WO2019009708A1

WO2019009708A1 - Method and system for production of natural polyelectrolytes such as microbial extracellular polymeric substances

Info

Publication number: WO2019009708A1
Application number: PCT/NL2018/050428
Authority: WO
Inventors: Bernhard Gerhard TEMMINK; Victor Olusola AJAO
Original assignee: Stichting Wetsus, European Center Of Excellence For Sustainable Water Technology
Priority date: 2017-07-04
Filing date: 2018-07-03
Publication date: 2019-01-10
Also published as: NL2019171B1

Abstract

The present invention relates to a method and system for the production of natural, biodegradable polymeric floccuiants from a fluid, such as a waste fluid, such floccuiants and their use. The method comprising the steps of: providing a reactor comprising at least one reactor vessel, one fluid inlet, one fluid outlet, one flocculant outlet, and at least one membrane provided in the reactor vessel between the fluid inlet and the fluid outlet; inoculation of the reactor; providing feed to the at least one fluid inlet; forming the floccuiants; separating the floccuiants using the membrane; and providing the floccuiants at the flocculant outlet.

Description

METHOD AND SYSTEM FOR PRODUCTION OF NATURAL POLYELECTROLYTES SUCH AS MICROBIAL EXTRACELLULAR POLYMERIC SUBSTANCES

The invention relates to a method for the production of natural, biodegradable polymeric flocculants from a fluid, for example a waste fluid. For example, the method discloses the manufacturing of microbial Extracellular Polymeric Substances (EPS) that can be applied as flocculants for waste fluid treatment or other fluids containing (colloidal) particles.

It is known from practice to use carboxyl cellulose flocculants and (biodegradable) flocculants. Conventional production methods for these flocculants include EPS production strategies involving identification and isolation of EPS producing microbial strains with single organic substrate feeding. Conventional EPS production uses pure cultures which need to be fed with expensive and unsustainable carbon sources as well as with other valuable nutrients.

From practice it is also known to use inorganic coagulants that are cheap and easy to use. However, inorganic coagulants do not flocculate efficiently at low dosages, are non-specific, leave residual metal pieces in treated water, and produce toxic sludge (metal hydroxide). This makes disposal of this sludge difficult, expensive and non-reusable, for example in agriculture and, therefore, the associated food chain. Synthetic organic polyelectrolytes (PEs)/flocculants, have higher flocculating efficiency, lower dosage requirements, ability to dewater sludge more effectively, and can form strong and dense floes. Nonetheless, they suffer non-negligible drawbacks of slow biodegradability and generation of toxic degradation products/monomer residues that may enter the food chain and cause severe neurotoxic or carcinogenic effects.

Besides, unreacted chemicals used to synthesize the monomer units, such as formaldehyde, epichlorohydrin and dimethylamine, are toxic and have been found as sources of contaminants in 'treated' water. Hence, the use of synthetic flocculants can hardly be considered a sustainable wastewater treatment approach, especially in open systems such as dredging and mining applications.

An objective of the present invention is to provide a method for the production of natural, biodegradable polymeric flocculants from a waste fluid that obviates or at least reduces the aforementioned problems and/or is more efficient as compared to conventional methods.

The objective is achieved with the method for the production of natural, biodegradable polymeric flocculants from a fluid, such as a waste fluid, according to the invention, the method comprising the steps of:

- providing a reactor comprising:

- at least one reactor vessel;

- at least one fluid inlet;

- at least one fluid outlet; - at least one fiocculant outlet; and

- at least one membrane provided in the reactor vessel between the fluid inlet and the fluid outlet;

- inoculation of the reactor;

- providing feed to the at least one fluid inlet;

- forming the flocculants;

- separating the flocculants using the membrane; and

- providing the flocculants at the fiocculant outlet.

Production of natural, biodegradable polymeric flocculants from a fluid, such as a waste fluid enables cost effective manufacturing. This is achieved by the use of readily available raw material for the manufacturing process. The flocculants can be used for waste fluid treatment and/or treatment of other fluids containing (colloidal) particles. This use of the flocculants will therefore be cost effective and cheap to use. hi presently preferred embodiments the fluid relates to fluids such as a waste fluid.

