Wastewater treatment method

Abstract

The present invention pertains to: a method for treating drainage water containing a furan compound; a microbial preparation used in said method; a high-density culturing method for microorganisms used in said method; and a method for producing a microbial preparation using said culturing method.

Description

[Technical Field]

[0001] Related Applications: This specification includes the contents described in the specifications of Japanese Patent Application No. 2020-052011 (filed March 24, 2020) and Japanese Patent Application No. 2021-031309 (filed March 1, 2021), which are the basis of the priority of this application. Technical fields: The present invention relates to a method for treating wastewater containing furan compounds, a microbial preparation for use in the method, a method for high-density cultivation of microorganisms for use in the method, and a method for producing a microbial preparation using the cultivation method. [Background technology]

[0002] Furan compounds are cyclic ether compounds, such as furfural, 2-methylfuran, 3-methylfuran, furan, dihydrofuran, furfuryl alcohol, tetrahydrofuran, tetrahydrofurfuryl alcohol, and hydroxymethylfurfural, and are useful petrochemical derivatives. Traditionally, furan compounds have been produced from petroleum, but in recent years, with increasing awareness of environmental issues, the production of furan compounds from biomass resources has been considered.

[0003] Wastewater generated during the production of furan compounds contains furan aldehydes such as furfural and tetrahydrofurfural. Furan compounds, especially furan aldehyde, inhibit the growth and metabolism of microorganisms and are highly toxic to activated sludge, making biological treatment difficult. Regulatory values ​​for wastewater containing furan compounds are strict in various countries, and efficient treatment technologies for wastewater containing furan compounds are needed.

[0004] Recently, microorganisms that specifically decompose furan compounds have been discovered, and biological treatment methods for furan compounds utilizing these microorganisms have been reported (Patent Documents 1 and 2, Non-Patent Document 1).

[0005] Patent Document 1 discloses a method for decomposing hydroxymethylfurfural and furfural using the fungus Paecilomyces sp. FA13 strain, but its use is limited to compost production, and from the standpoints of environmental impact and energy cost, it is not suitable for application to wastewater treatment containing large amounts of furan compounds. Patent Document 2 discloses a method for decomposing cyclic ether compounds including furan using Pseudonocardia sp. RM31 strain, but while this method reduces 1,4-dioxane in wastewater, it is ineffective against furan compounds.

[0006] In addition to the wastewater treatment mentioned above, microorganisms are currently used for various purposes, such as producing compounds using enzymes produced by microorganisms and adding them to soil after harvesting crops as soil conditioners.

[0007] Therefore, there are situations where a large amount of microorganisms is required, and there is a demand for a technique for culturing microorganisms at high density (high concentration) (see Patent Documents 3 and 4). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-67288 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-42097 [Patent Document 3] Japanese Patent Application Publication No. 2019-30292 [Patent Document 4] Japanese Patent Application Publication No. 4-234981 [Non-patent literature]

[0009] [Non-Patent Document 1] Wierckx el al., Appl Microbiol Biotechnol (2011) 92:1095-1105 Summary of the Invention [Problem to be solved by the invention]

[0010] The first object of the present invention is to provide a novel method for efficiently treating wastewater containing furan compounds. The second object of the present invention is to provide a technique for culturing microorganisms at high concentrations in order to obtain a large amount of microorganisms to be used in wastewater treatment and the like. [Means for solving the problem]

[0011] The present inventors have conducted extensive research in light of the problems of the prior art and have found that in the biological treatment of wastewater containing furan compounds, the degradation of the activity of activated sludge and the like can be prevented and wastewater treatment can be performed efficiently by bringing the compounds into contact with microorganisms having hydroxymethylfurfural and / or furfural oxidase activity to decompose the compounds, thereby completing the present invention.

[0012] That is, the present invention provides the following [1] to

[21] . [1] A microbial preparation for treating wastewater containing furan compounds, comprising at least one microorganism selected from the group consisting of microorganisms belonging to the genus Commamonas, microorganisms belonging to the genus Burkholderia, microorganisms belonging to the genus Paraburkholderia, and microorganisms belonging to the genus Pseudomonas. [2] The microbial preparation according to [1], wherein the microorganism belonging to the genus Commamonas is Commamonas testosteroni and / or Commamonas thiooxydans, the microorganism belonging to the genus Burkholderia is Burkholderia multivorans, the microorganism belonging to the genus Paraburkholderia is Paraburkholderia xenovorans, and the microorganism belonging to the genus Pseudomonas is Pseudomonas putida or Pseudomonas oryzihabitans. [3] The microbial preparation according to [1] or [2], wherein the furan compound is furan aldehyde. [4] A wastewater treatment method, comprising a step of contacting wastewater containing furan compounds with at least one microorganism selected from the group consisting of microorganisms belonging to the genus Commamonas, microorganisms belonging to the genus Burkholderia, microorganisms belonging to the genus Paraburkholderia, and microorganisms belonging to the genus Pseudomonas. [5] The wastewater treatment method according to [4], wherein the microorganisms belonging to the genus Commamonas are Commamonas testosteroni and / or Commamonas thiooxydans, the microorganisms belonging to the genus Burkholderia are Burkholderia multivorans, the microorganisms belonging to the genus Paraburkholderia are Paraburkholderia xenovorans, and the microorganisms belonging to the genus Pseudomonas are Pseudomonas putida or Pseudomonas oryzihabitans. [6] The wastewater treatment method according to [4] or [5], wherein the furan compound is furan aldehyde. [7] A wastewater treatment method, comprising a step of contacting wastewater containing furan compounds with at least one microorganism selected from the group consisting of microorganisms belonging to the genus Commamonas, microorganisms belonging to the genus Burkholderia, microorganisms belonging to the genus Paraburkholderia, and microorganisms belonging to the genus Pseudomonas, in the presence of a membrane separation device. [8] (1) A process for contacting wastewater containing furan compounds with at least one microorganism selected from the group consisting of microorganisms belonging to the genus Commamonas, microorganisms belonging to the genus Burkholderia, microorganisms belonging to the genus Paraburkholderia, and microorganisms belonging to the genus Pseudomonas; and (2) A wastewater treatment method comprising a step of contacting the wastewater obtained in the step (1) with at least one selected from activated carbon, a Fenton catalyst, and a polycyclic aromatic hydrocarbon-degrading enzyme. [9] The wastewater treatment method according to any one of [4] to [8], wherein the CODcr value of the resulting wastewater is 500 ppm or less.

[10] A method for culturing a microorganism belonging to the genus Commamonas, a microorganism belonging to the genus Burkholderia, or a microorganism belonging to the genus Paraburkholderia, comprising the step of culturing the microorganism in a medium containing gluconic acid.

[11] A method for culturing a microorganism belonging to the genus Commamonas, comprising the step of culturing the microorganism in a medium containing at least one selected from gluconic acid, ethanol, and succinic acid.

[12] The method according to

[10] or

[11] , wherein the optical density of the culture medium containing the microorganism at a wavelength of 660 nm is 15 or more 24 hours after the start of culture.

[13] The method according to

[10] or

[11] , wherein the optical density of the culture medium containing the microorganism at a wavelength of 660 nm is 20 or more 48 hours after the start of culture.

[14] The method according to any one of

[10] to

[13] , wherein the concentrations of gluconic acid, ethanol, and succinic acid present in the medium are all 10 g / L or less.

[15] The method according to any one of

[10] to

[14] , wherein the growth rate of the microorganism is 0.2 g / L / hr or more in terms of dry cell weight.

[16] The method according to any one of

[10] to

[15] , wherein the culture is carried out by fed-batch culture.

[17] The method according to any one of

[10] to

[16] , which comprises a step of aerating a gas containing 90% v / v or more oxygen into the culture solution.

[18] The method according to

[17] , wherein the aeration is carried out at an aeration rate of 6 to 5 vvm.

[19] A method for producing a microbial preparation according to any one of [1] to [3], comprising a step of freeze-drying a composition comprising a microorganism obtained by the method according to any one of

[10] to

[18] and a cryoprotectant in an amount 1 to 10 times the dry weight of the microorganism.

[20] The method according to

[19] , wherein the cryoprotectant is at least one selected from trehalose, skim milk, and glutamic acid.

[21] A microbial preparation according to any one of [1] to [3], which is produced by the method described in

[19] or

[20] . [Effects of the Invention]

[0013] According to the present invention, it is possible to suppress a decrease in the effectiveness of activated sludge and efficiently decompose furan compounds contained in wastewater. Furthermore, according to the present invention, in addition to furan compounds, acid components such as formic acid and acetic acid, and sugars such as xylose contained in wastewater can also be decomposed. Furthermore, by combining treatment with activated carbon, a Fenton catalyst, and a polycyclic aromatic hydrocarbon-degrading enzyme after microbial treatment, it is possible to reduce the CODcr value of the wastewater and achieve wastewater treatment that complies with strict wastewater treatment standards. Furthermore, according to the present invention, it is possible to high-density culture microorganisms belonging to the genus Commamonas, the genus Burkholderia, or the genus Paraburkholderia, which are used in wastewater treatment. This allows the microorganisms to be obtained in large quantities in a short period of time. [Brief explanation of the drawings]

[0014] [Figure 1] Decomposition of furfural, formic acid, and acetic acid by Commamonas testosteroni NBRC12047 (changes in concentration over 48 hours). Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). [Figure 2] Decomposition of furfural, formic acid, and acetic acid by Commamonas thiooxydans NBRC 110656 (changes in concentration over 48 hours) is shown. Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). [Figure 3] Decomposition of furfural, formic acid, and acetic acid by Paraburkholderia xenovorans DSM17367 (changes in concentration over 48 hours). Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). [Figure 4]Decomposition of furfural, formic acid, and acetic acid by Pseudomonas putida NBRC3738 (change in concentration over 48 hours). Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). [Figure 5] This shows the change over time in the CODcr removal rate of treated water using Commamonas testosteroni NBRC12047 strain when a membrane separation device is used. Vertical axis (CODcr removal rate % (treated water / raw water)), horizontal axis (number of days) [Figure 6] Decomposition of furfural, formic acid, and acetic acid by Burkholderia multivorans strain NBRC102086 (changes in concentration over 48 hours). Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). [Figure 7] Decomposition of furfural, formic acid, and acetic acid by Paraburkholderia caledonica strain NBRC102488 (change in concentration over 48 hours). Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). [Figure 8] Decomposition of furfural, formic acid, and acetic acid by Pseudomonas oryzihabitans NBRC10219ans (changes in concentration over 48 hours) is shown. Vertical axis (concentration [g / L]), horizontal axis (time [h]). Dotted line: ▲ (FRL: furfural), solid line: ◆ (FA: formic acid), dashed line: ■ (ACE: acetic acid). DETAILED DESCRIPTION OF THE INVENTION

[0015] 1. Wastewater treatment method The present invention relates to a wastewater treatment method for biologically treating wastewater containing furan compounds, characterized in that the wastewater is brought into contact with microorganisms having hydroxymethylfurfural and / or furfural oxidase activity.

