CONTAINER WITH BIOFILM FORMATION-INHIBITING MICROORGANISMS IMMOBILIZED THEREIN AND MEMBRANE WATER TREATMENT APPARATUS USING THE SAME

Abstract

The present disclosure relates to a technique for inhibiting biofouling of the surface of a membrane caused by a biofilm, through immobilizing biofilm formation-inhibiting microorganisms to a container in a membrane water treatment process. The present disclosure provides a non-hollow/hollow columnar or sheet-like permeable carrier with flowability owing to submerged aeration and a container with biofilm formation-inhibiting microorganisms immobilized therein, comprising biofilm formation-inhibiting microorganisms immobilized in the carrier. The present disclosure also provides a membrane water treatment apparatus comprising a reactor accommodating water to be treated, a membrane module for water treatment and a container with biofilm formation-inhibiting microorganisms immobilized therein placed in the reactor.

Claims

1. A membrane bio-reactor (MBR) process for water treatment, comprising: contacting water to be treated with microorganisms for biological water treatment; and filtering the biologically treated water through the membrane in the presence of a container comprising a permeable carrier and biofilm formation-inhibiting microorganisms, other than the microorganisms for biological water treatment, immobilized in the carrier, wherein the biofilm formation-inhibiting microorganisms inhibit quorum sensing of the microorganisms for biological water treatment responsible for biofilm formation on a surface of the membrane; and wherein the permeable carrier comprises a hydrogel and has fluidisability through submerged aeration.

2. A membrane bio-reactor (MBR) process for water treatment according to claim 1, wherein the permeable carrier is a spherical in shape.

3. A membrane bio-reactor (MBR) process for water treatment according to claim 1, wherein the permeable carrier is a columnar or sheet-like in shape.

4. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the permeable carrier is a circular columnar in shape.

5. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the permeable carrier is a hollow columnar in shape.

6. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the columnar permeable carrier has an aspect ratio of around 5-500, the aspect ratio being a ratio of a maximum diameter of a cross-section to length thereof.

7. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the sheet-like permeable carrier has a ratio of surface area to volume (SAN) of around 5-1,000 mm.sup.1.

8. A membrane bio-reactor (MBR) process for water treatment according to claim 1, wherein the permeable carrier comprises at least one material selected from a group consisting of alginate-based, PVA-based, polyethylene glycol-based and polyurethane-based.

9. A membrane bio-reactor (MBR) process for water treatment according to claim 1, wherein the permeable carrier has a 3-dimensional network structure through internal chemical crosslinking.

10. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the permeable carrier further comprises a carbonaceous additive.

11. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the permeable carrier further comprises a bio-inspired adhesive polymer additive.

12. A membrane bio-reactor (MBR) process for water treatment according to claim 1, wherein the biofilm formation-inhibiting microorganisms are recombinant microorganisms or natural microorganisms capable of producing enzymes for inhibiting biofilm formation.

13. A membrane bio-reactor (MBR) process for water treatment according to claim 1, wherein the biofilm formation-inhibiting microorganisms are capable of producing enzymes for inhibiting quorum sensing.

14. A membrane bio-reactor (MBR) process for water treatment according to claim 13, wherein the enzyme for inhibiting quorum sensing comprises lactonase or acylase.

15. A membrane bio-reactor (MBR) process for water treatment according to claim 3, wherein the biofilm formation-inhibiting microorganisms are capable of producing substance for inhibiting quorum sensing.

16. A membrane bio-reactor (MBR) process for water treatment according to claim 15, wherein the substance for inhibiting quorum sensing comprises farnesol.

Description

DESCRIPTION OF DRAWINGS

[0049] FIGS. 1a-1d show schematic diagrams and photographs of a container for inhibiting biofilm formation with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 1 of the present disclosure (FIGS. 1a-1b: both ends sealed; FIGS. 1c-1d: one end sealed).

[0050] FIG. 2 schematically shows a process of preparing the container for inhibiting biofilm formation according to Embodiment 1 of the present disclosure.

[0051] FIG. 3 shows a schematic diagram of a membrane bioreactor process using a membrane bioreactor apparatus for water treatment accommodating the container for inhibiting biofilm formation according to Embodiment 1 of the present disclosure.

[0052] FIG. 4 shows increase of transmembrane pressure (increase of membrane biofouling) in Example 2A according to Embodiment 1 of the present disclosure and in Comparative Example 2A.

[0053] FIG. 5 shows signal molecule decomposition activity of the container for inhibiting biofilm formation according to Embodiment 1 of the present disclosure.

[0054] FIG. 6 shows that the signal molecule decomposition activity of the container for inhibiting biofilm formation according to Embodiment 1 of the present disclosure is maintained for a long period of time.

[0055] FIG. 7 shows increase of transmembrane pressure (increase of membrane biofouling) in Example 4A according to Embodiment 1 of the present disclosure and in Comparative Example 4A.

[0056] FIG. 8 shows increase of transmembrane pressure (increase of membrane biofouling) in Example 5A according to Embodiment 1 of the present disclosure and in Comparative Example 5A.

[0057] FIGS. 9a and 9b show a schematic diagram of a container (comprising spherical fluidisable carrier) for inhibiting biofilm formation with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure and photographs of a realistically prepared container (fluidisable carrier).

[0058] FIGS. 10a and 10b show photographs of a bioreactor including the container for inhibiting biofilm formation according to Embodiment 2 of the present disclosure (FIG. 10a: without aeration; FIG. 10b: with aeration).

