ES2469741A9 - Procedure and system for eliminating organic microcontaminants in wastewater by means of an enzymatic ceramic membrane reactor - Google Patents

Abstract

Procedure and system for the elimination of organic microcontaminants in wastewater by means of an enzymatic ceramic membrane reactor. The invention relates to a method and system for eliminating organic microcontaminants present in secondary effluents of WWTP or industrial effluents by ligninolytic enzymes, of the peroxidase or laccase type, in ultra-filtration ceramic membrane (REM) enzymatic reactors nanofiltration The water to be treated, after a pretreatment of filtration to minimize the concentration of colloids, is introduced into the reactor, where the enzyme: peroxidase or laccase, is found in free form. The reactor outlet is pumped to the ceramic membrane, separating into two streams. The retention stream, which contains the enzyme, is recycled to the reactor. The permeate stream, which constitutes the effluent of REM, is free of microcontaminants and can be discharged into the aquatic environment.

Description

Procedure and system for eliminating organic microcontaminants in wastewater by means of an enzymatic ceramic membrane reactor

TECHNICAL SECTOR OF THE INVENTION

The present invention relates to a method and system for the elimination of organic microcontaminants of the type of pharmaceutical and personal care compounds in addition to endocrine disrupting compounds present in effluents from wastewater treatment plants after secondary treatment, as well as in industrial effluents .

STATE OF THE TECHNIQUE

The discharge into the aquatic environment of organic microcontaminants from wastewater treatment plants (WWTPs) constitutes a major environmental problem due to the possible harmful effects that these chemical compounds have on living beings, even at trace concentrations (μg / L). Highlights include the Endocrine Disruptor Compounds (CDE), a group of substances of natural or anthropogenic origin (for example drugs, pesticides, cosmetic and personal care products, flame retardants, hormones and other industrial chemicals) with the ability to alter endocrine system functions and, consequently, cause adverse effects on an organism or its progeny. These compounds reach the WWTP from various sources: domestic, industrial wastewater, runoff water, etc. Since conventional wastewater treatment systems are not designed to eliminate these types of compounds, their disposal in treatment plants is partial, releasing the environment continuously. In those cases in which the aquatic environment also serves as a drain for sewage effluents and as a source for water purification, it is especially important to avoid the discharge of these microcontaminants.

Also, in the effluents of chemical or pharmaceutical companies it is very characteristic, even after a conventional treatment system, the presence of organic microcontaminants in low concentrations but sufficient to exert a potential effect on the aquatic environment.

In recent years, different treatments or post-treatments have been developed for the elimination of organic microcontaminants based on advanced oxidation processes such as photodegradation, photocatalysis, ozonation, hypochlorite or chlorine oxides, ultrasound, etc. However, these procedures are generally expensive, have low specificity (Esplugas S. et al., 2002; Water Res 36: 1034-1042), and sometimes generate byproducts that can potentially be more harmful or react with other components of the aqueous matrix, causing compounds with a similar or even greater estrogenicity than the starting one (Shappell NW et al., 2008; Environ Sci Technol 42: 1296-1300).

Enzymatic treatment is proposed as an alternative for the transformation of these microcontaminants into less toxic products, either because polymerization is favored or because the transformation is carried out in molecules without pharmacological action or endocrine disruptor. Eventually, they can be transformed into more easily degradable products (Galliker P. et al., 2010; J Colloid Interface Sci 349: 98105).

The oxidative enzymes produced by white rot fungi have been successfully applied to carry out the elimination of different endocrine disrupting compounds, such as bisphenol A, nonylphenol, triclosan, as well as the elimination of the estrogenic activity associated with them. (Cabana H. et al., 2007; Eng Life Sci

7: 429-456). Extracellular enzymes secreted by fungi can belong to two types: peroxidases, such as manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) or oxidases, such as laccase (Lac).

Peroxidases are hemo-proteins that require the presence of hydrogen peroxide as an electron acceptor to carry out the oxidation of the substrates. They have oxidation potentials of up to 1.51 V. Lignin peroxidase (LiP, EC 1.11.1.13) is capable of oxidizing both phenolic and non-phenolic aromatic substrates, with veratrolic alcohol being one of the most common substrates of the enzyme. Manganese peroxidase enzyme (MnP, EC 1.11.1.14) requires Mn2 + to close its catalytic cycle by oxidizing it to Mn3 + (E0 1.54 V), which diffuses and is capable of oxidizing both the phenolic and non-phenolic units. phenolic through lipid peroxidation reactions. In order to increase the stability of the Mn3 + ions in the aqueous phase, organic acids are added to form the Mn3 + -organic acid complex (E0 0.9-1.2 V). Versatile peroxidase (VP, EC 1.11.1.16) combines the catalytic properties of the two previous enzymes, since it efficiently oxidizes non-phenolic compounds of high redox potential, such as veratrolic alcohol (used by LiP) or Mn2 + ( used by the MnP) (Ruiz-Dueñas FJ et al., 2009; J Exp Bot 60 (2): 441-52). In addition, it oxidizes phenols in the absence of

Mn2 +.