In the context of the present invention waste fluids include fresh and saline waste fluids, for example. The waste fluid could be obtained as domestic waste fluid and/or industrial waste fluids. Examples of industrial waste fluids are waste fluids from chemical industry, agriculture industry, food processing industry, pharmaceutical industry and health care industry.

In the context of the present invention EPS relates to Extracellular Polymeric Substances and includes organic matter such as flocculants.

Microbial Extracellular Polymeric Substances (EPS) applied as flocculants are generally considered as biopolymers excreted by micro-organisms. These microbial EPS are products of biochemical secretion and/or cell lysis.

The production of flocculants is performed in a reactor vessel. Preferably, the reactor vessel comprises a volume such that the hydraulic retention time is below 6 hours, more preferably below 4 hours and most preferably below 2 hours. The solids retention time is significantly below 10 days, preferably below 7 days, more preferably below 4 days, even more preferably below 3 days. In an embodiment of the invention the solids retention time is preferably below 2 days and more preferably below 1 day. The reactor vessel involves at least one fluid inlet including a pipe, a tube, a valve, for example. The reactor vessel also involves a fluid outlet and/or fiocculant outlet. These outlets may involve a pipe, a tube, an overspill, a container, a valve, for example.

According to the method of the invention the reactor vessel is inoculated. Preferably, inoculation involves micro-organisms that preferably originate from a biological waste water treatment plant. This enables to recover valuable resources, for example polyelectrolytes. These micro-organisms are readily available and can be used in a non-sterile environment. As compared to conventional sterile pure micro-organism cultures that need to be fed with non-sustainable carbon sources as well as with other valuable nutrients, the present invention enables the use of non-sterile micro-organisms that are less demanding in nutrients source and cheaper as compared to sterile micro-organisms. This renders the manufacturing method of the invention more cost effective and more sustainable.

After inoculation the flocculants are formed comprising organic microbial EPS. The EPS is separated by a membrane and collected at the flocculant outlet. The flocculants are preferably formed in the reactor vessel volume. It will be understood that according to the invention another separation technique can also be applied in addition to or as an alternative for the membrane separation.

The method of the invention uses waste fluid, such as a waste water, as feedstock. Such feedstock typically involves biodegradable and preferably water soluble organic pollutants. In one of the embodiments of the invention the waste water flows comprises fresh and saline waste water conditions containing ethanol and glycerol, glucose and fructose, acetate, for example. This enables an effective manufacturing process.

As a further effect of forming of the flocc lants in accordance with the method of the invention less exploited natural flocculants comprising organic microbial EPS are provided. These flocculants are products of biochemical secretions and/or cell lysis, and can make up as much as 50-90% of the organic matter content of microbial aggregates. These microbial EPS include high molecular weight substances, for example polysaccharides, proteins, lipids, and combinations thereof. Examples of combinations are liposaccharides, glycoproteins and lipoproteins.

Flocculants produced by the method of the invention can show comparable or better flocculation properties as compared to conventional flocculants. For example, the natural flocculants produced according to the method of the invention appear to be less shear sensitive as compared to conventional flocculants from synthetic polymers achieving a larger operation window, specifically a wider range of shear conditions.

In a preferred embodiment of the invention the feedstock has a chemical oxygen demand to nitrogen ratio above 5, preferably above 8, and most preferably above 10 and/or a chemical oxygen demand to phosphorous ratio above 10, preferably above 15, and most preferably above 20.

Further experiments have shown that a feedstock containing a chemical oxygen demand to nitrogen ratio above 15, preferably above 50, more preferably above 90, and most preferably above 100 provides an even better yield for obtaining flocculants.

A natural or industrial waste stream can be used as input fluid for the production of EPS. The method provides a dual function, i.e. production of EPS and/or waste water treatment.

