in the form of discrete pieces comprising an inert carrier body having a diameter in the range of 0.2-30 cm or wherein the BGS is provided in the form of a layer comprising a sheet of inert carrier.

2. Process according to claim 1 , wherein, prior to the harvesting step, the BGS comprising the EPS-containing biofilm is separated from the aqueous stream.

3. Process according to claim 1 or 2, wherein the inert carrier body has a diameter in the range of 0.3-20 cm.

4. Process according to any one of the preceding claims, wherein the aqueous stream percolates through a bed of the BGS pieces.

5. Process according to any one of the preceding claims, wherein pieces of BGS comprising the EPS-containing biofilm are removed from the bottom of the bed of BGS pieces while simultaneously introducing inert carrier bodies to the top of the bed of BGS pieces.

6. Process according to any one of the preceding claims, wherein the inert carrier is selected from synthetic polymer, ceramic, minerals, metal and combinations thereof.

7. Process according to any one of the preceding claims, wherein the aqueous stream has a COD of 500 to 75,000 mg / L.

8. Process according to any one of the preceding claims, wherein the carbon source is an organic compound that contains 1 to 18 C atoms.

9. Process according to any one of the preceding claims, wherein the aqueous stream is nutrient and / or oxygen limited.

10. Process according to any one of the preceding claims, wherein the aqueous stream has a dissolved oxygen content of at least 0.01 mg / L.

11. Process according to any one of the preceding claims, wherein the retention time of the BGS is 0.5 to 5 days.

12. Process according to any one of the preceding claims, wherein EPS is extracted from the EPS- containing biofilm during or after separation of the biofilm from the inert carrier.

13. Process according to claim 12, wherein the separation of the biofilm and extraction of EPS from the biofilm are carried out simultaneously by contacting the BGS comprising the EPS-containing biofilm with an extraction liquid to detach the biofilm from the inter carrier into the extraction liquid and to extract the EPS from the biofilm into the extraction liquid.

14. An EPS-containing biofilm obtained by a process according to any one of claims 1-11 or an EPS extract obtained by a process according to claim 12 or 13.

15. Use of the EPS-containing biofilm or the EPS extract according to claim 14 as a flocculant, as a soil improver or in bioremediation.

Images