The ability of peroxidases to degrade endocrine disrupting compounds has been demonstrated in a large number of jobs. As examples it can be noted that Hirano et al. carried out the degradation of Bisphenol A (BPA) using the enzyme MnP (Hirano T. et al., 2000; Biosci Biotechnol Biochem 64: 1958-1962). This same enzyme was used successfully to eliminate the hormone estrone and the associated estrogenic activity (Tamagawa Y. et al., 2006; Chemosphere 65: 97-101). Taboada et al. they used the versatile peroxidase enzyme immobilized in CLEAs to remove bisphenol A, nonylphenol, triclosan and 17 a-estradiol (Taboada-Puig R. et al., 2011; Bioresour Technol 102: 6593-6599).

Laccase (EC.1.10.3.2) is an oxidase that catalyzes the monoelectronic oxidation of phenolic substrates or aromatic amines, and transforms them into the corresponding radicals with the consequent reduction of molecular oxygen to water. The low substrate specificity, the use of oxygen as the final electron acceptor (instead of the H2O2 used by peroxidases) and the generation of water as the sole byproduct of the reaction (Paice MG et al., 1995; J Pulp Paper Sci 21 : 280-284), give the house a high applicability in various biotechnological processes. Although they have a maximum E0 of 0.8 V, lower than ligninolytic peroxidases, the presence of low molecular weight substrates, called mediators, allows the oxidation of a wide range of phenolic and non-phenolic compounds. Helices (Morozova O. et al., 2007; Appl Biochem Microbiol

43: 523-535 | Ca�as A.I. et al., 2009; Biotechnol Adv 28: 694-705).

Several studies have shown the ability of the enzyme lacasa to eliminate microcontaminants. As examples it can be noted that Auriol et al. they used the Trametes versicolor house to eliminate the estrogenic activity associated with natural and synthetic hormones (Auriol M. et al., 2008; Chemosphere 70: 445-452). Lloret et al. they used the Myceliophthora thermophila laccase to eliminate various microcontaminants from the anti-inflammatory group: naproxen, diclofenac and various hormones (Lloret L. et al., 2010; Bioch Eng Journal 51: 124131). This same laccase was also successfully applied to degrade bisphenol A, nonylphenol and triclosan (Cabana H. et al., 2007; Chemosphere 67 (4): 770-778).

However, the use of enzyme in free form in a reactor operated continuously as a tertiary treatment is not economically viable due to the high consumption of enzyme, since it would leave the reactor with the treated effluent. As an alternative, the use of immobilized enzyme can be considered, although it entails a series of disadvantages such as the decrease in enzymatic activity, limitations on the transfer of matter, extra cost, increase in catalyst volume and the need to find a suitable method of immobilization (Bornscheuer UT, 2003; Angew Chem Int Ed 42: 3336-3337).

The enzyme in free form can be used if a membrane unit is coupled to the reactor in order to separate and retain the enzyme. This configuration of Membrane Enzyme Reactor (REM) allows a retention of the biocatalyst in the system while degradation products and other permeable compounds leave it with the permeate stream, which constitutes the effluent of REM.

The membrane enzyme reactor system based on the use of immobilized or free manganese peroxidase has been successfully applied to the removal of industrial dyes (López C. et al., 2002; J Biotechnol 99: 249-257) and Results are reflected in the patent application (Lema et al. 2004, EP1457463 A1). The configuration of REM using a polymeric membrane (polyethersulfone) of cut size of 10 kDa has been used in an effective way for the elimination of estrogens present in the effluent of the secondary treatment of WWTP by the free house of Myceliophthora thermophila ( Lloret L. et al., 2013; Environ Sci Technol 47: 4536-4543). However, it has not been successfully applied for the elimination of other CDEs such as bisphenol A, nonylphenol or triclosan due to their high degree of adsorption to the polymeric material of the membrane. It has been shown that bisphenol A and other organic microcontaminants are retained by polymeric membranes (Agenson KO et al., 2003; J Membr Sci 225: 91-103 | Kimura K. et al., 2004; J Membr Sci 245: 71-78 | Zhang Y. et al., 2006; Water Res 40: 3793-3799) and the microcontaminant retained in the membrane could eventually be expelled again with the permeate, especially in those cases in which the concentration of microcontaminants in the influent to REM shows high variability (Zhang Y. et al., 2006; Water Res 40: 3793-3799).