[0016] (1) Wastewater to be treated In the present invention, the wastewater to be treated is wastewater containing furan compounds. The origin of the wastewater is not limited as long as it contains furan compounds. Examples include wastewater generated in a process for producing furan compounds from petroleum feedstocks, and wastewater generated in the production of furfural from C5 sugars extracted from biomass (e.g., bagasse, the residue left after squeezing sugarcane).

[0017] In addition to furan compounds, wastewater may contain acid components such as formic acid, acetic acid, and lactic acid, and sugars such as xylose. The wastewater generated in the process of producing furan compounds from biomass also contains these acid components and sugars. These acid components and sugars, like furan compounds, also make biological wastewater treatment difficult, so they are preferably removed from the wastewater.

[0018] The wastewater may be pretreated as needed before being subjected to microbial treatment. For example, if the wastewater is excessively acidic due to a high content of acid components, an alkaline component can be added to adjust the pH to 5 to 9, preferably 5.5 to 8.5, and more preferably 6 to 8. There are no limitations on the alkaline component used, and for example, NaOH, KOH, etc. can be used in solid or liquid form.

[0019] (2) Furan compounds Furan compounds are compounds having a furan skeleton, such as furfural, hydroxymethylfurfural, 2-methylfuran, 3-methylfuran, furan, dihydrofuran, furfuryl alcohol, tetrahydrofuran, and tetrahydrofurfuryl alcohol. In the present invention, the target furan compounds are not particularly limited, but furan aldehydes such as furfural and tetrahydrofurfural, which are highly toxic to microorganisms and activated sludge, are particularly desirable targets for treatment (decomposition).

[0020] (3) Microorganisms used in wastewater treatment The microorganism used in the present invention is a "microorganism having hydroxymethylfurfural and / or furfural oxidase activity." "Hydroxymethylfurfural and / or furfural oxidase activity" refers to an enzyme that oxidizes hydroxymethylfurfural and / or furfural to convert it into the corresponding carboxylic acid. Specifically, the enzyme oxidizes hydroxymethylfurfural (also known as 5-hydroxymethylfurfural) to 5-formyl-2-furoic acid and further to 2,5-furandicarboxylic acid, and furfural to 2-furoic acid. Decomposition of hydroxymethylfurfural or oxidation of hydroxymethylfurfural to 5-formyl-2-furoic acid and further to 2,5-furandicarboxylic acid can be confirmed by known means such as HPLC. Similarly, decomposition of furfural or oxidation of furfural to 2-furoic acid can also be confirmed by known means such as HPLC.

[0021] Examples of the microorganism include microorganisms belonging to the genus Commamonas, Burkholderia, Paraburkholderia, and Pseudomonas. One type of microorganism may be used alone, or two or more types may be used in combination.

[0022] Preferred microorganisms belonging to the genus Commamonas include Commamonas acidovorans, Commamonas composti, Commamonas guangdongensis, Commamonas terrae, Commamonas testosteroni, and Commamonas thiooxydans, with Commamonas testosteroni and Commamonas thiooxydans being more preferred, and Commamonas testosteroni being particularly preferred.

[0023] Examples of Commamonas testosteroni that can be used include, but are not limited to, the NBRC 12047 strain, the NBRC 12048 strain, the NBRC 14951T strain, the NBRC 100989 strain, the NBRC 109938 strain, the NBRC 110673 strain, the ATCC 700441 strain, the ATCC 13474 strain, the ATCC 700441D-5 strain, the ATCC 55744 strain, the ATCC 49249 strain, the ATCC 33083 strain, the ATCC 17510 strain, the ATCC 17409 strain, the ATCC 15666 strain, the ATCC 15667 strain, the ATCC 39523 strain, the ATCC 53716 strain, the ATCC 25094 strain, the TA441 strain, the TK102 strain, and the like.

[0024] Examples of Commamonas thiooxydans that can be used include, but are not limited to, the NBRC 110656 strain, the S23 strain, and the CNB-1 strain.

[0025] Preferred examples of microorganisms belonging to the genus Burkholderia include Burkholderia vietnamiensis, Burkholderia lata, Burkholderia cenocepacia, Burkholderia ambifaria, Burkholderia multivorans, Burkholderia cepacia, Burkholderia dolosa, Burkholderia pyrrocinia, Burkholderia contaminans, Burkholderia ubonensis, Burkholderia diffusa, Burkholderia latens, Burkholderia territorii, Burkholderia seminalis, Burkholderia pseudomultivorans, Burkholderia metallica, Burkholderia stagnalis, Burkholderia stabilis, Burkholderia glumae, Burkholderia gladioli, Burkholderia insecticola, and Burkholderia plantarii, with Burkholderia multivorans being more preferred.

[0026] Examples of Burkholderia multivorans that can be used include, but are not limited to, the NBRC 102086 strain, the ATCC 17616D-5 strain, the ATCC 17616 strain, and the ATCC BAA-247 strain.

[0027] Preferred microorganisms belonging to the genus Paraburkholderia include Paraburkholderia xenovorans, Paraburkholderia phymatum, Paraburkholderia phenoliruptrix, Paraburkholderia phenoliruptrix, Paraburkholderia phytofirmans, Paraburkholderia fungorum, Paraburkholderia caribensis, Paraburkholderia sprentiae, Paraburkholderia aromaticivorans, Paraburkholderia hospita, Paraburkholderia terrae, Paraburkholderia graminis, Paraburkholderia caledonica, and Paraburkholderia terricola, with Paraburkholderia xenovorans being more preferred.

[0028] Examples of Paraburkholderia xenovorans that can be used include, but are not limited to, the DSM 17367 strain and the LB400 strain.

[0029] Microorganisms belonging to the genus Pseudomonas include Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas resinovorans, Pseudomonas alcaligenes, Pseudomonas citronellolis, Pseudomonas putida, Pseudomonas fulva, Pseudomonas monteilii, Pseudomonas soli, Pseudomonas plecoglossicida, Pseudomonas oryzihabitans, Pseudomonas syringae, Pseudomonas savastanoi, Pseudomonas amygdali, Pseudomonas cichorii, Pseudomonas avellanae, Pseudomonas protegens, Pseudomonas fluorescens, Pseudomonas poae, Pseudomonas synxantha, Pseudomonas mandelii, Pseudomonas trivialis, Pseudomonas corrugata, Pseudomonas veronii, Pseudomonas azotoformans, Pseudomonas orientalis, Pseudomonas simiae, Pseudomonas lurida, Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas balearica, Pseudomonas brassicacearum, Pseudomonas knackmussii, Pseudomonas chlororaphis, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas alkylphenolica, Pseudomonas rhizosphaerae, Pseudomonas cremoricolorata, Pseudomonas parafulva, Pseudomonas versuta, Pseudomonas koreensis, PseudomonasPreferred are Pseudomonas frederiksbergensis, Pseudomonas antarctica, Pseudomonas psychrotolerans, Pseudomonas silesiensis, Pseudomonas yamanorum, Pseudomonas kribbensis, Pseudomonas anguilliseptica, Pseudomonas deceptionensis, Pseudomonas denitrificans, Pseudomonas nitroreducens, Pseudomonas pavonaceae, Pseudomonas testosteroni, etc., and among these, Pseudomonas putida, Pseudomonas testosteroni, etc. are more preferred.

[0030] Examples of Pseudomonas putida include, but are not limited to, ATCC 3738 strain, ATCC 12653 strain, ATCC 12668 strain, ATCC 12996 strain, ATCC 13696 strain, ATCC 14164T strain, ATCC 14671 strain, ATCC 14796 strain, ATCC 15366 strain, ATCC 100650 strain, ATCC 100651 strain, ATCC 100988 strain, ATCC 101019 strain, ATCC 101020 strain, ATCC 102090 strain, ATCC 102092 strain, ATCC 102093 strain, ATCC 109109 strain, ATCC 109110 strain, ATCC 109347 strain, ATCC 109348 strain, ATCC ATCC 109349 strain, ATCC 109350 strain, ATCC 110474 strain, ATCC 110475 strain, ATCC 110476 strain, ATCC 110477 strain, ATCC 110482 strain, ATCC 110654 strain, ATCC 110666 strain, ATCC 110667 strain, etc. can be used.

[0031] The microorganisms used may be commercially available microorganisms, for example, from the American Type Culture Collection (ATCC) or the NITE Biological Resource Center (NBRC), or may be microorganisms collected from soil, wastewater, etc. Furthermore, the microorganisms used may be wild-type microorganisms or genetically modified microorganisms.

[0032] (4) Wastewater treatment method (4-1) Treatment with Microorganisms The wastewater can be treated by contacting the wastewater with the microorganisms. The method for contacting the wastewater with the microorganisms is not particularly limited, and the microorganisms may be added to the wastewater, or the wastewater may be added to the microorganisms.

[0033] The wastewater treatment using microorganisms may be a continuous reaction or a batch reaction, which can be appropriately selected by those skilled in the art depending on the amount and type of wastewater.

[0034] The amount of microorganisms to be added is not limited and can be appropriately set depending on the amount and quality of the wastewater to be treated, etc. The microorganisms may be added all at once at the start of the reaction, or may be added multiple times.

[0035] When the microorganisms are added multiple times, they may be added at a constant pace, or may be added as needed while observing the wastewater treatment rate, etc.

[0036] The treatment time with the microorganisms (the time during which the microorganisms are in contact with the wastewater) is not particularly limited, and for example, the treatment can be continued until the compound to be decomposed is reduced to below the detection limit.

[0037] The temperature of the wastewater during microbial treatment is not limited as long as it allows efficient microbial treatment, and may be, for example, 15 to 60°C, preferably 20 to 50°C, and more preferably 25 to 45°C.

[0038] The pH of the wastewater to be treated can be appropriately set so that the treatment by microorganisms can be carried out efficiently.

[0039] (4-2) Use of membrane separation equipment The wastewater treatment method of the present invention can also use (or use in combination with) a membrane separation method. Membrane separation refers to a method in which water (treated water) treated by microorganisms is separated using a separation membrane (in the presence of a membrane separation device). Among these, a preferred embodiment of the present invention is a membrane bioreactor (MBR). MBR is a type of activated sludge process in which treated water and activated sludge are separated using a microfiltration membrane (MF membrane) or an ultrafiltration membrane (UF membrane). Treating wastewater with microorganisms having hydroxymethylfurfural and / or furfural oxidase activity decomposes furan compounds in the wastewater, suppressing a decline in the capacity of activated sludge and enabling efficient wastewater treatment.

