JP2020500698A - Biobeads for biofouling control in membrane bioreactors - Google Patents

Abstract

Disclosed herein is a composite material comprising quorum quenching (QQ) bacteria retained within a cross-linked polymeric matrix material, such as calcium alginate, for reducing and / or preventing membrane biofouling in wastewater treatment. You. The composite may further include a biocompatible absorbent material, such as activated carbon, and a further polymeric surface coating, such as polysulfone. The composite materials disclosed herein may also be particularly suitable for use in a membrane bioreactor, certain QQ bacterial consortiums are suitable for aerobic membrane bioreactors, others are anaerobic membrane bioreactors. Suitable for bioreactors.

Description

  The present invention relates to a composite material for use in a membrane bioreactor to reduce and / or prevent membrane biofouling.

  A listing or reference to a document as set forth in this specification should not necessarily be taken as an acknowledgment that the document is part of the state of the art or is common general knowledge.

  Membrane bioreactors (MBR), a combination of biodegradation and membrane filtration, are widely recognized as advanced wastewater treatment technologies and are gaining increasing popularity worldwide in wastewater reclamation and reuse [J. Membr Sci. 284 (2006) 17-53]. Compared to the conventional activated sludge process, MBR has unique advantages including high treatment efficiency, low sludge production, small footprint and better effluent quality [Desalination. 306 (2012) 35-40]. However, membrane fouling (or biofouling) leaves a major challenge of significantly reducing flow and consequently increasing operating and maintenance costs. Thus, membrane fouling remains an obstacle to the growth of the MBR market due to the high energy demands and operating costs associated with fouling control.

  Anaerobic treatment of low-intensity municipal wastewater in anaerobic membrane bioreactors (AnMBR) has increased in recent years due to neutral or even positive energy balance, low solids production, and a smaller footprint from conventional aerobic treatment. It is getting attention. However, like aerobic MBR, membrane biofouling remains the biggest obstacle to widespread application of AnMBR. Membrane biofouling reduces filtration flow and increases operating costs; biogas sparging and the most widely used biofouling control methods could cost as much as 60% of the total operating cost of a wastewater treatment plant. Therefore, the development of new antifouling membranes and fouling control technology is the focus of research on membrane technology.

Membrane biofouling is the three main stages in transmembrane pressure (TMP) profiles:
1. Initial rise due to pore blockage (stage 1);
2. Slow rise due to accumulation of extracellular polymeric substances (EPS) on the membrane surface; and Rapid jump due to biofilm formation (Stage 3)
Can be characterized by

  Fluctuations in hydrodynamic parameters (aeration rate and air diffuser position, relaxation / filtration mode and backwash), material pretreatment by coagulation / coagulation, chemical (acid, base and oxidant) cleaning, Various means, such as and the addition of polymer or powdered activated carbon (PAC), have been used in an attempt to identify and eradicate factors that cause membrane biofouling in aerobic systems. However, all of these approaches have limitations in mitigating membrane biofouling because they do not address the challenge of biofilm formation on the membrane surface-a natural process that leads to a sharp decrease in the critical flow of the membrane. I have.

  In the past few years, biological approaches-quorum quenching (QQ) have been employed to reduce cell-cell communication in bacteria, thereby delaying biofilm development on membranes. . QQ was either in liquid culture [J. Microbiol. Biotechnol. 24 (2014) 97-105] or immobilized in alginate beads [J. Membr. Sci. 411-412 (2012) 130-136]. Also introduces QQ enzyme producing bacteria into the MBR. Of the different approaches, the addition of QQ bacteria immobilized on alginate or polymer-coated alginate beads has been shown to be most effective for fouling control.

  N-acyl-homoserine lactone (AHL) mediates interactions within the same species in Gram-negative bacteria, while autoinducer-2 (AI-2) crosses both Gram-negative and Gram-positive bacteria. Mediating interactions between species, both contribute to the production of EPS and stimulate biofilm growth. As noted above, the most extensively studied aerobic QQ-based biofouling control technology involves enrichment of bacterial strains that quench AHL by immobilizing these bacteria in alginate beads. . When QQ-embedded alginate beads are added to the MBR, the QQ strain depletes the levels of AHL, thus preventing biofilm (cake layer) growth on the membrane. Also, biostimulation of endogenous QQ strains through continuous supplementation of stimuli has efficacy in controlling biofouling in aerobic MBR [Biotechnol. Bioeng. 113 (2016) 2624-2632]. Also, recently, the first autoinducer-2 (AI-2 targeted) QQ technology for aerobic systems utilizing fungal strains embedded in alginate beads has been developed for its control of fouling in aerobic MBR. Potential [Environ. Sci. Technol. 50 (2016) 10914-10922].

  In an early approach to aerobic systems, QQ bacteria were immobilized in calcium alginate beads. Furthermore, recently, these calcium alginate beads have been coated with polymers such as polyvinylidene fluoride, polysulfone, polyethersulfone to improve their stability.

  In addition, QQ-based biofouling control in anaerobic membrane bioreactors has not been studied. Only AHL-mediated quorum sensing (QS) characterized in methanogens shows that AHL in Methanosaeta harundinacea differs structurally and functionally from the previously described aerobic AHL in Methanosaeta harundinacea [ISME J. 6 (2012) 1336-1344].

  Thus, there is an urgent need for new bacterial fixation methods that are stable to harsh environmental conditions and are feasible for large-scale production of aerobic and anaerobic systems.

The present invention surprisingly provides a composite material that solves many of the problems encountered and described above. Surprisingly, it has been found that a composite material suitable for use in an anaerobic membrane bioreactor can be obtained. Thus, in a first aspect of the present invention there is provided a composite material for use in an anaerobic membrane bioreactor, wherein the composite material reduces and / or prevents membrane biofouling:
A cross-linked polymeric matrix material compatible with bacterial growth (bacterial growth); and a bacterial population (microbial population)
Including
The bacterial population is homogeneously mixed throughout the polymeric matrix material, and the bacterial population is substantially comprised of Microbacterium sp. QQ1 (KR084848); and the uniformly mixed bacterial population and the polymeric matrix material are It is coated with a coating material selected from one or more of the group consisting of vinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene.

  In embodiments of the first aspect of the present invention, the composite material may include a biocompatible adsorbent material that is uniformly mixed throughout the polymeric matrix material (eg, the biocompatible adsorbent material comprises For example, activated carbon may be selected from one or more of the group consisting of activated carbon, ion exchange resins, chitosan, zeolites and lignin).

It has also been surprisingly found that composite materials for aerobic systems containing biocompatible adsorbent materials have excellent properties (suitable biocompatible adsorbent materials are activated carbon , For example, activated carbon may be selected from one or more of the group consisting of: ion exchange resins, chitosan, zeolites and lignin). Thus, in a second aspect of the present invention, there is provided a composite material for use in a membrane bioreactor for reducing and / or preventing membrane biofouling, the composite material comprising:
A cross-linked polymeric matrix material compatible with bacterial growth;
A bacterial population; and a biocompatible adsorbent material;
The bacterial population and the adsorbent material are homogeneously mixed throughout the polymeric matrix material, and the bacterial population is composed of Microbacterium sp. QQ1 (KR058484), Enterobacter cloaca QQ13 (KR058544), Pseudomonas sp. QQ3 (KR05846). And one or more bacterial species selected from Rhodococcus species BH4.

In certain embodiments of the second aspect of the present invention:
(I) the bacterial population may further comprise E. coli ΔlsrRΔluxS;
(II) The homogeneously mixed bacterial population and the polymer matrix material are one or more of the group consisting of polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene. Can also be coated with a coating material selected from;
(III) The composite material has a Young's modulus value of 40,000 to 100,000 Pa, for example, 45,000 to 70,000 Pa, for example, 48,000 to 100,000, for example, 48,000 to 70,000. I can do it.

In embodiments of the first and second aspects of the invention:
(A) The crosslinked polymeric matrix material may be a polyanionic polymer crosslinked by divalent (2+) or trivalent (3+) metal ions (eg, the polyanionic polymer may be alginate or polyanionic cellulose). And / or the cross-linking metal ion can be a divalent metal ion selected from the group consisting of Ca ²⁺ and Mg ²⁺ , for example, the cross-linked polymeric matrix material is calcium alginate obtain);
(B) The ratio of bacterial population (dry weight) to crosslinked polymeric matrix material is from 1: 500 to 1: 2,000 w / w, for example, 1: 750 to 1: 1,500 w / w, for example, , About 1: 1,000 w / w;
(C) The ratio of bacterial population (dry weight) to biocompatible adsorbent material is from 1: 300 to 1: 800 w / w, for example 1: 400 to 1: 600 w / w, for example, about 1: Can be 500 w / w;
(D) the composite can be provided as spherical beads having a diameter of 2-3 mm;
(E) When the composite material is coated with a coating material, the coated composite material has a Young's of 200,000 to 400,000 Pa, for example, 250,000 to 350,000 Pa, for example, 300,000 to 310,000. It can have a rate value.

As noted above, the materials of the first aspect of the invention are particularly useful as part of an anaerobic membrane reactor. Accordingly, in a third aspect of the present invention, there is provided a method of using an anaerobic membrane bioreactor for treating wastewater, the method comprising:
(A) 0.5 to 10% v / v beads made from the technically perceptible combination of any of the composites and the embodiments according to the first aspect of the invention with membrane bio. Adding to the reactor; and (b) subsequently treating wastewater in said membrane bioreactor under anaerobic conditions.

The composite of the second aspect of the present invention may be particularly suitable for use in an aerobic membrane bioreactor. Accordingly, in a fourth aspect of the present invention, there is provided a method of using an aerobic membrane bioreactor for treating wastewater, the method comprising:
(A) 0.5 to 10% v / v beads made from a technically perceptible combination of the composite material according to the second aspect of the present invention and any of the embodiments thereof in a membrane bioreactor. And (b) subsequently treating wastewater in said membrane bioreactor under aerobic conditions.

  In embodiments of the third and fourth aspects of the invention, the beads may be provided in a 1% to 5% v / v amount, and / or the method may include a backwash step as part of step (b). May be included.

In a fifth aspect of the present invention, there is provided a method of manufacturing a composite material according to any technically perceptible combination of the second aspect of the present invention, and embodiments thereof, the method comprising:
(I) providing a first mixture comprising a bacterial population, an uncrosslinked polymeric material, and a biocompatible adsorbent material in a solvent;
(Ii) adding the first mixture to a second mixture comprising a crosslinking agent for the uncrosslinked polymeric material in a solvent to form a composite material;
The bacterial population consists essentially of one or more bacterial species selected from Microbacterium sp. QQ1 (KR058488), Enterobacter cloacae QQ13 (KR058854), Pseudomonas sp. QQ3 (KR058464) and Rhodococcus sp. BH4.

  In an embodiment of the fifth aspect of the present invention, the bacterial population may further comprise E. coli ΔlsrRΔluxS, and / or the method may comprise polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, The method may further include coating the composite of step (ii) with a coating material selected from one or more of the group consisting of polyacrylonitrile, polyethylene and polypropylene.

In a sixth aspect of the present invention, there is provided a method of manufacturing a composite material according to the technically perceptible combination of any of the first aspect of the present invention, and embodiments thereof, the method comprising:
(I) providing a first mixture comprising a bacterial population in a solvent, an uncrosslinked polymeric material, and an optional biocompatible adsorbent material;
(Ii) adding the first mixture to a second mixture comprising a crosslinking agent for the uncrosslinked polymeric material in a solvent to form a composite material; and (iii) polyvinyl chloride, polyvinylidene fluoride Coating the product of step (ii) with a coating material selected from one or more of the group consisting of: polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene;
The bacterial population consists essentially of Microbacterium sp. QQ1 (KR0848848).