[0059] FIG. 11 shows a process of preparing a container for inhibiting biofilm formation according to Embodiment 2 of the present disclosure.

[0060] FIG. 12 shows signal molecule decomposition activity of a container for inhibiting biofilm formation according to Embodiment 2 of the present disclosure.

[0061] FIG. 13 shows a schematic diagram of a membrane bioreactor apparatus accommodating the container for inhibiting biofilm formation according to Embodiment 2 of the present disclosure.

[0062] FIG. 14 shows increase of transmembrane pressure (increase of membrane biofouling) versus operation time, in Example 1B according to Embodiment 2 of the present disclosure and in Comparative Examples 1B and 2B.

[0063] FIG. 15 shows signal molecule decomposition activity (relative activity) of a fluidisable spherical carrier versus operation time, according to Embodiment 2 of the present disclosure in a membrane bioreactor apparatus.

[0064] FIG. 16 shows the degree of growth of biofilm formation-inhibiting microorganisms inside a spherical fluidisable carrier (as wet weight of the fluidisable carrier) versus operation time, according to Embodiment 2 of the present disclosure of a membrane bioreactor apparatus.

[0065] FIG. 17 shows an embodiment of preparing a container (comprising a spherical fluidisable carrier) with biofilm formation-inhibiting microorganisms (fungi) immobilized therein according to Embodiment 2 of the present disclosure.

[0066] FIG. 18 shows the assessment results of the activity for inhibiting quorum sensing of a container (comprising a spherical fluidisable carrier) with biofilm formation-inhibiting microorganisms (fungi) immobilized therein according to Embodiment 2 of the present disclosure.

[0067] FIG. 19 shows a schematic diagram of a membrane bioreactor apparatus for accommodating a container (comprising a spherical fluidisable carrier) with biofilm formation-inhibiting microorganisms (fungi) immobilized therein according to Embodiment 2 of the present disclosure in a bioreactor.

[0068] FIG. 20 shows increase of transmembrane pressure versus operation time in membrane bioreactor apparatuses, accommodating containers (comprising a spherical fluidisable carrier) with biofilm formation-inhibiting microorganisms (fungi) immobilized therein according to Example and Comparative Example of Embodiment 2 of the present disclosure.

[0069] FIGS. 21a and 21b show schematic diagrams of containers (comprising non-hollow and hollow circular columnar fluidisable carrier, respectively) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure, and FIG. 21c shows a schematic diagram of a container (comprising sheet-like fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure.

[0070] FIGS. 22a and 22b show an embodiment of preparing containers (comprising non-hollow or hollow circular columnar fluidisable carrier, respectively) with biofilm formation-inhibiting microorganisms immobilized therein according to an Embodiment 2 of the present disclosure, and FIG. 22c shows an embodiment of preparing a container with biofilm formation-inhibiting microorganisms immobilized therein (comprising sheet-like fluidisable carrier) according to Embodiment 2 of the present disclosure.

[0071] FIGS. 23a and 23b show photographs taken on containers (comprising non-hollow or hollow circular columnar fluidisable carrier, respectively) with biofilm formation-inhibiting microorganisms immobilized therein according to an Embodiment 2 of the present disclosure, and FIG. 23c shows a photograph taken on a container (comprising sheet-like fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure.

[0072] FIG. 24 shows the assessment results of the decomposition activity of signal molecules of a container (comprising non-hollow circular columnar fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure.

[0073] FIG. 25 shows the assessment results of the decomposition activity of signal molecules of a container (comprising hollow circular columnar fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure.

[0074] FIG. 26 shows a schematic diagram of a membrane bioreactor apparatus for accommodating containers (comprising non-hollow circular columnar fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure and operating.

[0075] FIG. 27 shows increase of transmembrane pressure versus operation time in membrane bioreactor apparatuses, accommodating containers (comprising non-hollow circular columnar fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Example and Comparative Example of Embodiment 2 of the present disclosure.

[0076] FIG. 28 shows increase of transmembrane pressure versus operation time in a membrane bioreactor apparatus, accommodating containers (comprising spherical fluidisable carrier and a non-hollow circular columnar fluidisable carrier, respectively) with biofilm formation-inhibiting microorganisms immobilized therein according to Examples of Embodiment 2 of the present disclosure.

[0077] FIG. 29 shows a schematic diagram of a membrane bioreactor apparatus for accommodating containers (comprising hollow circular columnar fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Embodiment 2 of the present disclosure.

[0078] FIG. 30 shows increase of transmembrane pressure versus operation time in membrane bioreactor apparatuses, accommodating containers (comprising hollow circular columnar fluidisable carrier) with biofilm formation-inhibiting microorganisms immobilized therein according to Example and Comparative Example of Embodiment 2 of the present disclosure.

MODE FOR INVENTION

[0079] Hereinafter, the present disclosure will be described in detail through examples. However, the present disclosure is not limited thereto.

Embodiment 1Hollow Porous Container with Biofilm Formation-Inhibiting Microorganisms (Genetically Recombined Microorganisms) Immobilized Therein

Preparation Example 1A: Preparation of a Container with Biofilm Formation-Inhibiting Microorganisms Immobilized Therein (Both Ends Sealed)

[0080] Genetically recombined E. coli capable of producing lactonase was used as biofilm formation-inhibiting microorganisms. Specifically, E. coli XL1-blue, which is commonly used in genetic recombination, was used and the aiiA gene from the Bacillus thuringiensis subsp. kurstaki was inserted therein through genetic recombination The aiiA gene codes for lactonase which decomposes signal molecules used in the quorum sensing mechanism.