[0040] In the present invention, the membrane separation device can be installed in a tank where wastewater is treated with microorganisms, or it can be installed in a tank separate from the tank where wastewater is treated with microorganisms, and treated water can be introduced into the separate tank for membrane separation. Multiple separate tanks can also be installed. By treating microorganisms in the presence of a membrane separation device, the efficiency of the microbial reaction can be improved. As a result, it is possible to reduce the amount of microorganisms required for wastewater treatment and shorten the treatment (reaction) time.

[0041] The type and size of the membrane separation device used in the present invention are not particularly limited, and can be appropriately selected depending on the size of the wastewater treatment facility, the amount of wastewater, etc.

[0042] The type of separation membrane used in the membrane separation device is preferably a microfiltration membrane (MF membrane) or an ultrafiltration membrane (UF membrane).

[0043] The shape of the separation membrane may be a hollow fiber membrane, a flat membrane, a tubular membrane, a bag-shaped membrane, etc. Among these, a hollow fiber membrane is preferred because it allows for a high degree of integration of the membrane area when compared on a volume basis.

[0044] Examples of materials for the separation membrane include organic materials (cellulose, cellulose acetate, polyolefins such as polyethylene and polypropylene, aromatic polyamides, polysulfones, polyvinyl alcohol, polymethyl methacrylate, polyvinylidene fluoride, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, polytetrafluoroethylene, etc.), metals (stainless steel, etc.), and inorganic materials (ceramics, etc.). The material for the separation membrane is selected appropriately depending on the properties of the wastewater, etc.

[0045] The pore size of the separation membrane may be selected appropriately depending on the purpose of treatment. In the membrane bioreactor (MBR) described below, the pore size of the separation membrane is usually preferably 0.001 to 3 μm. If the pore size is less than 0.001 μm, the resistance of the membrane is likely to increase. If the pore size exceeds 3 μm, the sludge cannot be completely separated, which may result in a deterioration in the quality of the treated water (permeate). The pore size of the separation membrane is more preferably 0.04 to 1.0 μm, which is the range of a microfiltration membrane.

[0046] In the present invention, the membrane separation device may be prepared using a commercially available separation membrane, or a commercially available membrane separation device may be used. For example, a hollow fiber membrane SADF (trade name "Sterapor SADF") manufactured by Mitsubishi Chemical Corporation may be used. TM "), a module using the DiaFellow membrane separation activated sludge process manufactured by Mitsubishi Chemical Corporation. TM AM, etc. In the wastewater treatment method of the present invention, one or more membrane separation devices may be installed in the wastewater treatment facility.

[0047] The amount of aeration when using a membrane separation device is not particularly limited, and can be appropriately selected depending on the amount and quality of wastewater, the type of microorganisms used, and the like.

[0048] (4-3) Primary treatment In the present invention, prior to treating wastewater with microorganisms, a process for removing solids from the wastewater (hereinafter referred to as "primary treatment") can be carried out. Examples of such a process include a screen that removes large debris with a net or fence, a grit basin that removes sand by settling, or a primary sedimentation basin that removes mud by settling.

[0049] (4-4) Secondary treatment In the present invention, a treatment for removing organic matter from wastewater using microorganisms such as activated sludge (hereinafter referred to as "secondary treatment") can be carried out.

[0050] (4-5) Tertiary treatment In the present invention, if there are compounds that have not been completely treated by the microorganisms, a further treatment (hereinafter referred to as "tertiary treatment") can be carried out after the microbial treatment in order to remove the compounds. By combining the tertiary treatment, the quality of the wastewater can be improved and the CODcr value, which will be described later, can be further reduced.

[0051] The type of tertiary treatment is not particularly limited and can be appropriately selected depending on the type of wastewater, the type of compounds that could not be treated by microorganisms, etc. Examples include treatment with activated carbon, treatment with a Fenton catalyst, and treatment with a polycyclic aromatic oxidation catalyst.

[0052] (4-3-1) Activated carbon treatment Activated carbon can remove compounds that have not been completely removed by microorganisms from wastewater by adsorbing them. The activated carbon used is preferably one obtained by carbonizing (heat treating) mineral raw materials such as petroleum pitch, coal, and coke, or plant raw materials such as wood and fruit shells such as coconut shells, or by subjecting them to heat treatment followed by activation. Commercially available activated carbon for liquid phase use can be used.

[0053] The method of treatment using activated carbon is not particularly limited, and can be carried out, for example, by filling a cylindrical object such as a column with activated carbon and passing the wastewater to be treated through it (activated carbon adsorption tower). The space velocity (SV) of the water passing through the activated carbon adsorption tower is not particularly limited. For example, the SV can be determined appropriately depending on the ease and amount of adsorption of components contained in the wastewater to activated carbon, the desired water quality requirements after treatment, the effluent standard values, etc. For example, if adsorption to activated carbon is difficult (the amount of adsorption is small) or if the water quality requirements for the wastewater are strict, the SV can be reduced and the contact time between the water to be treated (wastewater) and activated carbon can be extended.

[0054] (4-3-2) Fenton catalyst treatment The Fenton reaction is a reaction in which hydrogen peroxide reacts with ferrous ions (iron catalyst) to generate hydroxyl radicals. Hydroxy radicals have strong oxidizing power, and this oxidizing power can be used to decompose harmful substances and persistent pollutants, as well as for sterilization. The Fenton catalyst is the iron catalyst used in this Fenton reaction. The Fenton catalyst can remove compounds that could not be completely treated by microorganisms from wastewater by breaking them down with hydroxyl radicals.

[0055] The iron catalyst serving as the Fenton catalyst is not particularly limited as long as it dissolves in water to generate ferrous ions. For example, ferrous salts or ferrous oxides are preferred. Among them, ferrous sulfate or ferrous chloride are more preferred because they do not require management under wastewater standards and have excellent solubility.

[0056] The treatment method using the Fenton catalyst is not particularly limited as long as it can decompose the target compound. For example, an iron reagent is added to the wastewater to be treated while stirring the wastewater as needed, and then hydrogen peroxide is added to cause a reaction. The amount of iron reagent added, the amount of hydrogen peroxide added, the reaction time after adding both, and the like can be appropriately selected depending on the type of compound contained in the wastewater to be treated, the amount of wastewater, and the like.

[0057] (4-3-3) Polycyclic aromatic hydrocarbon degrading enzyme treatment Polycyclic aromatic hydrocarbon-degrading enzymes can decompose persistent polycyclic aromatic hydrocarbons that have not been completely treated by microorganisms and remove them from wastewater. The type of polycyclic aromatic hydrocarbon-degrading enzyme to be used is not particularly limited, but preferred is one that has the activity of catalyzing the oxidation and polymerization of persistent polycyclic aromatic hydrocarbons such as humins in the presence of hydrogen peroxide. Examples of such polycyclic aromatic hydrocarbon-degrading enzymes include peroxidase and laccase.

[0058] The peroxidase preferably used is derived from horseradish (Armorica rusticana) of the Brassicaceae family, and the laccase preferably used is derived from Trametes versicolor, Rhus vernicifera, Agaricus bisporus, Aspergillus sp., or the like.

[0059] The method of treatment with the polycyclic aromatic hydrocarbon-degrading enzyme is not particularly limited, and the enzyme may be added to the wastewater to be treated, or the enzyme may be immobilized and used. The amount of polycyclic aromatic hydrocarbon-degrading enzyme added to the wastewater is appropriately determined depending on the type and amount of the wastewater, and may be, for example, 0.1 to 300 ppm, preferably 0.5 to 200 ppm, and more preferably 1 to 100 ppm. The treatment time with the polycyclic aromatic hydrocarbon-degrading enzyme is also not particularly limited, and is appropriately determined depending on the amount and quality of the wastewater, the type and quality of the enzyme used, etc.

[0060] (5) Wastewater obtained by the present invention According to the present invention, furan compounds contained in wastewater can be efficiently decomposed. In addition to furan compounds, acid components such as formic acid and acetic acid, and sugars such as xylose can also be efficiently decomposed.

[0061] Furthermore, by treating the wastewater after microbial treatment with activated carbon, Fenton's catalyst, polycyclic aromatic hydrocarbon degrading enzymes, etc. (tertiary treatment), substances that could not be removed by microbial treatment can be removed, resulting in wastewater of higher quality.

[0062] COD (chemical oxygen demand) and BOD (biological oxygen demand) are commonly used as indicators of water quality. COD is the amount of oxygen required to oxidize the amount of oxidizable substances in water, while BOD is the amount of oxygen required to oxidize only biodegradable organic matter. COD is measured using potassium dichromate as an oxidizing agent (CODcr), potassium permanganate as an oxidizing agent (CODMn), and alkaline or potassium manganate (CODn). OH etc.

[0063] According to wastewater treatment standards, wastewater treated by the method of the present invention can produce high-quality wastewater in which not only furan compounds but also acid components such as formic acid and acetic acid and sugars such as xylose have been decomposed. The wastewater obtained by the method of the present invention has a CODcr value of less than 500 ppm, preferably less than 120 ppm, and complies with strict wastewater treatment standards.

[0064] 2. Microbial preparations for wastewater treatment The present invention also provides a microbial preparation for use in the wastewater treatment method described above. The target furan compounds are not particularly limited, but furan aldehydes such as furfural and tetrahydrofurfural, which are highly toxic to microorganisms and activated sludge, are particularly desirable targets for treatment (decomposition).

[0065] The microbial preparation of the present invention may be a liquid (suspension) or a solid. When the microbial preparation is used as a liquid, the microorganisms are cultured to a desired concentration, and then the preparation can be used as is or with the addition of additives such as preservatives and stabilizers. Furthermore, the microorganisms may be cultured to a desired concentration, and then, as necessary, separated, washed, purified, concentrated, or further suspended in an aqueous solution containing a buffer or the like, and then used as a microbial preparation.

[0066] When the microbial preparation is used as a solid, the microorganisms are cultured to a desired concentration, and then trehalose, sodium glutamate, skim milk, or the like is added as a freeze-drying protectant as needed, followed by freeze-drying. The freeze-dried cells can be used as is or in combination with various additives as the microbial preparation.

[0067] The microbial formulation of the present invention contains at least one microorganism selected from the group consisting of microorganisms belonging to the genus Commamonas, Burkholderia, Paraburkholderia, and Pseudomonas as an active ingredient, which has hydroxymethylfurfural and / or furfural oxidase activity.