In specific examples of the fifth and sixth aspects:
(Aa) the biocompatible adsorbent, if present, can be selected from one or more of the group consisting of activated carbon, chitosan, zeolite and lignin (eg, the biocompatible adsorbent can be activated carbon);
(Bb) The uncrosslinked polymeric material can be a polyanionic polymer salt when the salt is a monovalent (1+) metal ion (eg, the polyanionic polymer can be alginate or polyanionic cellulose, and And / or the monovalent metal ion can be selected from the group consisting of Na ⁺ and K ⁺ , for example, the uncrosslinked polymeric matrix material can be potassium alginate or sodium alginate;
(Cc) The crosslinker can be a divalent (2+) or trivalent (3+) metal ion salt (eg, the metal ion is a divalent (2+) metal selected from the group consisting of Ca ²⁺ and Mg ²⁺ Ion));
(Dd) The addition of the first mixture to the second mixture may be a drop-wise addition.

  Certain embodiments of the present disclosure are described more fully below with reference to the accompanying drawings.

FIG. 1 shows the transmembrane pressure (TMP) trend of MBR. The TMP profile of the MBR supplemented various beads. "MBR-C"-control MBR without any beads. “MBR-PAC _V ” —MBR with PAC alginate beads added. "MBR-PAC _QQ "-MBR supplemented with APQ beads. FIG. 2 shows the residual levels of signal molecules in all MBRs detected by LC-MS, (A) shows acyl-homoserine lactone (AHL) concentration in sludge, and (B) shows AHL in biocake. (C) shows the concentration of the autoinducer (AI-2) in the sludge. FIG. 3 shows the measured soluble microbial product (SMP) in the mixture when the MBR was initiated (0d) and completely contaminated (4, 8, or 19 days), (A) , Shows the concentration of hydrophobic and hydrophilic dissolved organic carbon (DOC), and (B) shows the distribution of proteins and polysaccharides in the MBR. The upper graph in FIG. 4 shows a plot of zeta potential (left axis) and conductivity (right axis), and the lower graph shows (A) MBR-C, (B) MBR-PAC _V and (C) MBR-. In PAC _QQ , excess extracellular macromolecules (EPS; left axis), bound EPS (first right axis) and protein-to-carbohydrate ratio (PN / C) (second right axis), respectively. . FIG. 5 shows microscopic images showing the sludge floc size observed in (A) MBR-C, (B) MBR-PAC _V and (C) MBR-PAC _QQ . FIG. 6 shows the average removal efficiency of five pharmaceutically active compounds (PhAC) at the permeate of MBR (bar, left axis) and their content adsorbed on sludge (dots, right axis). Error bars represent the standard deviation of multiple measurements (n = 5 for MBR-PAC _V , n = 6 for MBR-PAC _QQ ). FIG. 7 shows the distribution of how pharmaceutically active compounds (PhACs) in MBR-PAC _V and MBR-PAC _QQ are removed. FIG. 8 shows a Principal Coordinate Analysis (PCoA) plot of weighted UniFrac distances comparing variations in MBR communities. T1 to T12 are sample names. FIG. 9 shows the relative abundance of major bacterial and archaeal phyla (> 1%) in the biocake (BC), sludge and bead communities in the MBR. Each bar represents the mean of duplicate samples, except for "sludge -MBR-PAC _V", which is the result of only one sample. FIG. 10 shows the adsorption isotherms at various beads-to-volume ratios for A) trimethoprim, B) sulfamethoxazole (C) carbamazepine, D) diclofenac, E) triclosan: Q _e = PAC balanced load (mg pollutants / gPAC); _{C e} = equilibrium concentration in water (mg pollutant / L). FIG. 11 shows a schematic MBR configuration. FIG. 12 shows the residual AHL (C / C ₀ ) after anaerobic degradation (4 hours) by the QQ strain. FIG. 13 shows a comparison of TMP profiles in A) MBR1 and B) MBR2. FIG. 14 shows the variation in the EPS component of polysaccharide (PS) and protein (PN) in A) C-MBR and B) QQ1-MBR. Error bars represent one standard deviation of the measurement in triplicate AnMBR. FIG. 15 shows the AHL concentrations in C-MBR and QQ1-MBR when A) started and B) contaminated. Error bars represent one standard deviation of the measurement in triplicate MBR. C4-HL was not initially detected in QQ1-MBR. C8-HL was detected only in one of the triplicate MBRs in the final sample.

(Description)
The present invention relates to composite polymer particles containing a population of bacterial cells linked to a polymeric material. The particles may be coated with a polymer coating material in some cases to provide additional strength and stability to the particles. These composites may be suitable for use in anaerobic and / or aerobic membrane bioreactors treating wastewater.

Thus, disclosed are composite materials suitable for use in anaerobic membrane bioreactors that reduce and / or prevent membrane biofouling:
A crosslinked polymeric matrix material compatible with bacterial growth; and a bacterial population,
The bacterial population is uniformly mixed throughout the polymeric matrix material, and the bacterial population consists essentially of Microbacterium sp. QQ1 (KR0584848);
The uniformly mixed bacterial population and the polymeric matrix material are selected from one or more of the group consisting of polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene. Coated with a coating material. If desired, the composite material can further include a biocompatible adsorbent material that is uniformly mixed throughout the polymeric matrix material.

  It will be appreciated that while the composite material disclosed above may be particularly suitable for use in an anaerobic membrane bioreactor, it is not necessarily limited to this use alone. The term "anaerobic membrane bioreactor" as used herein refers to the use of a membrane bioreactor configured to operate under anaerobic conditions when treating wastewater.

  As used herein, “wastewater” may refer to sewage or contaminated water from an industrial plant. More specifically, "wastewater" may refer to sludge such as waste sludge, secondary sludge, oil sludge, industrial sludge or settled sludge.

Also disclosed is a composite for use in an aerobic membrane bioreactor that reduces and / or prevents membrane biofouling, wherein the composite comprises:
A cross-linked polymeric matrix material compatible with bacterial growth;
A bacterial population; and a biocompatible adsorbent material;
The bacterial population and the adsorbent material are homogeneously mixed throughout a polymeric matrix material, the bacterial population comprising Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464) and Rhodococcus sp. BH4 consists essentially of one or more bacterial species selected from:
Although not necessarily for functioning in an aerobic membrane bioreactor:
The bacterial population may further comprise E. coli ΔlsrRΔluxS; and / or the homogeneously mixed bacterial population and the polymeric matrix material may comprise polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone. , Polyacrylonitrile, polyethylene and polypropylene.

  It will be appreciated that the composite material disclosed immediately above may be particularly suitable for use in an aerobic membrane bioreactor, but is not necessarily limited to this use alone. The term “aerobic membrane bioreactor” as used herein refers to the use of a membrane bioreactor configured to operate under aerobic conditions when treating wastewater.

  In the embodiments of the present specification, the terms "comprising" and "comprises" may be construed as requiring the recited feature, but do not limit the presence of other features. Also, alternatively, the vocabulary “comprising” may pertain to situations in which only the listed components / features are intended to be present (eg, the vocabulary “comprising” may be Consisting of "or" consisting essentially of). It is expressly contemplated that both broader and narrower interpretations are applicable to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms can be replaced by the word "consisting of" or the word "consisting essentially" or their synonyms, and vice versa.

  As used herein, "bacterial population" refers to a population of bacterial cells that form part of the composite material disclosed herein. The cells of the bacterial population may be selected from the bacterial species described above.

The term "one or more bacterial species" means that the composite material used in the aerobic reactor is simply a composite of Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. It will be appreciated that one can also have one of the selected bacteriomas, which means that one can have two, three or all four of these species in any suitable combination. Also, these combinations can be supplemented by E. coli ΔlsrRΔluxS. For example, the bacterial population of the composite material used in the aerobic membrane bioreactor can be selected from:
(A) Microbacterium sp. QQ1 (KR058848);
(B) Enterobacter cloacae QQ13 (KR058854);
(C) Pseudomonas species QQ3 (KR058464);
(D) Rhodococcus sp. BH4;
(E) Microbacterium sp. QQ1 (KR058484) and Enterobacter cloacae QQ13 (KR058854);
(F) Microbacterium sp. QQ1 (KR058484) and Pseudomonas sp. QQ3 (KR058464);
(G) Microbacterium sp. QQ1 (KR058848) and Rhodococcus sp. BH4;
(H) Microbacterium sp. QQ1 (KR058848) and E. coli ΔlsrRΔluxS;
(I) Enterobacter cloacae QQ13 (KR058544) and Pseudomonas sp. QQ3 (KR084646);
(J) Enterobacter cloaca QQ13 (KR058544) and Rhodococcus sp. BH4;
(K) Enterobacter cloaca QQ13 (KR058544) and E. coli ΔlsrRΔluxS;
(L) Pseudomonas sp.QQ3 (KR058464) and Rhodococcus sp. BH4;
(M) Pseudomonas sp. QQ3 (KR058464) and E. coli ΔlsrRΔluxS;
(N) Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS;
(O) Microbacterium sp. QQ1 (KR058488), Enterobacter cloacae QQ13 (KR058544) and Pseudomonas sp. QQ3 (KR05846);
(P) Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR085854) and Rhodococcus sp. BH4;
(Q) Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR085854) and E. coli ΔlsrRΔluxS;
(R) Microbacterium sp. QQ1 (KR058484), Pseudomonas sp. QQ3 (KR058464) and Rhodococcus sp. BH4;
(S) Microbacterium sp. QQ1 (KR058488), Pseudomonas sp. QQ3 (KR058464) and E. coli ΔlsrRΔluxS;
(T) Microbacterium sp. QQ1 (KR058848), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS;
(U) Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR084646) and Rhodococcus sp. BH4;
(V) Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464) and E. coli ΔlsrRΔluxS;
(W) Enterobacter cloacae QQ13 (KR058854), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS;
(X) Pseudomonas sp. QQ3 (KR084646), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS;
(Y) Microbacterium sp. QQ1 (KR058488), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464) and Rhodococcus sp. BH4;
(Z) Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464) and E. coli ΔlsrRΔluxS;
(Aa) Microbacterium sp. QQ1 (KR0584848), Enterobacter cloacae QQ13 (KR058854), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS;
(Bb) Microbacterium sp. QQ1 (KR058484), Pseudomonas sp. QQ3 (KR058464), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS;
(Cc) Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS; KR084646), Rhodococcus sp. BH4 and E. coli ΔlsrRΔluxS.

  In certain embodiments of the invention which may be mentioned herein, the combination of bacterial species may be selected from the group of (e) to (dd). For example, a combination of bacterial species may be selected from the group of (o)-(dd), for example, (y)-(dd). In certain embodiments of the invention that may be mentioned herein, the combination of bacterial species may be selected from the group (y) or (dd).

  Without wishing to be bound by theory, it is believed that the bacteria that form part of the composite material are generally well connected or adhered to the polymeric matrix. That is, it is believed that because the bacterial cells are retained by the polymeric matrix, they cannot move freely, even if they are on the surface of the composite material.

  As used herein, the term "consists more substantially" means that the substance so identified is substantially made up of only the listed component materials, but may contain trace amounts of impurities. It is intended to mean. For example, the level of the impurities can be 0.00001 wt% to 5 wt%, such as 0.001 wt% to 1 wt%, such as 0.0001 wt% to 0.5 wt%. More particularly, "consisting essentially of Microbacterium sp. QQ1 (KR0848848)" or "Microbacterium sp. The term "consisting essentially of one or more selected bacteria", unless otherwise specified, means that the bacterial population is formed from the specified species of bacteria and that trace amounts of other bacteria are present (eg, due to contamination). Means to get.

  As will be appreciated, the coating material is applied (in certain embodiments and embodiments) to the surface of the composite material disclosed herein, and the composite material may generally be provided in particulate form. The particle morphology of the composite material can have any suitable shape and size. For example, the composite may be provided as spherical beads having a diameter of 2-3 mm.