Optionally, by introduction or addition of oxygen and/or nitrogen or phosphorous the chemical oxygen demand to nitrogen ratio and/or chemical oxygen demand to phosphorous ratio can be tuned. Therefore, the provided waste stream does not need further treatment to be used.

Experiments have shown that the method of the invention effectively provides the desired flocculants. In a presently preferred embodiment the forming and/or separating of the flocculants from the fluid preferably comprises the step of aerating.

Performing an aerating step contributes to the amount of dissolved oxygen in the reactor vessel. Therefore, aerating improves the activity of the micro-organisms that are present in the reactor. Under these conditions tests show that a mixed-culture of micro-organisms develops and mineralises a small fraction of the organic pollutants to carbon dioxide and water (typically less than 30%) and produces EPS. These may be used for the flocculants.

In a preferred embodiment of the invention a dissolved oxygen level of at least 0.5 mg 0₂/L. preferably at least 0.75 mg 0₂/L, and most preferably at least 1 mg 0₂/L is maintained to obtain further improvement of the yield of the flocculants formation.

Providing and maintaining the oxygen level in the ranges mentioned has a stimulating effect on the productivity of the micro-organisms, and thus on the manufacturing process of the flocculants.

In a further preferred embodiment of the invention the formation of the flocculants provides extracellular polymeric substances of polymers or proteins or a mixture of polymers and proteins.

Flocculants involving extracellular polymeric substances of polymers or proteins or a mixture of polymers and proteins provide an effective and efficient product. Preferably, the polymer fractions are polysaccharides and the charge density is 0.2 - 7 meq/g, preferably 0.35 - 6 meq/g, and most preferably 0.5 - 5 meq/g.

In experiments the polymer fractions have shown a charge density of 1.4 - 4.7 meq/g for carbon oxygen demand to nitrogen ratio of 100. This results in an even more effective and efficient product. A further effect of this charge density is the prevention of restabihsation of the flocculants at a higher polymer dosage.

It will be understood by the skilled person 0.1 - 1.0 mg EPS / gram suspended solid can be used as polymer dosage.

In one of the embodiments of the invention the waste fluid comprises one or more of fresh and saline waste waters comprising feedstock for the production of microbial EPS.

In experiments the saline and fresh water originating polymers gave a flocculation activity of 80% or above. This activity could even be improved by the addition free calcium ions in a concentration below 200 mg/L, preferably below 150 mg/L, more preferably below 100 mg/L, and most preferably below 50 mg/L. The addition of calcium results in the increase of the flocculation activity. In a preferred embodiment of the invention flocculants production is combined with biological waste water treatment, preferably comprising the treatment and production under fresh water and/or saline conditions.

The use of these condition results in an effective combination of purification of waste water and the recovery of valuable recourses. This provides efficient and cost-effective purification and recovery in a single operation.

The invention also relates to a system for the production of environmentally safe, biodegradable, organic microbial extracellular polymeric substances from a waste fluid, the system being capable of performing the method in one or more of the embodiments according the invention, wherein the system comprising:

- at least one reactor vessel;

- at least one fluid inlet;

- at least one fluid outlet;

- at least one flocculants outlet; and

- at least one membrane provided in the reactor vessel between the fluid inlet and the fluid outlet.

The system provides the same effects and advantages as those described for the method.

More specifically, the system enables an efficient and effective water treatment and flocculants production involving at least one reactor, at least one fluid inlet, at least one fluid outlet, at least one flocculants outlet and at least one membrane provided in the reactor vessel between the fluid inlet and the fluid outlet.

In further embodiments of the invention the system could be extended with different sensors, for example a pH sensor, a temperature sensor, an oxygen level sensor, and/or a level control sensor. It will be understood that further or alternative sensors may also be applied in accordance with the present invention. Preferably, the sensors are connected to a

controller/processor that enables monitoring and/or control of the production process. For example, this may involve maintaining the manufacturing within specific boundaries, such as dissolved oxygen level.

The invention also relates to flocculants that are produced by a method according to one of the embodiments of the invention.

The flocculants provide the same effects and advantages as those described for the method and/or system.