[0068] Specific examples of the microorganisms are as described in 1(3). Among them, preferred microorganisms belonging to the genus Commamonas include Commamonas testosteroni and Commamonas thiooxydans, preferred microorganisms belonging to the genus Burkholderia include Burkholderia multivorans, preferred microorganisms belonging to the genus Paraburkholderia include Paraburkholderia xenovorans, and preferred microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

[0069] The microbial preparation of the present invention contains the above-mentioned microorganisms and components necessary for maintaining the microorganisms, and is used by adding it to activated sludge. Alternatively, the microbial preparation may be prepared by mixing the microorganisms of the present invention with existing activated sludge. 3. Microbial preparations for decomposing furan compounds The present invention also provides the above-mentioned microbial preparation for decomposing furan compounds. The target furan compounds are not particularly limited, but furan aldehydes such as furfural and tetrahydrofurfural are preferred.

[0070] The microbial preparation for decomposing furan compounds of the present invention contains at least one of Commamonas testosteroni, Commamonas thiooxydans, and Paraburkholderia xenovorans, and components necessary for maintaining the microorganism, and is used for decomposing furan compounds.

[0071] 4. Use of microorganisms for wastewater treatment or degradation of furan compounds. The present invention also provides use of the microorganism described in (3) of 1 above for treating wastewater containing furan compounds or for differentiation of furan compounds. Such use can be carried out according to the description of 1 above.

[0072] 5. Methods for culturing microorganisms. The present invention also provides a method for high-density cultivation of microorganisms belonging to the genus Commamonas, microorganisms belonging to the genus Burkholderia, or microorganisms belonging to the genus Paraburkholderia.

[0073] As used herein, "cultivating at a high density" or "high-density culture" means culturing microorganisms at a high concentration. The term "high density" is not particularly limited, but may refer to, for example, the optical density (OD ) of a medium containing microorganisms at a wavelength of 660 nm 24 hours after the start of culture. 660 ) is 15 or more, preferably 16 or more, more preferably 18 or more, and even more preferably 20 or more. Alternatively, "high concentration (high density)" means that the optical density (OD ) at a wavelength of 660 nm of a medium containing a microorganism at 48 hours after the start of culture is 660 ) is 20 or more, preferably 21 or more, more preferably 22 or more, even more preferably 23 or more, and particularly preferably 25 or more.

[0074] OD 660 can be measured by diluting the culture medium and using an ultraviolet-visible spectrophotometer (UV-1280, Shimadzu Corporation).

[0075] In the culture method of the present invention, the growth rate of microorganisms is increased, thereby enabling the above-mentioned high-density culture.

[0076] The growth rate of a microorganism can be measured using known methods such as direct microscopic examination, plate culture, turbidity measurement, and gravimetric measurement. In the gravimetric method, the growth rate can be calculated by dividing the dry weight of the microorganisms by the culture time. The dry cell weight of a microorganism can be determined by placing a certain volume of washed culture medium on a pre-weighed weighing tube or aluminum dish, drying it, and then reweighing it to measure the dry weight per volume from the change in weight before and after.

[0077] According to the culture method of the present invention, the growth rate of the microorganism is 0.2 g / L / hr or more, preferably 0.3 g / L / hr or more, and more preferably 0.4 g / L / hr or more, based on gravimetric dry cell weight (g-DCW / L).

[0078] (1) Microorganisms The microorganisms that can be cultured by the method of the present invention are those belonging to the genus Commamonas, Burkholderia, or Paraburkholderia. Specific examples of these microorganisms are as described in 1(3).

[0079] (2) Cultivation of microorganisms (main culture) (2-1) Culture medium The medium refers to a substance that provides a growth environment for culturing microorganisms, and specifically refers to an aqueous medium such as water in which a carbon source, a nitrogen source, inorganic salts, etc. are dissolved.

[0080] The term "initial medium" refers to a medium into which a pre-culture (seed) is inoculated to start the culture (main culture) when culturing is performed by the fed-batch culture method described below.

[0081] The term "fed medium" refers to a medium that is added to the initial medium continuously or intermittently as the culture time elapses after the start of culture using the initial medium.

[0082] In the present invention, when a microorganism belonging to the genus Burkholderia or a microorganism belonging to the genus Paraburkholderia is cultured, the initial medium and the feed medium contain gluconic acid, and when a microorganism belonging to the genus Commamonas is cultured, the initial medium and the feed medium contain at least one selected from gluconic acid, ethanol, and succinic acid.

[0083] Both the initial medium and the feed medium are preferably sterilized before use in the culture. The method for sterilizing the medium is not limited as long as it can make the medium sterile and free of proliferative microorganisms. Examples of sterilization methods include pressure sterilization (autoclave; e.g., heat sterilization at 121°C for 20 minutes) and filtration sterilization (e.g., filtration through a filter with a pore size of 0.45 μm or 0.2 μm). If there is a concern about reactions between medium components during heat sterilization, one or more medium components may be sterilized separately from the other medium components, and the components may be mixed after sterilization.

[0084] (2-1-1) Initial medium The initial medium may be any medium that contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the microorganism and that allows the microorganism to be cultured at high density in a short period of time.

[0085] Carbon sources include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose, and starch; organic acids such as acetic acid, propionic acid, gluconic acid, and succinic acid; and alcohols such as ethanol and propanol.

[0086] As described above, the initial medium of the present invention contains at least one selected from gluconic acid, ethanol, and succinic acid, and preferably contains gluconic acid and / or ethanol.

[0087] Nitrogen sources that can be used include complex medium components derived from natural sources (microorganisms, plants, animal milk, animal meat, etc.); ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; ammonia; and other amino acids and nitrogen compounds.

[0088] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium carbonate, and ammonium molybdenum heptamolybdate.

[0089] If necessary, ingredients other than those mentioned above can be added to the initial medium, such as vitamins, or an antifoaming agent to prevent foaming of the medium during culture.

[0090] The initial medium includes, as a basal medium, for example, K1 mineral medium (JOURNAL OF BACTERIOLOGY, Dec. 2005, pp. 7996-8005), P. putida medium (Biotechnology and Bioengineering, Vol. 112, No. 4, April 2015), MM medium (dipotassium hydrogen phosphate 3.9 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate dihydrate 2.1 g / L, EDTA 10.0 mg / L, magnesium chloride hexahydrate 100 mg / L, zinc sulfate heptahydrate 2.0 mg / L, iron sulfate heptahydrate 5.0 mg / L, manganese chloride tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.2 mg / L, cobalt chloride hexahydrate 0.4 mg / L, Known inorganic media such as ammonium molybdenum heptamolybdate tetrahydrate 0.2 mg / L, calcium chloride dihydrate 1.0 mg / L, etc., can be used. Among these, MM medium is preferred.

[0091] In the present invention, when microorganisms belonging to the genus Burkholderia or Paraburkholderia are cultured, gluconic acid is added to the above-mentioned mineral medium.When microorganisms belonging to the genus Commamonas are cultured, at least one selected from gluconic acid, ethanol, and succinic acid, preferably gluconic acid and / or ethanol, is added to the above-mentioned mineral medium.

[0092] Gluconic acid may be used in the form of a salt thereof such as sodium gluconate or potassium gluconate. Succinic acid may be used in the form of a salt thereof such as sodium succinate or potassium succinate.

[0093] The amount of gluconic acid used is not particularly limited as long as it allows the microorganism to be cultured at a high concentration, and can be, for example, 50 g / L or less, preferably 30 g / L or less, and more preferably 10 g / L or less, based on the total volume of the medium at the start of the culture. By using a concentration of 10 g / L or less, the microorganism can be cultured at a higher concentration and with a higher yield. The reason for using a concentration of 110 g / L or less is that it becomes difficult to obtain the desired effect at a higher concentration.

[0094] The amount of ethanol used is not particularly limited as long as it allows the microorganisms to be cultured at a high concentration, and can be, for example, 30 g / L or less, preferably 20 g / L or less, and more preferably 10 g / L or less, based on the total volume of the medium at the start of the culture. By using an amount of 10 g / L or less, the microorganisms can be cultured at a higher concentration and with a higher yield. The reason for using an amount of 110 g / L or less is that it becomes difficult to obtain any effect at higher amounts.

[0095] The amount of succinic acid used is not particularly limited as long as it allows the microorganism to be cultured at a high concentration. For example, at the start of the culture, it can be 50 g / L or less, preferably 30 g / L or less, and more preferably 10 g / L or less, based on the total volume of the medium. By using 10 g / L, the microorganism can be cultured at a higher concentration and with a higher yield. The reason for using 110 g / L or less is that it becomes difficult to obtain the desired effect at a higher concentration.

[0096] (2-1-2) Fed-batch medium In the present invention, in order to culture microorganisms at high density, it is preferable to add a feed medium to the initial medium during the culture (in the culture process).

[0097] The components of the feed medium are not limited as long as the growth rate of the microorganism during culture is sufficiently maintained. In the present invention, the same components as those of the initial medium can be used.

[0098] When culturing a microorganism belonging to the genus Burkholderia or a microorganism belonging to the genus Paraburkholderia, the feed medium contains gluconic acid, and when culturing a microorganism belonging to the genus Commamonas, the feed medium contains at least one selected from gluconic acid, ethanol, and succinic acid.

[0099] The other ingredients and their amounts are not limited as long as the growth rate of the microorganisms during culture is maintained sufficiently, and can be appropriately selected by those skilled in the art.

[0100] The volume of the feed medium to be added is not limited as long as it allows sufficient growth of the microorganisms used in the present invention, and may be, for example, 0.1 to 1 times the volume of the initial medium, preferably 0.15 to 0.75 times the volume of the initial medium, and more preferably 0.2 to 0.5 times the volume of the initial medium. The reason for using a volume of 0.1 times or more is that it allows the microorganisms to be cultured at high density. The reason for using a volume of 300 g / L or less is that adding more than this to the medium makes it difficult to obtain the desired effect compared to the amount used.

[0101] (2-2)Culture method (2-2-1)Culture method Culture methods that can be used in the present invention include batch culture, fed-batch culture (semibatch culture, fed-batch culture), and continuous culture (perfusion culture), and among these, fed-batch culture is preferred.

[0102] The fed-batch culture method refers to a culture method in which a medium is continuously or intermittently fed (added) to a medium (e.g., an initial medium) during culture, and the medium is not removed from the vessel until the end of culture.

[0103] The feeding mode may be any method that allows recombinant microorganisms to be cultured to high densities in a short period of time and that allows efficient production of recombinant proteins, such as constant, exponential, stepwise increase, specific growth-rate control, pH-stat, DO-stat, glucose concentration control, acetate concentration monitoring, and pulse feed.

[0104] There are no limitations on the method of adding the feed medium as long as the growth and proliferation of the microorganisms during cultivation is maintained. For example, the constant flow feed method is a method in which the feed medium is added continuously or intermittently at a constant mass or volume flow rate. The flow rate is the amount of fluid moving per unit time.

[0105] The pulse feed method is a method in which dissolved oxygen is consumed as the substrate is decomposed, and then a constant mass flow rate or volume flow rate of the feed medium is added when the dissolved oxygen level rises again.