[0081] As a hollow porous container for immobilizing the biofilm formation-inhibiting microorganisms, a hollow fiber membrane (available from Econity Co., Ltd) was used. Since the hollow fiber membrane has a pore size of 0.4 m, the microorganisms cannot pass therethrough whereas water and signal molecules can easily pass therethrough and travel between the container and a reactor. A total of 55 strands of hollow fiber membranes were used to prepare a container with biofilm formation-inhibiting microorganisms immobilized therein having a length of 10 cm and a total membrane surface area of 112.31 cm.sup.2, with both ends sealed, as shown in FIGS. 1a and 1b.

[0082] After culturing for 24 hours, 200 mL of E. coli was centrifuged and the supernatant was discarded thereby removing the culture medium. The microorganisms were resuspended using Tris-HCl 50 mM buffer (pH 7.0) and then injected into the container using a pump, as shown in FIG. 2.

Preparation Example 2A: Preparation of a Container with Biofilm Formation-Inhibiting Microorganisms Immobilized Therein (One End Sealed)

[0083] A container with biofilm formation-inhibiting microorganisms immobilized therein was prepared in the same manner as in Preparation Example 1A, except that only one end of the container submerged in a reactor was sealed and the other end was communicated with the outside atmosphere via a filter member (PTFE, pore size 0.45 m) followed by a tube, and then biofilm formation-inhibiting microorganisms (E. coli) were injected (see FIGS. 1c, 1d and 2).

Example 1A: Measurement of Signal Molecule Decomposition Activity of a Container with Biofilm Formation-Inhibiting Microorganisms Immobilized Therein

[0084] Signal molecule (AHL) decomposition activity of the container with biofilm formation-inhibiting microorganisms immobilized therein was measured using N-octanoyl-L-homoserine lactone (OHL), which is one of representative signal molecules. After adding Tris-HCl 50 mM buffer (pH 7) to a test tube and then injecting OHL to a concentration of 0.2 M, the container with biofilm formation-inhibiting microorganisms immobilized therein was added thereto and the resulting mixture was reacted for 90 minutes in a shaking incubator of 30 C. at 200 rpm. As a result, about 60% of signal molecules were decomposed for 90 minutes (see FIG. 5).

Comparative Example 1A

[0085] The same procedure was repeated as Example 1A except that the microorganisms were not injected to the container. As a result, the signal molecules were hardly decomposed (see FIG. 5).

Example 2A: Application to Membrane Bioreactor Process (Genetically Recombined Microorganisms/Both Ends-Sealed Container)

[0086] The container with biofilm formation-inhibiting microorganisms immobilized therein, prepared in Preparation Example 1A, was applied to a laboratory-scale membrane bioreactor process (see FIG. 3). Specifically, 1.2 L of activated sludge was filled in a cylindrical reactor and diffuser stone was equipped at the bottom to maintain aeration of 1 L/min. A total of two pieces of containers with biofilm formation-inhibiting microorganisms immobilized therein were placed in the reactor symmetrically. For continuous operation, synthetic wastewater containing glucose as a main carbon source was introduced by an inflow pump. The chemical oxygen demand (COD) of the synthetic wastewater was about 550 ppm and hydraulic retention time was about 12 hours. The synthetic wastewater was filtered with a flux of about 18 L/m.sup.2 hr through a hollow fiber ultrafiltration membrane (Zeeweed 500, GE-Zenon, pore size 0.04 m) submerged in the reactor. The water level of the reactor was maintained by recirculating a part of the permeate using a level controller and a 3-way-valve. During the operation, mixed liquor suspended solids (MLSS) was maintained at 4500-5000 mg/L. The degree of membrane biofouling caused by biofilm formation on the membrane surface was represented with transmembrane pressure (TMP). The higher the transmembrane pressure, the larger is the degree of membrane biofouling. Even after operation for about 200 hours, the transmembrane pressure was no more than about 13 kPa (see FIG. 4).

Comparative Example 2A

[0087] The same procedure was repeated as Example 2A except that the microorganisms were not injected to the container. After operation for about 200 hours, the transmembrane pressure reached about 50 kPa (see FIG. 4).

Example 3A: Maintenance of Activity of a Container with Biofilm Formation-Inhibiting Microorganisms Immobilized Therein

[0088] It was investigated whether the signal molecule decomposition activity of the container with biofilm formation-inhibiting microorganisms immobilized therein is maintained for a long period of time. Specifically, after continuous operation for 25 days and 80 days, the container with biofilm formation-inhibiting microorganisms immobilized therein was taken out from the reactor and, followed by washing the outside of the container several times with distilled water, the same procedure as Example 1A was conducted (see FIG. 6). Even after operation for 80 days, the signal molecule decomposition activity was not significantly decreased.