The use of inorganic membranes, such as ceramics, is an alternative to polymers because its hydrophilic surface minimizes the adsorption effect. In addition, they are chemically inert, that is, resistant to the attack of chemical agents and corrosion, they have a greater range of operability against pH and temperature than polymers and their cleaning is easier. As a disadvantage, they generally have a higher cost of capital compared to polymeric membranes, although this factor can be compensated by the longer service life of inorganic membranes. Recently, other authors have proposed the use of a ceramic membrane in an enzymatic reactor to eliminate the tetracycline antibiotic in high concentrations (from Cazes M. et al., 2014; Catal Today, http://dx.doi.org/ 10.1016 / j.cattod. 2014.02.051). In said system, the membrane of pore size equal to 0.2 μm is only used as an immobilization support for the laccase enzyme. Due to the dimensions of said enzyme (6.5 x 5.5 x 4.5 nm) (Piontek K. et al., 2002; J Biol Chem 277: 37663-37669), this membrane is not able to retain the enzyme in free form, so that this system has the disadvantages of enzymatic immobilization.

DESCRIPTION OF THE INVENTION

In one aspect the present invention relates to a method of elimination of organic microcontaminants present in secondary effluents of WWTP or industrial effluents by ligninolytic enzymes, 5 peroxidases or lacases by the use of ultrafiltration ceramic membrane enzymatic reactors nanofiltration

The process of eliminating organic microcontaminants, present in secondary effluents from wastewater treatment plants (WWTP) or industrial effluents comprises:

10 a) apply a pretreatment that decreases the concentration of suspended solids and colloids

in the effluent; b) pump the effluent into a reaction vessel containing ligninolytic enzyme; c) control the enzymatic activity so that it is greater than a minimum threshold; d) pump the effluent into a ceramic membrane;

E) recirculating the retention current generated in the ceramic membrane and containing the enzyme to the reaction vessel; and f) discharge the permeate current from the membrane into the aquatic environment.

Pretreatment decreases the concentration of suspended solids and colloids in the water to be treated, in order

20 to maximize the enzymatic stability in the reactor as well as to minimize fouling of the ceramic membrane. In a particular embodiment of the invention, the pretreatment comprises circulating the effluent from the treatment plant through a bed of sand of granulometry of less than 1 mm. In a particular embodiment of the invention the pretreatment comprises using a microfiltration membrane, before introducing it into the REM.

In another aspect of the invention, the effluent flow rate introduced into the reaction vessel is determined as a function of the hydraulic residence time (HRT) in the reaction vessel, defined as the ratio between the reaction volume in the vessel and the flow rate. of the effluent that enters to be treated; and the percentage of contaminants that you want to remove.

30 In the event that the concentration of pollutants in the permeate stream is above a maximum set value, the HRT is increased, reducing the flow of effluent entering the reaction vessel or reactor.

In the reaction vessel is the ligninolytic enzyme, in free form. When an enzyme is used

The peroxidase activity is in the range 50-1000 U / L. The activity of the LiP enzyme is determined by the assay described by Tien and Kirk (Tien M. and Kirk TK, 1984; Proc Natl Acad Sci USA 81: 2280-2284) and the measurement of the MnP or VP activity is made using the trial collected by Taboada-Puig et al. (Taboada-Puig

R. et al., 2011; Biotechnol Prog 27 (3): 668-676). If a laccase enzyme is used, the enzymatic activity is in the range 100-1500 U / L, according to the measurement protocol detailed in the literature (Arca

40 Ramos A. et al., 2012; Process Biochem 47: 1115-1121). The operating temperature in the reactor should be in the range 10-40 ° C, preferably 25 ° C; and the pH should be between 3-8; preferably pH 4.5 in the case of peroxidases and pH 6-7 in the case of laccase.

In a particular embodiment the reaction vessel (reactor) comprises a stirred tank reactor.

The control of enzymatic activity comprises:

a) measure the enzymatic activity in the reaction vessel; b) if the enzymatic activity in the reaction vessel is less than a minimum value add enzyme in the form of pulses; and c) repeat steps a and b until the enzymatic activity in the reaction vessel is greater than the minimum set value.

During the operation the periodic measurement of the enzymatic activity in the vessel is carried out and when the activity drops below 50 U / L for peroxidases or 100 U / L in the case of lacasa, fresh enzyme should be added to the vessel. reactor.

In the case of using a peroxidase, hydrogen peroxide should be added to the reactor with a speed between 5-100 Imol / Lmin. If the peroxidase enzyme is manganese peroxidase (MnP) or versatile peroxidase (VP), in addition, dicarboxylic organic acid, such as maleic or oxic acid, is added continuously at a rate of 1-100 Imol / L� min.

In the event that the enzyme used is a peroxidase, the cofactors necessary to complete the catalytic cycle of the peroxidase: summer alcohol are introduced into the reactor, at a rate of between 0.5-1000 Imol / L�min in the if the enzyme is lignin peroxidase (LiP), and Mn + 2, at a rate between 0.5-100 Imol / L�min, in the case that the enzyme is manganese peroxidase (MnP) or versatile peroxidase ( VP).