  As used herein, the term "biocompatible material" refers to a material that does not kill the bacterial cells that form the bacterial population referred to herein. The term "sorbent material" as used herein refers to a material that absorbs another material on its accessible surface, which is accessible through pores or cracks or the like in the sorbent material. It may include a portion of the interior of the material. Suitable biocompatible adsorbent materials that may be mentioned herein include, but are not limited to, activated carbon, ion exchange resins, chitosan, zeolites and lignin. A particular biocompatible adsorbent material that may be mentioned herein is activated carbon.

As used herein, the term "crosslinked polymeric matrix material compatible with bacterial growth" is non-toxic to bacterial cells and at least completely inhibits the growth of bacterial cellular material within the polymeric matrix. Refers to a polymeric material that does not. Suitable polymeric matrix materials include, but are not limited to, polyanionic polymers cross-linked by divalent (2+) or trivalent (3+) metal ions. Suitable polyanionic polymeric materials include, but are not limited to, alginate or polyanionic cellulose. Suitable divalent (2+) or trivalent (3+) metal ions include, but are not limited to, Fe ²⁺ , Fe ³⁺ , or more specifically, Ca ²⁺ and Mg ²⁺ . In certain embodiments of the invention that may be mentioned herein, the crosslinked polymeric matrix material may be calcium alginate.

  The composite materials disclosed herein may use any suitable ratio of bacterial population (ie, dry weight of bacterial cells) to crosslinked polymeric matrix material. Suitable ratios include, but are not limited to, 1: 500 to 1: 2,000 w / w, for example, 1: 750 to 1: 1,500 w / w, for example, about 1: 1,000 w / w. Bacterial cell: a polymeric material. In further or alternative embodiments, the composite material disclosed herein comprises a bacterial population (ie, the dry weight of bacterial cells in the bacterial population) versus any suitable ratio of biocompatible adsorbent material. Can also be used. Suitable ratios include, but are not limited to, 1: 300 to 1: 800 w / w, eg, 1: 400 to 1: 600 w / w, eg, about 1: 500 w / w strain cell: adsorbent material. Is included.

  As will be appreciated, especially when placed in a membrane bioreactor, the composite requires at least some mechanical strength to retain the bond. In embodiments where the composite is not coated, the composite may be between 40,000 and 100,000 Pa, for example, 45,000 to 70,000 Pa, for example, 48,000 to 100,000 Pa, for example, 48,000 to 70,000 Pa. Young's modulus value. In embodiments where the composite material is coated with a coating material, the coated composite material has a Young's modulus of 200,000 to 400,000 Pa, for example, 250,000 to 350,000 Pa, for example, 300,000 to 310,000. May have a value. As shown, the coated composite may have an increase in mechanical strength as measured by Young's modulus, as compared to the uncoated material.

  Without wishing to be bound by theory, the coating materials described herein can increase the mechanical strength of the composite, but when placed in a membrane bioreactor environment, the coating material prevents chemical degradation of the composite Therefore, it is believed that the primary benefit of coating materials is that they provide increased chemical stability to the composite.

  The materials can provide unique and surprising properties when used in a membrane bioreactor to treat wastewater. These properties are first-in-class for operating under anaerobic conditions, and, surprisingly, are more durable and more effective than traditional systems used previously. And may be more efficient. In part, these properties may result from the ability of these composites to prevent membrane biofouling in bioreactors for extended periods of time, such as within six months.

Thus, there is also provided a method of using an anaerobic membrane bioreactor to treat wastewater, the method comprising:
(A) adding to the membrane bioreactor 0.5 to 10% v / v of beads made from said composite material, wherein the bacterial population consists substantially of Microbacterium sp. QQ1 (KR058848); and b) subsequently treating wastewater in said membrane bioreactor under anaerobic conditions.

  It will be appreciated that any suitable composite material disclosed herein in which the bacterial population consists essentially of Microbacterium sp. QQ1 (KR0848848) may be used in this method.

In an alternative embodiment, the method can involve a method using an aerobic membrane bioreactor to treat wastewater, the method comprising:
(A) the bacterial population is from one or more bacterial species selected from Microbacterium spp. Adding substantially 0.5 to 10% v / v of the beads made from the composite material to the membrane bioreactor; and (b) subsequently in the membrane bioreactor under aerobic conditions Including the step of treating wastewater.

  The present invention consists essentially of one or more bacterial species selected from Microbacterium sp. QQ1 (KR058488), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. It will be appreciated that any suitable composite material disclosed in the specification can be used in this method.

  The methods of wastewater treatment described above may use any suitable amount of composite material in bead form (the beads may take any suitable form as mentioned above, for example, spherical). A suitable amount of beads can be 0.5% to 10% v / v, for example 1% to 5% v / v, based on the volume of the membrane bioreactor.

  The wastewater treatment methods disclosed above can also benefit from the backwash step of step (b) of these processes. As used herein, the term "backwash" refers to "membrane backwash", in which permeate water (ie, purified water) is pumped back to the membrane and thus the pores of the membrane Through the feed channel, thereby removing internal and external contaminants.

  Further details of the above methods of use are provided in the experimental section below.

To prepare the above-mentioned composite material suitable for use in an anaerobic membrane bioreactor, the following method can be applied, which comprises:
(I) providing a first mixture comprising a bacterial population, an uncrosslinked polymeric material, and an optional biocompatible adsorbent material in a solvent;
(Ii) adding the first mixture to a second mixture comprising a crosslinking agent for the uncrosslinked polymeric material in a solvent to form a composite material; and (iii) polyvinyl chloride, polyvinyl fluoride. Coating the product of step (ii) with a coating material selected from one or more of the group consisting of vinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene, wherein the bacterial population Consists essentially of Microbacterium sp. QQ1 (KR058848).

To produce the above-mentioned composite material suitable for use in an aerobic membrane bioreactor, the following method can be applied, which comprises:
(I) Microbacterium sp. QQ1 (KR058488), Enterobacter cloacae QQ13 (KR058854), Pseudomonas sp. QQ3 (KR058464) and Rhodococcus sp. Providing a first mixture comprising a bacterial population substantially consisting of one or more bacterial species selected from a biocompatible adsorbent material; and (ii) crosslinking for uncrosslinked polymeric material in a solvent. Adding the first mixture to the second mixture comprising the agent to form a composite, and, optionally,
(Iii) coating the composite material with a coating material selected from one or more of the group consisting of polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene.

  As will be appreciated, certain components (eg, biocompatible adsorbents in composites suitable for use in anaerobic membrane bioreactors) and / or processes (eg, aerobic membrane bioreactors) The step of coating the composite material suitable for use) may be optional in one or the other of the methods, but the manufacturing methods are interrelated and share the same general process.

  In a first step of the process, a first mixture is provided containing an uncrosslinked polymeric material and an associated bacterial population, if present, in combination with a biocompatible adsorbent material in a solvent. .

  The bacterial population used herein can be provided in any suitable amount to a solution containing uncrosslinked polymeric material and biocompatible adsorbent material, if the biocompatible adsorbent material is present, As a result, it provides the desired dry weight of bacterial cells versus polymeric material as described herein above. For example, 5 mL of a 0.5 OD solution of bacteria (or 2.5 mL of 1 OD (optical density)) is added to 45 mL (or 47.5 mL) of a solution containing an uncrosslinked polymer material, and the resulting composite material is added. A suitable amount of bacterial cells therein may be provided. As will be appreciated, the bacteria can be combined with uncrosslinked polymeric material (and, if present, biocompatible adsorption) in a suitable solution, such as a biocompatible aqueous buffer (eg, a phosphate buffer). Material).

The solution containing the uncrosslinked polymeric material may use any suitable solvent, such as water. Since the uncrosslinked polymeric material must be in dissolved form, the uncrosslinked polymeric material will be soluble in the selected solvent. Conveniently, when the salt counterion is a monovalent (1+) metal ion, the uncrosslinked polymeric material can be a (eg, water-soluble) polyanionic polymeric salt. For example, any suitable polyanionic polymer from the group including, but not limited to, alkinates or polyanionic cellulose may be used. In addition, any suitable monovalent (1+) counterion can be used, which can include Na ⁺ and K ⁺ . Certain uncrosslinked polymeric matrix materials that may be mentioned herein for use in these manufacturing methods include potassium alginate and sodium alginate. The uncrosslinked polymeric matrix material may be present in the solution at a concentration of 0.5-10% w / v, for example 1-2% w / v, prior to the addition of the bacterial population.

  As noted above, the method may include the incorporation of a biocompatible absorbent material as defined herein above. Any suitable biocompatible sorbent material can be incorporated into the above-described manufacturing method. For example, when present, the biocompatible adsorbent material can include, but is not limited to, chitosan, zeolites, lignin, and more particularly, activated carbon. When present, the biocompatible adsorbent material is an uncrosslinked high cross-linked material in an amount of 0.1 to 3% w / v, for example 0.5 to 2% w / v, before addition of the bacterial population. It can be in a solution containing the molecular material.

The first mixture described above is then added to the second mixture in step (ii) of the process. The second mixture contains a cross-linking agent that cross-links with the uncross-linked polymeric material in the first mixture to form a cross-linked polymeric matrix and a composite of bacterial cells entrapped therein. The crosslinker can be a divalent (2+) and / or trivalent (3+) metal ion salt. The divalent (2+) and / or trivalent (3+) metal ions displace monovalent (1+) metal ions from more than one polymer chain in the first mixture, leading to crosslinking of the polymer chains. . Any suitable divalent (2+) and / or trivalent (3+) metal ions may be used in these methods. Suitable divalent (2+) and / or trivalent (3+) metal ions that may be mentioned herein include, but are not limited to, Ca ²⁺ and Mg ²⁺ . The crosslinker may have a concentration in the solvent of the second mixture of 1-10% w / v, such as 2-5% w / v, such as 4% w / v.

  Any suitable method of adding the first mixture to the second mixture may be used. However, the use of a drop addition process can be convenient. This process can result in the formation of substantially spherical particles of a crosslinked polymeric material that incorporate the bacterial cells within the polymeric matrix.

  As will be appreciated, the actual shape of the composite and the size of the beads can be influenced by the rate of flow rate of the first mixture into the second mixture, and the shape and size of the nozzle. For example, a slow flow rate that produces large droplets (eg, using drop addition) results in relatively large spherical beads of the composite, while a fast flow rate (which still produces droplets) results in a relatively large Will produce smaller beads. Similarly, if a continuous stream of the first mixture is added to the second mixture, then a tube / string-like composite material is created, provided that the first material is introduced into the second material. The flow rate is sufficiently controlled to allow crosslinking to occur before the first material begins to disperse. As described later in the specification in the Examples, an appropriate flow rate for obtaining the spherical beads of the composite material in Step (ii) in the above-mentioned production method is 1.0 mL / min using a nozzle having a diameter of 1 mm. possible. One of skill in the art can apply these variables as appropriate, depending on the size and / or shape of the composite material being obtained.

  As will be appreciated, the reaction product of the addition of the first mixture to the second mixture is a crosslinked polymeric composite that is not soluble in the solvent used. Thus, the composite material of step (ii) in the production method is easily separated from the liquid and then-either storage and / or use in the method of treating wastewater disclosed above, or any of the desired coating steps Can be washed and dried.

  Where necessary for a composite suitable for use in an anaerobic membrane bioreactor, or as required for a composite suitable for use in an aerobic membrane bioreactor, obtained in step (ii) of the above process. The crosslinked polymer composite can be further subjected to a coating step with a coating material. The process comprises subjecting the formed composite of step (ii) in the manufacturing step to a suitable concentration and a suitable amount of time (eg, from about 1 second to about 60 seconds, eg, from about 5 seconds to about 30 seconds, eg, For about 10 seconds). As mentioned above, the coating material is-any suitable polymeric material that can help to increase the chemical or mechanical stability of the resulting composite, especially with respect to the conditions found in the membrane bioreactor. Can be Suitable coating materials that may be mentioned include, but are not limited to, polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene. The concentration of the coating material in the solvent may range from 1% w / v to 50% w / v, for example, 2% w / v to 25% w / v, for example, 4% w / v to 8% in a suitable solvent. w / v. Any solvent capable of dissolving the coating material but not dissolving the composite material in step (ii) of the above manufacturing process may be used. A suitable solvent for this purpose that may be mentioned herein is N-methyl-2-pyrrolidone, while other solvents include the polymer used as the coating material and the polymer matrix material of step (ii). Equally suitable, depending on the polymer to be formed.