Example 4A: Application to Membrane Bioreactor Process (Natural Microorganisms/Both Ends-Sealed Container)

[0089] The microorganisms used in Example 2A were genetically modified by inserting the lactonase-producing gene into E. coli and they cannot survive in the actual wastewater environment for a long period of time. Therefore, in order to find microorganisms suitable to be applied to the actual water treatment process, microorganisms were isolated from sludge obtained from a sewage disposal plant located in Okcheon, Chungchengbuk-do, Korea. From the isolated microorganisms, the microorganisms of the genus Rhodococcus with excellent activity of decomposing signal molecules could be separated through enrichment culture. A container with biofilm formation-inhibiting microorganisms immobilized therein was prepared using these microorganisms, in the same manner as in Preparation Example 1A, and it was applied to a membrane bioreactor process under the same condition as Example 2A.

[0090] The container with biofilm formation-inhibiting microorganisms immobilized therein prepared above was applied to a laboratory-scale membrane bioreactor process. After operation for about 40 hours, transmembrane pressure reached about 24 kPa (see FIG. 7).

Comparative Example 4A

[0091] The same procedure was repeated as Example 4A except that the microorganisms were not injected to the container. After operation for about 40 hours, the transmembrane pressure reached about 50 kPa (see FIG. 7).

Example 5A: Application to Membrane Bioreactor Process (Natural Microorganisms/One End-Sealed Container)

[0092] A membrane bioreactor was operated under the same condition as Example 4A, except that 2.5 L of activated sludge used in Example 4A was filled in a cylindrical reactor, a total of four pieces of containers with biofilm formation-inhibiting microorganisms immobilized therein were placed in the reactor symmetrically, hydraulic retention time of glucose-containing synthetic wastewater was set to about 8 hours, the flux of the wastewater through the membrane was changed to about 30 L/m.sup.2 hr and MLSS was maintained at 7500-8500 mg/L.

[0093] After operation for about 50 hours, the transmembrane pressure reached about 22 kPa (see FIG. 8).

Comparative Example 5A

[0094] The same procedure was repeated as Example 5A except that the microorganisms were not injected to the container. After operation for about 40 hours, the transmembrane pressure reached about 64 kPa (see FIG. 8).

Embodiment 2

[0095] 1. Embodiment 2 Concerning Spherical Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganisms (Bacteria) Immobilized Therein

Preparation Example 1 B: Preparation of a Spherical Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganism Immobilized Therein and Measurement of Signal Molecule Decomposition Activity

[0096] As biofilm formation-inhibiting microorganisms, Rhodococcus qingshengii BH4, known to produce lactonase which is one of enzymes for inhibiting quorum sensing, that was isolated from sludge from the municipal wastewater treatment plant in the same manner described in Embodiment 1 was used.

[0097] As a spherical fluidisable carrier for immobilizing the biofilm formation-inhibiting microorganisms, the natural polymer of sodium alginate (Sigma Co.) was used.

[0098] Alginate is a typical material used to entrap microorganisms. A preliminary test was conducted in order to find out the alginate concentration that allows maintenance of physical strength in a membrane bioreactor for a long period of time. The concentration of alginate solution was adjusted to 4 wt % at the time of final injecting.

[0099] Rhodococcus qingshengii BH4 was proliferated by culturing for 24 hours in a shaking incubator. 200 mL of the culture was centrifuged and the supernatant was discarded thereby removing the culture medium. The remaining agglomerates of Rhodococcus qingshengii were washed with Tris-HCl 50 mM buffer (pH 7.0) and resuspended in ultrapure water. Subsequently, as shown in FIG. 11, the resuspended solution of the biofilm formation-inhibiting microorganisms was mixed with the alginate solution and injected to calcium chloride (CaCl.sub.2) solution. As a result, a spherical fluidisable carrier having a network structure allowing efficient mass transfer was prepared through chemical crosslinking. The concentration of the alginate solution at the time of the final injection was 4 wt % when preparing the spherical fluidisable carrier. After crosslinking in 2 wt % calcium chloride (CaCl.sub.2) solution for 1 hour, the prepared spherical fluidisable carrier was dried at room temperature for 20 hours in order to increase physical strength.

[0100] The signal molecule (AHL) decomposition activity of the spherical fluidisable carrier was measured using N-octanoyl-L-homoserine lactone (OHL) as in Embodiment 1. After adding 30 mL of Tris-HCl 50 mM buffer (pH 7) to a test tube and then injecting OHL to a concentration of 0.2 M, the spherical fluidisable carrier was added thereto and the resulting mixture was reacted for 60 minutes in a shaking incubator of 30 C. at 200 rpm. As a result, about 92% of signal molecules were decomposed for 90 minutes by the biofilm formation-inhibiting enzyme (lactonase) produced by the biofilm formation-inhibiting microorganisms (see FIG. 12).

Example 1B: Application to Membrane Bioreactor Apparatus

[0101] The spherical fluidisable carrier with biofilm formation-inhibiting microorganisms immobilized therein prepared in Preparation Example 1B was applied to a laboratory-scale membrane bioreactor process (see FIG. 13). Specifically, 1.6 L of activated sludge was filled in a cylindrical reactor and diffuser stone was equipped at the bottom to maintain aeration of 1 L/min. A total of 60 pieces of the spherical fluidisable carriers were placed in the reactor. For continuous operation, synthetic wastewater containing glucose as a main carbon source was introduced by an inflow pump. The chemical oxygen demand (COD) of the synthetic wastewater was about 560 ppm and hydraulic retention time was about 5.3 hours. The synthetic wastewater was filtered with a flux of about 28.7 L/m.sup.2 hr through a hollow fiber ultrafiltration membrane (Zeeweed 500, GE-Zenon, pore size 0.04 m) submerged in the reactor. The water level of the reactor was maintained by recirculating a part of the permeate using a level controller and a 3-way-valve. The degree of membrane biofouling caused by biofilm formation on the membrane surface was represented with transmembrane pressure (TMP). The higher the transmembrane pressure, the larger is the degree of membrane biofouling. Even after operation for about 77 hours, the transmembrane pressure was no more than about 5 kPa. After operation for about 400 hours, the transmembrane pressure reached about 70 kPa (see FIG. 14).