In the event that the enzyme used is laccase, the concentration of dissolved oxygen in the reactor is measured by a dissolved oxygen sensor such that when said concentration is less than 4 mg / L, aeration or oxygenation is carried out, either in continuous or by means of pulses through a diffuser, of the reactor. These pulses will be of sufficient duration for the concentration of dissolved oxygen to reach at least 90% of the oxygen concentration corresponding to saturation.

The flow of the reactor output current is pumped to the module where the ceramic membrane is located. A part of this current crosses the membrane, free of enzyme, constituting the effluent of the system or permeate. The flow rate of the permeate stream must match the flow rate of the influent to the reactor at steady state and is adjusted by means of a flow regulating valve located at the end of the membrane module. The current that does not pass through the membrane is the retentate, which contains the enzyme and is returned to the reactor in the recirculation stream. The transmembrane pressure must be systematically measured in order to determine the time to proceed with the chemical cleaning of the membrane. Likewise, periodic samples of the influent are taken to the REM and the permeate to determine the concentration of microcontaminants after the enzymatic treatment in the reactor. In the event that said concentration is higher than the preset, the hydraulic residence time in the reactor should be increased.

In another aspect, the invention relates to an elimination system of organic microcontaminants present in secondary effluents of WWTP or in industrial effluents by ligninolytic enzymes, peroxidases or lacases by the use of ultrafiltration ceramic membrane enzymatic reactors nanofiltration comprising:

a) a pretreatment system (102) that reduces the concentration of suspended solids and colloids in

the effluent;

b) a first pumping system (104);

c) a reaction vessel (106) or reactor comprising a solution of ligninolytic enzyme (108);

d) an enzyme activity control system (110) of the solution contained in the reaction vessel;

e) a second pumping system (112);

f) a ceramic membrane (114);

g) a recirculation system (116);

h) a microcontaminant measurement system in the enzyme reaction vessel and in the permeate of

the ceramic membrane.

In a particular embodiment, the pretreatment system comprises a microfiltration membrane filtration treatment system. In another particular embodiment the pretreatment system comprises a sand filter of granulometry less than 1 mm.

In the microcontaminant removal system, the first pumping system pumps the effluent from the pretreatment system to the reaction vessel.

In a particular embodiment, the reaction vessel or reactor comprises a stirred tank reactor.

The lignolytic enzyme introduced into the reactor is selected from peroxidase and laccase.

The enzyme activity control system comprises:

a) an enzyme activity sensor; Y

b) an enzyme pulse addition system.

The enzyme addition system adds enzyme to the reactor when the enzymatic activity is less than 50 U / L, in the case of using peroxidase, and less than 100 U / L, when the enzyme used is laccase.

The operating temperature of the reactor is in the range 10-40 ° C, preferably 25 ° C, and the pH of the mixture contained in the reactor is in the range 3 to 8, preferably pH 4.5 in the case of peroxidases, and pH in the range 6-7 in the case of lacasa.

In the event that the enzyme used is of the manganese peroxidase type (MnP) or of the versatile peroxidase type (VP), the microcontaminant removal system further comprises a continuous addition system (118) of hydrogen peroxide, which is added with a speed in the range 5-100 Imol / L�min. The microcontaminant removal system also comprises an addition system in

Continuous organic dicarboxylic acid when the enzyme used is manganese peroxidase, which is added with a speed in the range 1-100 Imol / L�min. In the event that the enzyme used is a peroxidase, the microcontaminant elimination system also includes a cofactor addition system necessary to complete the catalytic cycle. Said addition system incorporates veratrolic alcohol, when the enzyme used is lignin peroxidase (LiP), at a rate in the range 0.5-1000 Imol / Lmin, and Mn + 2 if the enzyme used is an enzyme of the manganese peroxidase (MnP) or versatile peroxidase (VP) type, at a speed in the range 0.5 - 100 Imol / L�min.

In the event that the enzyme used is a laccase, the reaction vessel further comprises an oxygen control system (122) dissolved in the effluent and enzyme mixture. Said control system (122) comprises:

a) a sensor for measuring the concentration of dissolved oxygen; Y

b) an aeration or oxygenation system.

When the concentration of dissolved oxygen is less than 4 mg / L, the aeration system acts by carrying out continuous aeration or by pulsing using a diffuser.

The second pumping system (112) pumps the reaction vessel mixture towards the ceramic membrane (114). The recirculation system (116) pumps the retention current, which contains the enzyme, into the reaction vessel. The transmembrane pressure measurement system (124) measures the transmembrane pressure in order to determine the time to proceed to chemical cleaning of the membrane. Likewise, the microcontaminant measurement system periodically takes samples of the influent to the REM and the permeate to determine the concentration of microcontaminants after enzymatic treatment in the reactor

In another aspect, the invention relates to the use of the method and system described above for the elimination of organic microcontaminants present in secondary effluents from wastewater treatment plants (WWTP) or industrial effluents.