  As will be appreciated, the coated composite is not soluble in the solvent used to apply the coating process, and thus is placed in water to recover the stored composite and thereby recover the surface of the composite. It is easy to provide a coating of the coating materials described above. Without wishing to be bound by theory, it is believed that the above-described coating process is obtained by precipitation of the coating material from its selected solvent with water. That is, when the composite of step (ii) is added to a solution of the coating material, residual water in the polymer matrix of the composite leads to some precipitation of the coating material on the surface of the composite. Subsequently, if the coated material is removed from the solution containing the coating material, it is placed in an aqueous environment prior to use and / or storage. The composite material removed from the solution containing the coating material has a layer of coating material deposited on the surface of the composite material of step (ii), and it has a layer of coating material solution on top of this layer. Including. When this layer of the coating material solution comes into contact with water, the polymer contained therein also deposits to complete the coating film.

  An alternative process for providing composite particles coated according to the present invention is described in Kim et al., Journal of Membrane Science 473 (2015) 109-117, which is hereby incorporated by reference in its entirety. (See page 2.3), section 2.3 (page 110).

  The coating layer, when present, can have a thickness of 1 to 100 μm, for example 10 to 100 μm, for example 60 to 100 μm. The coating layer may contain pores that allow for the transport of material from the outside to the crosslinked polymeric matrix containing the bacteria. The pores can have a pore size between 50 nm and 1 μm.

  Further non-limiting examples of the present invention will be described with reference to the following examples.

Materials and Methods Chemicals and QQ strains used for aerobic MBR Unlabeled SMX, CBZ, DCF and TCS were purchased from WAKO (Singapore), while TrMP was purchased from MP Biomedical (Singapore). Deuterium labeled _{TMP-d 3, SMX-d} 4, standard _{CBZ-d 10, DCF-d} 4 and _{TCS-d 3} was purchased from C / D / N Isotopes Inc. (Quebec, Canada).

  AI-2 degrading strain Escherichia coli ΔlsrRΔluxS [Cell Rep. 10 (2015) 1861-1871] and four AHL degrading strains isolated from activated sludge: Enterobacter cloacae (QQ13), Microbacterium species (QQ1) and Pseudomonas Species (QQ3) (16S rRNA gene accession numbers: KR085854, KR0584848 and KR058466, respectively), and Rhodococcus sp. BH4 [J. Membr. Sci. 473 (2015) 109-117; J. Membr. Sci. 483 (2015) 75 -83] from the QQ consortium. AHL-degraded strains are grown in Luria-Bertani (LB) medium at 30 ° C. for about 16 hours prior to immobilization in beads, while AI-2 degraded strains are grown at 37 ° C. for about 16 hours at 100 mg / L The cells were grown in LB medium containing streptomycin.

（式中、Ｒは透過抵抗（hydraulic resistance）（１／ｍ）、ΔＰはＴＭＰ上昇（Ｐａ）、Ｊは操作流れ（operational flux）（ｍ^３／ｍ^２／ｓ）、μは透過動粘度（dynamic viscosity）（Ｐａ・ｓ）である）に基づいて[Water Res. 30 (1996) 1771-1780]に記載された単純なレジスタンスインシリーズモデル（resistance in series model）を用いて評価した。ＭＢＲにおいて、総透過抵抗Ｒ_ｔは、ケーキ層抵抗（Ｒ_ｃ）、細孔閉塞抵抗（Ｒ_ｐ）および固有膜抵抗（Ｒ_ｍ）の合計である。 Membrane resistance analysis-Membrane resistance is Darcy's law:

(Where R is the hydraulic resistance (1 / m), ΔP is the TMP rise (Pa), J is the operational flux (m ³ / m ² / s), μ is the permeation kinematic viscosity ( dynamic viscosity) (Pa · s)) and evaluated using a simple resistance in series model described in [Water Res. 30 (1996) 1771-1780]. In MBR, the total permeation resistance _{R t} is the cake layer resistance _(R c), the sum of pore blockage resistance _{(R p)} and the intrinsic membrane resistance _{(R m).}

R _m is measured by allowing clean water to filter through a fresh or chemically purified membrane, while R _t is the flow across the membrane and the TMP when Milli-Q H ₂ O is filtered. Was measured at the end of the experiment by measuring. Then, the cake layer was removed tenderness sponge brush, film is submerged in Milli-Q _{H 2 O,} it was measured _R m + R _p. R _c is calculated by dividing the _R m + _{R p} from _{R t.}

  PhAC and AHL analysis-PhAC in aqueous samples from adsorption tests were analyzed directly without pretreatment. PhACs in aqueous samples from MBR experiments were determined using a 6-mL 30 μm Oasis® HLB solid phase extraction (SPE) cartridge (Water described previously in [Chem. Eng. J. 321 (2017) 335-345]). , Singapore). The amount of PhAC adsorbed on the sludge or beads was extracted from a 1 mL mixture sample or 10 randomly selected beads using liquid-liquid extraction. Beads were ground with a pestle before extraction. After pretreatment, PhAC was analyzed by liquid chromatography mass spectrometry (LC / MS / MS). AHL was extracted from biocake from MBR and activated sludge (50 mL) by the method described by Waheed et al. [Int. Biodeterior. Biodegrad. 113 (2016) 66-73]. The isolated AHL and AI-2 were analyzed by LC / MS / MS.

PhAC and AHL analysis-detailed method for LC / MS / MS Agilent 6460 (USA) QQQ triple quadrupole mass spectrometer (MS) equipped with AJS electrospray interface for analysis of PhAC, AHL and AI-2 Using. The instrument settings were optimized for all measurements as follows: interface voltage, 3.5 kV; dry gas temperature, 325 ° C .; dry gas (N ₂ ) flow rate, 8 L / min; sheath gas temperature, 300 ° C .; N ₂₎ flow rate, 11L / min. Agilent 1290 Infinity II HPLC was used for chromatographic separation. For both PhAC and AHL, a 100 × 2.1 mm Kinetex 2.6 μm biphenyl column (Phenomenex, USA) was used with 0.1% formic acid (A) as mobile phase and methanol with 0.1% formic acid (B). Gradient from 10% (5% per AHL) to 85% B within 10 minutes. For AI-2, a 2.1 × 100 mm Poroshell 120 2.7 μm SB-Aq column (Agilent, USA) was used as mobile phase with 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). With a gradient from 35% to 85% B within 5.5 minutes.

Chemicals and QQ strains used for anaerobic MBR N-butyryl-, N-hexanoyl-, N-heptanoyl-, N-octanoyl-, N-decanoyl-, N-dodecanoyl- and N-tetradecanoyl-DL-homoserine lactone (C4-, C6-, C7-, C8-, C10-, C12- or C14-HL); N- (3-oxohexanoyl)-, N- (3-oxooctanoyl)-, N- (3 AHL containing -oxododecanoyl)-and N- (3-oxotetradecanoyl) -L-homoserine lactone (3OC6-, 3OC8-, 3OC12-, 3OC14-HL) were purchased from Sigma-Aldrich (Singapore).

  Four QQ strains isolated from activated sludge, Microbacterium sp. QQ1, Pseudomonas plecogloschikida QQ2, Pseudomonas sp. QQ3 and Enterobacter cloacae QQ13 were used in this test [Chemosphere 182 (2017) 40-47]. They were kept on LB agar plates at 4 ° C. and freshly implanted every 30 days. Prior to use, single colonies of these strains were inoculated into 5 mL LB medium and cultured at 30 ° C. to refresh them.

Example 1 Optimization of Beads for PAC Bead Composition and Volume Ratio in the Absence of QQ Bacteria Batch-Scale Experiments of Five Independent Sets of Different Bead Compositions and Bead Dose (Bead to Reactor Volume Ratio) in Table 1 Was performed to optimize the adsorption of the pharmaceutically active compound (PhAC).

Methods PAC alginate beads were prepared by combining PAC (SAE2, Norit, The Netherlands) and Na alginate solution (2% w / v) at different ratios (1% w / v or 2% w / v, see Table 1). Mix and prepare by dripping into CaCl ₂ solution (4% w / v) via nozzle at a rate of 1 mL / min using a peristaltic pump; after soaking in CaCl ₂ solution for 3 hours, half the beads After immersion in a polysulfone solution (8% w / v in N-methyl-2-pyrrolidone) for 10 seconds, it was stored at 4 ° C. until use while the remaining beads were directly Milli-Q H ₂ O Moved to In addition, beads containing no PAC were prepared as a control.

  Five pharmaceutically active compounds were used collectively as target adsorbates. For each type of beads, five specific amounts of beads were added to a 150 mL serum bottle containing 50 mL of PhAC solution to provide five different volume loadings (1, 2, 5, 10, and 20%) (Table 1). ). The bottle was kept on a shaker at 150 rpm, 25 ° C. for 7 days. Samples were taken from each bottle at predetermined intervals and the time required to reach equilibrium was assessed.

Results The results shown in FIG. 10 indicate that the same adsorption of PhAC was achieved regardless of increasing PAC dose from 1% w / v (set 1) to 2% w / v (set 2); Increasing the bead volume ratio of / v significantly increased the adsorption of PhAC, but showed that no further increase was observed above 5% w / v. Therefore, a PAC dose of 1% w / v and a bead dose of 5% w / v were chosen for subsequent examples.

  FIG. 10 shows that uncoated beads show higher adsorption than coated beads, but uncoated beads were applied to membrane bioreactors, especially anaerobic systems, because they separated faster. In some cases, it shows poor stability. Thereby, the coated beads were chosen for the subsequent examples.

  The aqueous concentration of PhAC was monitored at regular intervals for 7 days to determine the time to reach adsorption equilibrium. The adsorption of the five PhACs increased over time, but within one day the main adsorption (> 95%) was completed (data not shown), and the high driving force of adsorption, namely PhAC and bio-yield It showed strong electrostatic attraction between the binding sites. A rapid decrease in the aqueous concentration of SMX was observed after 2 days, which was due to abiotic degradation. Thus, an equilibration time of one day was chosen for adsorption isotherm modeling.

Among the adsorption isotherm models, the Freundlich model has been widely applied in heterogeneous systems, especially for organic compounds or highly interacting species on activated carbon and molecular sieves (Foo and Hameed, 2010). Table 2 shows that when k is a constant indicating the relative adsorption capacity of the adsorbent and n is a constant indicating the intensity of adsorption (Hamdaoui and Naffrechoux, 2007), the equilibrium data was estimated by Freundlich model and regression estimation. Shows the results of fitting to a constant value. R ² (> 0.9) values for all PhACs in both sets 1 and 2 indicate a good fit of the Freundlich model. It is noted that PhACs exhibit varying degrees of adsorption strength (n values in Table 2 for PACs), which correspond well to their hydrophobicity. For further testing, set 1 was considered more favorable than set 2 due to the same removal efficiency of pollutants at lower amounts of PAC.

  It is noted that the addition of PAC to the alginate beads slightly increased their mechanical strength (see Table 3), but no clear change in size and shape was observed with the addition of the QQ strain.

  The Young's modulus of the beads was determined on an MTS Criterion model 43 mechanical tester as described by Ouwerx et al. 1998 (Polym. Gels Netw. 6, 393-408). For each type of bead, 10 randomly selected beads were tested in a 250 N compression cell; the piston moved continuously at 2 mm / min and stopped when 50% deformation was reached.