Comparative Example 1B

[0102] The same procedure was repeated as Example 1B except that 60 pieces of hydrogel spherical fluidisable carriers without any microorganisms immobilized (prepared by not immobilizing the biofilm formation-inhibiting microorganisms in Preparation Example 1B) were placed in the reactor instead of the spherical fluidisable carriers with biofilm formation-inhibiting microorganisms immobilized therein. After operation for about 77 hours, the transmembrane pressure reached about 70 kPa (see FIG. 14).

Comparative Example 2B

[0103] The same procedure was repeated as Example 1B except that the spherical fluidisable carriers were not placed in the membrane bioreactor. After operation for about 43 hours, the transmembrane pressure reached about 70 kPa (see FIG. 14).

[0104] From Example 1B and Comparative Examples 1B-2B, it can be seen that the membrane bioreactor apparatus in which the spherical fluidisable carriers with biofilm formation-inhibiting microorganisms immobilized therein of the present disclosure are placed (Example 1B) exhibits remarkably decreased biofouling on the membrane surface as compared to when the spherical fluidisable carriers without the microorganisms immobilized are placed (Comparative Example 1B) or no spherical fluidisable carrier is placed (Comparative Example 2B). This is thought of as a synergic effect of molecular biological effect of inhibiting biofilm formation by the biofilm formation-inhibiting microorganisms stably immobilized in the spherical fluidisable carrier and removal of biofilms on the membrane surface by physical washing owing to the carrier having fluidisability through submerged aeration.

Example 2B: Maintenance of Activity of the Spherical Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganisms Immobilized Therein

[0105] It was investigated whether the signal molecule decomposition activity of the biofilm formation-inhibiting microorganisms inside the spherical fluidisable carrier is maintained for a long period of time. Specifically, after continuous operation for 0, 1, 3, 5, 7, 10, 13, 15, 17, 20, 23, 25, 27 and 30 days in the Example 1B, the spherical fluidisable carrier was taken out from the reactor and, followed by washing the outside of the fluidisable carrier several times with distilled water, the signal molecule decomposition activity of the biofilm formation-inhibiting microorganisms was measured according to the same procedure as Preparation Example 1B. Relative activity was measured relative to the activity of the spherical fluidisable carrier on day 0 as 100%. Even after operation for 20 days, the signal molecule decomposition activity of the spherical fluidisable carrier did not decrease but increased slightly as compared to the initial (day 0) activity (FIG. 15).

Example 3B: Growth of Biofilm Formation-Inhibiting Microorganisms Inside the Spherical Fluidisable Carrier

[0106] The degree of growth of the biofilm formation-inhibiting microorganisms was investigated after the spherical fluidisable carrier was placed in a membrane bioreactor and operated for a long period of time.

[0107] Specifically, while operating the reactor for 25 days after placing the spherical fluidisable carrier, 10 pieces of the spherical fluidisable carriers were recovered every 24 hours and, followed by washing the outside of the fluidisable carrier several times with distilled water, and wet weight was measured (Average was taken for 5 repeated measurements). 25 days later, the wet weight was increased as compared to that of the initially (day 0) entrapped biofilm formation-inhibiting microorganisms (FIG. 16).

Comparative Example 3B

[0108] The same procedure was repeated as Example 3B except that alginate fluidisable carrier with no biofilm formation-inhibiting microorganisms immobilized was used. There was almost no change in wet weight (FIG. 16).

[0109] From Examples 2B-3B and Comparative Example 3B, it can be seen that the biofilm formation-inhibiting microorganisms immobilized in the spherical fluidisable carrier of the present disclosure grow inside the fluidisable carrier and lead to increased wet weight. This explains why the signal molecule decomposition activity does not decrease but increase slightly.

[0110] 2. Embodiment 2 Concerning a Spherical Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganisms (Fungi) Immobilized Therein

Preparation Example 1CPreparation of a Spherical Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganisms (Fungi) Immobilized Therein and Measurement of Inhibition Activity of Quorum Sensing

[0111] Genetically recombined Candida albicans, one of the fungi of genus Candida, capable of secreting excessive farnesol which is a substance involved in the inhibition of AI-2 quorum sensing mechanism was used as the biofilm formation-inhibiting microorganisms.

[0112] A mixture of sodium alginate (produced by Sigma Co.) which is a typical natural polymer used for entrapping microorganisms and polyvinyl alcohol (produced by Sigma Co.) was used as a raw material of a fluidisable carrier for immobilizing the biofilm formation-inhibiting microorganisms. A resuspended solution of Candida albicans was prepared by proliferating through culturing in a shaking incubator for 24 hours, centrifugating 200 ml of the shaken culture, discarding the supernatant, removing the culture medium, and thereafter resuspending remaining agglomerates of Candida albicans in ultrapure water. As shown in FIG. 17, the resuspended solution of Candida albicans and the raw material mixture of carrier of sodium alginate/polyvinyl alcohol were mixed to prepare a carrier solution (1 wt % of sodium alginate and 10 wt % of polyvinyl alcohol), and the carrier solution was injected into an aqueous mixture solution of calcium chloride (CaCl2, 4 wt %) and boric acid (H3B03, 7 wt %) as a crosslinking solution to perform first solidification for 1 hour. Then, second solidification was performed in a 0.5 M aqueous sodium sulfate solution for 12 hours to finally prepare a spherical fluidisable carrier (average diameter: 4 mm) having a network structure allowing efficient mass transfer through internal chemical crosslinking.