[0106] The addition of dissolved oxygen can be started when 80 to 99.9% of the dissolved oxygen in the initial medium has been consumed, preferably when 85 to 99.5% has been consumed, and more preferably when 90 to 99% has been consumed and the dissolved oxygen level starts to increase again. From the viewpoints of workload and improving bacterial yield, the pulse feed method is preferred, as this allows microorganisms to be cultured at high density in a short period of time by adding the feed medium.

[0107] The amount and rate of addition of the feed medium are not limited as long as the above concentrations of gluconic acid, ethanol, and succinic acid are maintained in the medium during culture and the growth and proliferation of the microorganisms are sufficiently maintained. For example, increasing the concentrations of the medium components is preferable because it requires less amount of feed medium to be added.

[0108] (2-2-2) Culture temperature The culture temperature is not limited as long as the microorganisms used in the present invention grow and proliferate sufficiently. For example, the temperature can be 10°C to 45°C, preferably 15°C to 40°C, and more preferably 20°C to 37°C. The temperature can also be changed during culture as needed. By setting the culture temperature at 10°C or higher, microorganisms can be cultured at a high concentration, and by setting the culture temperature at 45°C, a decrease in the culture rate can be suppressed.

[0109] (2-2-3) pH The pH of the medium during culture is not limited as long as the microorganisms used in the present invention grow and proliferate sufficiently. For example, the pH of the medium can be set to 3 to 9, preferably 3.5 to 8.5, and more preferably 5 to 8. This is because culturing at a pH within this range allows microorganisms to be cultured at high density in a short period of time.

[0110] To control the pH during cultivation, inorganic or organic acids or alkaline solutions can be used. The preferred acids are inorganic acids, such as sulfuric acid, phosphoric acid, hydrochloric acid, and nitric acid. Alkalis include potassium hydroxide, sodium hydroxide, and ammonia.

[0111] (2-2-4) Ventilation It is also possible to aerate the culture solution during cultivation. For example, when culturing the microorganisms used in the present invention at high concentrations, it is preferable to aerate a gas containing a higher concentration of oxygen. The concentration of oxygen contained in the gas to be aerated can be selected appropriately depending on the type of microorganism to be cultured, the culture conditions, etc., and can be, for example, 20% v / v or more, preferably 50% v / v or more, and more preferably 90% v / v or more. This is because aeration of a gas containing 90% v / v or more of oxygen promotes the growth of the microorganisms.

[0112] The aeration rate can be appropriately selected depending on the culture conditions, such as the size of the culture tank, the type of microorganism to be cultured, and the like, and can be, for example, 0.6 to 10 vvm (1.2 to 20 L / min), preferably 0.8 to 8 vvm (1.6 to 16 L / min), and more preferably 1 to 5 vvm (2 to 10 L / min). By setting the aeration rate to 0.6 vvm or more, microorganisms can be cultured at a high concentration. The reason for setting the aeration rate to 10 vvm or less is that even if the aeration rate is increased beyond this, it is difficult to achieve the above-mentioned effects.

[0113] (2-2-5) Pressure The pressure during cultivation is not particularly limited. Cultivation can be carried out at atmospheric pressure, or, if necessary, under increased pressure. The pressure can be, for example, 0 to 0.5 MPa, preferably 0.01 to 0.3 MPa, and more preferably 0.02 to 0.2 MPa. By increasing the pressure, the dissolved oxygen concentration in the medium increases, allowing the microorganisms to be cultivated at a higher concentration.

[0114] (2-2-6) Mixing In the present invention, the culture medium can be cultured while being stirred as needed. The stirring speed can also be appropriately selected depending on the culture conditions and the type of microorganism. For example, it can be 10 to 2500 rpm, preferably 20 to 2000 rpm, and more preferably 30 to 1500 rpm. By setting the stirring speed to 10 rpm or higher, microorganisms can be cultured at a high concentration. By setting the stirring speed to 3000 rpm or lower, stress on the microorganisms can be reduced.

[0115] (2-2-7)Culture time The culture time is not limited as long as the microorganisms grow and proliferate to a sufficiently high concentration. For example, the culture time may be about 5 to 120 hours, preferably about 10 to 100 hours, more preferably about 15 to 80 hours, and even more preferably about 20 to 60 hours. There are also no particular limitations on when the culture should end, and the culture may be ended once the desired concentration (amount) of microorganisms has been obtained.

[0116] (2-2-8) Other conditions In the present invention, pre-culture can also be carried out as needed. Pre-culture is a culture for preparing seeds to be inoculated into a culture (main culture) in which microorganisms are to be cultured at high density. By carrying out the pre-culture appropriately, the amount of bacterial cells required as seeds for the main culture can be secured.

[0117] The medium used for pre-culture is not particularly limited as long as it does not inhibit high-density cultivation of microorganisms in the main culture. For example, it may contain the same carbon source, nitrogen source, and inorganic salts as the initial medium for the main culture, and other components may also be added as necessary. The culture temperature, pH, pressure, and culture time during pre-culture may also be conditions that do not inhibit the growth of microorganisms in the main culture.

[0118] The culture temperature may be, for example, 10°C to 45°C, preferably 15°C to 45°C, and more preferably 20°C to 37°C. The pH of the medium during culture may be controlled within a certain range using an acid or alkali, as in the main culture, but this is not necessarily required. For example, the pH of the medium may be adjusted to 3 to 9, preferably 3.5 to 8.5, and more preferably 5 to 8, during culture preparation, and the culture may be performed without pH control. The culture may be performed at atmospheric pressure, or, if necessary, at a pressure of 0 to 0.1 MPa, preferably 0.01 to 0.05 MPa. The culture time is not particularly limited, and may be the time required to obtain the amount of bacterial cells necessary for high-density culture of the microorganisms in the main culture. For example, the culture time may be 0.5 to 48 hours, preferably 1 to 30 hours, and more preferably 3 to 24 hours.

[0119] Since the culture medium obtained by the culture method of the present invention contains microorganisms at a high concentration, the culture medium can be used as a microbial preparation as is.

[0120] The microorganisms may be used after washing, further concentration, or addition of additives such as preservatives and stabilizers, if necessary.

[0121] The microorganisms can be freeze-dried by adding trehalose, sodium glutamate, skim milk, etc. as a freeze-protectant, if necessary. The freeze-dried cells can be used as they are or in combination with various additives as a microbial preparation.

[0122] The microorganisms used in the present invention can decompose furfural, acetic acid, formic acid, etc., and therefore the microbial preparation can be used for the wastewater treatment described in 1. [Example]

[0123] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0124] [PART I] [Test Example 1] Decomposition of furan compounds in wastewater over time by Commamonas testosteroni NBRC12047 strain Commamonas testosteroni NBRC12047 strain was added to 20 mL of liquid medium containing 100 ppm, 1000 ppm, and 1300 ppm furfural, formic acid, and acetic acid in a 200 mL Erlenmeyer flask to a final OD660 of 0.005. The flask was plugged with cotton and shaken at 230 rpm at 30°C. Samples were taken at 0, 7, 24, and 48 hours. The suspended solids in the samples were filtered through a 0.45 μm filter, and the concentrations of furfural, formic acid, and acetic acid in the samples were measured using HPLC.

[0125] The measurement conditions were as follows: for furfural, a Nakarai 5C18-MS-II (4.6 ID x 250 mm) column was used. Measurement was performed for 20 minutes using a 20 mM formic acid to methanol ratio of 20:80. For formic acid, acetic acid, and xylose, an ULTRON PS-80H (ID 8.0 mm x 300 mm) column was used. Measurement was performed for 20 minutes using 0.108% perchloric acid.

[0126] The results are shown in Figure 1. As shown in Figure 1, it was confirmed that furfural present at 100 ppm was decomposed to 0 ppm, formic acid present at 1000 ppm was decomposed to 130 ppm, and acetic acid present at 1300 ppm was decomposed to 0 ppm in 48 hours.

[0127] [Test Example 2] Decomposition test of furan compounds in wastewater over time using Commamonas thiooxydans NBRC 110656 strain The same experiment as in Test Example 1 was carried out, except that Commamonas thiooxydans NBRC 110656 strain was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid had been adjusted to 100 ppm, 1800 ppm, and 2500 ppm, respectively, so that the final OD660 was 0.005. The results are shown in Figure 2. As shown in Figure 2, it was confirmed that, within 48 hours, furfural present at 100 ppm had decomposed to 0 ppm, formic acid present at 1800 ppm had decomposed to 220 ppm, and acetic acid present at 2500 ppm had decomposed to 0 ppm.

[0128] [Test Example 3] Decomposition test of wastewater components containing furan compounds over time using Paraburkholderia xenovorans DSM17367 The same experiment as in Test Example 1 was carried out, except that Paraburkholderia xenovorans DSM17367 strain was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid had been adjusted to 100 ppm, 900 ppm, and 1300 ppm, respectively, so that the final OD660 was 0.005. The results are shown in Figure 3. As shown in Figure 3, it was confirmed that, within 48 hours, furfural present at 100 ppm was decomposed to 0 ppm, formic acid present at 900 ppm was decomposed to 200 ppm, and acetic acid present at 1300 ppm was decomposed to 180 ppm.

[0129] [Test Example 4] Decomposition test of wastewater components containing furan compounds over time using Pseudomonas putida NBRC3738 strain The same experiment as in Test Example 1 was carried out, except that Pseudomonas putida NBRC3738 strain was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid had been adjusted to 100 ppm, 900 ppm, and 1200 ppm, respectively, so that the final OD660 was 0.005. The results are shown in Figure 5. As shown in Figure 5, it was confirmed that, within 48 hours, furfural present at 100 ppm was decomposed to 0 ppm, formic acid present at 900 ppm was decomposed to 0 ppm, and acetic acid present at 1200 ppm was decomposed to 0 ppm.

[0130] [Test Example 5] Decomposition test of furan compounds in wastewater over time using Burkholderia multivorans NBRC 102086 strain The same procedure as in Test Example 1 was carried out, except that Burkholderia multivorans NBRC 102086 was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid were adjusted to 100 ppm, 1800 ppm, and 2500 ppm, respectively, so that the final OD660 was 0.005.

[0131] [Test Example 6] Time-dependent treatment of wastewater containing furan compounds using Commamonas testosteroni NBRC12047 strain in a membrane separation device An unblocked membrane module (effective membrane length 125 mm, membrane area 0.022 m) was prepared by uniformly arranging 20 hollow fiber microfiltration membranes (Mitsubishi Chemical Corporation, polyvinylidene fluoride "SADF membrane") in a width of 105 mm and connecting both ends of the hollow fiber microfiltration membranes to annular supports. 2 The membrane module was installed above an aeration pipe inside an aeration tank (10 cm wide, 12 cm deep, 35 cm high) with the membrane length aligned vertically to form a membrane bioreactor.