BRIEF DESCRIPTION OF THE FIGURES

The modalities detailed in the figures are illustrated by way of example and not by way of limitation:

Figure 1. Schematic diagram of the ceramic membrane enzyme reactor system and its application in the removal of microcontaminants. The influent to the reactor, with a certain concentration of microcontaminants, is continuously pumped to the reaction vessel, where the enzyme is in solution. Said enzyme is added to the system in periodic pulses when the activity is lower than required. The reactor output current is pumped into the ceramic membrane module. Oxygen, pH and temperature sensors and a level controller are available in the reactor. In the membrane module there are two gauges for measuring transmembrane pressure. Two currents leave the membrane: the retention current, which contains the enzyme and that is recirculated to the reactor, and the permeate one, whose flow rate is set to be identical to that of the influent to the reactor, and which can be discharged to the aquatic environment Being free of microcontaminants.

Figure 2. Concentration profiles of BPA (0) and NP (�) in the influent to REM, laccase activity (

) in the stirred tank reactor and concentration of BPA (e) and NP (�) at the output of the REM.

Figure 3. Concentration profiles (percentage with respect to the concentration in the input current) of BPA

(e) in the output current of the REM and the activity (percentage with respect to the activity in time 0 h) of the house (

) in the stirred tank reactor for the continuous treatment of BPA (85 μg / L) per laccase with HRT of 5 h.

Figure 4. Concentration profiles (percentage with respect to the concentration in the input current) of BPA

(e) in the output current of the REM and the activity (percentage with respect to the activity in time 0 h) of the house (

)

 in the stirred tank reactor for the continuous treatment of BPA (85 μg / L) per laccase with HRT of 7.5

h.

Figure 5. Concentration profiles (percentage with respect to the concentration in the input current) of BPA

(e) in the output current of the REM and the activity (percentage with respect to the activity in time 0 h) of the house (

) in the stirred tank reactor for continuous treatment of BPA (85 μg / L) per laccase with HRT of 10 h.

EXAMPLES

Example 1

The procedure described above was applied for the treatment of the compounds: bisphenol A (BPA) and

5 nonylphenol (NP) by free laccase in a stirred tank reactor with a useful volume of 1.5 L coupled to a ceramic nanofiltration membrane with a pore size of 1 nm in order to retain the enzyme and recirculate it to the reactor according to the scheme shown in Figure 1. The average enzyme concentration was 400 U / L and the concentrations of BPA and NP in the influent were 64 μg / L and 48 μg / L, respectively. The operating conditions are detailed below: temperature, 25�C; pH 6; stirring, 250 rpm; reason for

10 recirculation (recirculation / effluent), 3: 1; residence time, 2 h.

The disappearance of BPA and NP was determined by gas chromatography coupled to mass spectrometry. Enzyme activity was determined spectrophotometrically. The results show that in the steady state, more than 80% of the BPA and more than 97% of NP are eliminated (Figure 2).

15 Example 2

The procedure described above for the removal of bisphenol A by free laccase was applied in a stirred tank reactor with a useful volume of 1.5 L coupled to a ceramic nanofiltration membrane with a pore size of 1 nm for the purpose of retain the enzyme and recirculate it to the reactor (Figure 1). The initial enzyme concentration was 1,000 U / L and the contaminant concentration in the REM influent was 85 μg / L.

20 The operating conditions were as follows: temperature, 25�C; pH 6; stirring, 250 rpm; Recirculation ratio (recirculation / effluent), 4: 1.

Experiments were performed at different times of hydraulic residence (HRT) 5 h, 7.5 h and 10 h. Before the start of the tests it was found that the concentration of BPA in the input and output current of the REM in the absence of enzyme were identical, which allows confirming that the elimination of BPA is exclusively due

25 to enzymatic action.

The removal of BPA was determined by gas chromatography coupled to mass spectrometry. Enzyme activity was determined spectrophotometrically.

The results (Figures 3, 4 and 5) show that the elimination percentage is greater than 92% operating at all HRT considered and increases with HRT such that for 10 h the elimination of BPA was

30 complete (Figure 5). Also, the enzyme showed high stability.

Claims (58)

1. Procedure for the elimination of organic microcontaminants, present in secondary effluents from wastewater treatment plants (WWTP) or industrial effluents comprising:

to.

apply a pretreatment that decreases the concentration of suspended solids and colloids in the effluent;

b.

pump the effluent into a reaction vessel containing ligninolytic enzyme;

C.

control enzymatic activity so that it is greater than a minimum threshold;

d.

pump the effluent into a ceramic membrane;

and.

recirculating the retention current that is generated in the ceramic membrane and which contains the enzyme towards the reaction vessel; Y

F.

discharge the permeate current from the membrane into the aquatic environment.