[0113] The effect of inhibition of quorum sensing for certain microorganisms (bacteria) for water treatment by farnesol secreted by the biofilm formation-inhibiting microorganisms, i.e., Candida albicans is thought to be attributed to the inhibition of quorum sensing mechanism using AI-2 signal molecules, which was indirectly assessed by means of Vibrio harveyi BB152, a bacteria producing only AI-2, and Vibrio harveyi BB170, bacteria allowing bioluminescence by specifically accepting only AI-2. Particularly, Vibrio harveyi BB152 producing only AI-2 signal molecules was inoculated in an AB medium (Autoinducer Bioassay medium, Tega et al., 2011) and cultivated to a certain degree of optical density (O.D..sub.600) of 0.1-0.3. Farnesol was injected to attain final concentration of 800 M (and no farnesol was injected for its comparison), each sample was taken after reaction for 90 minutes, and the each sample was reacted with Vibrio harveyi BB170 to measure bioluminescence. As a result, the bioluminescence of Vibrio harveyi BB170 was decreased by about 42 percents for the case of not injecting the farnesol (designated as Control) when compared to the case of injecting the farnesol (see FIG. 18). This result was supposed to be obtained, because the farnesol inhibited the generation of AI-2 which is one kind of signal molecules for quorum sensing of certain microorganisms (bacteria for water treatment).

Example 1CApplication to Membrane Bioreactor Apparatus

[0114] The spherical fluidisable carrier with biofilm formation-inhibiting microorganisms (Candida albicans) immobilized therein prepared in Preparation Example 1C was applied to a laboratory-scale membrane bioreactor apparatus (see FIG. 19).

[0115] Particularly, 2.5 L of activated sludge was filled in a cylindrical reactor, and a diffuser stone was equipped at the bottom to maintain an aeration of 1.5 L/min. The spherical fluidisable carrier with Candida albicans immobilized therein was injected into the reactor by 0.5 v/v % of the reactor volume (corresponds to about 200-250 pieces). Municipal sewage (wastewater from the cafeteria of Seoul National University, COD: about 100-200 ppm) was injected into a reactor via an inflow pump and was operated with hydraulic retention time of about 10 hours. A submerged-type hollow fiber ultrafiltration membrane module (Zeeweed 10, GE-Zenon, pore size 0.04 m) was installed in the reactor, and the flux of the permeate passing through the membrane was kept on about 30 L/m.sup.2.Math.hr. The water level of the reactor was maintained by recirculating a part of the permeate using a level controller and a 3-way-valve. During the operation, a biofilm was formed on the membrane surface, and the decrease of the water permeability of the membrane due to the increase of membrane biofouling was represented by the increase of transmembrane pressure (TMP). According to the experiment results, the transmembrane pressure was merely less than about 15 kPa even after operation for 2 days and finally reached about 40 kPa after operation for 4.2 days (see FIG. 20).

Comparative Example 1C

[0116] The same procedure was repeated as Example 1C except for injecting a spherical fluidisable carrier with no microorganisms immobilized therein (a carrier prepared without immobilizing biofilm formation-inhibiting microorganisms therein in Preparation Example 1C) by 0.5 v/v % of the reactor volume (corresponding to about 200-250 pieces) instead of the spherical fluidisable carrier with the biofilm formation-inhibiting microorganisms (Candida albicans) immobilized therein. After operation for merely about 2 days out of 4.2 days of total operation days, the transmembrane pressure reached about 40 kPa (see FIG. 20).

[0117] That is, according to the results above of Example 1C and Comparative Example 1C, similar to the case of the spherical fluidisable carrier with biofilm formation-inhibiting microorganisms (bacteria) immobilized therein in Example 1 B, the membrane biofouling in the membrane bioreactor apparatus due to the biofilm formation on the membrane surface was remarkably relieved in the case of using the fluidisable spherical carrier with the biofilm formation-inhibiting microorganisms (fungi) immobilized therein (Example 1C) when compared to the case of the spherical fluidisable carrier with no biofilm formation-inhibiting microorganisms (fungi) immobilized therein (Comparative Example 1C). The results are thought to be obtained because the biofilm formation due to the microorganisms for water treatment on the membrane surface was restrained by the substance for inhibiting quorum sensing secreted by stably immobilized biofilm formation-inhibiting microorganisms (Candida albicans) inside the fluidisable carrier.