In a presently preferred embodiment the flocculants produced by the method in the disclosed system, wherein the EPS comprising a molecular weight, ranging between 2 kDa and 10000 kDa, preferably ranging between 3 kDa and 8000 kDa, more preferably ranging between 5 kDa and 6000 kDa, and most preferably ranging between 10 kDa and 2000 kDa. An effect of (maintaining) the EPS in these range(s) is the effective recovery and regaining of valuable recourses, preferably in combination with the cost effective treatment of waste fluid.

In experiments the polymer EPS have shown a molecular weight ranging between 20 kDa and 4000 kDa. Such a mixture of high, medium and low (high: > 10⁶ Da, medium: 10⁶ - 10³ Da, low: < 10⁵ Da) molecular weights in EPS could make them more resilient under practical conditions such as varying particle type, size, and concentration.

Furthermore, the experiments have shown a carbon to oxygen demand removal of > 95%. An effect of (maintaining) the EPS in these range(s) is the even more effective recovery and regaining of valuable resources, preferably in combination with the cost effective treatment of waste fluid.

Effective regaining of the valuable resources is mainly achieved when EPS are applied. In a presently preferred embodiment the flocculanl produced by the method in the disclosed system, wherein the EPS comprises capability to:

- binding and/or removal of ammonium and/or heavy metals; and/or

- settleability of at most 1.7 mL/min.

An effect of the EPS is that they can be applied in a wide variety of applications. For example, binding and/or removal of 1.3 gram of lead per gram of EPS. Furthermore, the EPS have a settleability of 1.7 ml/min, which is faster compared to the control (3 mL/min, where the control is 50 g kaolin/L and a density of 50 - 150 g kaolin/L). The EPS also produce supernatant of lower turbidity, up to 79% turbidity removal after settling for 30 min.

An even further effect is that the EPS can be applied in an effective range of dosage of 0.1 - 1.0 mg of EPS/g of solids.

Therefore, the mixture of EPS produced by the method in the disclosed system have better flocculation performance than single-type EPS.

It will be understood by the skilled person that mixed EPS refers to EPS with a mixture of polysaccharides and proteins and/or EPS with a mixture of different molecular weight fractions (high: > 10⁶ Da, medium: 10⁶ - 10⁵ Da, low: < 10⁵ Da) and single-type EPS refers to EPS that mainly consist of either polysaccharide or proteins with one molecular weight fraction.

The waste fluid has a COD of 1000 ± 25 mg/L, mainly provided from glycerol and ethanol (1 : 1 ratio), simulating biodiesel and bioethanol wastewater. The main nitrogen source was NH₄C1, and its concentration varied based on the utilised COD/N ratio. The mineral medium composition per litre of tap water comprised: 200 mg MgCl₂-6H₂0, 150 mg CaCl₂-2H₂0, 15 mg K₂HP0₄, 25 mg KH₂P0₄, and 2 mL trace elements solution.

From a waste fluid produced EPS can be used to bind metals, inorganics, large organic molecules, for example residues of medicines. The invention further also relates to a use of the environmentally safe, biodegradable, organic microbial extracellular polymeric substances from a waste fluid for the production of flocculants.

The use provides the same effects and advantages as those described for the method, the system and/or the flocculants.

In a presently preferred embodiment the use of the flocculants is cheap and cost effective.

Further advantages, features and details of the invention are elucidated on the basis of preferred embodiments thereof, wherein reference is made to the accompanying drawings, in which:

- Figure 1 shows a schematic overview of the method according to the present

invention; and

- Figure 2 shows an embodiment of the system capable of performing the method described in the description.

Method 2 (Figure 1) for the production of environmentally safe, biodegradable, organic microbial extracellular polymeric substances, preferably from a waste fluid, follows a sequence of different steps.