2.

The method according to claim 1, characterized in that the pretreatment comprises a filtration treatment.

3.

The method according to claims 1 and 2, characterized in that the pretreatment comprises the use of a microfiltration membrane or a sand filter with a particle size of less than 1 mm.

Four.

The method according to claim 1, characterized in that the flow rate of the effluent introduced into the reaction vessel is determined as a function of the hydraulic residence time (HRT) in the reaction vessel, defined as the ratio between the reaction volume in the vessel and the effluent flow that enters to be treated; and the percentage of contaminants that you want to remove.

5.

The method according to claim 4, characterized in that if the concentration of contaminants in the permeate stream is above a set maximum value, the HRT is increased, reducing the flow of the effluent entering the reaction vessel.

6.

The method according to claim 1, characterized in that the lignolytic enzyme is selected from peroxidases or lacases.

7.

The method according to claim 6, characterized in that the activity of the enzyme peroxidase in free form is in the range 50-1000 U / L.

8.

The method according to claim 6, characterized in that the activity of the enzyme laccase in free form is in the range 100-1500 U / L.

9.

The method according to claims 1 and 4 to 8, characterized in that the reaction vessel comprises a stirred tank reactor.

10.

The method according to claims 1 and 4 to 8, characterized in that the operating temperature of the reaction vessel is in the range 10 to 40 ° C.

eleven.

The process according to claims 1 and 4 to 8, characterized in that the pH of the effluent and enzyme mixture in the reaction vessel is in the range 3 to 8.

12.

The process according to claims 1 and 4 to 11, characterized in that the control of the enzymatic activity comprises:

to.

measure the enzymatic activity in the reaction vessel;

b.

add enzyme in the form of pulses if the enzymatic activity in the reaction vessel is less than a minimum value; Y

C.

Repeat steps a and b until the enzymatic activity in the reaction vessel is greater than the minimum set value.

13.

The method according to claim 12 characterized in that the minimum value of enzymatic activity is 50 U / L in the case where the enzyme is peroxidase.

14.

The method according to claim 12, characterized in that the minimum value of enzymatic activity is 100 U / L in the event that the enzyme is laccase.

fifteen.

The process according to any of claims 1 and 4-14, characterized in that if the enzyme used is an enzyme of the peroxidase type, it is continuously added to the hydrogen peroxide reaction vessel.

16.

The process according to claim 15, characterized in that the hydrogen peroxide is added with a speed between 5 and 100 Imol / Lmin.

17.

The process according to claims 1 and 4 to 16, characterized in that if the enzyme used is manganese peroxidase (MnP) or versatile peroxidase (VP), in addition, dicarboxylic organic acid is added continuously.

18.

The process according to claim 17, characterized in that the organic dicarboxylic acid is added with a rate between 1 and 100 Imol / Lmin.

19.

The method according to any of claims 1 and 4-18, characterized in that the cofactor necessary to complete the catalytic cycle of peroxidase is also added in the reaction vessel.

twenty.

The method according to claim 19, characterized in that the cofactor comprises veratrolic alcohol in the case where the enzyme used is a lignin peroxidase (LiP) type peroxidase.

twenty-one.

The method according to claim 20, characterized in that the veratrolic alcohol is introduced at a rate in the range 0.5-1000 Imol / Lmin

22

The method according to claim 19, characterized in that the cofactor comprises Mn + 2 in the case where the enzyme used is peroxidase of the manganese peroxidase type (MnP) or of the versatile peroxidase type (VP).

2. 3.

The method according to claim 22, characterized in that the Mn + 2 is introduced at a speed in the range 0.5-100 Imol / L�min.

24.

The method, according to any of the preceding claims, characterized in that it further comprises measuring the concentration of dissolved oxygen in the reaction vessel when the enzyme used is laccase type.

25.

The method according to claim 24, characterized in that if the oxygen concentration is less than 4 mg / L, aeration or oxygenation is carried out, either continuously or by pulsing through a diffuser.

26.

The method according to the preceding claims, characterized in that the flow rate of the current pumped into the ceramic membrane is greater than the flow rate of the effluent introduced into the reaction vessel.

27.

The process according to claim 1, characterized in that the retention current containing the enzyme is recirculated to the reactor.

28.

The method according to any of claims 1-14, characterized in that the transmembrane pressure is measured.

29.

The process, according to any of the preceding claims, characterized in that the concentration of microcontaminants in the enzyme reaction vessel and in the permeate of the ceramic membrane is periodically measured.

30

An organic microcontaminant disposal system, present in secondary effluents (100) of wastewater treatment plants (WWTP) or industrial effluents comprising:

to.

a pretreatment system (102) that reduces the concentration of suspended solids and colloids in the effluent;

b.

a first pumping system (104);

C.

a reaction vessel (106) comprising a solution of ligninolytic enzyme (108);

d.

an enzyme activity control system (110) of the solution contained in the reaction vessel;

and.

a second pumping system (112);

F.

a ceramic membrane (114);

g.

a recirculation system (116); Y

h.

a microcontaminant measurement system in the enzyme reaction vessel and in the permeate of the ceramic membrane.