[0118] 3. Embodiment 2 Concerning a Columnar Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganisms (Bacteria) Immobilized Therein

Preparation Example 1DPreparation of Non-Hollow and Hollow Circular Columnar Fluidisable Carrier with Biofilm Formation-Inhibiting Microorganisms (Bacteria) Immobilized Therein and Measurement of Decomposition Activity of Signal Molecules

[0119] As biofilm formation-inhibiting microorganisms, Rhodococcus qingshengii BH4, isolated from sludge from the municipal wastewater treatment plant was used in the same manner described in Preparation Example 1B. A mixture of sodium alginate (produced by Sigma Co.) which is a typical natural polymer used for entrapping microorganisms and polyvinyl alcohol (produced by Sigma Co.) was used as a raw material of a fluidisable carrier for immobilizing the biofilm formation-inhibiting microorganisms. Rhodococcus qingshengii BH4 was proliferated by culturing for 24 hours in a shaking incubator. 200 mL of the culture was centrifuged and the supernatant was discarded thereby removing the culture medium. The remaining agglomerates of Rhodococcus qingshengii BH4 were washed with Tris-HCl 50 mM buffer (pH 7.0) and resuspended in ultrapure water. Subsequently, as shown in FIGS. 22a and 22b, the resuspended solution of Rhodococcus qingshengii BH4 was mixed with the raw material mixture of carrier of sodium alginate/PVA to prepare a carrier solution (1 wt % of sodium alginate and 10 wt % of polyvinyl alcohol), and the carrier solution was injected to an aqueous mixture solution of calcium chloride (CaCl2, 4 wt %) and boric acid (H3B03, 7 wt %) as a crosslinking solution to perform first solidification for 1 hour. Then, second solidification was performed in 0.5 M aqueous sodium sulfate (Na2SO4) solution for 4 hours to finally prepare non-hollow and hollow circular columnar fluidisable carriers having a network structure allowing efficient mass transfer through internal chemical crosslinking {corresponding to Preparation Example 1D(i) and Preparation Example 1D(ii), respectively}. As a result, non-hollow and hollow circular columnar fluidisable carriers with various diameters/lengths were prepared as shown in FIGS. 23a and 23b.

[0120] The signal molecule (AHL) decomposition activity of the columnar fluidisable carrier with biofilm formation-inhibiting microorganisms immobilized therein was measured using N-octanoyl-L-homoserine lactone (OHL) as in Preparation Example 1B. After adding 30 mL of Tris-HCl 50 mM buffer (pH 7.0) to a test tube and then injecting OHL to a concentration of 1 M, the non-hollow and hollow columnar fluidisable carriers with biofilm formation-inhibiting microorganisms (Rhodoccocus qingshengii) immobilized therein were added thereto and the resulting mixture was reacted for 60 minutes in a shaking incubator of 30 C. at 200 rpm. The activity was measured from the amount of the signal molecule decomposed for 60 minutes by biofilm formation-inhibiting enzyme (lactonase) produced from the biofilm formation-inhibiting microorganisms (Rhodococcus qingshengii BH4). For comparison, a spherical fluidisable carrier with the same microorganisms immobilized therein was additionally prepared (hereinafter Preparation Example 1BD), and the decomposition activity of the signal molecule was measured {corresponding to Preparation Example 1BD(i) and Preparation Example 1BD(ii), respectively} (see FIGS. 24 and 25, respectively).

Example 1D(i)(a)Application to Membrane Bioreactor Apparatus

[0121] The non-hollow circular columnar fluidisable carrier with the biofilm formation-inhibiting microorganisms immobilized therein prepared in Preparation Example 1D(i) was applied to a laboratory-scale membrane bioreactor apparatus (see FIG. 26). Particularly, 4.5 L of activated sludge was filled in a rectangular reactor, and a diffuser stone was equipped at the bottom to maintain an aeration of 2 L/min. 120 pieces of non-hollow circular columnar fluidisable carriers with biofilm formation-inhibiting microorganisms immobilized therein (diameter 1-1.5 mm, length 20-23 mm, corresponding to 0.5 vol % of reactor) was injected into the reactor. For continuous operation, synthetic wastewater (COD of about 600 ppm) containing glucose as a main carbon source was introduced by an inflow pump, and operated with hydraulic retention time of about 8 hours. The synthetic wastewater was filtered with a flux of the permeate of about 37 L/m.sup.2 hr through a submerged-type flat-sheet microfiltration membrane module (C-PVC, Pure-envitech Co., pore size 0.4 m). The water level of the reactor was maintained by recirculating a part of the permeate using a level controller and a 3-way-valve. As a result, the transmembrane pressure after operation even for about 7 days (about 168 hours) was merely about 10 kPa, and the transmembrane pressure reached about 25 kPa after operation about 14 days (about 320 hours) (see FIG. 27).

Comparative Example 1D(i)(a)

[0122] The same procedure was repeated as Example 1D(i)(a) except that the columnar fluidisable carrier was not injected into the membrane bioreactor. As a result, the transmembrane pressure reached about 25 kPa after operation for about 6 days (about 140 hours) (see FIG. 27).

Example 1D(i)(b)Application to Membrane Bioreactor Apparatus

[0123] The same procedure was repeated as Example 1D(i)(a) except that the flux of the permeate in the membrane bioreactor was kept on about 29 Um.sup.2 hr and the dimension and the number of the columnar fluidisable carrier were changed (diameter 1-1.5 mm, length 8-12 mm, 198 pieces). As a result, the transmembrane pressure after operation even for about 23 days (about 534 hours) was merely about 10 kPa, and the transmembrane pressure reached about 25 kPa after operation about 43 days (about 1044 hours) (see FIG. 28).