[0132] Activated sludge (pH 7.0) collected from the wastewater treatment facility at the Mitsubishi Chemical Corporation Science & Innovation Center was loaded into a membrane bioreactor (2000 mL) to achieve a MLSS of 4000 mg / L. The sludge was then heated at 30°C with an aeration rate of 10 L / min and a flow rate of 720 mL / day. Commamonas testosteroni NBRC12047, containing furfural, formic acid, acetic acid, and xylose at concentrations of 100 ppm, 2000 ppm, 200 ppm, and 550 ppm, respectively, was added to the wastewater to achieve a final OD660 of 0.005. The membrane-filtered water was sampled daily. Total organic carbon (TOC) during sampling was measured using a TOC analyzer (Shimadzu Corporation, Total Organic Carbon Analyzer: TOC-V SCN). This measurement was carried out using the combustion oxidation-infrared TOC analysis method in accordance with "JIS K-0102 Industrial Wastewater Testing Method." The CODcr in the samples was determined by multiplying the TOC of each sample by the correlation coefficient between samples with known TOC and CODcr. The results are shown in Figure 5. As shown in Figure 5, it was confirmed that the CODcr removal rate of the treated water was maintained at approximately 90% or more for 27 days.

[0133] [Test Example 7] Activated carbon treatment test of wastewater obtained after treatment of furan compounds with Commamonas testosteroni NBRC12047 strain and activated sludge The wastewater obtained in Test Example 6 was packed into a column of activated carbon (Mitsubishi Chemical Aqua Solutions Co., Ltd.) to a packing height of 35 cm, and the water was passed through the column by gravity for 2 hours at a linear velocity of 1.3 m / h and a space velocity of 41 / h. The CODcr before the water passage was 620 ppm, but after the water passage, the CODcr decreased to 30 ppm.

[0134] [Test Example 8] Fenton catalyst treatment test of wastewater obtained after treatment of furan compounds using Commamonas testosteroni NBRC12047 strain and activated sludge The wastewater obtained in Test Example 6 was placed in a 50 mL Falcon tube, and iron sulfate heptahydrate and hydrogen peroxide were added in molar amounts twice the TOC content of the wastewater. The tube was then left at room temperature (25°C) for one hour. The supernatant of the sample containing the precipitate was filtered through a 0.45 μm filter. The CODcr value before treatment was 520 ppm, but after Fenton treatment, it was reduced to 102 ppm.

[0135] [Test Example 9] Peroxidase and laccase treatment test of wastewater obtained after treatment of wastewater containing furan compounds using Commamonas testosteroni NBRC12047 strain and activated sludge The wastewater obtained in Test Example 6 was placed in a 50 mL Falcon tube, and hydrogen peroxide and peroxidase (derived from horseradish (Armorica rusticana), Fujifilm Wako Pure Chemical Industries, Ltd.) or laccase (derived from Trametes versicolor, Sigma-Aldrich) were added at concentrations of 10 ppm, 5 ppm, and 5 ppm, respectively, and the tubes were then left at room temperature for 24 hours. The supernatant of the sample, in which a precipitate had formed, was filtered through a 0.45 μm filter. The CODcr (CODcr) of 620 ppm before treatment was reduced to 110 ppm after treatment with hydrogen peroxide and peroxidase. Similarly, the CODcr was reduced to 100 ppm after treatment with laccase.

[0136] [Test Example 10] Decomposition test of furan compounds in wastewater over time using Burkholderia multivorans NBRC102086 strain The same experiment as in Test Example 1 was carried out, except that Burkholderia multivorans NBRC 102086 strain was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid had been adjusted to 100 ppm, 200 ppm, and 2000 ppm, respectively, so that the final OD660 was 0.1. The results are shown in Figure 6. As shown in Figure 6, it was confirmed that furfural present at 100 ppm was decomposed to 0 ppm, formic acid present at 200 ppm was decomposed to 0 ppm, and acetic acid present at 2000 ppm was decomposed to 0 ppm within 48 hours.

[0137] [Test Example 11] Decomposition test of furan compounds in wastewater over time using Paraburkholderia caledonica NBRC102488 strain The same procedure as in Test Example 1 was carried out, except that Paraburkholderia caledonica NBRC102488 strain was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid had been adjusted to 100 ppm, 200 ppm, and 2000 ppm, respectively, so that the final OD660 was 0.1. As shown in Figure 7, it was confirmed that, within 48 hours, furfural present at 100 ppm was decomposed to 0 ppm, formic acid present at 200 ppm was decomposed to 0 ppm, and acetic acid present at 2000 ppm was decomposed to 0 ppm.

[0138] [Test Example 12] Decomposition test of furan compounds in wastewater over time using Pseudomonas oryzihabitans NBRC102199 strain The same procedure as in Test Example 1 was carried out, except that Pseudomonas oryzihabitans NBRC102199 strain was added to a liquid medium in which the concentrations of furfural, formic acid, and acetic acid had been adjusted to 100 ppm, 200 ppm, and 2000 ppm, respectively, so that the final OD660 was 0.1. This is shown in Figure 8. As shown in Figure 8, it was confirmed that, in 120 hours, furfural present at 100 ppm was decomposed to 0 ppm, formic acid present at 200 ppm was decomposed to 0 ppm, and acetic acid present at 2000 ppm was decomposed to 0 ppm.

[0139] [PART II] 1. Identifying the optimal carbon source using test tube culture Example 1 (Main culture) A colony of Commamonas testosteroni NBRC 12047 strain was inoculated into 3 mL of main culture medium prepared in a 15 mL test tube, and cultured with shaking at 30°C and 200 rpm for 16 hours.

[0140] The main culture medium (sodium gluconate 10 g / L, dipotassium hydrogen phosphate 3.9 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate dihydrate 2.1 g / L, EDTA 10.0 mg / L, magnesium chloride hexahydrate 100 mg / L, zinc sulfate heptahydrate 2.0 mg / L, ferrous sulfate heptahydrate 5.0 mg / L, manganese chloride tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.2 mg / L, cobalt chloride hexahydrate 0.4 mg / L, ammonium molybdenum heptamolybdate tetrahydrate 0.2 mg / L, calcium chloride dihydrate 1.0 mg / L) was dissolved in water, diluted to a measuring cup, and then heat sterilized (121°C, 20 min) to prepare the medium.

[0141] The bacterial concentration was measured by measuring the absorbance at 660 nm (OD660). The results are shown in Table 1.

[0142] <Example 2> Cultivation was carried out in the same manner as in Example 1, except that 10 g / L of disodium succinate was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 1.

[0143] Example 3 Except for using 10 g / L of glycerol in place of sodium gluconate in the main culture medium, the culture was carried out in the same manner as in Example 1. The results are also shown in Table 1.

[0144] Example 4 Except for using 10 g / L of ethanol in place of sodium gluconate in the main culture medium, the culture was carried out in the same manner as in Example 1. The results are also shown in Table 1.

[0145] <Comparative Example 1> Except for using 10 g / L of glucose in place of sodium gluconate in the main culture medium, the culture was carried out in the same manner as in Example 1. The results are also shown in Table 1.

[0146] <Comparative Example 2> Except for using 10 g / L of xylose in place of sodium gluconate in the main culture medium, the culture was carried out in the same manner as in Example 1. The results are also shown in Table 1.

[0147] <Comparative Example 3> Cultivation was carried out in the same manner as in Example 1, except that 10 g / L of sucrose was used in place of sodium gluconate in the main culture medium.

[0148] [Table 1]

[0149] Table 1 confirms that the Commamonas testosteroni NBRC 12047 strain had a high cell concentration after 16 hours of culture when sodium gluconate, disodium succinate, ethanol, or glycerol was used as the carbon source, whereas it did not grow when glucose, xylose, or sucrose was used as the carbon source.

[0150] <Example 5> Except for using Burkholderia multivorans NBRC 102086 strain as the microorganism, cultivation was carried out in the same manner as in Example 1. The results are shown in Table 2.

[0151] Example 6 Cultivation was carried out in the same manner as in Example 5, except that 10 g / L of disodium succinate was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 2.

[0152] Example 7 Cultivation was carried out in the same manner as in Example 5, except that 10 g / L of glycerol was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 2.

[0153] Example 8 Cultivation was carried out in the same manner as in Example 5, except that 10 g / L of ethanol was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 2.

[0154] <Comparative Example 4> Cultivation was carried out in the same manner as in Example 5, except that 10 g / L of glucose was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 2.

[0155] <Comparative Example 5> Cultivation was carried out in the same manner as in Example 5, except that 10 g / L of xylose was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 2.

[0156] <Comparative Example 6> Cultivation was carried out in the same manner as in Example 5, except that 10 g / L of sucrose was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 2.

[0157] [Table 2]

[0158] Example 9 Except for using Paraburkholderia caledonica NBRC102488 strain as the microorganism, culture was carried out in the same manner as in Example 1. The results are shown in Table 3.

[0159] Example 10 Cultivation was carried out in the same manner as in Example 9, except that 10 g / L of disodium succinate was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 3.

[0160] Example 11 Cultivation was carried out in the same manner as in Example 9, except that 10 g / L of glycerol was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 3.

[0161] Example 12 Cultivation was carried out in the same manner as in Example 9, except that 10 g / L of ethanol was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 3.

[0162] <Comparative Example 7> Cultivation was carried out in the same manner as in Example 9, except that 10 g / L of glucose was used in place of sodium gluconate in the main culture medium. The results are also shown in Table 3.

[0163] <Comparative Example 8> Cultivation was carried out in the same manner as in Example 9, except that 10 g / L of xylose was used instead of sodium gluconate as the main culture medium. The results are also shown in Table 3.

[0164] <Comparative Example 9> Cultivation was carried out in the same manner as in Example 9, except that 10 g / L of sucrose was used in place of sodium gluconate as the main culture medium. The results are also shown in Table 3.

[0165] [Table 3]

[0166] 2. Optimization of the initial concentration of sodium gluconate in the main culture medium using 1L jar culture Example 13 (pre-culture) A colony of the Commamonas testosteroni NBRC 12047 strain was inoculated into 3 mL of pre-preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 15 mL test tubes, and the mixture was shaken at 30°C and 230 rpm for 8 hours.

[0167] The pre-preculture medium was prepared by dissolving 25 g of LB Broth Miller Novagen in water, measuring up to 1000 mL, and then heat sterilizing (121°C, 20 minutes).

[0168] (preculture) 3 mL of the obtained pre-preculture solution was inoculated into 100 mL of preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in a 500 mL flask, and cultured with shaking at 30°C and 230 rpm for 16 hours.

[0169] The pre-preculture medium was prepared by dissolving 2.5 g of LB Broth Miller Novagen in water, measuring up to 100 mL, and then heat sterilizing (121°C, 20 minutes).