31.

The system according to claim 30, characterized in that the pretreatment system comprises a microfiltration membrane filtration treatment system.

32

The system according to claim 30, characterized in that the pretreatment system comprises a sand filter of particle size of less than 1 mm.

33.

The system according to claim 30, characterized in that the first pumping system pumps the effluent from the pretreatment system to the reaction vessel.

3. 4.

The system according to claim 30, characterized in that the reaction vessel comprises a stirred tank reactor.

35

The system according to claim 30, characterized in that the lignolytic enzyme is selected from peroxidase and laccase.

36.

The system according to claim 30, characterized in that the enzyme activity control system comprises:

to.

an enzyme activity sensor; Y

b.

an enzyme pulse addition system.

37.

The system according to claim 36, characterized in that the enzyme addition system adds enzyme when the enzymatic activity is less than a minimum value of enzymatic activity.

38.

The system according to claim 37, characterized in that the minimum value of enzymatic activity is 50 U / L when the enzyme used is peroxidase.

39.

The system according to claim 37, characterized in that the minimum value of enzymatic activity is 100 U / L when the enzyme used is laccase.

40

The system according to claims 30 and 33 to 39, characterized in that the operating temperature of the reaction vessel is in the range 10 to 40 ° C.

41.

The system according to claims 30 and 33 to 39, characterized in that the pH of the effluent and enzyme mixture of the reaction vessel is in the range 3 to 8.

42

The system according to claims 30 and 33 to 41, characterized in that the reaction vessel further comprises a continuous addition system (118) of hydrogen peroxide when the enzyme used is of the manganese peroxidase type (MnP) or of the versatile peroxidase type (VP).

43

The system according to claim 42, characterized in that the addition system adds hydrogen peroxide with a speed in the range 5-100 Imol / Lmin.

44.

The system according to claim 42, characterized in that the reaction vessel further comprises a continuous addition system of organic dicarboxylic acid when the enzyme used is manganese peroxidase.

Four. Five.

The system according to claim 44, characterized in that the addition system adds dicarboxylic organic acid with a speed in the range 1-100 Imol / Lmin.

46.

The system according to claim 30, characterized in that the reaction vessel further comprises a cofactor addition system (120) necessary to complete the catalytic cycle of peroxidase.

47

The system according to claim 46, characterized in that the cofactor comprises veratrolic alcohol if the enzyme used is lignin peroxidase (LiP).

48.

The system according to claim 46, characterized in that the veratrolic alcohol is added at a speed in the range 0.5-1000 Imol / Lmin.

The system according to claim 46, characterized in that the cofactor comprises Mn + 2 if the enzyme used is an enzyme of the manganese peroxidase (MnP) or versatile peroxidase (VP) type.

fifty.

The system according to claim 49, characterized in that the Mn + 2 is added at a speed in the range 0.5-100 Imol / L�min.

51.

The system according to claims 30 and 33-50, characterized in that the reaction vessel comprises

10 also an oxygen control system (122) dissolved in the effluent and enzyme mixture in the event that the enzyme used is laccase. 52. The system according to claim 51, characterized in that the oxygen control system comprises: to. a sensor for measuring the concentration of dissolved oxygen; and 15 b. an aeration or oxygenation system.

53.

The system according to claim 52, characterized in that the aeration or oxygenation system aerates the mixture when the concentration of dissolved oxygen is less than 4 mg / L.

54

The system according to claim 52, characterized in that the aeration is carried out continuously.

55. The system according to claim 52, characterized in that the aeration is carried out by means of 20 pulses using a diffuser.

56.

The system according to claim 30, characterized in that the second pumping system pumps the reaction vessel mixture towards the ceramic membrane.

57.

The system according to claim 30, characterized in that the recirculation system pumps the retention current that the enzyme contains into the reaction vessel.

The system according to claim 30, characterized in that it further comprises a transmembrane pressure measurement system (124) in the ceramic membrane. 59. Use of the method according to claims 1 to 29, and of the system according to claims 30 to 58, for the elimination of organic microcontaminants present in secondary effluents from wastewater treatment plants (WWTP) or industrial effluents. FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 SPANISH OFFICE OF THE PATENTS AND BRAND Application No.: 201430762 SPAIN Date of submission of the application: 05.23.2014 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C02F3 / 34 (2006.01)  RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

LLORET et al. Degradation of estrogens by laccase from Myceliophthora in fed-batch and enzymatic membrane reactors. Journal of Hazardous Materials, 2012, Vol. 213-214, pages 175-183, section 2.3.2 1-59