In the illustrated embodiment this starts with providing 4 a reactor and a feedstock. The reactor is inoculated in inoculation step 6 and supplied 8 with microorganisms if these are not already present in the feedstock. As a next step, after inoculation 6, forming 12 of flocculants occurs from the waste fluid. The flocculants are separated from the remaining fluid in separation step 14, preferably using a membrane such as an ultrafiltration membrane. Flocculants are collected in collecting stepl8 resulting in a valuable product and treated waste water. The left over fluid could be recycled in recycling step 16 and returned to the reactor and mixed with the waste and/or recycle fluid.

In an illustrated embodiment of the invention, system 102 (Figure 2) produces environmentally safe, biodegradable, organic microbial extracellular polymeric substances from a waste fluid involving method steps of method 2.

System 102 comprises a feed tank 104 that can be filled with waste fluid 106. System 102 has outlet 108 connecting tank 104 to inlet pump 110. Pump 1 10 provides feedstock to inlet 1 12. In the illustrated embodiment inlet 112 is part of a so-called submerged membrane bioreactor 114. It will be understood that other reactor types and/or parts could also be applied in accordance with the invention.

In the illustrated system, in use, fluid 1 16 is present in reactor 114. In fluid 116 flocculants 1 18 are formed, specifically in compartment/reactor part 120. In compartment/reactor part 122 comprising (ultrafiltration) membrane 124 flocculants 118 are separated from fluid 116. Alternatively, a separation is achieved by centrifugation or by a sodium based cation-exchange resin, all followed by dialysis to remove low molecular weight compounds (<3 kDa).

Reactor 114 is provided with permeate outlet 126 that is connected to permeate pump 128 for further transport of permeate 130. Aeration base 132 enables aerating fluid 1 16. Waste outlet 134 is connected to waste pump 13 for further transport of waste 138. Sensors 140 enable monitoring and/or control of the production process.

Experiments have been performed with system 102. In the experiments parameters as mentioned in tables 1 and 2 are applied.

In the experiments, in the fresh water application the reactor was inoculated with aerobic sludge from a municipal waste water treatment plant and in the saline application the reactor was inoculated with aerobic sludge from a saline waste water treatment plant for chemical industries. The polymers were produced from a mimicked industrial waste water, mainly containing ethanol and glycerol, both under fresh and saline waste water conditions. The sheet membrane in the experiments had a nominal pore size of 0.2 μηι. After extraction using a ultrafiltration membrane, followed by dialysis to remove low molecular weight compounds (<3 kDa) and freeze -drying, solutions of these polymers were prepared and used to flocculate kaolin clay suspensions under fresh and saline water conditions and in the absence and presence of calcium ions. Kaolin clay was used to mimic usually negatively charged surface water and waste water particles.

Table 1 MBRs set-up and operation parameters

a COD: Chemical oxygen demand. TN: Total nitrogen - sum of all NH₃-N, X¾-N, N0₂-N, organic N

Table 2 Composition of synthetic waste water in both reactors Compound Concentration (mg/L) Nutrient solution Concentration (mg/L)

Glycerol 205 FeCl₃.6H₂0 1500

Ethanol 120 H₃B0₃ 150

NH₄CI 100 CoCl₂.6H₂0 150

Yeast extract 60 ZnSo₄.7H₂0 120

K₂HP0₄ 7.5 MnCl₂.2H₂0 120

KH₂P0₄ 12.5 Na₂Mo0₄.2H₂0 60

MgCl₂.6H₂0 100 CuS0₄.5H₂0 60

CaCl₂.2H₂0 150 Kl 30

NaCl^* 30000

Nutrient solution 2mL/L

* NaCl was fed only to the saline MBR

Flocculation tests show the applicability of the method according to the invention to produce flocculants. Furthermore, the results show an EPS yield after extraction, purification and lyophilisation as given in table 3.

Table 3 EPS yield after extraction, purification and lyophilisation.

In a further experiment performed with system 102, parameters as mentioned in tables 4 and 5 are applied.