[0170] (main culture) 200 mL of the resulting preculture solution was concentrated by centrifugation, and the concentrated bacterial cells were transferred to 500 mL of a main culture starter medium (sodium gluconate 10 g / L, dipotassium hydrogen phosphate 3.9 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate dihydrate 2.1 g / L, EDTA 10.0 mg / L, magnesium chloride hexahydrate 100 mg / L, zinc sulfate heptahydrate 2.0 mg / L, iron sulfate heptahydrate 5.0 mg / L, manganese chloride tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.2 mg / L, cobalt chloride hexahydrate 0.4 mg / L, ammonium molybdenum heptamolybdate tetrahydrate 0.2 mg / L, calcium chloride dihydrate 1.0 mg / L) prepared in a 1 L jar fermenter so that the initial OD660 was 1.0. The bacteria were inoculated into 1 mg / L LG-294 (ADEKA Corporation, 1 g / L) and cultured at 30°C, 500 rpm, aerated with a gas containing 20% ​​v / v oxygen, 1.0 vvm, with a lower pH limit of 6.95 and an upper pH limit of 7.05 (using 1 N potassium hydroxide solution).

[0171] The initial medium for this culture was prepared by dissolving the required amount of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate in water, measuring up to 2000 mL, and then heat-sterilizing (121°C, 20 minutes). This was mixed under sterile conditions with aqueous solutions of each component except for sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0172] The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration was measured in the culture medium obtained after 47 hours of culture. The bacterial concentration was measured by measuring the absorbance at 660 nm (OD660). The results are shown in Table 4.

[0173] <Comparative Example 10> Cultivation was carried out in the same manner as in Example 13, except that the amount of sodium gluconate in the main culture medium was 20 g / L. The results are also shown in Table 4.

[0174] <Comparative Example 11> Cultivation was carried out in the same manner as in Example 13, except that the cells were inoculated so that the initial OD660 was 0.5 and the main culture was recovered after 23 hours of cultivation. The results are also shown in Table 4.

[0175] <Comparative Example 12> Cultivation was carried out in the same manner as in Example 13, except that the amount of sodium gluconate in the main culture medium was 50 g / L. The results are also shown in Table 4. <Comparative Example 13> Cultivation was carried out in the same manner as in Example 13, except that the amount of sodium gluconate in the main culture medium was 100 g / L. The results are also shown in Table 3.

[0176] [Table 4]

[0177] Optimizing aeration volume using 3.5L jar culture Example 14 (pre-culture) A colony of the Commamonas testosteroni NBRC 12047 strain was inoculated into 3 mL of pre-preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 15 mL test tubes, and the mixture was shaken at 30°C and 230 rpm for 8 hours.

[0178] The pre-preculture medium was prepared by dissolving 25 g of LB Broth Miller Novagen in water, measuring up to 1000 mL, and then heat sterilizing (121°C, 20 minutes).

[0179] (preculture) 3 mL of the obtained pre-preculture solution was inoculated into 100 mL of preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 500 mL flasks, and cultured with shaking at 30°C and 230 rpm for 16 hours.

[0180] The pre-preculture medium was prepared by dissolving 2.5 g of LB Broth Miller Novagen in water, measuring up to 100 mL, and then heat sterilizing (121°C, 20 minutes).

[0181] (Main culture) 800 mL of the resulting preculture solution was concentrated using a centrifuge, and the concentrated bacterial cells were added to a 5-L jar fermenter to prepare 2 L of a main culture medium (sodium gluconate 10 g / L, dipotassium hydrogen phosphate 3.9 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate dihydrate 2.1 g / L, EDTA 10.0 mg / L, magnesium chloride hexahydrate 100 mg / L, zinc sulfate heptahydrate 2.0 mg / L, iron sulfate heptahydrate 5.0 mg / L, manganese chloride tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.2 mg / L, cobalt chloride hexahydrate 0.4 mg / L, ammonium molybdenum heptamolybdate tetrahydrate 0.2 mg / L, calcium chloride dihydrate 1.0 mg / L). The bacteria were inoculated into 1 mg / L LG-294 (ADEKA Corporation, 1 g / L) and cultured at a temperature of 30°C, a rotation speed of 200 rpm, aeration with a gas containing 90% v / v or more oxygen, 2.0 vvm, pressure of 0.05 MPa, a lower limit pH of 6.95, and an upper limit pH of 7.05 (using 1 N potassium sodium hydroxide solution).

[0182] The initial medium for this culture was prepared by dissolving the required amount of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate in water, measuring up to 2000 mL, and then heat-sterilizing (121°C, 20 minutes). This was mixed under sterile conditions with aqueous solutions of each component except for sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0183] (Addition of feed medium) After 90-99% of the dissolved oxygen in the initial medium was consumed, when the dissolved oxygen began to increase again, the main culture feed medium (sodium gluconate 259 g / L, dipotassium hydrogen phosphate 1.12 g / L, ammonium sulfate 1.12 g / L, potassium dihydrogen phosphate dihydrate 1.12 g / L, EDTA 0.02 mg / L, magnesium chloride hexahydrate 0.22 mg / L, zinc sulfate heptahydrate 0.22 mg / L, ferrous sulfate heptahydrate 0.22 mg / L) was added at a constant rate using the pulse-feed method.

[0184] The main culture feed medium was prepared by mixing under sterile conditions the required amounts of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate diluted to the above concentrations, dissolving them in water, diluting to 1000 mL, and heat sterilizing (121°C, 20 minutes) with aqueous solutions of the components other than sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0185] The culture was continued for a total of 48 hours, with sampling being performed at appropriate times. The fed-batch medium was added at a constant flow rate of 2.6 mL / min from the start of the culture until approximately 48 hours into the culture. The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration of the resulting culture medium was measured. The bacterial concentration was measured by measuring the absorbance at 660 nm (OD660). The results are shown in Table 5.

[0186] Example 15 Cultivation was carried out in the same manner as in Example 14, except that the aeration rate was limited to 0.5 vvm. The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration of the obtained culture solution was measured in the same manner as in Example 1. The results are also shown in Table 5.

[0187] Example 16 Cultivation was carried out in the same manner as in Example 14, except that the aeration rate was limited to 1.0 vvm. The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration of the obtained culture solution was measured in the same manner as in Example 1. The results are also shown in Table 5.

[0188] [Table 5]

[0189] Optimization of oxygen concentration in aerating gas using 4.5L jar culture Example 17 (pre-culture) A colony of the Commamonas testosteroni NBRC 12047 strain was inoculated into 3 mL of pre-preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 15 mL test tubes, and the mixture was shaken at 30°C and 230 rpm for 8 hours.

[0190] The pre-preculture medium was prepared by dissolving 25 g of LB Broth Miller Novagen in water, measuring up to 1000 mL, and then heat sterilizing (121°C, 20 minutes).

[0191] (preculture) 3 mL of the obtained pre-preculture solution was inoculated into 100 mL of preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 500 mL flasks, and cultured with shaking at 30°C and 230 rpm for 16 hours.

[0192] The pre-preculture medium was prepared by dissolving 2.5 g of LB Broth Miller Novagen in water, measuring up to 100 mL, and then heat sterilizing (121°C, 20 minutes).

[0193] (Main culture) 800 mL of the resulting preculture solution was concentrated by centrifugation, and the concentrated bacterial cells were transferred to a 5-L jar fermenter containing 2 L of a starting medium for the main culture (sodium gluconate 10 g / L, dipotassium hydrogen phosphate 3.9 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate dihydrate 2.1 g / L, EDTA 10.0 mg / L, magnesium chloride hexahydrate 100 mg / L, zinc sulfate heptahydrate 2.0 mg / L, iron sulfate heptahydrate 5.0 mg / L, manganese chloride tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.2 mg / L, cobalt chloride hexahydrate 0.4 mg / L, ammonium molybdenum heptamolybdate tetrahydrate 0.2 mg / L, calcium chloride dihydrate 1.0 mg / L). The bacteria were inoculated into 1 mg / L LG-294 (ADEKA Corporation, 1 g / L) and cultured at a temperature of 30°C, a rotation speed of 200 rpm, aeration with a gas containing 90% v / v or more oxygen, 1.0 vvm, a pressure of 0.05 MPa, a lower limit pH of 6.95, and an upper limit pH of 7.05 (using 1N potassium sodium hydroxide solution).

[0194] The initial medium for this culture was prepared by dissolving the required amount of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate in water, measuring up to 2000 mL, and then heat-sterilizing (121°C, 20 minutes). This was mixed under sterile conditions with aqueous solutions of each component except for sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0195] (Addition of feed medium) After 90-99% of the dissolved oxygen in the initial medium was consumed, when the dissolved oxygen began to increase again, the main culture feed medium (sodium gluconate 259 g / L, dipotassium hydrogen phosphate 1.12 g / L, ammonium sulfate 1.12 g / L, potassium dihydrogen phosphate dihydrate 1.12 g / L, EDTA 0.02 mg / L, magnesium chloride hexahydrate 0.22 mg / L, zinc sulfate heptahydrate 0.22 mg / L, ferrous sulfate heptahydrate 0.22 mg / L) was added at a constant rate using the pulse-feed method.

[0196] The main culture feed medium was prepared by mixing under sterile conditions the required amounts of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate diluted to the above concentrations, dissolving them in water, diluting to 1000 mL, and heat sterilizing (121°C, 20 minutes) with aqueous solutions of the components other than sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0197] The culture was continued for a total of 25 hours, with sampling being performed at appropriate times. The fed-batch medium was added at a constant flow rate of 2.6 mL / min from the start of the culture until approximately 48 hours into the culture. The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration of the resulting culture medium was measured. The bacterial concentration was measured by measuring the absorbance at 660 nm (OD660). The results are shown in Table 6.

[0198] Example 18 Cultivation was carried out in the same manner as in Example 17, except that a gas containing 20% ​​v / v oxygen was aerated at 1.0 vvm. The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration of the obtained culture solution was measured in the same manner as in Example 1. The results are also shown in Table 6.

[0199] [Table 6]

[0200] Optimizing the dosing method using a 5.5L jar Example 19 (pre-culture) A colony of the Commamonas testosteroni NBRC 12047 strain was inoculated into 3 mL of pre-preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 15 mL test tubes, and the mixture was shaken at 30°C and 230 rpm for 8 hours.

[0201] The pre-preculture medium was prepared by dissolving 25 g of LB Broth Miller Novagen in water, measuring up to 1000 mL, and then heat sterilizing (121°C, 20 minutes).

[0202] (preculture) 3 mL of the obtained pre-preculture solution was inoculated into 100 mL of preculture medium (LB Broth, Miller Novagen, catalog number: 71753-5CN) prepared in eight 500 mL flasks, and cultured with shaking at 30°C and 230 rpm for 16 hours.