TO

TABOADA R. Removal of Endocrine Disrupting Chemicals by the Ligninolytic Enzyme Versatile Peroxidase. Doctoral thesis, University of Santiago de Compostela, December 2012, chapter 7 1-59

TO

KRAWCZYK et al. Combined membrane filtration and enzymatic treatment for recovery of high molecular mass hemicelluloses from chemithermomechanical pulp process water. Chemical Engineering Journal, 2013, Vol. 225, pages 292-299, section 2 1-59

TO

CASTILLO et al. Degradation of estrogens by laccase from Trametes versicolor in enzymatic membrane reactors. XV National Congress of Biotechnology and Bioengineering, Canc�n, Mexico, 06.27.2013 1-59

TO

LLORET et al. Continuous Biotransformation of Estrogens by Lacasse in an Enzymatic Membrane Reactor. Chemical Engineering Transactions, 2012, Vol. 27 1-59

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 09.06.2014

Examiner A. Rua Aguete Page 1/5

SPANISH OFFICE OF THE PATENTS AND BRAND Application No.: 201430762 SPAIN Date of submission of the application: 05.23.2014 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C02F3 / 34 (2006.01)  RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

A

US 2011/0110894 A1 (DRAHOS ET AL) 12.05.2011, lines 133-144; figure 1 EP 2489639 (MIMAKI ENGINEERING CO, LTD) 22.08.2012, figure 1, paragraphs [30-44] 1-59 1-59

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 09.06.2014

Examiner A. Rua Aguete Page 2/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201430762 Minimum documentation sought (classification system followed by classification symbols) C02F Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, XPESP, CAPLUS State of the Art Report Page 3/5  WRITTEN OPINION Application number: 201430762 Date of Written Opinion: 09.06.2014 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-59 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-59 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 4/5  WRITTEN OPINION Application number: 201430762  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

LLORET et al. Degradation of estrogens by laccase from Myceliophthora in fed-batch and enzymatic membrane reactors. Journal of Hazardous Materials, 2012, Vol. 213-214, pages 175-183, section 2.3.2

D02

TABOADA R. Removal of Endocrine Disrupting Chemicals by the Ligninolytic Enzyme Versatile Peroxidase. Doctoral thesis, University of Santiago de Compostela, December 2012, chapter 7

D03

KRAWCZYK et al. Combined membrane filtration and enzymatic treatment for recovery of high molecular mass hemicelluloses from chemithermomechanical pulp process water. Chemical Engineering Journal, 2013, Vol. 225, pages 292-299, section 2

D04

CASTILLO et al. Degradation of estrogens by laccase from Trametes versicolor in enzymatic membrane reactors. XV National Congress of Biotechnology and Bioengineering, Canc�n, Mexico, 06.27.2013

 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention is a method and system for the elimination of organic microcontaminants present in secondary effluents from sewage treatment plants or industrial effluents comprising a reactor containing lignolytic enzyme of the laccase and peroxidase type, a control system for the enzymatic activity in the reactor and a ceramic membrane coupled to the reactor for the retention and recirculation of the enzyme. Document D1 discloses a process and system for the degradation of estrogens present in wastewater by treatment with laccase in an enzymatic membrane reactor system. The system comprises a stirred tank reactor coupled to a polyether sulfone polymer membrane. The reactor is equipped with an enzyme activity control system. To increase the efficiency of the system, the contact time between the substrate and the enzyme is increased, reducing the frequency of addition of the enzyme in the form of pulses, thus saving the consumed enzyme. The polymeric membrane retains the enzyme efficiently, which is considered optimal for treatment. (See sections 2.3.1 and 2.3.2). Document D2 discloses an enzymatic ultrafiltration system for the elimination of endocrine disrupting compounds by means of a stirred tank reactor to which peroxidase coupled to a polyether sulfone ultrafiltration membrane is added for recycling the enzyme to the reactor. (See figure 7.2). Document D3 discloses a system and procedure for the recovery of hemicelluloses from the papermaking process waters by passing through a microfiltration polymeric membrane and an ultrafiltration ceramic membrane prior to enzymatic treatment in a stirred tank reactor to which lacasa is added and subsequent passage through a polymer ultrafiltration membrane. (See section 2.4.3). Document D4 discloses a system for the degradation of estrogens in a membrane enzyme reactor system in which a ceramic membrane is used as a support for immobilization of the enzyme lacasa. (See introduction). None of the documents D1 to D4 cited or any relevant combination thereof discloses a system and procedure for the removal of organic microcontaminants in wastewater by lignolytic enzymes through the use of nanofiltration ultrafiltration ceramic membrane enzymatic reactors �n, in which a ceramic and non-polymeric membrane is used for the retention and recycling of the enzyme to the reactor. Therefore, the invention as set forth in claims 1 to 59 of the application is new and involves inventive activity. (Art. 6 and 8 of the LP). State of the Art Report Page 5/5