In the further experiments, in the fresh water application the reactor was inoculated with aerobic sludge from a municipal waste water treatment plant and in the saline application the reactor was inoculated with aerobic sludge from a saline waste water treatment plant for chemical industries. The polymers were produced from a mimicked industrial waste water, mainly containing ethanol and glycerol, both under fresh and saline waste water conditions. The sheet membrane in the experiments had a nominal pore size of 0.2 pm. After extraction using a ultrafiltration membrane, followed by dialysis to remove low molecular weight compounds (<3 kDa) and freeze-drying, solutions of these polymers were prepared and used to flocculate kaolin clay suspensions under fresh and saline water conditions and in the absence and presence of calcium ions. Kaolin clay was used to mimic usually negatively charged surface water and waste water particles.

Inlet TN (mg/L) does not have a set minimum and/or maximum value. The ratio

COD/TN is of importance, as a result of an increase of the COD value, the TN should increase accordingly.

Table 4 MBRs set-up and operation parameters

Parameter Saline and freshwater MBRs

Reactor type Submerged membrane bioreactor

Effective volume (L) 3.3

Temperature (°C) 20 + 1

PH 7.5 + 0.1

Solids retention time, SRT (d) 3

Hydraulic retention time, HRT (h) 4

Dissolved oxygen concentration (mg/L) 4.0 + 1.0

Operation time (d) 50

Inlet COD¹¹ (mg/L) 1000 mg/L

Inlet TN^b (mg/L) variable

COD/N 5, 20, 60, 100

a COD: Chemical oxygen demand. TN: Total nitrogen - sum of all NH N, NO₃-N, N0₂-

N, organic N

Table 5 Composition of synthetic waste water in both reactors

Compound Concentration (mg/L) Nutrient solution Concentration (mg/L)

Glycerol 410 FeCl₃.6H₂0 1500

Ethanol 240 H₃B0₃ 150

NH₄C1 variable CoCl₂.6H₂0 150

Yeast extract 100 ZnSo₄.7H₂0 120

K₂HP0₄ 15 MnCl₂.2H₂0 120

KH₂P0₄ 12.5 Na₂Mo0₄.2H₂0 60

MgCl₂.6H₂0 100 CuS0₄.5H₂0 60

CaCl₂.2H₂0 150 Kl 30

NaCI^* 30000

Nutrient solution 2mL/L

* NaCI was fed only to the saline MBR Flocculation tests show the applicability of the method according to the invention to produce flocculants. Furthermore, the results of the further experiment show an EPS yield after extraction, purification and lyophilisation as given in table 6.

Table 6 EPS yield after extraction, purification and lyophilisation.

EPS yield in g EPS-COD/g of wastewater COD

COD/N ratio J fresh wastewater saline wastewater

5 < 8 < 8

20 5.2 7.3

60 27 28

100 54 36

The skilled person would understand the Inlet COD (mg/L) mentioned in table 4 is an approximate amount, which is also shown in table 1.

In a further experiment binding and/or removal of metal was performed. The following conditions were applied:

100 mL Erlenmeyer flask, containing 100 mL of lead solution (50 mg/L) and 5 mL of crude (non-lyophilised) EPS-solution. The crude EPS solution, placed in a 12 - 14 kDa molecular weight cut-off dialysis bag, was kept in the flask. The content of the flask was stirred at 600 rpm at room temperature for 4 - 5 hours. Samples were taken at 10, 30, 60, 120, 180, 240 and 300 minutes and analysed using inductively coupled plasma - optical emission spectrometry (table 7).

It was found binding and/or removal of 1.3 gram of lead per gram of EPS was possible with the set-up.

Table 7 lead absorption

Data is expressed as mean ± standard deviation of duplicate extraction (VSS: volatile suspended solids; COD;^: Chemical Oxygen Demand of inlet waste water; COD_removed: Chemical Oxygen Demand removed during waste water treatment). Tests and experiments performed with system 102 and parameters as mentioned in tables 1 and 2, 4 and 5 resulted in a mixture of polymers of variable molecular weight ranging between l O kDa and 2000 kDa.

An effect of (maintaining) the EPS in these range(s) is the effective recovery and regaining of valuable recourses, preferably in combination with the cost effective treatment of waste fluid.

A further effect of this charge density is the prevention of restabilisation of the flocculants at a higher polymer dosage.