[0203] The pre-preculture medium was prepared by dissolving 2.5 g of LB Broth Miller Novagen in water, measuring up to 100 mL, and then heat sterilizing (121°C, 20 minutes).

[0204] (main culture) 800 mL of the resulting preculture solution was concentrated using a centrifuge, and the concentrated bacterial cells were transferred to a 5-L jar fermenter containing 2 L of a starting medium for the main culture (sodium gluconate 10 g / L, dipotassium hydrogen phosphate 3.9 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate dihydrate 2.1 g / L, EDTA 10.0 mg / L, magnesium chloride hexahydrate 100 mg / L, zinc sulfate heptahydrate 2.0 mg / L, iron sulfate heptahydrate 5.0 mg / L, manganese chloride tetrahydrate 10 mg / L, copper sulfate pentahydrate 0.2 mg / L, cobalt chloride hexahydrate 0.4 mg / L, ammonium molybdenum heptamolybdate tetrahydrate 0.2 mg / L, calcium chloride dihydrate 1.0 mg / L). The bacteria were inoculated into 1 mg / L LG-294 (ADEKA Corporation, 1 g / L) and cultured at a temperature of 30°C, a rotation speed of 200 rpm, aeration with a gas containing 20% ​​v / v or more oxygen, 1.0 vvm, a pressure of 0.05 MPa, a lower limit pH of 6.95, and an upper limit pH of 7.05 (using 1N potassium sodium hydroxide solution).

[0205] The initial medium for this culture was prepared by dissolving the required amount of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate in water, measuring up to 2000 mL, and then heat-sterilizing (121°C, 20 minutes). This was mixed under sterile conditions with aqueous solutions of each component except for sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0206] (Addition of feed medium) After 90-99% of the dissolved oxygen in the initial medium was consumed, when the dissolved oxygen began to increase again, the main culture feed medium (sodium gluconate 259 g / L, dipotassium hydrogen phosphate 1.12 g / L, ammonium sulfate 1.12 g / L, potassium dihydrogen phosphate dihydrate 1.12 g / L, EDTA 0.02 mg / L, magnesium chloride hexahydrate 0.22 mg / L, zinc sulfate heptahydrate 0.22 mg / L, ferrous sulfate heptahydrate 0.22 mg / L) was added at a constant rate using the pulse-feed method.

[0207] The main culture feed medium was prepared by mixing under sterile conditions the required amounts of sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate diluted to the above concentrations, dissolving them in water, diluting to 1000 mL, and heat sterilizing (121°C, 20 minutes) with aqueous solutions of the components other than sodium gluconate, dipotassium hydrogen phosphate, ammonium sulfate, and potassium dihydrogen phosphate dihydrate, which had been previously filtered and sterilized using a 0.20 μm filter.

[0208] The pH of the culture was maintained at approximately 7.0 until the end of the culture. The bacterial concentration was measured for the culture medium obtained after 48 hours of culture. The bacterial concentration was measured by measuring the absorbance at 660 nm (OD660). The amount of gluconic acid added (the sum of the amount of gluconic acid in the initial medium and the amount of gluconic acid added) was 80 g.

[0209] The yield of bacterial cells relative to gluconic acid was calculated by dividing the bacterial cell concentration by the amount of gluconic acid charged. The results are shown in Table 7.

[0210] Example 20 Cultivation was carried out in the same manner as in Example 1, except that a gas containing 20% ​​v / v oxygen was aerated at 2 vvm, the fed-batch medium was added at a constant flow rate of 0.1 mL / min, and the cultivation was continued for 96 hours. The amount of gluconic acid charged was 136 g. The pH of the culture was maintained at approximately 7.0 until the end of the cultivation. The bacterial concentration of the obtained culture broth was measured in the same manner as in Example 1. The results are also shown in Table 7.

[0211] [Table 7]

[0212] 6. Production method of freeze-dried bacterial cells Example 21 Five mL of the culture medium was added to a 15 mL Falcon tube and centrifuged to concentrate the bacterial cells 10-fold (80 g DCW / L). Trehalose was added as a cryoprotectant in an amount equal to the bacterial weight, mixed, and frozen at -80°C for 17 hours in an ultra-low temperature freezer (CLN-50UW, Nippon Freezer Co., Ltd.). Freeze-drying was carried out in a freeze dryer (FDU-2110, Tokyo Rikakikai Co., Ltd.) at a vacuum of 3.7 Pa, a drying temperature of 30°C, a drying time of 24 hours, and a trap temperature of -45.5°C. The resulting freeze-dried bacterial cells were refrigerated for 40 days and then suspended in 20 mM phosphate buffer. Viable cell count evaluation and a furan compound wastewater component degradation test were performed as follows.

[0213] <Evaluation of viable bacteria count> After refrigeration for 40 days, the freeze-dried cells were suspended in 20 mM phosphate buffer, and then 10 4 Five microliters of the diluted solution was applied to an LB agar medium (LB Broth Miller Novagen, catalog number 71753-5CN, adjusted to 1.5% agar) and cultured at 30°C for 2 days. The number of colonies that appeared was counted visually and used as the viable bacterial count (CFU: Colony forming unit).

[0214] <Furan compound wastewater component decomposition test> Commamonas testosteroni NBRC12047 strain was added to 20 mL of MM liquid medium, adjusted to a furfural concentration of 100 ppm, in a 200 mL Erlenmeyer flask to a final OD660 of 0.1. The flask was plugged with cotton and shaken at 30°C and 230 rpm for 72 hours. The suspended solids in the sample were filtered through a 0.45 μm filter, and the furfural concentration in the sample was measured using HPLC.

[0215] The measurement conditions for furfural were as follows: a Nakarai 5C18-MS-II (4.6 ID x 250 mm) column was used. The ratio of 20 mM formic acid to methanol was 20:80, and the measurement was performed for 20 minutes. The results are shown in Table 8.

[0216] Example 22 The experiment was carried out in the same manner as in Example 21, except that trehalose was added in an amount five times the weight of the cells. The results are also shown in Table 8.

[0217] Example 23 The experiment was carried out in the same manner as in Example 21, except that skim milk was added in an amount equal to the weight of the bacteria. The results are also shown in Table 8.

[0218] Example 24 The experiment was carried out in the same manner as in Example 21, except that skim milk was added in an amount five times the weight of the bacteria. The results are also shown in Table 8.

[0219] Example 25 The experiment was carried out in the same manner as in Example 21, except that sodium glutamate was added in an amount equal to the bacterial weight. The results are also shown in Table 8.

[0220] Example 26 The experiment was carried out in the same manner as in Example 21, except that sodium glutamate was added in an amount five times the weight of the cells. The results are also shown in Table 8.

[0221] <Comparative Example 14> The cells concentrated 10 times by centrifugation were not freeze-dried, and the viable cell count and decomposition test for furan compound wastewater components were carried out in the same manner as in Example 21. The results are also shown in Table 8.

[0222] <Comparative Example 15> Immediately after freeze-drying, without adding any cryoprotectant, a viable cell count evaluation and a furan compound wastewater component decomposition test were carried out in the same manner as in Example 21. The results are also shown in Table 8.

[0223] [Table 8] [Industrial Applicability]

[0224] According to the present invention, in the biological treatment of wastewater containing furan compounds, a decrease in the treatment capacity can be prevented and furan compounds, acid components, and sugars in the wastewater can be efficiently removed. The treated water obtained by the present invention is of high quality, with a CODcr of 500 ppm or less, and can comply with strict wastewater regulations.

[0225] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A microbial preparation for treating wastewater containing furan aldehyde, comprising at least one selected from Commamonas thiooxydans, Burkholderia multivorans, Paraburkholderia xenovorans, Paraburkholderia caledonica, and Pseudomonas oryzihabitans.

2. A microbial preparation as described in claim 1, containing Commamonas thiooxydans.

3. A microbial preparation as described in claim 1, containing Burkholderia multivorans.

4. A microbial preparation as described in claim 1, containing Paraburkholderia xenovorans.

5. A microbial preparation as described in claim 1, containing Paraburkholderia caledonica.

6. A microbial preparation as described in claim 1, containing Pseudomonas oryzihabitans.

7. A method for treating wastewater, comprising a step of contacting wastewater containing furan aldehyde with at least one species selected from Commamonas thiooxydans, Burkholderia multivorans, Paraburkholderia xenovorans, Paraburkholderia caledonica, and Pseudomonas oryzihabitans.

8. A wastewater treatment method as described in claim 7, wherein the wastewater further contains formic acid and acetic acid in addition to furanaldehyde.

9. A wastewater treatment method comprising a step of contacting wastewater containing furan aldehyde with at least one species selected from Commamonas thiooxydans, Burkholderia multivorans, Paraburkholderia xenovorans, Paraburkholderia caledonica, and Pseudomonas oryzihabitans in the presence of a membrane separation device.

10. (1) contacting wastewater containing furan aldehyde with at least one selected from Commamonas thiooxydans, Burkholderia multivorans, Paraburkholderia xenovorans, Paraburkholderia caledonica, and Pseudomonas oryzihabitans; and (2) A wastewater treatment method comprising a step of contacting the wastewater obtained in the step (1) with at least one selected from activated carbon, a Fenton catalyst, and a polycyclic aromatic hydrocarbon-degrading enzyme.

11. The wastewater treatment method according to any one of claims 7 to 10, wherein the CODcr value of the resulting wastewater is 500 ppm or less.

12. A method for culturing microorganisms, comprising a step of culturing a microorganism belonging to the genus Burkholderia or a microorganism belonging to the genus Paraburkholderia in a medium containing gluconic acid.

13. A method for culturing a microorganism, comprising a step of culturing Commamonas testosteroni in a medium containing at least one selected from gluconic acid and succinic acid.

14. The method according to claim 12 or 13, wherein the optical density of the culture medium containing the microorganism at a wavelength of 660 nm is 15 or more 24 hours after the start of culture.

15. The method according to claim 12 or 13, wherein the optical density of the culture medium containing the microorganism at a wavelength of 660 nm is 20 or more 48 hours after the start of culture.

16. The method according to claim 13, and any one of claims 14 and 15 which cite claim 13, wherein the concentrations of gluconic acid and succinic acid present in the medium are both 10 g / L or less.

17. The method according to any one of claims 12 to 16, wherein the growth rate of the microorganism is 0.2 g / L / hr or more in terms of dry cell weight.

18. The method according to any one of claims 12 to 17, wherein the culture is carried out by fed-batch culture.

19. The method according to any one of claims 12 to 18, comprising a step of aerating the culture solution with a gas containing 90% v / v or more oxygen.

20. 20. The method of claim 19, wherein the aeration is carried out at an aeration rate of 6 to 5 vvm.

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