Example 2: Preparation procedure of QQ-PAC beads QQ-PAC beads for biofouling control and removal of organic contaminants (TrOC) in MBR were prepared by the following procedure.
1. QQ bacteria were separated from the activated sludge and stored at -80C. Prior to bead preparation, QQ bacteria were revived in Luria-Bertani (LB) medium. 50 μL of the revived culture (OD> 0.5) was then used to inoculate 5 mL of LB medium, and the culture was cultured overnight at 30 ° C. before use.
2.5 mL of the overnight culture (OD 0.5) was collected by centrifugation and resuspended in 5 mL of 1 × phosphate buffer (PBS). 1 × PBS is, 10mM _PO ⁴ 3-, had a final concentration of 137 mM NaCl and 2.7 mM KCl. The bacterial suspension is then added to 45 mL of a 2% (w / v; 45 mL solution) of sodium alginate solution containing 1% (w / v) PAC (SAE2, Norit, The Netherlands) and mixed with stirring. I let it. 1% PAC (w / v) was established as an optimized ratio based on the batch adsorption test detailed in Example 1.
3. While maintaining agitation, the mixture was pumped with a peristaltic pump at a rate of about 1.0 mL / min and added dropwise (1 drop / sec) to a 4% (w / v) calcium chloride solution via a 1 mm nozzle. Thus, spherical beads were formed.
4. The APQ beads were kept in the calcium chloride solution for 2-3 hours to mature the beads. Then, after soaking the beads in 10 seconds polysulfone solution (N- methyl -2- 8% w / v of the pyrrolidone) and transferred them to Milli-Q _{H 2} O. Beads can be stored in H ₂ O within one week prior to application in MBR. The beads can be stored in 4 ° C. H ₂ O for long term storage. These beads will have to be revived via overnight immersion in LB medium.

QQ strains were captured separately in beads and added equally to MBR to avoid heterogeneous mixtures.
The mass ratio of QQ bacteria (dry weight): PAC: alginate is estimated to be 1: 500: 1000.

The volume of culture (V) depends on the OD and final volume of the culture _{(V F).} The V _F, pumped with a peristaltic pump, means the volume of the final mixture to be calculated by the equation _{V = 0.1 * V F * 0.5} / OD.

Example 3 Procedure for Preparing Empty PAC Beads Empty PAC beads were prepared by mixing PAC (SAE2, Norit, Netherlands) and Na alginate solution (2% w / v) at a dose of 1% w / v, and a peristaltic pump. Was prepared by dripping into a CaCl ₂ solution (4% w / v) via a nozzle at a rate of 1 mL / min using. After immersion in CaCl ₂ solution for 3 hours, the beads were immersed in polysulfone solution (8% w / v in N-methyl-2-pyrrolidone) for 10 seconds, and the beads were transferred to Milli-Q H ₂ O and used for 4 hours. Stored at ° C.

Example 4: Effect of PAC and QQ on MBR performance Laboratory scale MBR configuration and operation Hollow fiber ultrafiltration PVDF membrane module with 0.04 μm pore size and 0.047 m ² (immersion) surface area (ZeeWeed, A cylindrical acrylic acid reactor (3.2 L working volume) equipped with a conventional MBR (MBR-C), MBR containing empty PAC beads prepared according to Example 3 (MBR-PAC _V ) equipped with GE (USA, GE) was run. MBRs containing QQ-PAC beads prepared according to Example 2 (MBR-PAC _QQ ) were sequentially operated (FIG. 11). For the latter two modes with beads inside, the membrane was backwashed with effluent in the same stream at the point of filtration for 5 minutes every 2 hours. Each operating cycle ended when the TMP value reached 30 kPa. The membrane is then removed from the reactor and immersed in sodium hypochlorite solution (2%) for 1 hour, thorough with tap water prior to the next trial, except for MBR-C when unused membrane is used. I rinsed it. Synthetic wastewater (see Table 4 below) was used as the feed for MBR-C, but five PhACs were supplemented to the feed at a concentration of 2 μg / L for the others.

Sludge (Ulu Pandan Water Reclamation Plant, collected from Singapore) was used as seed sludge after acclimating to synthetic wastewater for one month. The mixed liquid suspended solids (MLSS) concentration was maintained in the typical range of MBR (5.7 ± 0.5 g / l) throughout the study. Solid retention time (SRT) and hydraulic retention time (HRT) is at an initial flow of 15L / ^{m 2} / Time (LMH), respectively, and maintained for 30 days and 6 hours. Beads were added to the MBR at a 5% volume ratio (established based on the adsorption test detailed in Example 1).

  QQ-PAC beads can be added to the MBR at a dose of 1-10% (ratio of the bulk volume of the beads to the effective volume of the reactor) per MBR fouling control. Also, if necessary, higher doses can be used to create fluidized MBR, which will allow efficient removal of TrOCs for very long periods of time.

Results FIG. 1 shows the time course of transmembrane pressure (TMP) in MBR-C, MBR-PAC _V and MBR-PAC _QQ . A fresh membrane was used for MBR-C, which was fouled after 4 days of operation; the overall TMP rise was 3 and 6.5 times faster than MBR-PAC _V and MBR-PAC _QQ , respectively. In MBR-PAC _QQ , TMP gradually increased until reaching 8 kPa on day 16, then increased sharply. This sharp rise was delayed by 450% and 150%, respectively, as compared to MBR-C and MBR-PAC _V. Overall, the average fouling rates (ΔTMP / Δt) for MBR-C, MBR-PAC _V and MBR-PAC _QQ were 0.3, 0.16 and 0.07 kPa / hour, respectively. In an earlier test, the addition of PAC alone resulted in a 25% increase in filtration time [Water Res. 105 (2016) 65-75], but a 100% increase in filtration time was achieved by a combination of PAC and backwash. Achieved in the test. Upon addition of QQ, the filtration time was further increased by 450% in MBR-PAC _QQ . This clearly shows that the combination of QQ bacteria with PAC is useful in preventing membrane biofouling.

To elucidate the effect of the PAC and QQ mechanisms on membrane fouling control, a membrane resistance analysis was performed after each MBR trial (see the section under “Membrane Resistance Analysis” for calculations). It was observed a slight increase in intrinsic membrane resistance (R _m) after any chemical cleaning (Table 5). For MBR-C, the cake resistance (R _c ) contributed significantly to 86% of the total resistance (R _t ), indicating strong cake layer formation on the membrane surface within 4 days of operation. Contribution of R _c for R _t is reduced to 50% in the MBR-PAC _V, which is the combined effect of the surface polishing by air PAC beads and backwashing is to exhibit reducing bacterial adhesion to the film surface . Without wishing to be bound by theory, it is believed that the PAC particles can alter the structure of the cake layer, making it thicker but more permeable and less compact. Even when PAC particles were used at lower doses, they alleviated biofouling by increasing sludge floc strength and reducing pollutant emissions. As disclosed in this example, the PAC particles were entrapped in alginate beads and greatly enhanced the mechanical acidification effect while preventing adsorption of foulants. Addition of QQ bacteria to PAC beads, as compared to the MBR-PAC _V, reduced an additional 50% of relative contribution of _{R c.}

Since control of pore blocking is essential for minimizing biofouling associated with MBR, backwashing was introduced to reduce pore blocking resistance (R _p ) in MBR-PAC _V. Backwashing partially reduces physically reversible pore blockage but does not reduce irreversible pore blockage. Compared to that of MBR-C achieved a significant increase in _{R p} of 17% of the MBR-PAC _V. However, a more long filtration times, the pore clogging irreversible become more significant; its contribution to _{R t} was maximum (32%) in the _{MBR-PAC QQ.} These results indicate that the integration of the QQ mechanism in PAC and backwashing effectively reduced biocake formation and physically reversible pore occlusion, thus greatly extending membrane cycle time. .

Example 5: Quenching of QS Signaling Molecules The role of QQ bacteria was confirmed in this example by monitoring the concentration of two types of signal molecules, AHL and AI-2 using LC-MS / MS.

Methods AHL was extracted from biocake from MBR and activated sludge (50 mL) by the method described by Waheed et al. [Int. Biodeterior. Biodegrad. 113 (2016) 66-73]. AHL and AI-2 were analyzed by LC / MS / MS (see "Materials and Methods" for detailed methods).

Results FIG. 2A shows the concentration of AHL in sludge and biocake of all three MBRs. It is noted that the composition of AHL was quite different in the three reactors. _C 6-HSL is dominant AHL in both MBR-C and _{MBR-PAC QQ} was detected in MBR-PAC _{V. 3OC} 12 -HSL a major constituent of AHL in MBR-PAC _V and _{MBR-PAC QQ} was detected in MBR-C. C _4-HSL, most of the AHL in the MBR-PAC _V, including C 10-HSL and _C 14-HSL was not detected in the other two MBR. In addition, the biocake of each reactor showed a different AHL profile than the supernatant of that reactor. The most widely detected AHL in MBR, C8-HSL, was generally present at very low concentrations in sludge, but was an important AHL in both MBR-C and MBR-PAC _V biocakes . AHL was not detected in the MBR-PAC _{QQ biocake} . The AHL concentration in the sludge was 0.5-1 ng / mg VSS for C6-HSL and 3OC8-HSL in MBR-C, and C4-HSL, C10-HSL, 3OC12-HSL and C14-HSL in MBR-PAC _V. It ranges from 0.25 to 0.5 ng / mg, and 0.2 to 0.4 ng / mg for C6-HSL and 3OC12-HSL in MBR-PAC _QQ . All of these are two orders of magnitude higher than the other detected AHLs. Differences in microbial community between MBRs (as detailed in Example 9) could also result in different AHL-mediated quorum sensing molecules. Despite the separate AHL profiles between the reactors, the total AHL concentration in the sludge of MBR-C and MBR-PAC _V was the same (about 1.5 ng / mg VSS), but it was _higher in MBR-PAC _QQ . Less than half of that (approximately 0.6 ng / mg VSS). The total AHL concentration in bio cake MBR-C and MBR-PAC _V are each a 3900ng / ^{m 2} and 6000 ng / ^{m 2,} they were significantly higher than those found in the _{MBR-PAC QQ} (Figure 2B) . These results suggest that in the biocake, QQ partially reduced AHL in bulk sludge, but greatly depleted AHL, especially C8-HSL.

The effectiveness of the QQ bacteria in reducing the AI-2 signal molecule is shown in FIG. 2C, with MBR-PAC _QQ having the lowest concentration of AI-2 on day 4. However, a subsequent gradual increase in AI-2 levels suggests that the AI-2-reduced strain in the QQ consortium has lost its quenching activity in long-term manipulation.

Example 6: Effect of PAC and QQ on SMP production and its composition The production of soluble microbial products (SMPs) in the MBR process depends on their biofouling and drainage if MBR infiltration is reused for various applications This is a significant concern as it directly contributes to the chemical oxygen demand (COD) of the liquid. In this example, SMP was characterized using LC-OCD-OND.

  SMP analysis-Soluble microbial product in mixed liquor of all MBRs using size exclusion chromatography combined with organic carbon and nitrogen detection (LC-OCD-OND) (DOC-LABOR, Germany) (SMP) was characterized and quantified. Briefly, organic compounds: biopolymers, hydrophilic dissolved organic carbon (DOC), hydrophobic DOC, high molecular weight (HMW) proteins, building blocks, low molecular weight (LMW) neutrals, LMW acids, HMW polysaccharides have been identified and are described in detail in Xiao et al. [Chemosphere. 170 (2017) 233-241]. Software from the manufacturer was used for data collection and processing [S.A. Huber, A. Balz, M. Abert, W. Pronk, Water Res. 45 (2011) 879-885].