[0124] For comparison, the transmembrane pressure of the same membrane module was observed by applying a spherical fluidisable carrier (average diameter: 4 mm) prepared in Preparation Example 1BD to a membrane bioreactor apparatus {corresponding to Example 1BD(i)(b)}. More particularly, the same operation procedure was repeated as Example 1D(i)(b) except for injecting a spherical fluidisable carrier with the same biofilm formation-inhibiting microorganisms (bacteria) immobilized therein (700 pieces, 0.5 vol % relative to reactor volume) into a membrane bioreactor in Example 1D(ii). As a result, the transmembrane pressure reached about 25 kPa after operation for about 26 days (about 630 hours) (see FIG. 28).

Example 1D(ii)Application to Membrane Bioreactor Apparatus

[0125] The hollow circular columnar fluidisable carrier with the biofilm formation-inhibiting microorganisms immobilized therein prepared in Preparation Example 1D(ii) was applied to a laboratory-scale membrane bioreactor apparatus (see FIG. 29). Particularly, 3 L of activated sludge was filled in a cylindrical reactor, and a diffuser stone was equipped at the bottom to maintain an aeration of 2 L/min. 280 pieces of hollow circular columnar fluidisable carriers with biofilm formation-inhibiting microorganisms immobilized therein (outer diameter 3.2 mm, inner diameter 2.0 mm, length 20 mm) was injected into a reactor. For continuous operation, synthetic wastewater containing glucose as a main carbon source (COD of about 600 ppm) was introduced by an inflow pump, and operation was performed with hydraulic retention time of about 8 hours. The synthetic wastewater was filtered with a flux of the permeate of about 21 L/m.sup.2 hr through a submerged-type hollow fiber ultrafiltration membrane module (Zeeweed 500, GE-Zenon Co., pore size 0.04 m) installed in the reactor. The water level of the reactor was maintained by recirculating a part of the permeate using a level controller and a 3-way-valve. As a result, the transmembrane pressure after operation even for about 5 days was maintained less than or equal to about 6 kPa (see FIG. 30).

Comparative Example 1D(ii)

[0126] The same procedure was repeated as Example 1D(ii) except for not injecting the fluidisable carrier with the biofilm formation-inhibiting microorganisms immobilized therein into the membrane bioreactor. As a result, the transmembrane pressure after operation for about 5 days reached about 47 kPa (see FIG. 30).

[0127] That is, according to the results above of Examples 1D(i)-(ii) and Comparative Examples 1D(i)-(ii), the membrane biofouling due to the biofilm formation on the membrane surface was remarkably relieved in the case of using the columnar fluidisable carrier with the biofilm formation-inhibiting microorganisms immobilized therein was injected (Examples 1D(i) and 1D(ii)) when compared to the cases of not injecting the columnar fluidisable carrier (Comparative Examples 1D(i)(a) and 1D(ii)). The results are thought to be obtained because the biofilm formation-inhibiting microorganisms were stably immobilized in the columnar fluidisable carrier with high surface area per carrier volume, mass transfer across the carrier surface was more efficiently enhanced, and thereby molecular biological effects of efficient inhibition of the biofilm formation mechanism were attained. In addition, the effect of relieving biofouling due to physical washing of the membrane surface by movement of the columnar fluidisable carrier through submerged aeration was also supposed to be attained.

[0128] In addition, according to the results above of Examples 1D(i)(b) and 1BD(i)(b), the water permeability of the membrane was further improved for the case of using the membrane bioreactor apparatus in which the columnar fluidisable carrier of the present disclosure was injected (Example 1D(i)(b)) when compared to the spherical fluidisable carrier of the present disclosure (Example 1BD(i)(b)). It is thought to be synergistically obtained, because substantial area (surface area per carrier volume) for contact with water to be treated for the columnar fluidisable carrier was remarkably higher than that for the spherical fluidisable carrier, thereby remarkably increasing the inhibiting efficiency of the biofilm formation by more readily contacting with bacteria responsible for the biofilm formation, and the columnar fluidisable carrier has fluidisability through submerged aeration and larger substantial area for contact with the membrane surface (surface area per carrier volume), thereby expediting additional decrease of biofouling due to the detachment of the biofilm (through physical washing) on the membrane surface.

INDUSTRIAL APPLICABILITY

[0129] When applied to an actual membrane water treatment process, the container with biofilm formation-inhibiting microorganisms immobilized therein of the present disclosure can inhibit the formation of biofilms on the membrane surface molecular biologically and, optionally, can provide an effect of physically removing membrane biofouling. As a result, decrease of permeability can be prevented, membrane cleaning cycle is lengthened, consumption of cleansers can be reduced, and lifespan of the membrane can be increased. Particularly, in the columnar or sheet-like permeable fluidisable carrier which has even larger surface area per carrier volume, biofilm formation-inhibiting microorganisms may be efficiently immobilized in the carrier (as a matrix). Thus, mass transfer across the carrier surface may be enhanced, and the formation of the biofilm may be effectively inhibited in view of the molecular biological perspective. The columnar or sheet-like permeable fluidisable carrier is not readily to be trapped even when inserted in a certain type of membrane module, and may secure a sufficient area for contact with membrane surface, thereby expediting the removal of the biofilm by efficient physical blow onto the membrane surface. Therefore, the columnar or sheet-like permeable fluidisable carrier may maximize the inhibiting/removing effect of the biofouling of the membrane surface.

[0130] And, when compared with the conventional magnetic carrier with biofilm formation-inhibiting enzyme immobilized thereon, the present disclosure is economically superior since the procedure of extracting and immobilizing enzymes is unnecessary and the apparatus for recovering the magnetic carrier is also unnecessary.