Further tests and experiments performed with system 102 and parameters as mentioned in tables 1 and 2, 4 and 5 resulted in a mixture of polymers of variable molecular weight ranging between 20 kDa and 4000 kDa.

An effect of (maintaining) the EPS in these range(s) is the even more effective recovery and regaining of valuable recourses, preferably in combination with the cost effective treatment of waste fluid. Advantageously, EPS can be formed using a low chemical oxygen demand to nitrogen ratio and chemical oxygen demand to phosphorus ratio. For example, a chemical oxygen demand to nitrogen ratio of 5 and chemical oxygen demand to phosphorus ratio of 10.

The present invention is by no means limited to the above described and preferred embodiments thereof. The rights sought are defined by the following claims within the scope of which many modifications can be envisaged.

Claims

1. Method for production of natural, biodegradable polymeric flocculants from a fluid, such as a waste fluid, the method comprising the steps of:

- providing a reactor comprising:

- at least one reactor vessel;

- at least one fluid inlet;

- at least one fluid outlet;

- at least one flocculant outlet; and

- inoculation of the reactor;

- providing feed to the at least one fluid inlet;

- forming the flocculants;

- separating the flocculants using the membrane; and

- providing the flocculants at the flocculant outlet.

2. Method according to claim 1 , wherein the fluid comprises biodegradable and soluble organic pollutants.

3. Method according to claim 2, wherein the pollutants comprises a chemical oxygen demand to nitrogen ratio above 5, preferably above 8, and most preferably above 10 and/or a chemical oxygen demand to phosphorous ratio above 10, preferably above 15, and most preferably above 20.

4. Method according to claim 1 , 2 or 3, wherein the pollutants comprises a chemical oxygen demand to nitrogen ratio above 15, preferably above 50, more preferably above 90, and most preferably above 100.

5. Method according to one or more of the foregoing claims, wherein inoculation of the reactor comprises inoculation with nucro-organisms from a biological waste water treatment plant.

6. Method according to one or more of the foregoing claims, separating and/or wherein forming the flocculants comprises the step of aerating.

7. Method according to claim 6, wherein forming and/or separating a dissolved oxygen level of at least 0.5 mg 0₂/L, preferably at least 0.75 mg O2/L, and most preferably at least 1 mg 0₂/L is maintained.

8. Method by process according to one of the foregoing claims, wherein the formation of the flocculants provides extracellular polymeric substances of polymers or proteins or a mixture of polymers and proteins.

9. Method according to claim 6, wherein the polymer fractions are preferably polysaccharides, and the charge density is 0.2 - 7 meq/g, preferably 0.35 - 6 meq/g, and most preferably 0.5 - 5 meq/g.

10. Method by process according to one or more of the foregoing claims, wherein the waste fluid comprises one or more of fresh and/or saline waste waters comprising microbial extracellular polymeric substances.

1 1. Method according to one or more of the foregoing claims, further comprising the combined biological waste water treatment and flocculants production, comprising the treatment and production under fresh water and/or saline conditions.

12. System for the production of biodegradable, organic microbial extracellular polymeric substances, the system comprising:

- at least one reactor vessel;

- at least one fluid inlet;

- at least one fluid outlet;

- at least one flocculants outlet; and

13. System according to claim 12, further comprising two independent chambers.

14. Flocculants produced by the method and/or in the system of one or more of the foregoing claims, wherein the flocculants comprising a flocculation activity of 80% or above.

15. Flocculants of claim 14, wherein the extracellular polymeric substances comprising a molecular weight, ranging between 2 kDa and 10000 kDa, preferably ranging between 3 kDa and 8000 kDa, more preferably ranging between 5 kDa and 6000 kDa, and most preferably ranging between 10 kDa and 2000 kDa.

16. Flocculants of claim 14 or 15. wherein the EPS comprises the capability to:

- binding and/or removal of ammonium and/or heavy metals; and/or

- seattleability of at most 1.7 mL/min.

17. Use of microbial extracellular polymeric substances for the production of flocculants from a waste fluid.