  EPS extraction and analysis-Thermal extraction method [X.Y. Li, S.F. Yang, Water Res. 41 (2007) 1022-1030] was used for the extraction of extracellular polymeric substances (EPS). Briefly, 15 mL of sludge was collected from the MBR and centrifuged at 4000 rpm and 4 ° C. for 30 minutes (and unless otherwise noted, these conditions were used in the following centrifugation steps) and the supernatant was dissolved in soluble EPS. Stored as The sludge pellet was resuspended in 0.05% (w / v) NaCl; the samples were then centrifuged (15 min at 4000 rpm at 4 ° C.) and the supernatant was stored as loosely bound (LB) EPS. did. The sludge pellet was resuspended in the same NaCl solution and incubated at 60 ° C. for 1 hour; after centrifugation, the supernatant was stored as tightly bound (TB) EPS.

For protein (PN) analysis, the absorbance at 750 nm was measured using a Lowry method [J. Biol. Chem. 193 (1951) 265-275] using a folin-thiocartophenolic acid reagent, with a spectrophotometer (UV-2600, Shimadzu) Manufacture, Singapore). For the quantification of polysaccharide (PS), the absorbance at 490 nm was measured using the phenol sulfate method [Anal. Chem. 28 (1956) 350-356].
.

  Total protein and polysaccharide concentrations were determined by the modified Lowry method [B. Frolund, R. Palmgren, K. Keiding, P.H. Nielsen, Water Res. 30 (1996) 1749-1758] and the phenol-sulfuric acid method (ibid.). The LMW protein and LMW polysaccharide concentrations were determined by dividing the HMW protein and HMW polysaccharide from the total protein and polysaccharide concentrations tested by spectroscopy, respectively.

Results The change in dissolved organic carbon (DOC) content relative to the initial level when quantifying biopolymers, building blocks, low molecular weight (LMW) acids and neutrals as part of the hydrophilic (HI) DOC content, Each MBR was compared (FIG. 3A).

By the time the membrane was completely fouled, total DOC concentrations in MBR-C, MBR-PAC _V and MBR-PAC _QQ were all increased compared to their initial values. The PAC was able to initially adsorb a certain amount of DOC up to the saturation point of the PAC in MBR-PAC _V and MBR-PAC _QQ , but longer operating time resulted in higher DOC concentration in the final stage. Interestingly, the DOC composition in MBR-PAC _QQ was significantly different from the other two. In MBR-PAC _QQ , DOC consisted mainly of LMW neutrals and hydrophobic (HB) DOCs, which contributed to 76% and 20% of the total DOC content. In contrast, the sum of LMW neutrals and HB DOC was less than 50% in MBR-C and MBR-PAC _V. In addition, building blocks, biopolymers and LMW acids were all significantly reduced in MBR-PAC _QQ , but all of them were increased in the other two MBRs. Reduction of high MW organic compounds (eg, biopolymers and building blocks) in MBR-PAC _QQ may be due to the breakdown of these molecules into low MW organics as a result of prolonged suspension of PAC _QQ beads. did it. These results suggest that, in addition to QS molecules, microbial variability in MBR-PAC _QQ can biodegrade these building blocks, biopolymers and LMW acids. However, final fouling in the MBR-PAC _QQ can be due to (i) a decrease in PAC adsorption capacity over time due to partial obstruction of the pores by microorganisms or macromolecules, and / or (ii) long term operation May be due to a decrease in AI-2 quenching activity (Figure 2C).

In addition, the presence of aromatics in SMP for MBR-PAC _V and MBR-PAC _QQ was significantly reduced by 74 and 36%, respectively, which is attributed to the strong affinity of the aromatics for PAC.

At the end of the run, the total protein and polysaccharide concentrations in MBR-C, MBR-PAC _V and MBR-PAC _QQ were found to be 27, 51 and 5 mg / L, respectively (FIG. 3B). Significantly lower concentrations of these substances in MBR-PAC _QQ indicated a role for the QQ mechanism in membrane biofouling. Polysaccharide concentrations were higher than LMW and HMW proteins in all MBR mixtures except for MBR-PAC _V. These results, together with the DOC composition, indicate that the polysaccharide (biopolymer = polysaccharide + protein (HMW + LMW)) contributes significantly to the cake layer formation, but LMW organics partially or completely enter the pores of the membrane and The result suggests that a hydrologically irreversible membrane biofouling occurs. This is particularly important in _{MBR-PAC QQ,} 2.4 times the longer operating time, compared with the MBR-PAC _V, it increased 1.6 fold in _{R p.}

FIG. 4 (bottom of graphs AC) shows the ratio of protein to carbohydrate of bound EPS (PN / C) and the concentration of bound EPS at different time intervals in all MBRs. Bound EPS increased for both MBR-C and MBR-PAC _V and remained at about 70 mg / g VSS if the membrane was fouled. This ratio also increased in MBR-PAC _QQ within the first 4 days, but then decreased gradually, maintaining about 50 mg / g VSS if the membrane was fouled. It is widely accepted that EPS helps maintain the spatial arrangement of the biofilm matrix on the membrane surface. Without wishing to be bound by theory, it is believed that QQ beads reduce the release of EPS and thus disrupt biofilm layer formation.

Example 7: Change of sludge properties Performance and sludge characterization. Chemical oxygen demand (COD), sludge volume index (SVI), mixed liquid suspended solids (MLSS) and volatile suspended solids (VSS) were analyzed by standard analytical methods [American Public Health Association (APHA), American Water. Works Association (AWWA), Water Environment Federation (WEF), Standard Methods for the Examination of Water and Wastewater, 21st ed., Washington, DC, 2012]. Dissolved oxygen (DO) and intrinsic oxygen uptake rate (SOUR) were analyzed using a multimeter (XL600, Fisher Scientific, USA). Floc size distribution was monitored using a particle size analyzer (SALD-3101, Shimadzu, Singapore). The sludge floc was occasionally imaged using a fluorescence microscope (Nikon-ECLIPSE, Japan). Sludge dewaterability with respect to capillary suction time (CST) was evaluated using a CST device (Ofite 294-50, OFI Testing Equipment Inc., USA). The zeta potential and conductivity (EC) of the mixture were measured by zetasizer (MRK570-01, Malvern, UK).

Intrinsic oxygen uptake rate (SOUR) was frequently used to characterize respiratory activity and to infer the physiological state of cells [Biochem. Eng. J. 49 (2010) 289-307]. The SOUR was the same in MBR-C and MBR-PAC _V , as shown in Table 6, but doubled at the end of the experiment in MBR-PAC _QQ . This indicates that QQ events or perturbations during bacterial signaling may also have a direct effect on the metabolic activity of microbial cells, which may be related to deficiency conditions that appear during interruptions in bacterial communication.

Floc size distribution was monitored throughout the study. As shown in Table 6, the floc size in MBR-C did not change significantly, but increased 59% and 128% in MBR-PAC _V and MBR-PAC _QQ , respectively, until the end of the run. Microscopic images (FIG. 5) clearly show a tendency to increase sludge floc size from MBR-C to MBR-PAC _V and further to MBR-PAC _QQ . Reduction of sludge floc size has been reported in MBR supplemented with QQ [H. Waheed, Y. Xiao, I. Hashmi, D. Stuckey, Y. Zhou, Insights into quorum quenching mechanisms to control membrane biofouling under changing. organic loading rates, Chemosphere. 182 (2017) 40-47], thus such an increase appears to be due to PACs in the QQ beads. The addition of PAC enhances biological stability, sludge settling, and improves the effectiveness of the activated sludge process due to its larger surface area, which acts as a support medium and provides a suitable environment for bacteria. The smaller sludge floc in MBR-PAC _V compared to MBR-PAC _QQ could be due to shorter contact time of PAC with sludge. Larger flocs in MBR-PAC _QQ may contribute to better dehydration, as evidenced by the lowest CST value of all MBRs (Table 6).

The zeta potential of the sludge from MBR-C and MBR-PAC _V remained relatively stable at about -8 and -14 mV, respectively (FIG. 4. Graph top, AC). Since the individual EPS components, such as proteins, humic acids, uronic acids and nucleic acids in EPS, generally carry abundant ionizable functionalities that are more surface charge sources than polysaccharides, the increase in absolute zeta potential is PN / C This may be due to an increase in the ratio. Thereby, changing the composition of the EPS by QQ can also affect the zeta potential of the obtained sludge. Typically, a more negative zeta potential carries more negative charge, which increases repulsive electrostatic interactions and affects the aggregation process. As indicated above, the addition of PAC increased the sludge floc size; thereby, a sudden drop to less than -18 mV in the MBR-PAC _QQ after an initial stabilization of about -12 mV caused the larger floc size to decrease. It did not offset the increased dehydration.

Example 8: MBR performance and micropollutant removal The effect of the addition of PAC or QQ on the COD removal efficiency of selected pharmaceutical compounds in MBR was studied.

Methods Chemical oxygen demand (COD), sludge volume index (SVI), mixed liquid suspended solids (MLSS) and volatile suspended solids (VSS) were analyzed by standard analytical methods [American Public Health Association (APHA), American Water Works Association (AWWA), Water Environment Federation (WEF), Standard Methods for the Examination of Water and Wastewater, 21st ed., Washington, DC, 2012].

  PhACs in aqueous samples from MBR experiments were determined using a 6-mL 30-μm Oasis® HLB solid phase as previously described by Xiao et al. [Chem. Eng. J. 321 (2017) 335-345]. Concentration was performed using an extraction (SPE) cartridge (Waters, Singapore). The amount of PhAC adsorbed on the sludge or beads was extracted from a 1 mL mixture sample or 10 randomly selected beads using liquid-liquid extraction. Beads were ground with a pestle before extraction. After the pretreatment, PhAC was analyzed using a liquid chromatography mass spectrometer (LC / MS / MS).

An Agilent 6460 (USA) QQQ triple quadrupole mass spectrometer (MS) equipped with an AJS electrospray interface was used for PhAC analysis. Instrument settings were optimized for all measurements as follows: interface voltage, 3.5 kV; dry gas temperature, 325 ° C .; dry gas (N ₂ ) flow rate, 8 L / min; sheath gas temperature, 300 ° C .; sheath gas (N ₂ ) Flow rate, 11 L / min. Agilent 1290 Infinity II HPLC was used for chromatographic separation. A 100 × 2.1 mm Kinetex 2.6 μm biphenyl column (Phenomenex, USA) was used with 0.1% formic acid (A) and methanol containing 0.1% formic acid (B) within 10 minutes within 10% (per AHL). 5%) to 85% B.

Results Similar COD removal efficiencies (93.0 ± 2.5, 95 ± 4 and 92.1 ± 1.5%) were achieved for MBR-C, MBR-PAC _V and MBR-PAC _QQ , respectively. Addition of PAC or QQ did not result in any significant changes in COD removal.

FIG. 6 shows the elimination efficiencies of selected pharmaceutical compounds in the MBR, which were trimethoprim (TrMP), sulfamethoxazole (SMX), carbamazepine (CBZ), diclofenac (DCF) and triclosan (TCS). there were. The removal efficiencies of TrMP, SMX, CBZ, DCF and TCS were 71 ± 15, 80 ± 7, 63 ± 21, 82 ± 8 and 67 ± 22% in MBR-PAC _V , respectively; -Slight increase for all PhACs in PAC _QQ . CBZ showed the lowest removal efficiency among all PhACs. However, no statistically significant difference in PhAC removal efficiency between MBR-PAC _V and MBR-PAC _QQ was observed (p> 0.05 for all). PhAC adsorbed on the sludge was analyzed for both MBRs (FIG. 7). Lower concentrations of relatively low concentrations of SMX, DCF and TCS are found in the sludge, and thus these components can be considered highly degradable. Mean TrMP concentrations ranged from 0.18 to 0.91 μg / g VSS, while values for CBZ ranged from 0.88 to 1.28 μg / g VSS for MBR-PAC _QQ . Thus, the removal of TrMP and CBZ may be by adsorption rather than biodegradation. On the other hand, the p-values of the t-test for the TrMP, SMX, CBZ, DCF, TCS adsorbed in the beads were 0.425, 0.119, 0.428, 0.159 and 0.617, respectively. . No statistically significant difference in adsorbed PhAC between MBR-PAC _V and MBR-PAC _QQ was observed (p> 0.05 for all).

The fate of all PhACs was calculated and is shown in FIG. This figure clearly shows that the contribution of biodegradation to the fate of SMX, DCF and TCS was increased with the addition of the QQ strain. This is particularly noticeable with DCF, which was removed in PAC _V via biodegradation (33%) and adsorption (42%) on sludge, whereas in PAC _QQ it was mainly removed via biodegradation (86%). Was done. The consistency of the biodegradability of all PhACs adsorbed on QQ beads indicates the effectiveness of PAC-QQ beads. Furthermore, the amount of pollutants adsorbed on the PAC beads was quantified and shown in Table 7. Regardless of the better removal efficiency achieved in MBR-PAC _QQ permeation and the same concentration of adsorbate in the sludge of both MBRs, the concentration of PhAC adsorbed on the QQ bacterial capture beads is much higher than empty PAC beads. it was high. It is well known that the uptake or adsorption of micropollutants generally depends on the effectiveness of the adsorption site on the adsorbent. Therefore, it can be assumed that the contaminants adsorbed on the PAC are further utilized by the bacterial strain as a carbon source, thus regenerating some of the adsorption sites and increasing the overall removal efficiency.
These observations suggest that simultaneous adsorption and biodegradation may be possible via PAC-QQ beads.

Example 9: Variation in microbial community For a better understanding of the effects of QQ or PAC in MBR, the microbial community was analyzed using Illumina MiSeq high performance sequencing.

RNA extraction and microbial community analysis Duplicate samples of bulk sludge, biocake and beads from MBR were immediately frozen at -80 <0> C when the operation was completed. Genomic DNA and RNA were extracted from frozen samples using the method of Towe et al. [J. Microbiol. Methods. 84 (2011) 406-412]. The mixture was treated with RQ1 RNase-free DNase (Promega, USA) according to the manufacturer's instructions to obtain RNA. cDNA was synthesized from RNA using the GoScript Reverse Transcription System (Promega, USA) in a total volume of 40 μl according to the manufacturer's protocol. The cDNA was used for sequencing on the Illumina Miseq platform at Research Technology Support Facility, Michigan State University, USA. A dual indexed Illumina compatibility library was prepared using the primers described by Takahashi et al. [PLOS ONE. 9 (2014)], which was used to generate V3 of 16S rRNA from a wide range of bacterial and archaeal species. The ~ V4 region was amplified. Following PCR, the reaction products were normalized using an Invitrogen Sequal Prep DNA standardization plate and the recovered DNA was pooled. The final pool was cleaned up using AmpureXP. Library pools were validated and quantified using a combination of the Qubit dsDNA assay, Caliper LabChipGX and Kapa Illumina Library Quantification qPCR kit. Each pool was loaded onto an Illumina MiSeq standard v2 flow cell and sequencing was performed using a v2 500 cycle reagent cartridge in a 2x250bp paired end format.

  Sequencing data was analyzed using the QIIME 1 pipeline [Nat. Methods. 7 (2010) 335-336]. The paired end data were linked using the join_paired_ends script with a min_overlap of 50 bp and a perc_max_diff of 15%, and then QIIME [NA Bokulich, et al., Nat. Methods. 10 (2013) 57 -59] using the default strategy. Sequences were clustered into operational taxonomic units (OTUs) using the open-reference OUT picking protocol [PeerJ. 2 (2014)]. Diversity was analyzed using a core diversity analysis script, and the results were visualized in principal coordinate analysis (PCoA) plots with the Emperor tool [Y. Vazquez-baeza et al., GigaScience. 2 (2013) 16].

The PCoA plot (FIG. 8) shows that there were significant differences in the microbial community between the three MBR biocakes and the sludges of MBR-PAC _V and MBR-PAC _QQ . Proteobacterial phylum members were the most dominant group in all communities (FIG. 9), with OUT counts ranging from 34-55%. What followed this bacterium was Actinobacteria (19-36%) in all sludge or biocake communities. The candidate TM7 member was a dominant group (26-30%) in the MBR-C biocake, but it was barely present in other communities, and cyanobacteria (11-22%) in MBR-PAC _V. Bacteroides (24-25%) were greatly replaced in the biocake of the members of MBR-PAC _QQ . Fluid forces generated by the beads led to differences in microbial community between MBR-C and MBR-PAC _V , as similar effects on microbial communities due to shear forces were also observed in reverse osmosis membranes. Could be a major factor. However, PACs could also contribute to a shift in the microbial community. The difference in microbial community between MBR-PAC _V and MBR-PAC _QQ could be attributed to QQ activity. These differences in microbial community between MBRs have resulted in distinct AHL profiles in MBRs (FIG. 2).

It is worth noting that the microbial community of the MBR-PAC _{QQ biocake} was different from that of the sludge in the same reactor. Although the abundance of Bacteroides was greatly reduced to 3-5% in the sludge, the composition of Proteobacteria and Actinobacterium was 8-50% and 32-36% in the sludge and 39-36% in the biocake, respectively. ４１41% and 19-21%. This difference in microbial community between biocake and bulk sludge in the MBR is widely observed in full-scale MBR, and HRT, F / M (food-to-microorganism ratio) or MLSS fluctuations indicate that biocake formation A selective pressure was created. However, in laboratory tests performed by the inventors and interested parties, the microbial community in the biocake was very similar to that in bulk sludge. Unidentified genera of the Spirosoma and Comamondaceae families are the two predominant varieties in the biocake, accounting for about 22% and about 14%, respectively, of the total OUT count, but of the Methylocysticaceae and Streptomyces families. The two unidentified types were the predominant types in bulk sludge, with abundances of about 17% and about 10%, respectively. Spirosoma members were observed to grow only as fresh river biofilms under defined continuous stages [Microb. Ecol. 50 (2005) 589-601]. Bacteroides are also an important part of the anaerobic MBR biocake under sonication, which increases the production of protein EPS [Z. Yu, X. Wen, M. Xu, X. Huang, Characteristics of extracellular polymeric substances and bacterial communities in an anaerobic membrane bioreactor coupled with online ultrasound equipment, Bioresour. Technol. 117 (2012) 333-340]. Thus, with a major suppression of polysaccharide production in EPS, QQ activity disrupts the microbial community balance in the MBR-PAC _QQ biocake and favors the growth of spirosomes.

8 and 9 also show that very distinct microbial communities were formed in PAC _V and PAC _QQ beads. Proteobacteria were still the dominant phylum in these beads (46-55%), but ureaquiota methanogens displaced most of the actinobacteria and PAC _V and PAC _QQ beads. Formed the second (15-21%) and third (13-15%) most dominant phyla, respectively. Methanosaeta was the sole (> 99.5%) member of the ureakiota in almost both beads. In addition, Firmicutes members increased to 8-13% and 5-7% in PAC _V and PAC _QQ beads, respectively. Both Methanosaeta and Firmicutes are generally known to be present in methanation reactors. These results indicate that oxygen cannot diffuse into the alginate beads, resulting in the formation of depletion conditions inside the beads and methane formation may be the active route of metabolism inside the beads of PAC _V and PAC _QQ. Suggests. Methane produced by methanogens in the MBR may be utilized in the MBR by members of Methylocytis, a family that includes a large number of methane-oxidizing bacteria. The presence of both oxidizing and reducing conditions can contribute to the high biodegradation of PhAC inside both reactors.

Members of the same type of QQ strain, except for the AI-2 degraded E. coli strain, were all present in the PAC _QQ beads. However, their abundance was very low (<0.5% of total OUT count). The presence of these microorganisms does not guarantee the viability and activity of the QQ strain, as these species except Microbacterium were also present in PAC _V beads (<0.5% of the total OUT count). Thus, the microbial community in the beads was mainly affected by the sludge in the reactor. The absence of E. coli in the microbial community of PAC _QQ beads accounts for the increase in AI-2 levels in the MBR-PAC _QQ reactor (FIG. 2C).

Example 10: Anaerobic degradation of AHL by QQ strains The ability of four QQ strains to degrade AHL under anaerobic conditions was examined in a batch test. All four QQ strains tested in this example exhibited high AHL degradation efficiency aerobically. The four QQ strains are Microbacterium sp. QQ1, Pseudomonas plecogloschikida QQ2, Pseudomonas sp. QQ3, and Enterobacter cloacae QQ13 isolated from activated sludge.

A mixture of AHLs containing C4-, C6-, C7-, C8-, C10-, 3OC6-, and 3OC8-HL (each about 100 μg / L) were prepared anaerobically in PBS buffer. 5 ml each of an overnight QQ cultures (5 mL) were washed, resuspended in 1mL of PBS was flushed with _{N 2,} it was added to Hungate tubes containing AHL solution 4 mL. A mixture having a final volume of 5mL was flushed and sealed with N _2. The tubes were then incubated at 35 ° C. for 4 hours at 100 rpm in a rotary shaker. Samples were taken at designated intervals (see below) for AHL analysis by liquid-liquid extraction and LC / MS / MS (see "Materials and Methods" above for detailed methods used herein). ).

Results When the four QQ strains were grown aerobically and transferred to anaerobic conditions, washed cells of the QQ2 and QQ3 strains were unable to degrade AHL and the residual AHL was larger than the control (see FIG. 12). ). In contrast, significant degradation of all AHL was achieved with the washed cells of QQ1 and QQ13 strains, especially the QQ1 strain, degrading AHL (except 3OC8-HL) by more than 20-30% than control. . Both QQ1 and QQ13 decomposed 3OC8-HL almost completely within 4 hours.

A 30 minute lag phase was observed for degradation of C4-HL and 3OC6-HL by strain QQ1. However, no lag phase was observed for other AHLs (data not shown). The propensity of all AHLs to degrade by the QQ1 strain is well compatible with the pseudo-first order reaction (R ² > 0.86), which is quite unlike the zero order kinetics of spontaneous AHL decay in the control (data not shown). different. These results suggest that the QQ enzyme of the QQ1 strain was still active in anaerobic conditions.

Example 11: Anaerobic MBR manipulation and transmembrane pressure (TMP) trends Four QQ strains (Microbacterium sp. QQ1, Pseudomonas plecogloschikida QQ2, Pseudomonas sp. Immobilized on alginate beads and added to triplicate MBR. When the TMP reached 30 kPa, the operation was terminated.

Bacterial immobilization The QQ strain was immobilized in alginate beads and coated with a polymer membrane. Briefly, the QQ strain is harvested from a fresh grown culture by extraction of a 5 mL culture, then centrifuged to provide a bacterial pellet, and then a 5 mL NaCl solution (0.05% w / v NaCl concentration). The mixture was then added (10% v / v) to a sodium alginate solution (2% w / v) and a CaCl ₂ solution (4% w / v) was passed through a nozzle at a rate of 1 mL / min using a peristaltic pump. After dipping in CaCl ₂ solution for 3 hours, beads were dipped in polysulfone solution (8% w / v in N-methyl-2-pyrrolidone) for 10 seconds and then transferred to Milli-Q H ₂ O. And stored at 4 ° C. until use. The beads prepared here are hereinafter referred to as QQ beads.

Empty beads were made following the same procedure as above (dropping Na alginate solution into CaCl ₂ solution) but without the addition of QQ in the NaCl solution.

MBR operation Using the same four laboratory scale immersion AnMBRs (0.5 L effective volume) equipped with a handmade PVDF hollow fiber membrane module (0.1 μm pore size and 0.007 m ² surface area), Synthetic wastewater (COD about 500 mg / L) composed of glucose, peptone, meat extract and other minerals was treated [Chem. Eng. J. 321 (2017) 335-345]. AnMBR was sprinkled in the laboratory on anaerobic digested sludge maintained with the same feed and operated at 35 ° C. for 9 hours with HRT and a constant flow of 8 LMH. For each QQ strain, three MBRs were subsequently run with the addition of empty beads (C-MBR) and QQ beads (QQ-MBR). Transmembrane pressure (TMP) was continuously monitored and recorded with a pressure sensor and a data logger.

Results The triplicate MBR performed very differently than the empty beads therein and stained them for MBR1, MBR2 and MBR3 at 26, 114 and 41 hours, respectively. The handmade membrane modules in their MBR had similar permeation resistance (7.3 ± 0.5 × 10 ¹¹ m ⁻¹ ) and gas sparging rate (0.41 ± 0.04 L / min). Thus, the difference in fouling rate was due to the uneven distribution of gas bubbles in the sparger, which resulted in a difference in effective polishing.

  The addition of QQ2, QQ3 and QQ13 did not show any beneficial effect on biofouling control, and their fouling rates in QQ-MBR were almost the same (QQ2) or faster than C-MBR ( QQ3 and QQ13) (data not shown). In contrast, QQ1 significantly delayed biofouling, with filtration times extended 3.6 and 3.0 fold in MBR1 and MBR2, respectively (FIG. 13). Impairment in the gas porous dispersion tube in MBR3 impaired its performance and increased the filtration time by a factor of 1.6 (data not shown).

  Over 70% removal of CODs containing 60% methane content in biogas was achieved in all QQ1-MBRs, which was the same as C-MBR. Thus, the performance of MBR for COD removal and methane production was not affected by the addition of QQ1.

Example 12: Effect of QQ on EPS production and AHL degradation Analysis AHL in 85 mL of MBR supernatant was extracted twice with 100 mL of ethyl acetate and concentrated in 100 μL of acetonitrile. They were then analyzed using an Agilent 6460 (USA) QQQ triple quadrupole mass spectrometer (MS) equipped with an AJS electrospray interface (Waheed, H. et al., Chemosphere 182, 40-47). .

  EPS in the sludge was extracted and classified into soluble (S), loosely bound (LB) and tightly bound (TB) categories. The polysaccharide and protein content in each part was determined by the phenol sulfate method and the Lowry method. Details have been previously described (Waheed, H. et al., Chemosphere 182, 40-47).

The membrane permeation resistance for the intrinsic membrane (R _m ), pore occlusion (R _p ) and cake layer (R _c ) is determined by Darcy's law by measuring the TMP of filtration of ultrapure water through the membrane at a specific flow rate (Waheed, H. et al., Chemosphere 182, 40-47). COD was measured by a standard analysis method (American Public Health Association, American Water Works Association, Water Environment Federation, 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed). Biogas composition was analyzed using a Shimadzu GC 2010 Plus gas chromatograph (Chem. Eng. J. 321, 335-345).

Results To assess the mechanism of fouling control by QQ1 in AnMBR, the initial (start of operation) and final (end of operation) EPS levels in C-MBR and QQ1-MBR were monitored and plotted in FIG. . Protein was the predominant component in both MBRs, as the ratio of protein to polysaccharide was 5-20 for LB-EPS and TB-EPS, and 6-40 for S-EPS. Polysaccharide levels in LB and TB-EPS in both reactors were reduced compared to their initial levels, but they were increased in S-EPS. This may have resulted from bead polishing and may have dissociated the polysaccharide bound to the aqueous phase into the aqueous phase. In contrast, the final protein level in all three fractions of EPS remained the same as the initial one, except for LB-EPS in QQ1-MBR, which was significantly reduced by 44%. These results suggest that QQ1 greatly suppressed LB-EPS production of proteins in AnMBRs.

 Of the eleven quantifiable AHLs, only C4-, C6-, C8-HL and C10-HL were detected in the AnMBR. At concentrations of 19-43 ng / L, C4-HL had the highest concentration among them. The concentrations of C6-HL and C8-HL ranged from 1 to 20 ng / L, which was similar to that in aerobic MBR. C10-HL was detected at a minimum level of 0.7-0.9 ng / L. When AnMBR was just begun, C4-HL was about 24 ng / L in C-MBR, but not detected in QQ1-MBR (FIG. 15); C6- and C8-HL in QQ1-MBR were Even significantly lower than in the MBR. However, when the AnMBR was fouled, the concentrations of the three AHLs were not significantly different between the two AnMBRs. These results suggest that QQ beads could have their highest activity at the beginning of the run, but activity decreased over time. No significant difference between the C10-HL concentrations in the two AnMBRs was observed at any stage, which contrasted with the results of the batch test (FIG. 12). The polymer coating on the beads might have blocked the diffusion of C10-HL.

It should be noted that QQ activity (biofilm formation) is triggered by the threshold concentration of AHL; thus, it is not proportional to the concentration of AHL. The concentration of C4-HL, C6-HL and C8-HL in QQ1-MBR, when they are started, can be below the threshold concentration, which can lead to a delay in the TMP jump.

Claims (31)

A composite material for use in an anaerobic membrane bioreactor that reduces and / or prevents membrane biofouling,
A crosslinked polymeric matrix material compatible with bacterial growth; and a bacterial population,
The bacterial population is homogeneously mixed throughout the polymeric matrix material, the bacterial population consists essentially of Microbacterium sp. QQ1 (KR0848848); and the homogeneously mixed bacterial population and the polymeric matrix material are: Said composite material coated with a coating material selected from the group consisting of: polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene.   The composite of claim 1, wherein the composite further comprises a biocompatible adsorbent material that is uniformly mixed throughout the polymeric matrix material. A composite material for use in an aerobic membrane bioreactor that reduces and / or prevents membrane biofouling,
A cross-linked polymeric matrix material compatible with bacterial growth;
A bacterial population; and a biocompatible adsorbent material;
The bacterial population and the adsorbent material are homogeneously mixed throughout a polymeric matrix material, the bacterial population comprising Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464) and Such a composite material consisting essentially of one or more bacterial species selected from Rhodococcus species BH4.   The composite of claim 3, wherein the bacterial population further comprises Escherichia coli ΔlsrRΔluxS.   The homogeneously mixed bacterial population and the polymeric matrix material are selected from one or more of the group consisting of polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene. 5. The composite material according to claim 3, which is coated with a coating material.   The composite material according to any one of claims 2 to 5, wherein the biocompatible adsorption material is selected from one or more of the group consisting of activated carbon, ion exchange resin, chitosan, zeolite and lignin.   7. The composite of claim 6, wherein said biocompatible adsorbent is activated carbon.   The composite material according to any one of claims 1 to 7, wherein the crosslinked polymer matrix material is a polyanionic polymer crosslinked by divalent (2+) or trivalent (3+) metal ions. The composite according to claim 8, wherein the polyanionic polymer is alginate or polyanionic cellulose, and / or the metal ion to be crosslinked is a divalent metal ion selected from the group consisting of Ca ²⁺ and Mg ^2+. material.   The composite of claim 9, wherein the crosslinked polymeric matrix material is calcium alginate.   The ratio of the bacterial population (dry weight) to the crosslinked polymeric matrix material is from 1: 500 to 1: 2,000 w / w, for example, 1: 750 to 1: 1,500 w / w, for example, The composite of any one of claims 1 to 10, wherein the composite is about 1: 1,000 w / w.   The ratio of the bacterial population (dry weight) to the biocompatible adsorbent material is from 1: 300 to 1: 800 w / w, for example, from 1: 400 to 1: 600 w / w, for example, about 1: 500 w / w. The composite material according to any one of claims 1 to 11, wherein w is w.   13. A composite according to any one of the preceding claims, provided as spherical beads having a diameter of 2-3 mm.   Claims 3 to 4 and claims having a Young's modulus value of 40,000 to 100,000 Pa, such as 45,000 to 70,000 Pa, such as 48,000 to 100,000 Pa, such as 48,000 to 70,000 Pa. The composite material according to any one of claims 6 to 13, which is dependent on item 3 or 4.   Claims: When coated with a coating material, the coated composite material has a Young's modulus value of 200,000 to 400,000 Pa, such as 250,000 to 350,000 Pa, such as 300,000 to 310,000. 15. The composite material according to any one of 1 to 14. Use of an aerobic membrane bioreactor for treating wastewater, comprising:
(A) adding to the membrane bioreactor 0.5 to 10% v / v of beads made from the composite material according to any one of claims 3 to 15 which is dependent on any of claims 3 to 5; And b) subsequently treating wastewater in the membrane bioreactor under aerobic conditions. A method of using an anaerobic membrane bioreactor to treat wastewater, comprising:
(A) 0.5 to 10% v / v of a bead made from the composite material according to any one of claims 1 to 2 or any one of claims 6 to 15 depending on claim 1 or 2 as a membrane bioreactor. And (b) subsequently treating wastewater with said membrane bioreactor under anaerobic conditions.   18. Use according to claim 16 or 17, wherein the beads are provided in an amount of 1% to 5% v / v.   The method according to any one of claims 16 to 18, further comprising a backwashing step as a part of the step (b). The method for producing a composite material according to any one of claims 3 to 15, which is dependent on any one of claims 3 to 5,
(I) providing a first mixture comprising a bacterial population, an uncrosslinked polymeric material, and a biocompatible adsorbent material in a solvent;
(Ii) adding the first mixture to a second mixture comprising a crosslinking agent for the uncrosslinked polymeric material in a solvent to form a composite material;
The bacterial population consists essentially of one or more bacterial species selected from Microbacterium sp. QQ1 (KR058484), Enterobacter cloacae QQ13 (KR058544), Pseudomonas sp. QQ3 (KR058464) and Rhodococcus sp. BH4. The manufacturing method described above.   The method according to claim 20, wherein the bacterial population further comprises Escherichia coli ΔlsrRΔluxS.   The composite material of step (ii) in claim 20 with a coating material selected from one or more of the group consisting of polyvinyl chloride, polyvinylidene fluoride, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene. 22. The method according to claim 20, further comprising a step of coating. The method for producing a composite material according to any one of claims 6 to 15, wherein the composite material is dependent on claim 1 or 2 or claim 1 or 2.
(I) providing a first mixture comprising a bacterial population, an uncrosslinked polymeric material, and an optional biocompatible adsorbent material in a solvent;
(Ii) adding the first mixture to a second mixture comprising a crosslinking agent for the uncrosslinked polymeric material in a solvent to form a composite; and (iii) polyvinyl chloride, polyfluorinated. Coating the product of step (ii) with a coating material selected from one or more of the group consisting of vinylidene, polytetrafluoroethylene, polysulfone, polyethersulfone, polyacrylonitrile, polyethylene and polypropylene;
The method according to any of the preceding claims, wherein the bacterial population consists essentially of Microbacterium sp. QQ1 (KR084848).   24. The method according to any one of claims 20 to 23, wherein the biocompatible adsorbent, if present, is selected from one or more of the group consisting of activated carbon, chitosan, zeolite and lignin.   The method according to claim 24, wherein the biocompatible adsorption material is activated carbon.   The method according to any one of claims 20 to 25, wherein the uncrosslinked polymer material is a polyanionic polymer salt, and a counter ion of the salt is a monovalent (1+) metal ion. 27. The method according to claim 26, wherein the polyanionic polymer is alginate or polyanionic cellulose and / or the monovalent metal ion is selected from the group consisting of Na ⁺ and K ⁺ .   The method according to claim 27, wherein the uncrosslinked polymer matrix material is potassium alginate or sodium alginate.   The method according to any one of claims 20 to 28, wherein the crosslinking agent is a divalent (2+) or trivalent (3+) metal ion salt. The method according to claim 29, wherein the metal ion is a divalent (2+) metal ion selected from the group consisting of Ca ²⁺ and Mg ²⁺ .   The method according to any one of claims 20 to 30, wherein the addition of the first mixture to the second mixture is dropwise addition.

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